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Feature Papers in Molecular Biophysics

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A topical collection in International Journal of Molecular Sciences (ISSN 1422-0067). This collection belongs to the section "Molecular Biophysics".

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Prof. Dr. Ian A. Nicholls

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Collection Editor

Department of Chemistry and Biomedical Sciences, Linnaeus University, Kalmar, Sweden
Interests: biomimetic systems; complex mixture modelling and spectroscopic studies of biological; synthetic and hybrid systems; biosensors
Special Issues, Collections and Topics in MDPI journals

Prof. Dr. Vladimir N. Uversky

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Collection Editor

Department of Molecular Medicine, USF Health Byrd Alzheimer’s Research Institute, Morsani College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd, MDC07, Tampa, FL 33612, USA
Interests: intrinsically disordered proteins; protein folding; protein misfolding; partially folded proteins; protein aggregation; protein structure; protein function; protein stability; protein biophysics; protein bioinformatics; conformational diseases; protein–ligand interactions; protein–protein interactions; liquid-liquid phase transitions
Special Issues, Collections and Topics in MDPI journals

Topical Collection Information

Dear Colleagues,

As follows from the title, this Topical Collection “Feature Papers in Molecular Biophysics” aims to collect high quality research articles, short communications, and review articles in all the fields of molecular biophysics. Since the aim of this Topical Collection is to illustrate, through selected works, frontier research in molecular biophysics, we encourage Editorial Board Members of the Molecular Biophysics Section of the International Journal of Molecular Sciences to contribute papers reflecting the latest progress in their research field, or to invite relevant experts and colleagues to do so.

Topics include, but are not limited to:

molecular structure and dynamics
nucleic acid structure and dynamics
protein structure and dynamics
membrane structure and dynamics
biomimetic material structure and dynamics
molecular simulations
molecular modeling
single molecule biophysics
biophysical techniques in the study of biomacromolecular and biomimetic systems
biomolecular interactions
biomimetic material interactions
macromolecular structure determination or prediction
characterization of disordered proteins and their interactions
computational biophysics
bioinformatics
biophysical and computational approaches to drug design and development
advances in molecular biophysical methodologies as well as imaging techniques and data analysis
application of biophysical methods

Prof. Dr. Ian A. Nicholls
Prof. Dr. Vladimir N. Uversky
Guest Editors

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

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Published Papers (137 papers)

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2024

Jump to: 2023, 2022, 2021, 2020, 2019

24 pages, 703 KiB

Open AccessReview

Transmission-Blocking Vaccines against Schistosomiasis Japonica

by Chika P. Zumuk, Malcolm K. Jones, Severine Navarro, Darren J. Gray and Hong You

Int. J. Mol. Sci. 2024, 25(3), 1707; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms25031707 - 30 Jan 2024

Viewed by 1091

Control of schistosomiasis japonica, endemic in Asia, including the Philippines, China, and Indonesia, is extremely challenging. Schistosoma japonicum is a highly pathogenic helminth parasite, with disease arising predominantly from an immune reaction to entrapped parasite eggs in tissues. Females of this species can [...] Read more.

Control of schistosomiasis japonica, endemic in Asia, including the Philippines, China, and Indonesia, is extremely challenging. Schistosoma japonicum is a highly pathogenic helminth parasite, with disease arising predominantly from an immune reaction to entrapped parasite eggs in tissues. Females of this species can generate 1000–2200 eggs per day, which is about 3- to 15-fold greater than the egg output of other schistosome species. Bovines (water buffalo and cattle) are the predominant definitive hosts and are estimated to generate up to 90% of parasite eggs released into the environment in rural endemic areas where these hosts and humans are present. Here, we highlight the necessity of developing veterinary transmission-blocking vaccines for bovines to better control the disease and review potential vaccine candidates. We also point out that the approach to producing efficacious transmission-blocking animal-based vaccines before moving on to human vaccines is crucial. This will result in effective and feasible public health outcomes in agreement with the One Health concept to achieve optimum health for people, animals, and the environment. Indeed, incorporating a veterinary-based transmission vaccine, coupled with interventions such as human mass drug administration, improved sanitation and hygiene, health education, and snail control, would be invaluable to eliminating zoonotic schistosomiasis. Full article

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2023

Jump to: 2024, 2022, 2021, 2020, 2019

13 pages, 2946 KiB

Open AccessArticle

Simultaneous Improvement in the Thermostability and Catalytic Activity of Epoxidase Lsd18 for the Synthesis of Lasalocid A

by Ning Liu, Hongli Xiao, Yongjian Zang, Longji Zhou, Jun Mencius, Zhiwei Yang, Shu Quan and Xi Chen

Int. J. Mol. Sci. 2023, 24(23), 16795; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms242316795 - 27 Nov 2023

Viewed by 757

Enzymes used in the synthesis of natural products are potent catalysts, capable of efficient and stereoselective chemical transformations. Lsd18 catalyzes two sequential epoxidations during the biosynthesis of lasalocid A, a polyether polyketide natural product. We performed protein engineering on Lsd18 to improve its [...] Read more.

Enzymes used in the synthesis of natural products are potent catalysts, capable of efficient and stereoselective chemical transformations. Lsd18 catalyzes two sequential epoxidations during the biosynthesis of lasalocid A, a polyether polyketide natural product. We performed protein engineering on Lsd18 to improve its thermostability and catalytic activity. Utilizing structure-guided methods of FoldX and Rosetta-ddG, we designed 15 mutants of Lsd18. Screening of these mutants using thermal shift assay identified stabilized variants Lsd18-T189M, Lsd18-S195M, and the double mutant Lsd18-T189M-S195M. Trypsin digestion, molecular dynamic simulation, circular dichroism (CD) spectroscopy, and X-ray crystallography provided insights into the molecular basis for the improved enzyme properties. Notably, enhanced hydrophobic interaction within the enzyme core and interaction of the protein with the FAD cofactor appear to be responsible for its better thermostability. Full article

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17 pages, 1929 KiB

Open AccessArticle

Biophysical Insights into the Antitumoral Activity of Crotalicidin against Breast Cancer Model Membranes

by Maria C. Klaiss-Luna, Juan M. Giraldo-Lorza, Małgorzata Jemioła-Rzemińska, Kazimierz Strzałka and Marcela Manrique-Moreno

Int. J. Mol. Sci. 2023, 24(22), 16226; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms242216226 - 12 Nov 2023

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Bioactive peptides have emerged as promising therapeutic agents with antimicrobial, antifungal, antiparasitic, and, recently, antitumoral properties with a mechanism of action based on membrane destabilization and cell death, often involving a conformational change in the peptide. This biophysical study aims to provide preliminary [...] Read more.

Bioactive peptides have emerged as promising therapeutic agents with antimicrobial, antifungal, antiparasitic, and, recently, antitumoral properties with a mechanism of action based on membrane destabilization and cell death, often involving a conformational change in the peptide. This biophysical study aims to provide preliminary insights into the membrane-level antitumoral mode of action of crotalicidin, a cationic host defense peptide from rattlesnake venom, toward breast cancer cell lines. The lipid composition of breast cancer cell lines was obtained after lipid extraction and quantification to prepare representative cell membrane models. Membrane–peptide interaction studies were performed using differential scanning calorimetry and Fourier-transform infrared spectroscopy. The outcome evidences the potential antitumoral activity and selectivity of crotalicidin toward breast cancer cell lines and suggests a mechanism initiated by the electrostatic interaction of the peptide with the lipid bilayer surface and posterior conformation change with membrane intercalation between the acyl chains in negatively charged lipid systems. This research provides valuable information that clears up the antitumoral mode of action of crotalicidin. Full article

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17 pages, 2475 KiB

Open AccessArticle

K⁺-Dependent Photocycle and Photocurrent Reveal the Uptake of K⁺ in Light-Driven Sodium Pump

by Jikang Xu, Qifan Yang, Baofu Ma, Longjie Li, Fei Kong, Lan Xiao and Deliang Chen

Int. J. Mol. Sci. 2023, 24(19), 14414; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms241914414 - 22 Sep 2023

Cited by 2 | Viewed by 881

Engineering light-controlled K⁺ pumps from Na⁺-pumping rhodopsins (NaR) greatly expands the scope of optogenetic applications. However, the limited knowledge regarding the kinetic and selective mechanism of K⁺ uptake has significantly impeded the modification and design of light-controlled K⁺ [...] Read more.

Engineering light-controlled K⁺ pumps from Na⁺-pumping rhodopsins (NaR) greatly expands the scope of optogenetic applications. However, the limited knowledge regarding the kinetic and selective mechanism of K⁺ uptake has significantly impeded the modification and design of light-controlled K⁺ pumps, as well as their practical applications in various fields, including neuroscience. In this study, we presented K⁺-dependent photocycle kinetics and photocurrent of a light-driven Na⁺ pump called Nonlabens dokdonensis rhodopsin 2 (NdR2). As the concentration of K⁺ increased, we observed the accelerated decay of M intermediate in the wild type (WT) through flash photolysis. In 100 mM KCl, the lifetime of the M decay was approximately 1.0 s, which shortened to around 0.6 s in 1 M KCl. Additionally, the K⁺-dependent M decay kinetics were also observed in the G263W/N61P mutant, which transports K⁺. In 100 mM KCl, the lifetime of the M decay was approximately 2.5 s, which shortened to around 0.2 s in 1 M KCl. According to the competitive model, in high KCl, K⁺ may be taken up from the cytoplasmic surface, competing with Na⁺ or H⁺ during M decay. This was further confirmed by the K⁺-dependent photocurrent of WT liposome. As the concentration of K⁺ increased to 500 mM, the amplitude of peak current significantly dropped to approximately ~60%. Titration experiments revealed that the ratio of the rate constant of H⁺ uptake (k_H) to that of K⁺ uptake (k_K) is >10⁸. Compared to the WT, the G263W/N61P mutant exhibited a decrease of approximately 40-fold in k_H/k_K. Previous studies focused on transforming NaR into K⁺ pumps have primarily targeted the intracellular ion uptake region of Krokinobacter eikastus rhodopsin 2 (KR2) to enhance K⁺ uptake. However, our results demonstrate that the naturally occurring WT NdR2 is capable of intracellular K⁺ uptake without requiring structural modifications on the intracellular region. This discovery provides diverse options for future K⁺ pump designs. Furthermore, we propose a novel photocurrent-based approach to evaluate K⁺ uptake, which can serve as a reference for similar studies on other ion pumps. In conclusion, our research not only provides new insights into the mechanism of K⁺ uptake but also offers a valuable point of reference for the development of optogenetic tools and other applications in this field. Full article

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14 pages, 2778 KiB

Open AccessArticle

A High-Throughput Small-Angle X-ray Scattering Assay to Determine the Conformational Change of Plasminogen

by Adam J. Quek, Nathan P. Cowieson, Tom T. Caradoc-Davies, Paul J. Conroy, James C. Whisstock and Ruby H. P. Law

Int. J. Mol. Sci. 2023, 24(18), 14258; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms241814258 - 19 Sep 2023

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Plasminogen (Plg) is the inactive form of plasmin (Plm) that exists in two major glycoforms, referred to as glycoforms I and II (GI and GII). In the circulation, Plg assumes an activation-resistant “closed” conformation via interdomain interactions and is mediated by the lysine [...] Read more.

Plasminogen (Plg) is the inactive form of plasmin (Plm) that exists in two major glycoforms, referred to as glycoforms I and II (GI and GII). In the circulation, Plg assumes an activation-resistant “closed” conformation via interdomain interactions and is mediated by the lysine binding site (LBS) on the kringle (KR) domains. These inter-domain interactions can be readily disrupted when Plg binds to lysine/arginine residues on protein targets or free L-lysine and analogues. This causes Plg to convert into an “open” form, which is crucial for activation by host activators. In this study, we investigated how various ligands affect the kinetics of Plg conformational change using small-angle X-ray scattering (SAXS). We began by examining the open and closed conformations of Plg using size-exclusion chromatography (SEC) coupled with SAXS. Next, we developed a high-throughput (HTP) 96-well SAXS assay to study the conformational change of Plg. This method enables us to determine the K_open value, which is used to directly compare the effect of different ligands on Plg conformation. Based on our analysis using Plg GII, we have found that the K_open of ε-aminocaproic acid (EACA) is approximately three times greater than that of tranexamic acid (TXA), which is widely recognized as a highly effective ligand. We demonstrated further that Plg undergoes a conformational change when it binds to the C-terminal peptides of the inhibitor α2-antiplasmin (α2AP) and receptor Plg–R_KT. Our findings suggest that in addition to the C-terminal lysine, internal lysine(s) are also necessary for the formation of open Plg. Finally, we compared the conformational changes of Plg GI and GII directly and found that the closed form of GI, which has an N-linked glycosylation, is less stable. To summarize, we have successfully determined the response of Plg to various ligand/receptor peptides by directly measuring the kinetics of its conformational changes. Full article

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17 pages, 2938 KiB

Open AccessArticle

Evaluating the Cysteine-Rich and Catalytic Subdomains of Human Tyrosinase and OCA1-Related Mutants Using 1 μs Molecular Dynamics Simulation

by Taariq Woods and Yuri V. Sergeev

Int. J. Mol. Sci. 2023, 24(17), 13032; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms241713032 - 22 Aug 2023

Cited by 1 | Viewed by 849

The inherited disorder oculocutaneous albinism type 1 (OCA1) is caused by mutations in the TYR gene encoding tyrosinase (Tyr), an enzyme essential to producing pigments throughout the human body. The intramelanosomal domain of Tyr consists of the cysteine-rich and tyrosinase catalytic subdomains, which [...] Read more.

The inherited disorder oculocutaneous albinism type 1 (OCA1) is caused by mutations in the TYR gene encoding tyrosinase (Tyr), an enzyme essential to producing pigments throughout the human body. The intramelanosomal domain of Tyr consists of the cysteine-rich and tyrosinase catalytic subdomains, which are essential for enzymatic activity. In protein unfolding, the roles of these subdomains are not well established. Here, we performed six molecular dynamics simulations at room temperature for Tyr and OCA1-related mutant variants P406L and R402Q intramelanosomal domains. The proteins were simulated for 1 μs in water and urea to induce unfolding. In urea, we observed increases in surface area, decreases in intramolecular hydrogen bonding, and decreases in hydrophobic interactions, suggesting a ‘molten globule’ state for each protein. Between all conditions, the cysteine-rich subdomain remains stable, whereas the catalytic subdomain shows increased flexibility. This flexibility is intensified by the P406L mutation, while R402Q increases the catalytic domain’s rigidity. The cysteine-rich subdomain is rigid, preventing the protein from unfolding, whereas the flexibility of the catalytic subdomain accommodates mutational changes that could inhibit activity. These findings match the conclusions from our experimental work suggesting the function alteration by the P406L mutation, and the potential role of R402Q as a polymorphism. Full article

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19 pages, 11780 KiB

Open AccessArticle

Elucidating the Racemization Mechanism of Aliphatic and Aromatic Amino Acids by In Silico Tools

by Mateo S. Andino, José R. Mora, José L. Paz, Edgar A. Márquez, Yunierkis Perez-Castillo and Guillermin Agüero-Chapin

Int. J. Mol. Sci. 2023, 24(15), 11877; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms241511877 - 25 Jul 2023

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The racemization of biomolecules in the active site can reduce the biological activity of drugs, and the mechanism involved in this process is still not fully comprehended. The present study investigates the impact of aromaticity on racemization using advanced theoretical techniques based on [...] Read more.

The racemization of biomolecules in the active site can reduce the biological activity of drugs, and the mechanism involved in this process is still not fully comprehended. The present study investigates the impact of aromaticity on racemization using advanced theoretical techniques based on density functional theory. Calculations were performed at the ωb97xd/6-311++g(d,p) level of theory. A compelling explanation for the observed aromatic stabilization via resonance is put forward, involving a carbanion intermediate. The analysis, employing Hammett’s parameters, convincingly supports the presence of a negative charge within the transition state of aromatic compounds. Moreover, the combined utilization of natural bond orbital (NBO) analysis and intrinsic reaction coordinate (IRC) calculations confirms the pronounced stabilization of electron distribution within the carbanion intermediate. To enhance our understanding of the racemization process, a thorough examination of the evolution of NBO charges and Wiberg bond indices (WBIs) at all points along the IRC profile is performed. This approach offers valuable insights into the synchronicity parameters governing the racemization reactions. Full article

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16 pages, 2722 KiB

Open AccessArticle

I-Shaped Dimers of a Plant Chloroplast F_OF₁-ATP Synthase in Response to Changes in Ionic Strength

by Stepan D. Osipov, Yury L. Ryzhykau, Egor V. Zinovev, Andronika V. Minaeva, Sergey D. Ivashchenko, Dmitry P. Verteletskiy, Vsevolod V. Sudarev, Daria D. Kuklina, Mikhail Yu. Nikolaev, Yury S. Semenov, Yuliya A. Zagryadskaya, Ivan S. Okhrimenko, Margarita S. Gette, Elizaveta A. Dronova, Aleksei Yu. Shishkin, Norbert A. Dencher, Alexander I. Kuklin, Valentin Ivanovich, Vladimir N. Uversky and Alexey V. Vlasov

Int. J. Mol. Sci. 2023, 24(13), 10720; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms241310720 - 27 Jun 2023

Cited by 2 | Viewed by 1165

F-type ATP synthases play a key role in oxidative and photophosphorylation processes generating adenosine triphosphate (ATP) for most biochemical reactions in living organisms. In contrast to the mitochondrial F_OF₁-ATP synthases, those of chloroplasts are known to be mostly monomers [...] Read more.

F-type ATP synthases play a key role in oxidative and photophosphorylation processes generating adenosine triphosphate (ATP) for most biochemical reactions in living organisms. In contrast to the mitochondrial F_OF₁-ATP synthases, those of chloroplasts are known to be mostly monomers with approx. 15% fraction of oligomers interacting presumably non-specifically in a thylakoid membrane. To shed light on the nature of this difference we studied interactions of the chloroplast ATP synthases using small-angle X-ray scattering (SAXS) method. Here, we report evidence of I-shaped dimerization of solubilized F_OF₁-ATP synthases from spinach chloroplasts at different ionic strengths. The structural data were obtained by SAXS and demonstrated dimerization in response to ionic strength. The best model describing SAXS data was two ATP-synthases connected through F₁/F₁′ parts, presumably via their δ-subunits, forming “I” shape dimers. Such I-shaped dimers might possibly connect the neighboring lamellae in thylakoid stacks assuming that the F_OF₁ monomers comprising such dimers are embedded in parallel opposing stacked thylakoid membrane areas. If this type of dimerization exists in nature, it might be one of the pathways of inhibition of chloroplast F_OF₁-ATP synthase for preventing ATP hydrolysis in the dark, when ionic strength in plant chloroplasts is rising. Together with a redox switch inserted into a γ-subunit of chloroplast F_OF₁ and lateral oligomerization, an I-shaped dimerization might comprise a subtle regulatory process of ATP synthesis and stabilize the structure of thylakoid stacks in chloroplasts. Full article

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14 pages, 5035 KiB

Open AccessReview

On the Roles of the Nuclear Non-Coding RNA-Dependent Membrane-Less Organelles in the Cellular Stress Response

by Anastasia A. Gavrilova, Anna S. Fefilova, Innokentii E. Vishnyakov, Irina M. Kuznetsova, Konstantin K. Turoverov, Vladimir N. Uversky and Alexander V. Fonin

Int. J. Mol. Sci. 2023, 24(9), 8108; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24098108 - 30 Apr 2023

Viewed by 1479

At the beginning of the 21st century, it became obvious that radical changes had taken place in the concept of living matter and, in particular, in the concept of the organization of intracellular space. The accumulated data testify to the essential importance of [...] Read more.

At the beginning of the 21st century, it became obvious that radical changes had taken place in the concept of living matter and, in particular, in the concept of the organization of intracellular space. The accumulated data testify to the essential importance of phase transitions of biopolymers (first of all, intrinsically disordered proteins and RNA) in the spatiotemporal organization of the intracellular space. Of particular interest is the stress-induced reorganization of the intracellular space. Examples of organelles formed in response to stress are nuclear A-bodies and nuclear stress bodies. The formation of these organelles is based on liquid–liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) and non-coding RNA. Despite their overlapping composition and similar mechanism of formation, these organelles have different functional activities and physical properties. In this review, we will focus our attention on these membrane-less organelles (MLOs) and describe their functions, structure, and mechanism of formation. Full article

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15 pages, 2205 KiB

Open AccessArticle

Oligomeric State and Holding Activity of Hsp60

by Celeste Caruso Bavisotto, Alessia Provenzano, Rosa Passantino, Antonella Marino Gammazza, Francesco Cappello, Pier Luigi San Biagio and Donatella Bulone

Int. J. Mol. Sci. 2023, 24(9), 7847; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24097847 - 25 Apr 2023

Viewed by 1241

Similar to its bacterial homolog GroEL, Hsp60 in oligomeric conformation is known to work as a folding machine, with the assistance of co-chaperonin Hsp10 and ATP. However, recent results have evidenced that Hsp60 can stabilize aggregation-prone molecules in the absence of Hsp10 and [...] Read more.

Similar to its bacterial homolog GroEL, Hsp60 in oligomeric conformation is known to work as a folding machine, with the assistance of co-chaperonin Hsp10 and ATP. However, recent results have evidenced that Hsp60 can stabilize aggregation-prone molecules in the absence of Hsp10 and ATP by a different, “holding-like” mechanism. Here, we investigated the relationship between the oligomeric conformation of Hsp60 and its ability to inhibit fibrillization of the Ab40 peptide. The monomeric or tetradecameric form of the protein was isolated, and its effect on beta-amyloid aggregation was separately tested. The structural stability of the two forms of Hsp60 was also investigated using differential scanning calorimetry (DSC), light scattering, and circular dichroism. The results showed that the protein in monomeric form is less stable, but more effective against amyloid fibrillization. This greater functionality is attributed to the disordered nature of the domains involved in subunit contacts. Full article

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36 pages, 18751 KiB

Open AccessReview

FDA-Approved Fluorinated Heterocyclic Drugs from 2016 to 2022

by Carla Rizzo, Sara Amata, Ivana Pibiri, Andrea Pace, Silvestre Buscemi and Antonio Palumbo Piccionello

Int. J. Mol. Sci. 2023, 24(9), 7728; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24097728 - 23 Apr 2023

Cited by 19 | Viewed by 3543

The inclusion of fluorine atoms or heterocyclic moiety into drug structures represents a recurrent motif in medicinal chemistry. The combination of these two features is constantly appearing in new molecular entities with various biological activities. This is demonstrated by the increasing number of [...] Read more.

The inclusion of fluorine atoms or heterocyclic moiety into drug structures represents a recurrent motif in medicinal chemistry. The combination of these two features is constantly appearing in new molecular entities with various biological activities. This is demonstrated by the increasing number of newly synthesized fluorinated heterocyclic compounds among the Food and Drug Administration FDA-approved drugs. In this review, the biological activity, as well as the synthetic aspects, of 33 recently FDA-approved fluorinated heterocyclic drugs from 2016 to 2022 are highlighted. Full article

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23 pages, 6113 KiB

Open AccessArticle

A Novel Ambroxol-Derived Tetrahydroquinazoline with a Potency against SARS-CoV-2 Proteins

by Alena I. Krysantieva, Julia K. Voronina and Damir A. Safin

Int. J. Mol. Sci. 2023, 24(5), 4660; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24054660 - 28 Feb 2023

Cited by 1 | Viewed by 1853

We report synthesis of a novel 1,2,3,4-tetrahydroquinazoline derivative, named 2-(6,8-dibromo-3-(4-hydroxycyclohexyl)-1,2,3,4-tetrahydroquinazolin-2-yl)phenol (1), which was obtained from the hydrochloride of 4-((2-amino-3,5-dibromobenzyl)amino)cyclohexan-1-ol (ambroxol hydrochloride) and salicylaldehyde in EtOH. The resulting compound was produced in the form of colorless crystals of the composition 1∙0.5EtOH. [...] Read more.

We report synthesis of a novel 1,2,3,4-tetrahydroquinazoline derivative, named 2-(6,8-dibromo-3-(4-hydroxycyclohexyl)-1,2,3,4-tetrahydroquinazolin-2-yl)phenol (1), which was obtained from the hydrochloride of 4-((2-amino-3,5-dibromobenzyl)amino)cyclohexan-1-ol (ambroxol hydrochloride) and salicylaldehyde in EtOH. The resulting compound was produced in the form of colorless crystals of the composition 1∙0.5EtOH. The formation of the single product was confirmed by the IR and ¹H spectroscopy, single-crystal and powder X-ray diffraction, and elemental analysis. The molecule of 1 contains a chiral tertiary carbon of the 1,2,3,4-tetrahydropyrimidine fragment and the crystal structure of 1∙0.5EtOH is a racemate. Optical properties of 1∙0.5EtOH were revealed by UV-vis spectroscopy in MeOH and it was established that the compound absorbs exclusively in the UV region up to about 350 nm. 1∙0.5EtOH in MeOH exhibits dual emission and the emission spectra contains bands at about 340 and 446 nm upon excitation at 300 and 360 nm, respectively. The DFT calculations were performed to verify the structure as well as electronic and optical properties of 1. ADMET properties of the R-isomer of 1 were evaluated using the SwissADME, BOILED-Egg, and ProTox-II tools. As evidenced from the blue dot position in the BOILED-Egg plot, both human blood–brain barrier penetration and gastrointestinal absorption properties are positive with the positive PGP effect on the molecule. Molecular docking was applied to examine the influence of the structures of both R-isomer and S-isomer of 1 on a series of the SARS-CoV-2 proteins. According to the docking analysis results, both isomers of 1 were found to be active against all the applied SARS-CoV-2 proteins with the best binding affinities with Papain-like protease (PLpro) and nonstructural protein 3 (Nsp3_range 207–379-AMP). Ligand efficiency scores for both isomers of 1 inside the binding sites of the applied proteins were also revealed and compared with the initial ligands. Molecular dynamics simulations were also applied to evaluate the stability of complexes of both isomers with Papain-like protease (PLpro) and nonstructural protein 3 (Nsp3_range 207–379-AMP). The complex of the S-isomer with Papain-like protease (PLpro) was found to be highly unstable, while the other complexes are stable. Full article

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19 pages, 4023 KiB

Open AccessArticle

Structural Characteristics of High-Mobility Group Proteins HMGB1 and HMGB2 and Their Interaction with DNA

by Tatiana Y. Starkova, Alexander M. Polyanichko, Tatiana O. Artamonova, Anna S. Tsimokha, Alexey N. Tomilin and Elena V. Chikhirzhina

Int. J. Mol. Sci. 2023, 24(4), 3577; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24043577 - 10 Feb 2023

Cited by 6 | Viewed by 1831

Non-histone nuclear proteins HMGB1 and HMGB2 (High Mobility Group) are involved in many biological processes, such as replication, transcription, and repair. The HMGB1 and HMGB2 proteins consist of a short N-terminal region, two DNA-binding domains, A and B, and a C-terminal sequence of [...] Read more.

Non-histone nuclear proteins HMGB1 and HMGB2 (High Mobility Group) are involved in many biological processes, such as replication, transcription, and repair. The HMGB1 and HMGB2 proteins consist of a short N-terminal region, two DNA-binding domains, A and B, and a C-terminal sequence of glutamic and aspartic acids. In this work, the structural organization of calf thymus HMGB1 and HMGB2 proteins and their complexes with DNA were studied using UV circular dichroism (CD) spectroscopy. Post-translational modifications (PTM) of HMGB1 and HMGB2 proteins were determined with MALDI mass spectrometry. We have shown that despite the similar primary structures of the HMGB1 and HMGB2 proteins, their post-translational modifications (PTMs) demonstrate quite different patterns. The HMGB1 PTMs are located predominantly in the DNA-binding A-domain and linker region connecting the A and B domains. On the contrary, HMGB2 PTMs are found mostly in the B-domain and within the linker region. It was also shown that, despite the high degree of homology between HMGB1 and HMGB2, the secondary structure of these proteins is also slightly different. We believe that the revealed structural properties might determine the difference in the functioning of the HMGB1 and HMGB2 as well as their protein partners. Full article

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19 pages, 5843 KiB

Open AccessArticle

Investigation of Multi-Subunit Mycobacterium tuberculosis DNA-Directed RNA Polymerase and Its Rifampicin Resistant Mutants

by Mokgerwa Zacharia Monama, Fisayo Olotu and Özlem Tastan Bishop

Int. J. Mol. Sci. 2023, 24(4), 3313; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24043313 - 07 Feb 2023

Cited by 4 | Viewed by 1830

Emerging Mycobacterium tuberculosis (Mtb) resistant strains have continued to limit the efficacies of existing antitubercular therapies. More specifically, mutations in the RNA replicative machinery of Mtb, RNA polymerase (RNAP), have been widely linked to rifampicin (RIF) resistance, which has led [...] Read more.

Emerging Mycobacterium tuberculosis (Mtb) resistant strains have continued to limit the efficacies of existing antitubercular therapies. More specifically, mutations in the RNA replicative machinery of Mtb, RNA polymerase (RNAP), have been widely linked to rifampicin (RIF) resistance, which has led to therapeutic failures in many clinical cases. Moreover, elusive details on the underlying mechanisms of RIF-resistance caused by Mtb-RNAP mutations have hampered the development of new and efficient drugs that are able to overcome this challenge. Therefore, in this study we attempt to resolve the molecular and structural events associated with RIF-resistance in nine clinically reported missense Mtb RNAP mutations. Our study, for the first time, investigated the multi-subunit Mtb RNAP complex and findings revealed that the mutations commonly disrupted structural–dynamical attributes that may be essential for the protein’s catalytic functions, particularly at the βfork loop 2, β’zinc-binding domain, the β’ trigger loop and β’jaw, which in line with previous experimental reports, are essential for RNAP processivity. Complementarily, the mutations considerably perturbed the RIF-BP, which led to alterations in the active orientation of RIF needed to obstruct RNA extension. Consequentially, essential interactions with RIF were lost due to the mutation-induced repositioning with corresponding reductions in the binding affinity of the drug observed in majority of the mutants. We believe these findings will significantly aid future efforts in the discovery of new treatment options with the potential to overcome antitubercular resistance. Full article

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21 pages, 5896 KiB

Open AccessArticle

Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme

by Alicja Dyzma, Beata Wielgus-Kutrowska, Agnieszka Girstun, Zoe Jelić Matošević, Krzysztof Staroń, Branimir Bertoša, Joanna Trylska and Agnieszka Bzowska

Int. J. Mol. Sci. 2023, 24(3), 2157; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24032157 - 21 Jan 2023

Cited by 1 | Viewed by 1322

Mammalian purine nucleoside phosphorylase (PNP) is biologically active as a homotrimer, in which each monomer catalyzes a reaction independently of the others. To answer the question of why the native PNP forms a trimeric structure, we constructed, in silico and in vitro, the [...] Read more.

Mammalian purine nucleoside phosphorylase (PNP) is biologically active as a homotrimer, in which each monomer catalyzes a reaction independently of the others. To answer the question of why the native PNP forms a trimeric structure, we constructed, in silico and in vitro, the monomeric form of the enzyme. Molecular dynamics simulations showed different geometries of the active site in the non-mutated trimeric and monomeric PNP forms, which suggested that the active site in the isolated monomer could be non-functional. To confirm this hypothesis, six amino acids located at the interface of the subunits were selected and mutated to alanines to disrupt the trimer and obtain a monomer (6Ala PNP). The effects of these mutations on the enzyme structure, stability, conformational dynamics, and activity were examined. The solution experiments confirmed that the 6Ala PNP mutant occurs mainly as a monomer, with a secondary structure almost identical to the wild type, WT PNP, and importantly, it shows no enzymatic activity. Simulations confirmed that, although the secondary structure of the 6Ala monomer is similar to the WT PNP, the positions of the amino acids building the 6Ala PNP active site significantly differ. These data suggest that a trimeric structure is necessary to stabilize the geometry of the active site of this enzyme. Full article

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18 pages, 10215 KiB

Open AccessArticle

Triacylglycerol Composition and Chemical-Physical Properties of Cocoa Butter and Its Derivatives: NMR, DSC, X-ray, Rheological Investigation

by Maria Francesca Colella, Nadia Marino, Cesare Oliviero Rossi, Lucia Seta, Paolino Caputo and Giuseppina De Luca

Int. J. Mol. Sci. 2023, 24(3), 2090; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24032090 - 20 Jan 2023

Cited by 3 | Viewed by 2346

In recent years, the food industry has become increasingly involved in researching vegetable fats and oils with appropriate mechanical properties (ease of transport, processing, and storage) and a specific lipidic composition to ensure healthy products for consumers. The chemical–physical behavior of these matrices [...] Read more.

In recent years, the food industry has become increasingly involved in researching vegetable fats and oils with appropriate mechanical properties (ease of transport, processing, and storage) and a specific lipidic composition to ensure healthy products for consumers. The chemical–physical behavior of these matrices depends on their composition in terms of single fatty acids (FA). However, as we demonstrate in this work, these properties, as well as the absorption, digestion and uptake in humans of specific FAs, are also largely determined by their regiosomerism within the TriAcylGlycerols (TAG) moieties (sn-1,2,3 positions). The goal of this work is to study for the first time vegetable fats obtained directly from a sample of natural cocoa butter (CB) through a process that manipulates the distribution of FAs but not their nature. Even if the initial percentage of each FA in the mixture remains the same, CB derivatives seem to show improved chemical–physical features. In order to understand which factors account for their physical and chemical characteristics, and to check whether or not the obtained new matrices could be considered as valid alternatives to other vegetable fats (e.g., palm oil (PO)), we carried out an experimental investigation at both the macroscopic and molecular level including: (i) Differential Scanning Calorimetry (DSC) analyses to examine thermal features; (ii) rheological testing to explore mechanical properties; (iii) powder X-ray diffraction (PXRD) to evaluate the solid-state phases of the obtained fats; and (iv) ¹H and ¹³C Nuclear Magnetic Resonance (NMR, 1D and 2D) spectroscopy to rapidly analyze fatty acid composition including regioisomeric distribution on the glycerol backbone. These last results open up the possibility of using NMR spectroscopy as an alternative to the chromatographic techniques routinely employed for the investigation of similar matrices. Full article

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12 pages, 2851 KiB

Open AccessArticle

Proof-of-Concept Method to Study Uncharacterized Methyltransferases Using PRDM15

by Li-Na Zhao, Ernesto Guccione and Philipp Kaldis

Int. J. Mol. Sci. 2023, 24(2), 1327; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24021327 - 10 Jan 2023

Viewed by 1397

The PRDM family of methyltransferases has been implicated in cellular proliferation and differentiation and is deregulated in human diseases, most notably in cancer. PRDMs are related to the SET domain family of methyltransferases; however, from the 19 PRDMs only a few PRDMs with [...] Read more.

The PRDM family of methyltransferases has been implicated in cellular proliferation and differentiation and is deregulated in human diseases, most notably in cancer. PRDMs are related to the SET domain family of methyltransferases; however, from the 19 PRDMs only a few PRDMs with defined enzymatic activities are known. PRDM15 is an uncharacterized transcriptional regulator, with significant structural disorder and lack of defined small-molecule binding pockets. Many aspects of PRDM15 are yet unknown, including its structure, substrates, reaction mechanism, and its methylation profile. Here, we employ a series of computational approaches for an exploratory investigation of its potential substrates and reaction mechanism. Using the knowledge of PRDM9 and current knowledge of PRDM15 as basis, we tried to identify genuine substrates of PRDM15. We start from histone-based peptides and learn that the native substrates of PRDM15 may be non-histone proteins. In the future, a combination of sequence-based approaches and signature motif analysis may provide new leads. In summary, our results provide new information about the uncharacterized methyltransferase, PRDM15. Full article

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10 pages, 2618 KiB

Open AccessArticle

Single–Molecule Study of DNAzyme Reveals Its Intrinsic Conformational Dynamics

by Yiming Zhang, Zongzhou Ji, Xin Wang, Yi Cao and Hai Pan

Int. J. Mol. Sci. 2023, 24(2), 1212; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24021212 - 07 Jan 2023

Cited by 1 | Viewed by 1531

DNAzyme is a class of DNA molecules that can perform catalytic functions with high selectivity towards specific metal ions. Due to its potential applications for biosensors and medical therapeutics, DNAzyme has been extensively studied to characterize the relationships between its biochemical properties and [...] Read more.

DNAzyme is a class of DNA molecules that can perform catalytic functions with high selectivity towards specific metal ions. Due to its potential applications for biosensors and medical therapeutics, DNAzyme has been extensively studied to characterize the relationships between its biochemical properties and functions. Similar to protein enzymes and ribozymes, DNAzymes have been found to undergo conformational changes in a metal–ion–dependent manner for catalysis. Despite the important role the conformation plays in the catalysis process, such structural and dynamic information might not be revealed by conventional approaches. Here, by using the single–molecule fluorescence resonance energy transfer (smFRET) technique, we were able to investigate the detailed conformational dynamics of a uranyl–specific DNAzyme 39E. We observed conformation switches of 39E to a folded state with the addition of Mg²⁺ and to an extended state with the addition of UO₂²⁺. Furthermore, 39E can switch to a more compact configuration with or without divalent metal ions. Our findings reveal that 39E can undergo conformational changes spontaneously between different configurations. Full article

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11 pages, 21247 KiB

Open AccessArticle

The Effect of Arginine on the Phase Stability of Aqueous Hen Egg-White Lysozyme Solutions

by Sandi Brudar and Barbara Hribar-Lee

Int. J. Mol. Sci. 2023, 24(2), 1197; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24021197 - 07 Jan 2023

Cited by 3 | Viewed by 1305

The effect of arginine on the phase stability of the hen egg-white lysozyme (HEWL) has been studied via molecular dynamics computer simulations, as well as experimentally via cloud-point temperature determination. The experiments show that the addition of arginine increases the stability of the [...] Read more.

The effect of arginine on the phase stability of the hen egg-white lysozyme (HEWL) has been studied via molecular dynamics computer simulations, as well as experimentally via cloud-point temperature determination. The experiments show that the addition of arginine increases the stability of the HEWL solutions. The computer simulation results indicate that arginine molecules tend to self-associate. If arginine residues are located on the protein surface, the free arginine molecules stay in their vicinity and prevent the way protein molecules “connect” through them to form clusters. The results are not sensitive to a particular force field and suggest a possible microscopic mechanism of the stabilizing role of arginine as an excipient. Full article

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14 pages, 2671 KiB

Open AccessArticle

An Overlooked Hepcidin–Cadmium Connection

by Dawid Płonka, Marta D. Wiśniewska, Manuel D. Peris-Díaz, Artur Krężel, Arkadiusz M. Bonna and Wojciech Bal

Int. J. Mol. Sci. 2022, 23(24), 15483; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232415483 - 07 Dec 2022

Cited by 1 | Viewed by 1211

Hepcidin (DTHFPICIFCCGCCHRSKCGMCCKT), an iron-regulatory hormone, is a 25-amino-acid peptide with four intramolecular disulfide bonds circulating in blood. Its hormonal activity is indirect and consists of marking ferroportin-1 (an iron exporter) for degradation. Hepcidin biosynthesis involves the N-terminally extended precursors prepro-hepcidin and pro-hepcidin, processed [...] Read more.

Hepcidin (DTHFPICIFCCGCCHRSKCGMCCKT), an iron-regulatory hormone, is a 25-amino-acid peptide with four intramolecular disulfide bonds circulating in blood. Its hormonal activity is indirect and consists of marking ferroportin-1 (an iron exporter) for degradation. Hepcidin biosynthesis involves the N-terminally extended precursors prepro-hepcidin and pro-hepcidin, processed by peptidases to the final 25-peptide form. A sequence-specific formation of disulfide bonds and export of the oxidized peptide to the bloodstream follows. In this study we considered the fact that prior to export, reduced hepcidin may function as an octathiol ligand bearing some resemblance to the N-terminal part of the α-domain of metallothioneins. Consequently, we studied its ability to bind Zn(II) and Cd(II) ions using the original peptide and a model for prohepcidin extended N-terminally with a stretch of five arginine residues (5R-hepcidin). We found that both form equivalent mononuclear complexes with two Zn(II) or Cd(II) ions saturating all eight Cys residues. The average affinity at pH 7.4, determined from pH-metric spectroscopic titrations, is 10^10.1 M⁻¹ for Zn(II) ions; Cd(II) ions bind with affinities of 10^15.2 M⁻¹ and 10^14.1 M⁻¹. Using mass spectrometry and 5R-hepcidin we demonstrated that hepcidin can compete for Cd(II) ions with metallothionein-2, a cellular cadmium target. This study enabled us to conclude that hepcidin binds Zn(II) and Cd(II) sufficiently strongly to participate in zinc physiology and cadmium toxicity under intracellular conditions. Full article

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27 pages, 6065 KiB

Open AccessArticle

Unveiling the Metal-Dependent Aggregation Properties of the C-terminal Region of Amyloidogenic Intrinsically Disordered Protein Isoforms DPF3b and DPF3a

by Tanguy Leyder, Julien Mignon, Denis Mottet and Catherine Michaux

Int. J. Mol. Sci. 2022, 23(23), 15291; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232315291 - 04 Dec 2022

Cited by 3 | Viewed by 1445

Double-PHD fingers 3 (DPF3) is a BAF-associated human epigenetic regulator, which is increasingly recognised as a major contributor to various pathological contexts, such as cardiac defects, cancer, and neurodegenerative diseases. Recently, we unveiled that its two isoforms (DPF3b and DPF3a) are amyloidogenic intrinsically [...] Read more.

Double-PHD fingers 3 (DPF3) is a BAF-associated human epigenetic regulator, which is increasingly recognised as a major contributor to various pathological contexts, such as cardiac defects, cancer, and neurodegenerative diseases. Recently, we unveiled that its two isoforms (DPF3b and DPF3a) are amyloidogenic intrinsically disordered proteins. DPF3 isoforms differ from their C-terminal region (C-TERb and C-TERa), containing zinc fingers and disordered domains. Herein, we investigated the disorder aggregation properties of C-TER isoforms. In agreement with the predictions, spectroscopy highlighted a lack of a highly ordered structure, especially for C-TERa. Over a few days, both C-TERs were shown to spontaneously assemble into similar antiparallel and parallel β-sheet-rich fibrils. Altered metal homeostasis being a neurodegeneration hallmark, we also assessed the influence of divalent metal cations, namely Cu²⁺, Mg²⁺, Ni²⁺, and Zn²⁺, on the C-TER aggregation pathway. Circular dichroism revealed that metal binding does not impair the formation of β-sheets, though metal-specific tertiary structure modifications were observed. Through intrinsic and extrinsic fluorescence, we found that metal cations differently affect C-TERb and C-TERa. Cu²⁺ and Ni²⁺ have a strong inhibitory effect on the aggregation of both isoforms, whereas Mg²⁺ impedes C-TERb fibrillation and, on the contrary, enhances that of C-TERa. Upon Zn²⁺ binding, C-TERb aggregation is also hindered, and the amyloid autofluorescence of C-TERa is remarkably red-shifted. Using electron microscopy, we confirmed that the metal-induced spectral changes are related to the morphological diversity of the aggregates. While metal-treated C-TERb formed breakable and fragmented filaments, C-TERa fibrils retained their flexibility and packing properties in the presence of Mg²⁺ and Zn²⁺ cations. Full article

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22 pages, 2506 KiB

Open AccessReview

How Functional Lipids Affect the Structure and Gating of Mechanosensitive MscS-like Channels

by Vanessa Judith Flegler, Tim Rasmussen and Bettina Böttcher

Int. J. Mol. Sci. 2022, 23(23), 15071; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232315071 - 01 Dec 2022

Cited by 4 | Viewed by 1949

The ability to cope with and adapt to changes in the environment is essential for all organisms. Osmotic pressure is a universal threat when environmental changes result in an imbalance of osmolytes inside and outside the cell which causes a deviation from the [...] Read more.

The ability to cope with and adapt to changes in the environment is essential for all organisms. Osmotic pressure is a universal threat when environmental changes result in an imbalance of osmolytes inside and outside the cell which causes a deviation from the normal turgor. Cells have developed a potent system to deal with this stress in the form of mechanosensitive ion channels. Channel opening releases solutes from the cell and relieves the stress immediately. In bacteria, these channels directly sense the increased membrane tension caused by the enhanced turgor levels upon hypoosmotic shock. The mechanosensitive channel of small conductance, MscS, from Escherichia coli is one of the most extensively studied examples of mechanically stimulated channels. Different conformational states of this channel were obtained in various detergents and membrane mimetics, highlighting an intimate connection between the channel and its lipidic environment. Associated lipids occupy distinct locations and determine the conformational states of MscS. Not all these features are preserved in the larger MscS-like homologues. Recent structures of homologues from bacteria and plants identify common features and differences. This review discusses the current structural and functional models for MscS opening, as well as the influence of certain membrane characteristics on gating. Full article

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15 pages, 3239 KiB

Open AccessArticle

Pharmaceutical Functionalization of Monomeric Ionic Liquid for the Preparation of Ionic Graft Polymer Conjugates

by Aleksy Mazur, Katarzyna Niesyto and Dorota Neugebauer

Int. J. Mol. Sci. 2022, 23(23), 14731; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232314731 - 25 Nov 2022

Cited by 5 | Viewed by 1275

Polymerizable choline-based ionic liquid (IL), i.e., [2-(methacryloyloxy)ethyl]-trimethylammonium (TMAMA/Cl¯), was functionalized by an ion exchange reaction with pharmaceutical anions, i.e., cloxacillin (CLX¯) and fusidate (FUS¯), as the antibacterial agents. The modified biocompatible IL monomers (TMAMA/CLX¯, TMAMA/FUS¯) were copolymerized with methyl methacrylate (MMA) to prepare [...] Read more.

Polymerizable choline-based ionic liquid (IL), i.e., [2-(methacryloyloxy)ethyl]-trimethylammonium (TMAMA/Cl¯), was functionalized by an ion exchange reaction with pharmaceutical anions, i.e., cloxacillin (CLX¯) and fusidate (FUS¯), as the antibacterial agents. The modified biocompatible IL monomers (TMAMA/CLX¯, TMAMA/FUS¯) were copolymerized with methyl methacrylate (MMA) to prepare the graft copolymers (19–50 mol% of TMAMA units) serving as the drug (co)delivery systems. The in vitro drug release, which was driven by the exchange reaction of the pharmaceutical anions to phosphate ones in PBS medium, was observed for 44% of CLX¯ (2.7 μg/mL) and 53% of FUS¯ (3.6 μg/mL) in the single systems. Similar amounts of released drugs were detected for the dual system, i.e., 41% of CLX¯ (2.2 μg/mL) and 33% of FUS¯ (2.0 μg/mL). The investigated drug ionic polymer conjugates were examined for their cytotoxicity by MTT test, showing a low toxic effect against human bronchial epithelial cells (BEAS-2B) and normal human dermal fibroblasts (NHDF) as the normal cell lines. The satisfactory drug contents and the release profiles attained for the well-defined graft polymers with ionically bonded pharmaceuticals in the side chains make them promising drug carriers in both separate and combined drug delivery systems. Full article

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19 pages, 1452 KiB

Open AccessArticle

Study of Membrane-Immobilized Oxidoreductases in Wastewater Treatment for Micropollutants Removal

by Agata Zdarta and Jakub Zdarta

Int. J. Mol. Sci. 2022, 23(22), 14086; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232214086 - 15 Nov 2022

Cited by 2 | Viewed by 1433

The development of efficient strategies for wastewater treatment to remove micropollutants is of the highest importance. Hence, in this study, we presented a rapid approach to the production of biocatalytic membranes based on commercially available cellulose membrane and oxidoreductase enzymes including laccase, tyrosinase, [...] Read more.

The development of efficient strategies for wastewater treatment to remove micropollutants is of the highest importance. Hence, in this study, we presented a rapid approach to the production of biocatalytic membranes based on commercially available cellulose membrane and oxidoreductase enzymes including laccase, tyrosinase, and horseradish peroxidase. Effective enzyme deposition was confirmed based on Fourier transform infrared spectra, whereas results of spectrophotometric measurements showed that immobilization yield for all proposed systems exceeded 80% followed by over 80% activity recovery, with the highest values (over 90%) noticed for the membrane-laccase system. Further, storage stability and reusability of the immobilized enzyme were improved, reaching over 75% after, respectively, 20 days of storage, and 10 repeated biocatalytic cycles. The key stage of the study concerned the use of produced membranes for the removal of hematoporphyrin, (2,4-dichlorophenoxy)acetic acid (2,4-D), 17α-ethynylestradiol, tetracycline, tert-amyl alcohol (anesthetic drug), and ketoprofen methyl ester from real wastewater sampling at various places in the wastewater treatment plant. Although produced membranes showed mixed removal rates, all of the analyzed compounds were at least partially removed from the wastewater. Obtained data clearly showed, however, that composition of the wastewater matrix, type of pollutants as well as type of enzyme strongly affect the efficiency of enzymatic treatment of wastewater. Full article

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15 pages, 2742 KiB

Open AccessArticle

Metal Binding to Sodium Heparin Monitored by Quadrupolar NMR

by Daniel Sieme, Christian Griesinger and Nasrollah Rezaei-Ghaleh

Int. J. Mol. Sci. 2022, 23(21), 13185; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232113185 - 29 Oct 2022

Cited by 1 | Viewed by 1497

Heparins and heparan sulfate polysaccharides are negatively charged glycosaminoglycans and play important roles in cell-to-matrix and cell-to-cell signaling processes. Metal ion binding to heparins alters the conformation of heparins and influences their function. Various experimental techniques have been used to investigate metal ion-heparin [...] Read more.

Heparins and heparan sulfate polysaccharides are negatively charged glycosaminoglycans and play important roles in cell-to-matrix and cell-to-cell signaling processes. Metal ion binding to heparins alters the conformation of heparins and influences their function. Various experimental techniques have been used to investigate metal ion-heparin interactions, frequently with inconsistent results. Exploiting the quadrupolar ²³Na nucleus, we herein develop a ²³Na NMR-based competition assay and monitor the binding of divalent Ca²⁺ and Mg²⁺ and trivalent Al³⁺ metal ions to sodium heparin and the consequent release of sodium ions from heparin. The ²³Na spin relaxation rates and translational diffusion coefficients are utilized to quantify the metal ion-induced release of sodium ions from heparin. In the case of the Al³⁺ ion, the complementary approach of ²⁷Al quadrupolar NMR is employed as a direct probe of ion binding to heparin. Our NMR results demonstrate at least two metal ion-binding sites with different affinities on heparin, potentially undergoing dynamic exchange. For the site with lower metal ion binding affinity, the order of Ca²⁺ > Mg²⁺ > Al³⁺ is obtained, in which even the weakly binding Al³⁺ ion is capable of displacing sodium ions from heparin. Overall, the multinuclear quadrupolar NMR approach employed here can monitor and quantify metal ion binding to heparin and capture different modes of metal ion-heparin binding. Full article

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17 pages, 965 KiB

Open AccessReview

Addressing Noise and Estimating Uncertainty in Biomedical Data through the Exploration of Chemical Space

by Enrique J. deAndrés-Galiana, Juan Luis Fernández-Martínez, Lucas Fernández-Brillet, Ana Cernea and Andrzej Kloczkowski

Int. J. Mol. Sci. 2022, 23(21), 12975; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232112975 - 26 Oct 2022

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Noise is a basic ingredient in data, since observed data are always contaminated by unwanted deviations, i.e., noise, which, in the case of overdetermined systems (with more data than model parameters), cause the corresponding linear system of equations to have an imperfect solution. [...] Read more.

Noise is a basic ingredient in data, since observed data are always contaminated by unwanted deviations, i.e., noise, which, in the case of overdetermined systems (with more data than model parameters), cause the corresponding linear system of equations to have an imperfect solution. In addition, in the case of highly underdetermined parameterization, noise can be absorbed by the model, generating spurious solutions. This is a very undesirable situation that might lead to incorrect conclusions. We presented mathematical formalism based on the inverse problem theory combined with artificial intelligence methodologies to perform an enhanced sampling of noisy biomedical data to improve the finding of meaningful solutions. Random sampling methods fail for high-dimensional biomedical problems. Sampling methods such as smart model parameterizations, forward surrogates, and parallel computing are better suited for such problems. We applied these methods to several important biomedical problems, such as phenotype prediction and a problem related to predicting the effects of protein mutations, i.e., if a given single residue mutation is neutral or deleterious, causing a disease. We also applied these methods to de novo drug discovery and drug repositioning (repurposing) through the enhanced exploration of huge chemical space. The purpose of these novel methods that address the problem of noise and uncertainty in biomedical data is to find new therapeutic solutions, perform drug repurposing, and accelerate and optimize drug discovery, thus reestablishing homeostasis. Finding the right target, the right compound, and the right patient are the three bottlenecks to running successful clinical trials from the correct analysis of preclinical models. Artificial intelligence can provide a solution to these problems, considering that the character of the data restricts the quality of the prediction, as in any modeling procedure in data analysis. The use of simple and plain methodologies is crucial to tackling these important and challenging problems, particularly drug repositioning/repurposing in rare diseases. Full article

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12 pages, 2636 KiB

Open AccessArticle

Discrimination of Brassica juncea Varieties Using Visible Near-Infrared (Vis-NIR) Spectroscopy and Chemometrics Methods

by Soo-In Sohn, Subramani Pandian, Young-Ju Oh, John-Lewis Zinia Zaukuu, Yong-Ho Lee and Eun-Kyoung Shin

Int. J. Mol. Sci. 2022, 23(21), 12809; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232112809 - 24 Oct 2022

Viewed by 1434

Brown mustard (Brassica juncea (L.) is an important oilseed crop that is mostly used to produce edible oils, industrial oils, modified lipids and biofuels in subtropical nations. Due to its higher level of commercial use, the species has a huge array of [...] Read more.

Brown mustard (Brassica juncea (L.) is an important oilseed crop that is mostly used to produce edible oils, industrial oils, modified lipids and biofuels in subtropical nations. Due to its higher level of commercial use, the species has a huge array of varieties/cultivars. The purpose of this study is to evaluate the use of visible near-infrared (Vis-NIR) spectroscopy in combination with multiple chemometric approaches for distinguishing four B. juncea varieties in Korea. The spectra from the leaves of four different growth stages of four B. juncea varieties were measured in the Vis-NIR range of 325–1075 nm with a stepping of 1.5 nm in reflectance mode. For effective discrimination, the spectral data were preprocessed using three distinct approaches, and eight different chemometric analyses were utilized. After the detection of outliers, the samples were split into two groups, one serving as a calibration set and the other as a validation set. When numerous preprocessing and chemometric approaches were applied for discriminating, the combination of standard normal variate and deep learning had the highest classification accuracy in all the growth stages achieved up to 100%. Similarly, few other chemometrics also yielded 100% classification accuracy, namely, support vector machine, generalized linear model, and the random forest. Of all the chemometric preprocessing methods, Savitzky–Golay filter smoothing provided the best and most convincing discrimination. The findings imply that chemometric methods combined with handheld Vis-NIR spectroscopy can be utilized as an efficient tool for differentiating B. juncea varieties in the field in all the growth stages. Full article

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9 pages, 1132 KiB

Open AccessArticle

Arrangement of Hydrogen Bonds in Aqueous Solutions of Different Globular Proteins

by Amber R. Titus, Pedro P. Madeira, Luisa A. Ferreira, Alexander I. Belgovskiy, Elizabeth K. Mann, Jay Adin Mann, Jr., William V. Meyer, Anthony E. Smart, Vladimir N. Uversky and Boris Y. Zaslavsky

Int. J. Mol. Sci. 2022, 23(19), 11381; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms231911381 - 27 Sep 2022

Cited by 1 | Viewed by 1608

This work presents the first evidence that dissolved globular proteins change the arrangement of hydrogen bonds in water, with different proteins showing quantitatively different effects. Using ATR-FTIR (attenuated total reflection—Fourier transform infrared) spectroscopic analysis of OH-stretch bands, we obtain quantitative estimates of the [...] Read more.

This work presents the first evidence that dissolved globular proteins change the arrangement of hydrogen bonds in water, with different proteins showing quantitatively different effects. Using ATR-FTIR (attenuated total reflection—Fourier transform infrared) spectroscopic analysis of OH-stretch bands, we obtain quantitative estimates of the relative amounts of the previously reported four subpopulations of water structures coexisting in a variety of aqueous solutions. Where solvatochromic dyes can measure the properties of solutions of non-ionic polymers, the results correlate well with ATR-FTIR measurements. In protein solutions to which solvatochromic dye probes cannot be applied, NMR (nuclear magnetic resonance) spectroscopy was used for the first time to estimate the hydrogen bond donor acidity of water. We found strong correlations between the solvent acidity and arrangement of hydrogen bonds in aqueous solutions for several globular proteins. Even quite similar proteins are found to change water properties in dramatically different ways. Full article

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12 pages, 4190 KiB

Open AccessArticle

Clustering Analysis, Structure Fingerprint Analysis, and Quantum Chemical Calculations of Compounds from Essential Oils of Sunflower (Helianthus annuus L.) Receptacles

by Yi He, Kaifeng Liu, Lu Han and Weiwei Han

Int. J. Mol. Sci. 2022, 23(17), 10169; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms231710169 - 05 Sep 2022

Cited by 2 | Viewed by 1496

Sunflower (Helianthus annuus L.) is an appropriate crop for current new patterns of green agriculture, so it is important to change sunflower receptacles from waste to useful resource. However, there is limited knowledge on the functions of compounds from the essential oils [...] Read more.

Sunflower (Helianthus annuus L.) is an appropriate crop for current new patterns of green agriculture, so it is important to change sunflower receptacles from waste to useful resource. However, there is limited knowledge on the functions of compounds from the essential oils of sunflower receptacles. In this study, a new method was created for chemical space network analysis and classification of small samples, and applied to 104 compounds. Here, t-SNE (t-Distributed Stochastic Neighbor Embedding) dimensions were used to reduce coordinates as node locations and edge connections of chemical space networks, respectively, and molecules were grouped according to whether the edges were connected and the proximity of the node coordinates. Through detailed analysis of the structural characteristics and fingerprints of each classified group, our classification method attained good accuracy. Targets were then identified using reverse docking methods, and the active centers of the same types of compounds were determined by quantum chemical calculation. The results indicated that these compounds can be divided into nine groups, according to their mean within-group similarity (MWGS) values. The three families with the most members, i.e., the d-limonene group (18), α-pinene group (10), and γ-maaliene group (nine members) determined the protein targets, using PharmMapper. Structure fingerprint analysis was employed to predict the binding mode of the ligands of four families of the protein targets. Thence, quantum chemical calculations were applied to the active group of the representative compounds of the four families. This study provides further scientific information to support the use of sunflower receptacles. Full article

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17 pages, 3047 KiB

Open AccessArticle

Albumin/Thiacalix[4]arene Nanoparticles as Potential Therapeutic Systems: Role of the Macrocycle for Stabilization of Monomeric Protein and Self-Assembly with Ciprofloxacin

by Luidmila Yakimova, Aisylu Kunafina, Olga Mostovaya, Pavel Padnya, Timur Mukhametzyanov, Alexandra Voloshina, Konstantin Petrov, Artur Boldyrev and Ivan Stoikov

Int. J. Mol. Sci. 2022, 23(17), 10040; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms231710040 - 02 Sep 2022

Cited by 3 | Viewed by 1759

The therapeutic application of serum albumin is determined by the relative content of the monomeric form compared to dimers, tetramers, hexamers, etc. In this paper, we propose and develop an approach to synthesize the cone stereoisomer of p-tert-butylthiacalix[4]arene with sulfobetaine fragments stabilization [...] Read more.

The therapeutic application of serum albumin is determined by the relative content of the monomeric form compared to dimers, tetramers, hexamers, etc. In this paper, we propose and develop an approach to synthesize the cone stereoisomer of p-tert-butylthiacalix[4]arene with sulfobetaine fragments stabilization of monomeric bovine serum albumin and preventing aggregation. Spectral methods (UV-vis, CD, fluorescent spectroscopy, and dynamic light scattering) established the influence of the synthesized compounds on the content of monomeric and aggregated forms of BSA even without the formation of stable thiacalixarene/protein associates. The effect of thiacalixarenes on the efficiency of protein binding with the antibiotic ciprofloxacin was shown by fluorescence spectroscopy. The binding constant increases in the presence of the macrocycles, likely due to the stabilization of monomeric forms of BSA. Our study clearly shows the potential of this macrocycle design as a platform for the development of the fundamentally new approaches for preventing aggregation. Full article

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9 pages, 3686 KiB

Open AccessArticle

A TD-DFT-Based Study on the Attack of the OH· Radical on a Guanine Nucleotide

by João Santiago, Jhaison C. de Faria, Miguel San-Miguel and Mario A. Bernal

Int. J. Mol. Sci. 2022, 23(17), 10007; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms231710007 - 02 Sep 2022

Cited by 1 | Viewed by 1770

Heavy charged particles induce severe damage in DNA, which is a radiobiological advantage when treating radioresistant tumors. However, these particles can also induce cancer in humans exposed to them, such as astronauts in space missions. This damage can be directly induced by the [...] Read more.

Heavy charged particles induce severe damage in DNA, which is a radiobiological advantage when treating radioresistant tumors. However, these particles can also induce cancer in humans exposed to them, such as astronauts in space missions. This damage can be directly induced by the radiation or indirectly by the attack of free radicals mainly produced by water radiolysis. We previously studied the impact of a proton on a DNA base pair, using the Time Dependent-Density Functional Theory (TD-DFT). In this work, we go a step further and study the attack of the OH· radical on the Guanine nucleotide to unveil how this molecule subsequently dissociates. The OH· attack on the H1′, H2′, H3′, and H5′ atoms in the guanine was investigated using the Ehrenfest dynamics within the TD-DFT framework. In all cases, the hydrogen abstraction succeeded, and the subsequent base pair dissociation was observed. The DNA dissociates in three major fragments: the phosphate group, the deoxyribose sugar, and the nitrogenous base, with slight differences, no matter which hydrogen atom was attacked. Hydrogen abstraction occurs at about 6 fs, and the nucleotide dissociation at about 100 fs, which agrees with our previous result for the direct proton impact on the DNA. These calculations may be a reference for adjusting reactive force fields so that more complex DNA structures can be studied using classical molecular dynamics, including both direct and indirect DNA damage. Full article

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17 pages, 4896 KiB

Open AccessArticle

Impact of Growth Conditions on Pseudomonas fluorescens Morphology Characterized by Atomic Force Microscopy

by Houssem Kahli, Laure Béven, Christine Grauby-Heywang, Nesrine Debez, Ibtissem Gammoudi, Fabien Moroté, Hana Sbartai and Touria Cohen-Bouhacina

Int. J. Mol. Sci. 2022, 23(17), 9579; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23179579 - 24 Aug 2022

Cited by 5 | Viewed by 4484

This work is dedicated to the characterization by Atomic Force Microscopy (AFM) of Pseudomonas fluorescens, bacteria having high potential in biotechnology. They were first studied first in optimal conditions in terms of culture medium and temperature. AFM revealed a more-or-less elongated morphology [...] Read more.

This work is dedicated to the characterization by Atomic Force Microscopy (AFM) of Pseudomonas fluorescens, bacteria having high potential in biotechnology. They were first studied first in optimal conditions in terms of culture medium and temperature. AFM revealed a more-or-less elongated morphology with typical dimensions in the micrometer range, and an organization of the outer membrane characterized by the presence of long and randomly distributed ripples, which are likely related to the organization of lipopolysaccharides (LPS). The outer membrane also presents invaginations, some of them showing a reorganization of ripples, which could be the first sign of a bacterial stress response. In a second step, bacteria grown under unfavorable conditions were characterized. The choice of the medium appeared to be more critical in the case of the second generation of cells, the less adapted medium inducing not only changes in the membrane organization but also larger damages in bacteria. An increased growth temperature affected both the usual “swollen” morphology and the organization of the outer membrane. Here also, LPS likely contribute to membrane remodelling, which makes them potential markers to track cell state changes. Full article

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19 pages, 42214 KiB

Open AccessArticle

RNA-As-Graphs Motif Atlas—Dual Graph Library of RNA Modules and Viral Frameshifting-Element Applications

by Qiyao Zhu, Louis Petingi and Tamar Schlick

Int. J. Mol. Sci. 2022, 23(16), 9249; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23169249 - 17 Aug 2022

Cited by 2 | Viewed by 1642

RNA motif classification is important for understanding structure/function connections and building phylogenetic relationships. Using our coarse-grained RNA-As-Graphs (RAG) representations, we identify recurrent dual graph motifs in experimentally solved RNA structures based on an improved search algorithm that finds and ranks independent RNA substructures. [...] Read more.

RNA motif classification is important for understanding structure/function connections and building phylogenetic relationships. Using our coarse-grained RNA-As-Graphs (RAG) representations, we identify recurrent dual graph motifs in experimentally solved RNA structures based on an improved search algorithm that finds and ranks independent RNA substructures. Our expanded list of 183 existing dual graph motifs reveals five common motifs found in transfer RNA, riboswitch, and ribosomal 5S RNA components. Moreover, we identify three motifs for available viral frameshifting RNA elements, suggesting a correlation between viral structural complexity and frameshifting efficiency. We further partition the RNA substructures into 1844 distinct submotifs, with pseudoknots and junctions retained intact. Common modules are internal loops and three-way junctions, and three submotifs are associated with riboswitches that bind nucleotides, ions, and signaling molecules. Together, our library of existing RNA motifs and submotifs adds to the growing universe of RNA modules, and provides a resource of structures and substructures for novel RNA design. Full article

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14 pages, 3373 KiB

Open AccessArticle

Time-Dependent DNA Origami Denaturation by Guanidinium Chloride, Guanidinium Sulfate, and Guanidinium Thiocyanate

by Marcel Hanke, Niklas Hansen, Emilia Tomm, Guido Grundmeier and Adrian Keller

Int. J. Mol. Sci. 2022, 23(15), 8547; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23158547 - 01 Aug 2022

Cited by 3 | Viewed by 2437

Guanidinium (Gdm) undergoes interactions with both hydrophilic and hydrophobic groups and, thus, is a highly potent denaturant of biomolecular structure. However, our molecular understanding of the interaction of Gdm with proteins and DNA is still rather limited. Here, we investigated the denaturation of [...] Read more.

Guanidinium (Gdm) undergoes interactions with both hydrophilic and hydrophobic groups and, thus, is a highly potent denaturant of biomolecular structure. However, our molecular understanding of the interaction of Gdm with proteins and DNA is still rather limited. Here, we investigated the denaturation of DNA origami nanostructures by three Gdm salts, i.e., guanidinium chloride (GdmCl), guanidinium sulfate (Gdm₂SO₄), and guanidinium thiocyanate (GdmSCN), at different temperatures and in dependence of incubation time. Using DNA origami nanostructures as sensors that translate small molecular transitions into nanostructural changes, the denaturing effects of the Gdm salts were directly visualized by atomic force microscopy. GdmSCN was the most potent DNA denaturant, which caused complete DNA origami denaturation at 50 °C already at a concentration of 2 M. Under such harsh conditions, denaturation occurred within the first 15 min of Gdm exposure, whereas much slower kinetics were observed for the more weakly denaturing salt Gdm₂SO₄ at 25 °C. Lastly, we observed a novel non-monotonous temperature dependence of DNA origami denaturation in Gdm₂SO₄ with the fraction of intact nanostructures having an intermediate minimum at about 40 °C. Our results, thus, provide further insights into the highly complex Gdm–DNA interaction and underscore the importance of the counteranion species. Full article

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17 pages, 28348 KiB

Open AccessArticle

Unravelling the Adaptation Mechanisms to High Pressure in Proteins

by Antonino Caliò, Cécile Dubois, Stéphane Fontanay, Michael Marek Koza, François Hoh, Christian Roumestand, Philippe Oger and Judith Peters

Int. J. Mol. Sci. 2022, 23(15), 8469; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23158469 - 30 Jul 2022

Cited by 5 | Viewed by 2379

Life is thought to have appeared in the depth of the sea under high hydrostatic pressure. Nowadays, it is known that the deep biosphere hosts a myriad of life forms thriving under high-pressure conditions. However, the evolutionary mechanisms leading to their adaptation are [...] Read more.

Life is thought to have appeared in the depth of the sea under high hydrostatic pressure. Nowadays, it is known that the deep biosphere hosts a myriad of life forms thriving under high-pressure conditions. However, the evolutionary mechanisms leading to their adaptation are still not known. Here, we show the molecular bases of these mechanisms through a joint structural and dynamical study of two orthologous proteins. We observed that pressure adaptation involves the decoupling of protein–water dynamics and the elimination of cavities in the protein core. This is achieved by rearranging the charged residues on the protein surface and using bulkier hydrophobic residues in the core. These findings will be the starting point in the search for a complete genomic model explaining high-pressure adaptation. Full article

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24 pages, 3610 KiB

Open AccessArticle

Synthesis, Physicochemical Characterization, and Antibacterial Performance of Silver—Lactoferrin Complexes

by Oleksandra Pryshchepa, Paweł Pomastowski, Katarzyna Rafińska, Adrian Gołębiowski, Agnieszka Rogowska, Maciej Monedeiro-Milanowski, Gulyaim Sagandykova, Bernhard Michalke, Philippe Schmitt-Kopplin, Michał Gloc, Renata Dobrucka, Krzysztof Kurzydłowski and Bogusław Buszewski

Int. J. Mol. Sci. 2022, 23(13), 7112; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23137112 - 26 Jun 2022

Cited by 7 | Viewed by 1993

Antibiotic-resistant bacteria pose one of the major threats to human health worldwide. The issue is fundamental in the case of chronic wound treatment. One of the latest trends to overcome the problem is the search for new antibacterial agents based on silver. Thus, [...] Read more.

Antibiotic-resistant bacteria pose one of the major threats to human health worldwide. The issue is fundamental in the case of chronic wound treatment. One of the latest trends to overcome the problem is the search for new antibacterial agents based on silver. Thus, the aim of this research was to synthesize the silver-lactoferrin complex as a new generation of substances for the treatment of infected wounds. Moreover, one of the tasks was to investigate the formation mechanisms of the respective complexes and the influence of different synthesis conditions on the features of final product. The batch-sorption study was performed by applying the Langmuir and Freundlich isotherm models for the process description. Characterization of the complexes was carried out by spectroscopy, spectrometry, and separation techniques, as well as with electron microscopy. Additionally, the biological properties of the complex were evaluated, i.e., the antibacterial activity against selected bacteria and the impact on L929 cell-line viability. The results indicate the formation of a heterogeneous silver–lactoferrin complex that comprises silver nanoparticles. The complex has higher antibacterial strength than both native bovine lactoferrin and Ag⁺, while being comparable to silver toxicity. Full article

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17 pages, 3372 KiB

Open AccessArticle

Exploration of Somatostatin Binding Mechanism to Somatostatin Receptor Subtype 4

by Rita Börzsei, Balázs Zoltán Zsidó, Mónika Bálint, Zsuzsanna Helyes, Erika Pintér and Csaba Hetényi

Int. J. Mol. Sci. 2022, 23(13), 6878; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23136878 - 21 Jun 2022

Cited by 4 | Viewed by 2418

Somatostatin (also named as growth hormone-inhibiting hormone or somatotropin release-inhibiting factor) is a regulatory peptide important for the proper functioning of the endocrine system, local inflammatory reactions, mood and motor coordination, and behavioral responses to stress. Somatostatin exerts its effects via binding to [...] Read more.

Somatostatin (also named as growth hormone-inhibiting hormone or somatotropin release-inhibiting factor) is a regulatory peptide important for the proper functioning of the endocrine system, local inflammatory reactions, mood and motor coordination, and behavioral responses to stress. Somatostatin exerts its effects via binding to G-protein-coupled somatostatin receptors of which the fourth subtype (SSTR4) is a particularly important receptor mediating analgesic, anti-inflammatory, and anti-depressant effects without endocrine actions. Thus, SSTR4 agonists are promising drug candidates. Although the knowledge of the atomic resolution-binding modes of SST would be essential for drug development, experimental elucidation of the structures of SSTR4 and its complexes is still awaiting. In the present study, structures of the somatostatin–SSTR4 complex were produced using an unbiased, blind docking approach. Beyond the static structures, the binding mechanism of SST was also elucidated in the explicit water molecular dynamics (MD) calculations, and key binding modes (external, intermediate, and internal) were distinguished. The most important residues on both receptor and SST sides were identified. An energetic comparison of SST binding to SSTR4 and 2 offered a residue-level explanation of receptor subtype selectivity. The calculated structures show good agreement with available experimental results and indicate that somatostatin binding is realized via prerequisite binding modes and an induced fit mechanism. The identified binding modes and the corresponding key residues provide useful information for future drug design targeting SSTR4. Full article

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24 pages, 5609 KiB

Open AccessArticle

Interactions between S100A9 and Alpha-Synuclein: Insight from NMR Spectroscopy

by Zigmantas Toleikis, Raitis Bobrovs, Agne Janoniene, Alons Lends, Mantas Ziaunys, Ieva Baronaite, Vytautas Petrauskas, Kristine Kitoka, Vytautas Smirnovas and Kristaps Jaudzems

Int. J. Mol. Sci. 2022, 23(12), 6781; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23126781 - 17 Jun 2022

Cited by 2 | Viewed by 2279

S100A9 is a pro-inflammatory protein that co-aggregates with other proteins in amyloid fibril plaques. S100A9 can influence the aggregation kinetics and amyloid fibril structure of alpha-synuclein (

α

-syn), which is involved in Parkinson’s disease. Currently, there are limited data regarding their cross-interaction [...] Read more.

S100A9 is a pro-inflammatory protein that co-aggregates with other proteins in amyloid fibril plaques. S100A9 can influence the aggregation kinetics and amyloid fibril structure of alpha-synuclein (

α

-syn), which is involved in Parkinson’s disease. Currently, there are limited data regarding their cross-interaction and how it influences the aggregation process. In this work, we analyzed this interaction using solution

^{19}

F and 2D ¹⁵N–¹H HSQC NMR spectroscopy and studied the aggregation properties of these two proteins. Here, we show that

α

-syn interacts with S100A9 at specific regions, which are also essential in the first step of aggregation. We also demonstrate that the 4-fluorophenylalanine label in alpha-synuclein is a sensitive probe to study interaction and aggregation using

^{19}

F NMR spectroscopy. Full article

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21 pages, 4986 KiB

Open AccessArticle

Molecular Dynamics of DHHC20 Acyltransferase Suggests Principles of Lipid and Protein Substrate Selectivity

by Irina Panina, Nikolay Krylov, Mohamed Rasheed Gadalla, Elena Aliper, Larisa Kordyukova, Michael Veit, Anton Chugunov and Roman Efremov

Int. J. Mol. Sci. 2022, 23(9), 5091; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23095091 - 03 May 2022

Cited by 5 | Viewed by 2931

Lipid modification of viral proteins with fatty acids of different lengths (S-acylation) is crucial for virus pathogenesis. The reaction is catalyzed by members of the DHHC family and proceeds in two steps: the autoacylation is followed by the acyl chain transfer onto protein [...] Read more.

Lipid modification of viral proteins with fatty acids of different lengths (S-acylation) is crucial for virus pathogenesis. The reaction is catalyzed by members of the DHHC family and proceeds in two steps: the autoacylation is followed by the acyl chain transfer onto protein substrates. The crystal structure of human DHHC20 (hDHHC20), an enzyme involved in the acylation of S-protein of SARS-CoV-2, revealed that the acyl chain may be inserted into a hydrophobic cavity formed by four transmembrane (TM) α-helices. To test this model, we used molecular dynamics of membrane-embedded hDHHC20 and its mutants either in the absence or presence of various acyl-CoAs. We found that among a range of acyl chain lengths probed only C16 adopts a conformation suitable for hDHHC20 autoacylation. This specificity is altered if the small or bulky residues at the cavity’s ceiling are exchanged, e.g., the V185G mutant obtains strong preferences for binding C18. Surprisingly, an unusual hydrophilic ridge was found in TM helix 4 of hDHHC20, and the responsive hydrophilic patch supposedly involved in association was found in the 3D model of the S-protein TM-domain trimer. Finally, the exchange of critical Thr and Ser residues in the spike led to a significant decrease in its S-acylation. Our data allow further development of peptide/lipid-based inhibitors of hDHHC20 that might impede replication of Corona- and other enveloped viruses. Full article

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15 pages, 1466 KiB

Open AccessReview

Lasers in Live Cell Microscopy

by Herbert Schneckenburger

Int. J. Mol. Sci. 2022, 23(9), 5015; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23095015 - 30 Apr 2022

Cited by 4 | Viewed by 2343

Due to their unique properties—coherent radiation, diffraction limited focusing, low spectral bandwidth and in many cases short light pulses—lasers play an increasing role in live cell microscopy. Lasers are indispensable tools in 3D microscopy, e.g., confocal, light sheet or total internal reflection microscopy, [...] Read more.

Due to their unique properties—coherent radiation, diffraction limited focusing, low spectral bandwidth and in many cases short light pulses—lasers play an increasing role in live cell microscopy. Lasers are indispensable tools in 3D microscopy, e.g., confocal, light sheet or total internal reflection microscopy, as well as in super-resolution microscopy using wide-field or confocal methods. Further techniques, e.g., spectral imaging or fluorescence lifetime imaging (FLIM) often depend on the well-defined spectral or temporal properties of lasers. Furthermore, laser microbeams are used increasingly for optical tweezers or micromanipulation of cells. Three exemplary laser applications in live cell biology are outlined. They include fluorescence diagnosis, in particular in combination with Förster Resonance Energy Transfer (FRET), photodynamic therapy as well as laser-assisted optoporation, and demonstrate the potential of lasers in cell biology and—more generally—in biomedicine. Full article

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16 pages, 1487 KiB

Open AccessReview

Stress-Induced Membraneless Organelles in Eukaryotes and Prokaryotes: Bird’s-Eye View

by Anna S. Fefilova, Alexander V. Fonin, Innokentii E. Vishnyakov, Irina M. Kuznetsova and Konstantin K. Turoverov

Int. J. Mol. Sci. 2022, 23(9), 5010; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23095010 - 30 Apr 2022

Cited by 7 | Viewed by 3372

Stress is an inevitable part of life. An organism is exposed to multiple stresses and overcomes their negative consequences throughout its entire existence. A correlation was established between life expectancy and resistance to stress, suggesting a relationship between aging and the ability to [...] Read more.

Stress is an inevitable part of life. An organism is exposed to multiple stresses and overcomes their negative consequences throughout its entire existence. A correlation was established between life expectancy and resistance to stress, suggesting a relationship between aging and the ability to respond to external adverse effects as well as quickly restore the normal regulation of biological processes. To combat stress, cells developed multiple pro-survival mechanisms, one of them is the assembly of special stress-induced membraneless organelles (MLOs). MLOs are formations that do not possess a lipid membrane but rather form as a result of the “liquid–liquid” phase separation (LLPS) of biopolymers. Stress-responsive MLOs were found in eukaryotes and prokaryotes, they form as a reaction to the acute environmental conditions and are dismantled after its termination. These compartments function to prevent damage to the genetic and protein material of the cell during stress. In this review, we discuss the characteristics of stress-induced MLO-like structures in eukaryotic and prokaryotic cells. Full article

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17 pages, 1278 KiB

Open AccessReview

Innovations in Genomics and Big Data Analytics for Personalized Medicine and Health Care: A Review

by Mubashir Hassan, Faryal Mehwish Awan, Anam Naz, Enrique J. deAndrés-Galiana, Oscar Alvarez, Ana Cernea, Lucas Fernández-Brillet, Juan Luis Fernández-Martínez and Andrzej Kloczkowski

Int. J. Mol. Sci. 2022, 23(9), 4645; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23094645 - 22 Apr 2022

Cited by 43 | Viewed by 8810

Big data in health care is a fast-growing field and a new paradigm that is transforming case-based studies to large-scale, data-driven research. As big data is dependent on the advancement of new data standards, technology, and relevant research, the future development of big [...] Read more.

Big data in health care is a fast-growing field and a new paradigm that is transforming case-based studies to large-scale, data-driven research. As big data is dependent on the advancement of new data standards, technology, and relevant research, the future development of big data applications holds foreseeable promise in the modern day health care revolution. Enormously large, rapidly growing collections of biomedical omics-data (genomics, proteomics, transcriptomics, metabolomics, glycomics, etc.) and clinical data create major challenges and opportunities for their analysis and interpretation and open new computational gateways to address these issues. The design of new robust algorithms that are most suitable to properly analyze this big data by taking into account individual variability in genes has enabled the creation of precision (personalized) medicine. We reviewed and highlighted the significance of big data analytics for personalized medicine and health care by focusing mostly on machine learning perspectives on personalized medicine, genomic data models with respect to personalized medicine, the application of data mining algorithms for personalized medicine as well as the challenges we are facing right now in big data analytics. Full article

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14 pages, 3437 KiB

Open AccessArticle

AlphaFold2: A Role for Disordered Protein/Region Prediction?

by Carter J. Wilson, Wing-Yiu Choy and Mikko Karttunen

Int. J. Mol. Sci. 2022, 23(9), 4591; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23094591 - 21 Apr 2022

Cited by 54 | Viewed by 4954

The development of AlphaFold2 marked a paradigm-shift in the structural biology community. Herein, we assess the ability of AlphaFold2 to predict disordered regions against traditional sequence-based disorder predictors. We find that AlphaFold2 performs well at discriminating disordered regions, but also note that the [...] Read more.

The development of AlphaFold2 marked a paradigm-shift in the structural biology community. Herein, we assess the ability of AlphaFold2 to predict disordered regions against traditional sequence-based disorder predictors. We find that AlphaFold2 performs well at discriminating disordered regions, but also note that the disorder predictor one constructs from an AlphaFold2 structure determines accuracy. In particular, a naïve, but non-trivial assumption that residues assigned to helices, strands, and H-bond stabilized turns are likely ordered and all other residues are disordered results in a dramatic overestimation in disorder; conversely, the predicted local distance difference test (pLDDT) provides an excellent measure of residue-wise disorder. Furthermore, by employing molecular dynamics (MD) simulations, we note an interesting relationship between the pLDDT and secondary structure, that may explain our observations and suggests a broader application of the pLDDT for characterizing the local dynamics of intrinsically disordered proteins and regions (IDPs/IDRs). Full article

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23 pages, 3419 KiB

Open AccessReview

Atomic-Resolution Structures and Mode of Action of Clinically Relevant Antimicrobial Peptides

by Surajit Bhattacharjya, Sk Abdul Mohid and Anirban Bhunia

Int. J. Mol. Sci. 2022, 23(9), 4558; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23094558 - 20 Apr 2022

Cited by 12 | Viewed by 2927

Global rise of infections and deaths caused by drug-resistant bacterial pathogens are among the unmet medical needs. In an age of drying pipeline of novel antibiotics to treat bacterial infections, antimicrobial peptides (AMPs) are proven to be valid therapeutics modalities. Direct in vivo [...] Read more.

Global rise of infections and deaths caused by drug-resistant bacterial pathogens are among the unmet medical needs. In an age of drying pipeline of novel antibiotics to treat bacterial infections, antimicrobial peptides (AMPs) are proven to be valid therapeutics modalities. Direct in vivo applications of many AMPs could be challenging; however, works are demonstrating encouraging results for some of them. In this review article, we discussed 3-D structures of potent AMPs e.g., polymyxin, thanatin, MSI, protegrin, OMPTA in complex with bacterial targets and their mode of actions. Studies on human peptide LL37 and de novo-designed peptides are also discussed. We have focused on AMPs which are effective against drug-resistant Gram-negative bacteria. Since treatment options for the infections caused by super bugs of Gram-negative bacteria are now extremely limited. We also summarize some of the pertinent challenges in the field of clinical trials of AMPs. Full article

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25 pages, 5345 KiB

Open AccessArticle

Computer Simulations and Network-Based Profiling of Binding and Allosteric Interactions of SARS-CoV-2 Spike Variant Complexes and the Host Receptor: Dissecting the Mechanistic Effects of the Delta and Omicron Mutations

by Gennady Verkhivker, Steve Agajanian, Ryan Kassab and Keerthi Krishnan

Int. J. Mol. Sci. 2022, 23(8), 4376; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23084376 - 15 Apr 2022

Cited by 13 | Viewed by 2355

In this study, we combine all-atom MD simulations and comprehensive mutational scanning of S-RBD complexes with the angiotensin-converting enzyme 2 (ACE2) host receptor in the native form as well as the S-RBD Delta and Omicron variants to (a) examine the differences in the [...] Read more.

In this study, we combine all-atom MD simulations and comprehensive mutational scanning of S-RBD complexes with the angiotensin-converting enzyme 2 (ACE2) host receptor in the native form as well as the S-RBD Delta and Omicron variants to (a) examine the differences in the dynamic signatures of the S-RBD complexes and (b) identify the critical binding hotspots and sensitivity of the mutational positions. We also examined the differences in allosteric interactions and communications in the S-RBD complexes for the Delta and Omicron variants. Through the perturbation-based scanning of the allosteric propensities of the SARS-CoV-2 S-RBD residues and dynamics-based network centrality and community analyses, we characterize the global mediating centers in the complexes and the nature of local stabilizing communities. We show that a constellation of mutational sites (G496S, Q498R, N501Y and Y505H) correspond to key binding energy hotspots and also contribute decisively to the key interfacial communities that mediate allosteric communications between S-RBD and ACE2. These Omicron mutations are responsible for both favorable local binding interactions and long-range allosteric interactions, providing key functional centers that mediate the high transmissibility of the virus. At the same time, our results show that other mutational sites could provide a “flexible shield” surrounding the stable community network, thereby allowing the Omicron virus to modulate immune evasion at different epitopes, while protecting the integrity of binding and allosteric interactions in the RBD–ACE2 complexes. This study suggests that the SARS-CoV-2 S protein may exploit the plasticity of the RBD to generate escape mutants, while engaging a small group of functional hotspots to mediate efficient local binding interactions and long-range allosteric communications with ACE2. Full article

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15 pages, 6251 KiB

Open AccessArticle

Conformational Changes in the BSA-LT4 Complex Induced by the Presence of Vitamins: Spectroscopic Approach and Molecular Docking

by Nicoleta Cazacu, Claudia G. Chilom, Melinda David and Monica Florescu

Int. J. Mol. Sci. 2022, 23(8), 4215; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23084215 - 11 Apr 2022

Cited by 3 | Viewed by 1934

Levothyroxine (LT4) is known for its use in various conditions including hypothyroidism. LT4 interaction with serum albumin may be influenced by the presence of vitamins. For this reason, we investigated the effect of vitamin C, vitamin B12, and folic acid on the complex [...] Read more.

Levothyroxine (LT4) is known for its use in various conditions including hypothyroidism. LT4 interaction with serum albumin may be influenced by the presence of vitamins. For this reason, we investigated the effect of vitamin C, vitamin B12, and folic acid on the complex of Bovine Serum Albumin with LT4 (BSA-LT4). UV-Vis spectroscopy was used to monitor the influence of vitamins on the BSA-LT4 complex. Fluorescence spectroscopy revealed a static quenching mechanism of the fluorescence of BSA-LT4 complex by the vitamin C and folic acid and a combined mechanism for vitamin B12. The interaction of vitamin C and folic acid with BSA-LT4 was moderate, while the binding of vitamin B12 was much stronger, extending the storage time of LT4 in blood plasma. Synchronous fluorescence found that the vitamins were closer to the vicinity of Trp than to Tyr and the effect was more pronounced for the binding of vitamin B12. The thermal stability of the BSA-LT4 complex was more evident, but no influence on the stability of BSA-LT4 complex was obtained for vitamin C. Molecular docking studies showed that vitamin C and folic acid bound the same site of the protein, while vitamin B12 bonded to a different site. Full article

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24 pages, 3871 KiB

Open AccessArticle

SARS-CoV-2 Membrane Protein: From Genomic Data to Structural New Insights

by Catarina Marques-Pereira, Manuel N. Pires, Raquel P. Gouveia, Nádia N. Pereira, Ana B. Caniceiro, Nícia Rosário-Ferreira and Irina S. Moreira

Int. J. Mol. Sci. 2022, 23(6), 2986; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23062986 - 10 Mar 2022

Cited by 16 | Viewed by 3711

Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) is composed of four structural proteins and several accessory non-structural proteins. SARS-CoV-2’s most abundant structural protein, Membrane (M) protein, has a pivotal role both during viral infection cycle and host interferon antagonism. This is a highly conserved [...] Read more.

Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) is composed of four structural proteins and several accessory non-structural proteins. SARS-CoV-2’s most abundant structural protein, Membrane (M) protein, has a pivotal role both during viral infection cycle and host interferon antagonism. This is a highly conserved viral protein, thus an interesting and suitable target for drug discovery. In this paper, we explain the structural nature of M protein homodimer. To do so, we developed and applied a detailed and robust in silico workflow to predict M protein dimeric structure, membrane orientation, and interface characterization. Single Nucleotide Polymorphisms (SNPs) in M protein were retrieved from over 1.2 M SARS-CoV-2 genomes and proteins from the Global Initiative on Sharing All Influenza Data (GISAID) database, 91 of which were located at the predicted dimer interface. Among those, we identified SNPs in Variants of Concern (VOC) and Variants of Interest (VOI). Binding free energy differences were evaluated for dimer interfacial SNPs to infer mutant protein stabilities. A few high-prevalent mutated residues were found to be especially relevant in VOC and VOI. This realization may be a game-changer to structure-driven formulation of new therapeutics for SARS-CoV-2. Full article

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24 pages, 3652 KiB

Open AccessArticle

The Study of Protein–Cyclitol Interactions

by Tetiana Dyrda-Terniuk, Mateusz Sugajski, Oleksandra Pryshchepa, Joanna Śliwiak, Magdalena Buszewska-Forajta, Paweł Pomastowski and Bogusław Buszewski

Int. J. Mol. Sci. 2022, 23(6), 2940; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23062940 - 09 Mar 2022

Cited by 6 | Viewed by 2227

Investigation of interactions between the target protein molecule and ligand allows for an understanding of the nature of the molecular recognition, functions, and biological activity of protein–ligand complexation. In the present work, non-specific interactions between a model protein (Bovine Serum Albumin) and four [...] Read more.

Investigation of interactions between the target protein molecule and ligand allows for an understanding of the nature of the molecular recognition, functions, and biological activity of protein–ligand complexation. In the present work, non-specific interactions between a model protein (Bovine Serum Albumin) and four cyclitols were investigated. D-sorbitol and adonitol represent the group of linear-structure cyclitols, while shikimic acid and D-(–)-quinic acid have cyclic-structure molecules. Various analytical methods, including chromatographic analysis (HPLC-MS/MS), electrophoretic analysis (SDS-PAGE), spectroscopic analysis (spectrofluorimetry, Fourier transform infrared spectroscopy, and Raman spectroscopy), and isothermal titration calorimetry (ITC), were applied for the description of protein–cyclitol interactions. Additionally, computational calculations were performed to predict the possible binding places. Kinetic studies allowed us to clarify interaction mechanisms that may take place during BSA and cyclitol interaction. The results allow us, among other things, to evaluate the impact of the cyclitol’s structure on the character of its interactions with the protein. Full article

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24 pages, 5154 KiB

Open AccessReview

Structural and Computational Studies of the SARS-CoV-2 Spike Protein Binding Mechanisms with Nanobodies: From Structure and Dynamics to Avidity-Driven Nanobody Engineering

by Gennady Verkhivker

Int. J. Mol. Sci. 2022, 23(6), 2928; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23062928 - 08 Mar 2022

Cited by 10 | Viewed by 4204

Nanobodies provide important advantages over traditional antibodies, including their smaller size and robust biochemical properties such as high thermal stability, high solubility, and the ability to be bioengineered into novel multivalent, multi-specific, and high-affinity molecules, making them a class of emerging powerful therapies [...] Read more.

Nanobodies provide important advantages over traditional antibodies, including their smaller size and robust biochemical properties such as high thermal stability, high solubility, and the ability to be bioengineered into novel multivalent, multi-specific, and high-affinity molecules, making them a class of emerging powerful therapies against SARS-CoV-2. Recent research efforts on the design, protein engineering, and structure-functional characterization of nanobodies and their binding with SARS-CoV-2 S proteins reflected a growing realization that nanobody combinations can exploit distinct binding epitopes and leverage the intrinsic plasticity of the conformational landscape for the SARS-CoV-2 S protein to produce efficient neutralizing and mutation resistant characteristics. Structural and computational studies have also been instrumental in quantifying the structure, dynamics, and energetics of the SARS-CoV-2 spike protein binding with nanobodies. In this review, a comprehensive analysis of the current structural, biophysical, and computational biology investigations of SARS-CoV-2 S proteins and their complexes with distinct classes of nanobodies targeting different binding sites is presented. The analysis of computational studies is supplemented by an in-depth examination of mutational scanning simulations and identification of binding energy hotspots for distinct nanobody classes. The review is focused on the analysis of mechanisms underlying synergistic binding of multivalent nanobodies that can be superior to single nanobodies and conventional nanobody cocktails in combating escape mutations by effectively leveraging binding avidity and allosteric cooperativity. We discuss how structural insights and protein engineering approaches together with computational biology tools can aid in the rational design of synergistic combinations that exhibit superior binding and neutralization characteristics owing to avidity-mediated mechanisms. Full article

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15 pages, 3784 KiB

Open AccessArticle

Salting-Out of DNA Origami Nanostructures by Ammonium Sulfate

by Marcel Hanke, Niklas Hansen, Ruiping Chen, Guido Grundmeier, Karim Fahmy and Adrian Keller

Int. J. Mol. Sci. 2022, 23(5), 2817; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23052817 - 04 Mar 2022

Cited by 8 | Viewed by 4307

DNA origami technology enables the folding of DNA strands into complex nanoscale shapes whose properties and interactions with molecular species often deviate significantly from that of genomic DNA. Here, we investigate the salting-out of different DNA origami shapes by the kosmotropic salt ammonium [...] Read more.

DNA origami technology enables the folding of DNA strands into complex nanoscale shapes whose properties and interactions with molecular species often deviate significantly from that of genomic DNA. Here, we investigate the salting-out of different DNA origami shapes by the kosmotropic salt ammonium sulfate that is routinely employed in protein precipitation. We find that centrifugation in the presence of 3 M ammonium sulfate results in notable precipitation of DNA origami nanostructures but not of double-stranded genomic DNA. The precipitated DNA origami nanostructures can be resuspended in ammonium sulfate-free buffer without apparent formation of aggregates or loss of structural integrity. Even though quasi-1D six-helix bundle DNA origami are slightly less susceptible toward salting-out than more compact DNA origami triangles and 24-helix bundles, precipitation and recovery yields appear to be mostly independent of DNA origami shape and superstructure. Exploiting the specificity of ammonium sulfate salting-out for DNA origami nanostructures, we further apply this method to separate DNA origami triangles from genomic DNA fragments in a complex mixture. Our results thus demonstrate the possibility of concentrating and purifying DNA origami nanostructures by ammonium sulfate-induced salting-out. Full article

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22 pages, 3745 KiB

Open AccessArticle

Cognate DNA Recognition by Engrailed Homeodomain Involves a Conformational Change Controlled via an Electrostatic-Spring-Loaded Latch

by Nicola D’Amelio, Benjamin Tanielian, Mourad Sadqi, Pilar López-Navajas and Victor Muñoz

Int. J. Mol. Sci. 2022, 23(5), 2412; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23052412 - 22 Feb 2022

Cited by 1 | Viewed by 2285

Transcription factors must scan genomic DNA, recognize the cognate sequence of their control element(s), and bind tightly to them. The DNA recognition process is primarily carried out by their DNA binding domains (DBD), which interact with the cognate site with high affinity and [...] Read more.

Transcription factors must scan genomic DNA, recognize the cognate sequence of their control element(s), and bind tightly to them. The DNA recognition process is primarily carried out by their DNA binding domains (DBD), which interact with the cognate site with high affinity and more weakly with any other DNA sequence. DBDs are generally thought to bind to their cognate DNA without changing conformation (lock-and-key). Here, we used nuclear magnetic resonance and circular dichroism to investigate the interplay between DNA recognition and DBD conformation in the engrailed homeodomain (enHD), as a model case for the homeodomain family of eukaryotic DBDs. We found that the conformational ensemble of enHD is rather flexible and becomes gradually more disordered as ionic strength decreases following a Debye–Hückel’s dependence. Our analysis indicates that enHD’s response to ionic strength is mediated by a built-in electrostatic spring-loaded latch that operates as a conformational transducer. We also found that, at moderate ionic strengths, enHD changes conformation upon binding to cognate DNA. This change is of larger amplitude and somewhat orthogonal to the response to ionic strength. As a consequence, very high ionic strengths (e.g., 700 mM) block the electrostatic-spring-loaded latch and binding to cognate DNA becomes lock-and-key. However, the interplay between enHD conformation and cognate DNA binding is robust across a range of ionic strengths (i.e., 45 to 300 mM) that covers the physiologically-relevant conditions. Therefore, our results demonstrate the presence of a mechanism for the conformational control of cognate DNA recognition on a eukaryotic DBD. This mechanism can function as a signal transducer that locks the DBD in place upon encountering the cognate site during active DNA scanning. The electrostatic-spring-loaded latch of enHD can also enable the fine control of DNA recognition in response to transient changes in local ionic strength induced by variate physiological processes. Full article

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16 pages, 3135 KiB

Open AccessArticle

Interferon-β Activity Is Affected by S100B Protein

by Alexey S. Kazakov, Alexander D. Sofin, Nadezhda V. Avkhacheva, Evgenia I. Deryusheva, Victoria A. Rastrygina, Maria E. Permyakova, Vladimir N. Uversky, Eugene A. Permyakov and Sergei E. Permyakov

Int. J. Mol. Sci. 2022, 23(4), 1997; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23041997 - 11 Feb 2022

Cited by 5 | Viewed by 1830

Interferon-β (IFN-β) is a pleiotropic cytokine secreted in response to various pathological conditions and is clinically used for therapy of multiple sclerosis. Its application for treatment of cancer, infections and pulmonary diseases is limited by incomplete understanding of regulatory mechanisms of its functioning. [...] Read more.

Interferon-β (IFN-β) is a pleiotropic cytokine secreted in response to various pathological conditions and is clinically used for therapy of multiple sclerosis. Its application for treatment of cancer, infections and pulmonary diseases is limited by incomplete understanding of regulatory mechanisms of its functioning. Recently, we reported that IFN-β activity is affected by interactions with S100A1, S100A4, S100A6, and S100P proteins, which are members of the S100 protein family of multifunctional Ca²⁺-binding proteins possessing cytokine-like activities (Int J Mol Sci. 2020;21(24):9473). Here we show that IFN-β interacts with one more representative of the S100 protein family, the S100B protein, involved in numerous oncological and neurological diseases. The use of chemical crosslinking, intrinsic fluorescence, and surface plasmon resonance spectroscopy revealed IFN-β binding to Ca²⁺-loaded dimeric and monomeric forms of the S100B protein. Calcium depletion blocks the S100B–IFN-β interaction. S100B monomerization increases its affinity to IFN-β by 2.7 orders of magnitude (equilibrium dissociation constant of the complex reaches 47 pM). Crystal violet assay demonstrated that combined application of IFN-β and S100B (5–25 nM) eliminates their inhibitory effects on MCF-7 cell viability. Bioinformatics analysis showed that the direct modulation of IFN-β activity by the S100B protein described here could be relevant to progression of multiple oncological and neurological diseases. Full article

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13 pages, 6389 KiB

Open AccessArticle

DMSO and TMAO—Differences in Interactions in Aqueous Solutions of the K-Peptide

by Julia Godlewska, Bartosz Cieśla, Jarosław Wawer and Piotr Bruździak

Int. J. Mol. Sci. 2022, 23(3), 1872; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23031872 - 07 Feb 2022

Cited by 5 | Viewed by 1807

Interactions between a solvent and their co-solute molecules in solutions of peptides are crucial for their stability and structure. The K-peptide is a synthetic fragment of a larger hen egg white lysozyme protein that is believed to be able to aggregate into amyloid [...] Read more.

Interactions between a solvent and their co-solute molecules in solutions of peptides are crucial for their stability and structure. The K-peptide is a synthetic fragment of a larger hen egg white lysozyme protein that is believed to be able to aggregate into amyloid structures. In this study, a complex experimental and theoretical approach is applied to study systems comprising the peptide, water, and two co-solutes: trimethylamide N-oxide (TMAO) or dimethyl sulfoxide (DMSO). Information about their interactions in solutions and on the stability of the K-peptide was obtained by FTIR spectroscopy and differential scanning microcalorimetry. The IR spectra of various osmolyte–water–model-peptide complexes were simulated with the DFT method (B3LYP/6-311++G(d,p)). The FTIR results indicate that both solutes are neutral for the K-peptide in solution. Both co-solutes affect the peptide to different degrees, as seen in the shape of its amide I band, and have different influences on its thermal stability. DFT calculations helped simplify the experimental data for easier interpretation. Full article

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24 pages, 54297 KiB

Open AccessArticle

Conformational Flexibility and Local Frustration in the Functional States of the SARS-CoV-2 Spike B.1.1.7 and B.1.351 Variants: Mutation-Induced Allosteric Modulation Mechanism of Functional Dynamics and Protein Stability

by Gennady Verkhivker

Int. J. Mol. Sci. 2022, 23(3), 1646; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23031646 - 31 Jan 2022

Cited by 2 | Viewed by 2489

Structural and functional studies of the SARS-CoV-2 spike proteins have recently determined distinct functional states of the B.1.1.7 and B.1.351 spike variants, providing a molecular framework for understanding the mechanisms that link the effect of mutations with the enhanced virus infectivity and transmissibility. [...] Read more.

Structural and functional studies of the SARS-CoV-2 spike proteins have recently determined distinct functional states of the B.1.1.7 and B.1.351 spike variants, providing a molecular framework for understanding the mechanisms that link the effect of mutations with the enhanced virus infectivity and transmissibility. A detailed dynamic and energetic analysis of these variants was undertaken in the present work to quantify the effects of different mutations on functional conformational changes and stability of the SARS-CoV-2 spike protein. We employed the efficient and accurate coarse-grained (CG) simulations of multiple functional states of the D614G mutant, B.1.1.7 and B.1.351 spike variants to characterize conformational dynamics of the SARS-CoV-2 spike proteins and identify dynamic signatures of the functional regions that regulate transitions between the closed and open forms. By combining molecular simulations with full atomistic reconstruction of the trajectories and the ensemble-based mutational frustration analysis, we characterized how the intrinsic flexibility of specific spike regions can control functional conformational changes required for binding with the host-cell receptor. Using the residue-based mutational scanning of protein stability, we determined protein stability hotspots and identified potential energetic drivers favoring the receptor-accessible open spike states for the B.1.1.7 and B.1.351 spike variants. The results suggested that modulation of the energetic frustration at the inter-protomer interfaces can serve as a mechanism for allosteric couplings between mutational sites and the inter-protomer hinges of functional motions. The proposed mechanism of mutation-induced energetic frustration may result in greater adaptability and the emergence of multiple conformational states in the open form. This study suggested that SARS-CoV-2 B.1.1.7 and B.1.351 variants may leverage the intrinsic plasticity of functional regions in the spike protein for mutation-induced modulation of protein dynamics and allosteric regulation to control binding with the host cell receptor. Full article

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13 pages, 3576 KiB

Open AccessArticle

New Evidence of the Importance of Weak Interactions in the Formation of PML-Bodies

by Alexander V. Fonin, Sergey A. Silonov, Anna S. Fefilova, Olesya V. Stepanenko, Anastasia A. Gavrilova, Alexey V. Petukhov, Anna E. Romanovich, Anna L. Modina, Tatiana S. Zueva, Evgeniy M. Nedelyaev, Nadejda M. Pleskach, Mirya L. Kuranova, Irina M. Kuznetsova, Vladimir N. Uversky and Konstantin K. Turoverov

Int. J. Mol. Sci. 2022, 23(3), 1613; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23031613 - 30 Jan 2022

Cited by 9 | Viewed by 3294

In this work, we performed a comparative study of the formation of PML bodies by full-length PML isoforms and their C-terminal domains in the presence and absence of endogenous PML. Based on the analysis of the distribution of intrinsic disorder predisposition in the [...] Read more.

In this work, we performed a comparative study of the formation of PML bodies by full-length PML isoforms and their C-terminal domains in the presence and absence of endogenous PML. Based on the analysis of the distribution of intrinsic disorder predisposition in the amino acid sequences of PML isoforms, regions starting from the amino acid residue 395 (i.e., sequences encoded by exons 4–6) were assigned as the C-terminal domains of these proteins. We demonstrate that each of the full-sized nuclear isoforms of PML is capable of forming nuclear liquid-droplet compartments in the absence of other PML isoforms. These droplets possess dynamic characteristics of the exchange with the nucleoplasm close to those observed in the wild-type cells. Only the C-terminal domains of the PML-II and PML-V isoforms are able to be included in the composition of the endogenous PML bodies, while being partially distributed in the nucleoplasm. The bodies formed by the C-terminal domain of the PML-II isoform are dynamic liquid droplet compartments, regardless of the presence or absence of endogenous PML. The C-terminal domain of PML-V forms dynamic liquid droplet compartments in the knockout cells (PML^−/−), but when the C-terminus of the PML-V isoform is inserted into the existing endogenous PML bodies, the molecules of this protein cease to exchange with the nucleoplasm. It was demonstrated that the K490R substitution, which disrupts the PML sumoylation, promotes diffuse distribution of the C-terminal domains of PML-II and PML-V isoforms in endogenous PML knockout HeLa cells, but not in the wild-type cells. These data indicate the ability of the C-terminal domains of the PML-II and PML-V isoforms to form dynamic liquid droplet-like compartments, regardless of the ordered N-terminal RBCC motifs of the PML. This indicates a significant role of the non-specific interactions between the mostly disordered C-terminal domains of PML isoforms for the initiation of liquid–liquid phase separation (LLPS) leading to the formation of PML bodies. Full article

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23 pages, 7205 KiB

Open AccessArticle

Insights to Human γD-Crystallin Unfolding by NMR Spectroscopy and Molecular Dynamics Simulations

by Shu-Shun Hsueh, S.-S. (Steven) Wang, Shu-Han Chen, Chia-Lin Wang, W. (Josephine) Wu and Ta-Hsien Lin

Int. J. Mol. Sci. 2022, 23(3), 1591; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23031591 - 29 Jan 2022

Cited by 1 | Viewed by 2243

Human γD-crystallin (HGDC) is an abundant lens protein residing in the nucleus of the human lens. Aggregation of this and other structural proteins within the lens leads to the development of cataract. Much has been explored on the stability and aggregation of HGDC [...] Read more.

Human γD-crystallin (HGDC) is an abundant lens protein residing in the nucleus of the human lens. Aggregation of this and other structural proteins within the lens leads to the development of cataract. Much has been explored on the stability and aggregation of HGDC and where detailed investigation at the atomic resolution was needed, the X-ray structure was used as an initial starting conformer for molecular modeling. In this study, we implemented NMR-solution HGDC structures as starting conformers for molecular dynamics simulations to provide the missing pieces of the puzzle on the very early stages of HGDC unfolding leading up to the domain swap theories proposed by past studies. The high-resolution details of the conformational dynamics also revealed additional insights to possible early intervention for cataractogenesis. Full article

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15 pages, 6329 KiB

Open AccessArticle

Slipknot or Crystallographic Error: A Computational Analysis of the Plasmodium falciparum DHFR Structural Folds

by Rolland B. Tata, Ali F. Alsulami, Olivier Sheik Amamuddy, Tom L. Blundell and Özlem Tastan Bishop

Int. J. Mol. Sci. 2022, 23(3), 1514; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23031514 - 28 Jan 2022

Cited by 2 | Viewed by 1873

The presence of protein structures with atypical folds in the Protein Data Bank (PDB) is rare and may result from naturally occurring knots or crystallographic errors. Proper characterisation of such folds is imperative to understanding the basis of naturally existing knots and correcting [...] Read more.

The presence of protein structures with atypical folds in the Protein Data Bank (PDB) is rare and may result from naturally occurring knots or crystallographic errors. Proper characterisation of such folds is imperative to understanding the basis of naturally existing knots and correcting crystallographic errors. If left uncorrected, such errors can frustrate downstream experiments that depend on the structures containing them. An atypical fold has been identified in P. falciparum dihydrofolate reductase (PfDHFR) between residues 20–51 (loop 1) and residues 191–205 (loop 2). This enzyme is key to drug discovery efforts in the parasite, necessitating a thorough characterisation of these folds. Using multiple sequence alignments (MSA), a unique insert was identified in loop 1 that exacerbates the appearance of the atypical fold-giving it a slipknot-like topology. However, PfDHFR has not been deposited in the knotted proteins database, and processing its structure failed to identify any knots within its folds. The application of protein homology modelling and molecular dynamics simulations on the DHFR domain of P. falciparum and those of two other organisms (E. coli and M. tuberculosis) that were used as molecular replacement templates in solving the PfDHFR structure revealed plausible unentangled or open conformations of these loops. These results will serve as guides for crystallographic experiments to provide further insights into the atypical folds identified. Full article

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18 pages, 4721 KiB

Open AccessArticle

Computational Analysis of Molnupiravir

by Artem V. Sharov, Tatyana M. Burkhanova, Tugba Taskın Tok, Maria G. Babashkina and Damir A. Safin

Int. J. Mol. Sci. 2022, 23(3), 1508; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23031508 - 28 Jan 2022

Cited by 27 | Viewed by 3700 | Correction

In this work, we report in-depth computational studies of three plausible tautomeric forms, generated through the migration of two acidic protons of the N⁴-hydroxylcytosine fragment, of molnupiravir, which is emerging as an efficient drug to treat COVID-19. The DFT calculations were [...] Read more.

In this work, we report in-depth computational studies of three plausible tautomeric forms, generated through the migration of two acidic protons of the N⁴-hydroxylcytosine fragment, of molnupiravir, which is emerging as an efficient drug to treat COVID-19. The DFT calculations were performed to verify the structure of these tautomers, as well as their electronic and optical properties. Molecular docking was applied to examine the influence of the structures of the keto-oxime, keto-hydroxylamine and hydroxyl-oxime tautomers on a series of the SARS-CoV-2 proteins. These tautomers exhibited the best affinity behavior (−9.90, −7.90, and −9.30 kcal/mol, respectively) towards RdRp-RTR and Nonstructural protein 3 (nsp3_range 207–379-MES). Full article

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16 pages, 3882 KiB

Open AccessArticle

Interaction between miR4749 and Human Serum Albumin as Revealed by Fluorescence, FRET, Atomic Force Spectroscopy and Computational Modelling

by Valentina Botti, Silvia Marrone, Salvatore Cannistraro and Anna Rita Bizzarri

Int. J. Mol. Sci. 2022, 23(3), 1291; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23031291 - 24 Jan 2022

Cited by 7 | Viewed by 1945

The interaction of Human Serum Albumin (HSA) with the microRNA, miR4749, was investigated by Atomic Force Spectrscopy (AFS), static and time-resolved fluorescence spectroscopy and by computational methods. The formation of a HSA/miR4749 complex with an affinity of about 10⁴ M⁻¹ has [...] Read more.

The interaction of Human Serum Albumin (HSA) with the microRNA, miR4749, was investigated by Atomic Force Spectrscopy (AFS), static and time-resolved fluorescence spectroscopy and by computational methods. The formation of a HSA/miR4749 complex with an affinity of about 10⁴ M⁻¹ has been assessed through a Stern–Volmer analysis of steady-state fluorescence quenching of the lone Trp residue (Trp214) emission of HSA. Förster Resonance Energy Transfer (FRET) measurements of fluorescence lifetime of the HSA/miR4749 complex were carried out in the absence and in the presence of an acceptor chromophore linked to miR4749. This allowed us to determine a distance of 4.3 ± 0.5 nm between the lone Trp of HSA and the dye bound to miR4749 5p-end. Such a distance was exploited for a screening of the possible binding sites between HSA and miR4749, as predicted by computational docking. Such an approach, further refined by binding free energy calculations, led us to the identification of a consistent model for the structure of the HSA/miR4749 complex in which a positively charged HSA pocket accommodates the negatively charged miRNA molecule. These results designate native HSA as a suitable miRNA carrier under physiological conditions for delivering to appropriate targets. Full article

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11 pages, 2029 KiB

Open AccessArticle

The Local Environment of Loop Switch 1 Modulates the Rate of ATP-Induced Dissociation of Human Cardiac Actomyosin

by Akhil Gargey and Yuri E. Nesmelov

Int. J. Mol. Sci. 2022, 23(3), 1220; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23031220 - 22 Jan 2022

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Two isoforms of human cardiac myosin, alpha and beta, share significant sequence similarities but show different kinetics. The alpha isoform is a faster motor; it spends less time being strongly bound to actin during the actomyosin cycle. With alpha isoform, actomyosin dissociates faster [...] Read more.

Two isoforms of human cardiac myosin, alpha and beta, share significant sequence similarities but show different kinetics. The alpha isoform is a faster motor; it spends less time being strongly bound to actin during the actomyosin cycle. With alpha isoform, actomyosin dissociates faster upon ATP binding, and the affinity of ADP to actomyosin is weaker. One can suggest that the isoform-specific actomyosin kinetics is regulated at the nucleotide binding site of human cardiac myosin. Myosin is a P-loop ATPase; the nucleotide-binding site consists of P-loop and loops switch 1 and 2. All three loops position MgATP for successful hydrolysis. Loops sequence is conserved in both myosin isoforms, and we hypothesize that the isoform-specific structural element near the active site regulates the rate of nucleotide binding and release. Previously we ran molecular dynamics simulations and found that loop S291-E317 near loop switch 1 is more compact and exhibits larger fluctuations of the position of amino acid residues in beta isoform than in alpha. In alpha isoform, the loop forms a salt bridge with loop switch 1, the bridge is not present in beta isoform. Two isoleucines I303 and I313 of loop S291-E317 are replaced with valines in alpha isoform. We introduced a double mutation I303V:I313V in beta isoform background and studied how the mutation affects the rate of ATP binding and ADP dissociation from actomyosin. We found that ATP-induced actomyosin dissociation occurs faster in the mutant, but the rate of ADP release remains the same as in the wild-type beta isoform. Due to the proximity of loop S291-E317 and loop switch 1, a faster rate of ATP-induced actomyosin dissociation indicates that loop S291-E317 affects structural dynamics of loop switch 1, and that loop switch 1 controls ATP binding to the active site. A similar rate of ADP dissociation from actomyosin in the mutant and wild-type myosin constructs indicates that loop switch 1 does not control ADP release from actomyosin. Full article

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21 pages, 5337 KiB

Open AccessArticle

Effect of Myosin Isoforms on Cardiac Muscle Twitch of Mice, Rats and Humans

by Momcilo Prodanovic, Michael A. Geeves, Corrado Poggesi, Michael Regnier and Srboljub M. Mijailovich

Int. J. Mol. Sci. 2022, 23(3), 1135; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23031135 - 20 Jan 2022

Cited by 9 | Viewed by 2447

To understand how pathology-induced changes in contractile protein isoforms modulate cardiac muscle function, it is necessary to quantify the temporal-mechanical properties of contractions that occur under various conditions. Pathological responses are much easier to study in animal model systems than in humans, but [...] Read more.

To understand how pathology-induced changes in contractile protein isoforms modulate cardiac muscle function, it is necessary to quantify the temporal-mechanical properties of contractions that occur under various conditions. Pathological responses are much easier to study in animal model systems than in humans, but extrapolation between species presents numerous challenges. Employing computational approaches can help elucidate relationships that are difficult to test experimentally by translating the observations from rats and mice, as model organisms, to the human heart. Here, we use the spatially explicit MUSICO platform to model twitch contractions from rodent and human trabeculae collected in a single laboratory. This approach allowed us to identify the variations in kinetic characteristics of α- and β-myosin isoforms across species and to quantify their effect on cardiac muscle contractile responses. The simulations showed how the twitch transient varied with the ratio of the two myosin isoforms. Particularly, the rate of tension rise was proportional to the fraction of α-myosin present, while the β-isoform dominated the rate of relaxation unless α-myosin was >50%. Moreover, both the myosin isoform and the Ca²⁺ transient contributed to the twitch tension transient, allowing two levels of regulation of twitch contraction. Full article

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8 pages, 1926 KiB

Open AccessArticle

Surface Engineering of Top7 to Facilitate Structure Determination

by Yuki Ito, Takuya Araki, Shota Shiga, Hiroyuki Konno and Koki Makabe

Int. J. Mol. Sci. 2022, 23(2), 701; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23020701 - 09 Jan 2022

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Top7 is a de novo designed protein whose amino acid sequence has no evolutional trace. Such a property makes Top7 a suitable scaffold for studying the pure nature of protein and protein engineering applications. To use Top7 as an engineering scaffold, we initially [...] Read more.

Top7 is a de novo designed protein whose amino acid sequence has no evolutional trace. Such a property makes Top7 a suitable scaffold for studying the pure nature of protein and protein engineering applications. To use Top7 as an engineering scaffold, we initially attempted structure determination and found that crystals of our construct, which lacked the terminal hexahistidine tag, showed weak diffraction in X-ray structure determination. Thus, we decided to introduce surface residue mutations to facilitate crystal structure determination. The resulting surface mutants, Top7sm1 and Top7sm2, crystallized easily and diffracted to the resolution around 1.7 Å. Despite the improved data, we could not finalize the structures due to high R values. Although we could not identify the origin of the high R values of the surface mutants, we found that all the structures shared common packing architecture with consecutive intermolecular β-sheet formation aligned in one direction. Thus, we mutated the intermolecular interface to disrupt the intermolecular β-sheet formation, expecting to form a new crystal packing. The resulting mutant, Top7sm2-I68R, formed new crystal packing interactions as intended and diffracted to the resolution of 1.4 Å. The surface mutations contributed to crystal packing and high resolution. We finalized the structure model with the R/R_free values of 0.20/0.24. Top7sm2-I68R can be a useful model protein due to its convenient structure determination. Full article

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13 pages, 2685 KiB

Open AccessArticle

Graphene Enhances Actin Filament Assembly Kinetics and Modulates NIH-3T3 Fibroblast Cell Spreading

by Jinho Park, Pavlo Kravchuk, Adithi Krishnaprasad, Tania Roy and Ellen Hyeran Kang

Int. J. Mol. Sci. 2022, 23(1), 509; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23010509 - 03 Jan 2022

Cited by 8 | Viewed by 2445

Actin plays critical roles in various cellular functions, including cell morphogenesis, differentiation, and movement. The assembly of actin monomers into double-helical filaments is regulated in surrounding microenvironments. Graphene is an attractive nanomaterial that has been used in various biomaterial applications, such as drug [...] Read more.

Actin plays critical roles in various cellular functions, including cell morphogenesis, differentiation, and movement. The assembly of actin monomers into double-helical filaments is regulated in surrounding microenvironments. Graphene is an attractive nanomaterial that has been used in various biomaterial applications, such as drug delivery cargo and scaffold for cells, due to its unique physical and chemical properties. Although several studies have shown the potential effects of graphene on actin at the cellular level, the direct influence of graphene on actin filament dynamics has not been studied. Here, we investigate the effects of graphene on actin assembly kinetics using spectroscopy and total internal reflection fluorescence microscopy. We demonstrate that graphene enhances the rates of actin filament growth in a concentration-dependent manner. Furthermore, cell morphology and spreading are modulated in mouse embryo fibroblast NIH-3T3 cultured on a graphene surface without significantly affecting cell viability. Taken together, these results suggest that graphene may have a direct impact on actin cytoskeleton remodeling. Full article

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2021

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15 pages, 6008 KiB

Open AccessArticle

Pulsed Electric Fields Alter Expression of NF-κB Promoter-Controlled Gene

by Justina Kavaliauskaitė, Auksė Kazlauskaitė, Juozas Rimantas Lazutka, Gatis Mozolevskis and Arūnas Stirkė

Int. J. Mol. Sci. 2022, 23(1), 451; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23010451 - 31 Dec 2021

Cited by 3 | Viewed by 2842

The possibility to artificially adjust and fine-tune gene expression is one of the key milestones in bioengineering, synthetic biology, and advanced medicine. Since the effects of proteins or other transgene products depend on the dosage, controlled gene expression is required for any applications, [...] Read more.

The possibility to artificially adjust and fine-tune gene expression is one of the key milestones in bioengineering, synthetic biology, and advanced medicine. Since the effects of proteins or other transgene products depend on the dosage, controlled gene expression is required for any applications, where even slight fluctuations of the transgene product impact its function or other critical cell parameters. In this context, physical techniques demonstrate optimistic perspectives, and pulsed electric field technology is a potential candidate for a noninvasive, biophysical gene regulator, exploiting an easily adjustable pulse generating device. We exposed mammalian cells, transfected with a NF-κB pathway-controlled transcription system, to a range of microsecond-duration pulsed electric field parameters. To prevent toxicity, we used protocols that would generate relatively mild physical stimulation. The present study, for the first time, proves the principle that microsecond-duration pulsed electric fields can alter single-gene expression in plasmid context in mammalian cells without significant damage to cell integrity or viability. Gene expression might be upregulated or downregulated depending on the cell line and parameters applied. This noninvasive, ligand-, cofactor-, nanoparticle-free approach enables easily controlled direct electrostimulation of the construct carrying the gene of interest; the discovery may contribute towards the path of simplification of the complexity of physical systems in gene regulation and create further synergies between electronics, synthetic biology, and medicine. Full article

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22 pages, 4053 KiB

Open AccessArticle

Integrative Study of the Structural and Dynamical Properties of a KirBac3.1 Mutant: Functional Implication of a Highly Conserved Tryptophan in the Transmembrane Domain

by Charline Fagnen, Ludovic Bannwarth, Iman Oubella, Dania Zuniga, Ahmed Haouz, Eric Forest, Rosa Scala, Saïd Bendahhou, Rita De Zorzi, David Perahia and Catherine Vénien-Bryan

Int. J. Mol. Sci. 2022, 23(1), 335; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23010335 - 29 Dec 2021

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ATP-sensitive potassium (K-ATP) channels are ubiquitously expressed on the plasma membrane of cells in several organs, including the heart, pancreas, and brain, and they govern a wide range of physiological processes. In pancreatic β-cells, K-ATP channels composed of Kir6.2 and SUR1 play a [...] Read more.

ATP-sensitive potassium (K-ATP) channels are ubiquitously expressed on the plasma membrane of cells in several organs, including the heart, pancreas, and brain, and they govern a wide range of physiological processes. In pancreatic β-cells, K-ATP channels composed of Kir6.2 and SUR1 play a key role in coupling blood glucose and insulin secretion. A tryptophan residue located at the cytosolic end of the transmembrane helix is highly conserved in eukaryote and prokaryote Kir channels. Any mutation on this amino acid causes a gain of function and neonatal diabetes mellitus. In this study, we have investigated the effect of mutation on this highly conserved residue on a KirBac channel (prokaryotic homolog of mammalian Kir6.2). We provide the crystal structure of the mutant KirBac3.1 W46R (equivalent to W68R in Kir6.2) and its conformational flexibility properties using HDX-MS. In addition, the detailed dynamical view of the mutant during the gating was investigated using the in silico method. Finally, functional assays have been performed. A comparison of important structural determinants for the gating mechanism between the wild type KirBac and the mutant W46R suggests interesting structural and dynamical clues and a mechanism of action of the mutation that leads to the gain of function. Full article

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15 pages, 5356 KiB

Open AccessArticle

Discrimination of Transgenic Canola (Brassica napus L.) and their Hybrids with B. rapa using Vis-NIR Spectroscopy and Machine Learning Methods

by Soo-In Sohn, Subramani Pandian, John-Lewis Zinia Zaukuu, Young-Ju Oh, Soo-Yun Park, Chae-Sun Na, Eun-Kyoung Shin, Hyeon-Jung Kang, Tae-Hun Ryu, Woo-Suk Cho and Youn-Sung Cho

Int. J. Mol. Sci. 2022, 23(1), 220; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23010220 - 25 Dec 2021

Cited by 10 | Viewed by 3897

In recent years, the rapid development of genetically modified (GM) technology has raised concerns about the safety of GM crops and foods for human health and the ecological environment. Gene flow from GM crops to other crops, especially in the Brassicaceae family, might [...] Read more.

In recent years, the rapid development of genetically modified (GM) technology has raised concerns about the safety of GM crops and foods for human health and the ecological environment. Gene flow from GM crops to other crops, especially in the Brassicaceae family, might pose a threat to the environment due to their weediness. Hence, finding reliable, quick, and low-cost methods to detect and monitor the presence of GM crops and crop products is important. In this study, we used visible near-infrared (Vis-NIR) spectroscopy for the effective discrimination of GM and non-GM Brassica napus, B. rapa, and F1 hybrids (B. rapa X GM B. napus). Initially, Vis-NIR spectra were collected from the plants, and the spectra were preprocessed. A combination of different preprocessing methods (four methods) and various modeling approaches (eight methods) was used for effective discrimination. Among the different combinations, the Savitzky-Golay and Support Vector Machine combination was found to be an optimal model in the discrimination of GM, non-GM, and hybrid plants with the highest accuracy rate (100%). The use of a Convolutional Neural Network with Normalization resulted in 98.9%. The same higher accuracy was found in the use of Gradient Boosted Trees and Fast Large Margin approaches. Later, phenolic acid concentration among the different plants was assessed using GC-MS analysis. Partial least squares regression analysis of Vis-NIR spectra and biochemical characteristics showed significant correlations in their respective changes. The results showed that handheld Vis-NIR spectroscopy combined with chemometric analyses could be used for the effective discrimination of GM and non-GM B. napus, B. rapa, and F1 hybrids. Biochemical composition analysis can also be combined with the Vis-NIR spectra for efficient discrimination. Full article

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11 pages, 4053 KiB

Open AccessArticle

Site-Search Process for Synaptic Protein-DNA Complexes

by Sridhar Vemulapalli, Mohtadin Hashemi and Yuri L. Lyubchenko

Int. J. Mol. Sci. 2022, 23(1), 212; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23010212 - 25 Dec 2021

Cited by 4 | Viewed by 2274

The assembly of synaptic protein-DNA complexes by specialized proteins is critical for bringing together two distant sites within a DNA molecule or bridging two DNA molecules. The assembly of such synaptosomes is needed in numerous genetic processes requiring the interactions of two or [...] Read more.

The assembly of synaptic protein-DNA complexes by specialized proteins is critical for bringing together two distant sites within a DNA molecule or bridging two DNA molecules. The assembly of such synaptosomes is needed in numerous genetic processes requiring the interactions of two or more sites. The molecular mechanisms by which the protein brings the sites together, enabling the assembly of synaptosomes, remain unknown. Such proteins can utilize sliding, jumping, and segmental transfer pathways proposed for the single-site search process, but none of these pathways explains how the synaptosome assembles. Here we used restriction enzyme SfiI, that requires the assembly of synaptosome for DNA cleavage, as our experimental system and applied time-lapse, high-speed AFM to directly visualize the site search process accomplished by the SfiI enzyme. For the single-site SfiI-DNA complexes, we were able to directly visualize such pathways as sliding, jumping, and segmental site transfer. However, within the synaptic looped complexes, we visualized the threading and site-bound segment transfer as the synaptosome-specific search pathways for SfiI. In addition, we visualized sliding and jumping pathways for the loop dissociated complexes. Based on our data, we propose the site-search model for synaptic protein-DNA systems. Full article

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35 pages, 7931 KiB

Open AccessArticle

Modifications of EHPDB Physical Properties through Doping with Fe₂O₃ Nanoparticles (Part II)

by Sebastian Lalik, Olaf Stefańczyk, Natalia Górska, Kunal Kumar, Shin-ichi Ohkoshi and Monika Marzec

Int. J. Mol. Sci. 2022, 23(1), 50; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23010050 - 21 Dec 2021

Cited by 1 | Viewed by 2517

The aim of our study was to analyze the influence of various concentrations of γ-Fe₂O₃ nanoparticles on the physical properties of the liquid crystalline ferroelectric SmC* phase, as well as to check the effect of introducing nanoparticles in the [...] Read more.

The aim of our study was to analyze the influence of various concentrations of γ-Fe₂O₃ nanoparticles on the physical properties of the liquid crystalline ferroelectric SmC* phase, as well as to check the effect of introducing nanoparticles in the LC matrix on their properties in the prepared five nanocomposites. UV-vis spectroscopy showed that the admixture reduced the absorption of nanocomposites in the UV range, additional absorption bands appeared, and all nanocomposites were transparent in the range of 500–850 nm. The molecular dynamics in particular phases of the nanocomposites were investigated by the dielectric spectroscopy method, and it was found that nanoparticles caused a significant increase in the dielectric constant at low frequencies, a strong modification of the dielectric processes in the SmC* phase, and the emergence of new relaxation processes for the highest dopant concentrations. SQUID magnetometry allowed us to determine the magnetic nature of the nanoparticles used, and to show that the blocked state of nanoparticles was preserved in nanocomposites (hysteresis loops were also registered in the ferroelectric SmC* phase). The dependence of the coercive field on the admixture concentration and the widening of the hysteresis loop in nanocomposites in relation to pure nanoparticles were also found. In turn, the FT-MIR spectroscopy method was used to check the influence of the impurity concentration on the formation/disappearance or modification of the absorption bands, and the modification of both the FWHM and the maximum positions for the four selected vibrations in the MIR range, as well as the discontinuous behavior of these parameters at the phase transitions, were found. Full article

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23 pages, 4065 KiB

Open AccessArticle

Aroylhydrazone Diorganotin Complexes Causes DNA Damage and Apoptotic Cell Death: From Chemical Synthesis to Biochemical Effects

by Wujiu Jiang, Yuxing Tan and Yiyuan Peng

Int. J. Mol. Sci. 2021, 22(24), 13525; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222413525 - 16 Dec 2021

Cited by 11 | Viewed by 2204

Under microwave irradiation, eighteen new aroylhydrazone diorganotin complexes (1a–9b) were produced through the reaction of aroylhydrazine, 2-ketobutyric acid, and the corresponding diorganotin. Fourier transform infrared spectroscopy, ¹H, ¹³C, and ¹¹⁹Sn nuclear magnetic resonance spectroscopies, high-resolution mass [...] Read more.

Under microwave irradiation, eighteen new aroylhydrazone diorganotin complexes (1a–9b) were produced through the reaction of aroylhydrazine, 2-ketobutyric acid, and the corresponding diorganotin. Fourier transform infrared spectroscopy, ¹H, ¹³C, and ¹¹⁹Sn nuclear magnetic resonance spectroscopies, high-resolution mass spectroscopy, X-ray crystallography, and thermogravimetric analysis (TGA) were performed to characterize the complexes. The in vitro anticancer activity for complexes were assessed using a CCK-8 assay on human cancer cells of HepG2, NCI-H460, and MCF-7. Complex 4b revealed more intensive anticancer activity against MCF-7 cells than the other complexes and cisplatin. Flow cytometry analysis and transmission electron microscope observation demonstrated that complex 4b mediated cell apoptosis of MCF-7 cells and arrested cell cycle in S phase. Western blotting analysis showed that 4b induced DNA damage in MCF-7 cells and led to apoptosis by the ATM-CHK2-p53 pathway. The single cell gel electrophoreses assay results showed that 4b induced DNA damage. The DNA binding activity of 4b was studied by UV–Visible absorption spectrometry, fluorescence competitive, viscosity measurements, gel electrophoresis, and molecular docking, and the results show that 4b can be well embedded in the groove and cleave DNA. Full article

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16 pages, 2833 KiB

Open AccessArticle

Evaluation of the Physico-Chemical Properties of Liposomes Assembled from Bioconjugates of Anisic Acid with Phosphatidylcholine

by Hanna Pruchnik, Anna Gliszczyńska and Aleksandra Włoch

Int. J. Mol. Sci. 2021, 22(23), 13146; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222313146 - 05 Dec 2021

Cited by 6 | Viewed by 2164

The aim of this work was the evaluation of the physico-chemical properties of a new type of liposomes that are composed of DPPC and bioconjugates of anisic acid with phosphatidylcholine. In particular, the impact of modified anisic acid phospholipids on the thermotropic parameters [...] Read more.

The aim of this work was the evaluation of the physico-chemical properties of a new type of liposomes that are composed of DPPC and bioconjugates of anisic acid with phosphatidylcholine. In particular, the impact of modified anisic acid phospholipids on the thermotropic parameters of liposomes was determined, which is crucial for using them as potential carriers of active substances in cancer therapies. Their properties were determined using three biophysical methods, namely differential scanning calorimetry (DSC), steady-state fluorimetry and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Moreover, temperature studies of liposomes composed of DPPC and bioconjugates of anisic acid with phosphatidylcholine provided information about the phase transition, fluidity regarding chain order, hydration and dynamics. The DSC results show that the main phase transition peak for conjugates of anisic acid with phosphatidylcholine molecules was broadened and shifted to a lower temperature in a concentration- and structure-dependent manner. The ATR-FTIR results and the results of measurements conducted using fluorescent probes located at different regions in the lipid bilayer are in line with DSC. The results show that the new bioconjugates with phosphatidylcholine have a significant impact on the physico-chemical properties of a membrane and cause a decrease in the temperature of the main phase transition. The consequence of this is greater fluidity of the lipid bilayer. Full article

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12 pages, 2301 KiB

Open AccessArticle

Validation of a Lab-on-Chip Assay for Measuring Sorafenib Effectiveness on HCC Cell Proliferation

by Emanuele Piccinno, Anna Grazia Monteduro, Francesco Dituri, Silvia Rizzato, Gianluigi Giannelli and Giuseppe Maruccio

Int. J. Mol. Sci. 2021, 22(23), 13090; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222313090 - 03 Dec 2021

Cited by 8 | Viewed by 1770

Hepatocellular carcinoma (HCC) is a highly lethal cancer, and although a few drugs are available for treatment, therapeutic effectiveness is still unsatisfactory. New drugs are urgently needed for hepatocellular carcinoma (HCC) patients. In this context, reliable preclinical assays are of paramount importance to [...] Read more.

Hepatocellular carcinoma (HCC) is a highly lethal cancer, and although a few drugs are available for treatment, therapeutic effectiveness is still unsatisfactory. New drugs are urgently needed for hepatocellular carcinoma (HCC) patients. In this context, reliable preclinical assays are of paramount importance to screen the effectiveness of new drugs and, in particular, measure their effects on HCC cell proliferation. However, cell proliferation measurement is a time-consuming and operator-dependent procedure. The aim of this study was to validate an engineered miniaturized on-chip platform for real-time, non-destructive cell proliferation assays and drug screening. The effectiveness of Sorafenib, the first-line drug mainly used for patients with advanced HCC, was tested in parallel, comparing the gold standard 96-well-plate assay and our new lab-on-chip platform. Results from the lab-on-chip are consistent in intra-assay replicates and comparable to the output of standard crystal violet proliferation assays for assessing Sorafenib effectiveness on HCC cell proliferation. The miniaturized platform presents several advantages in terms of lesser reagents consumption, operator time, and costs, as well as overcoming a number of technical and operator-dependent pitfalls. Moreover, the number of cells required is lower, a relevant issue when primary cell cultures are used. In conclusion, the availability of inexpensive on-chip assays can speed up drug development, especially by using patient-derived samples to take into account disease heterogeneity and patient-specific characteristics. Full article

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30 pages, 997 KiB

Open AccessReview

A Systematic Review on Nanoencapsulation Natural Antimicrobials in Foods: In Vitro versus In Situ Evaluation, Mechanisms of Action and Implications on Physical-Chemical Quality

by Carini Aparecida Lelis, Anna Paula Azevedo de Carvalho and Carlos Adam Conte Junior

Int. J. Mol. Sci. 2021, 22(21), 12055; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222112055 - 08 Nov 2021

Cited by 11 | Viewed by 2164

Natural antimicrobials (NA) have stood out in the last decade due to the growing demand for reducing chemical preservatives in food. Once solubility, stability, and changes in sensory attributes could limit their applications in foods, several studies were published suggesting micro-/nanoencapsulation to overcome [...] Read more.

Natural antimicrobials (NA) have stood out in the last decade due to the growing demand for reducing chemical preservatives in food. Once solubility, stability, and changes in sensory attributes could limit their applications in foods, several studies were published suggesting micro-/nanoencapsulation to overcome such challenges. Thus, for our systematic review the Science Direct, Web of Science, Scopus, and Pub Med databases were chosen to recover papers published from 2010 to 2020. After reviewing all titles/abstracts and keywords for the full-text papers, key data were extracted and synthesized. The systematic review proposed to compare the antimicrobial efficacy between nanoencapsulated NA (nNA) and its free form in vitro and in situ studies, since although in vitro studies are often used in studies, they present characteristics and properties that are different from those found in foods; providing a comprehensive understanding of primary mechanisms of action of the nNA in foods; and analyzing the effects on quality parameters of foods. Essential oils and nanoemulsions (10.9–100 nm) have received significant attention and showed higher antimicrobial efficacy without sensory impairments compared to free NA. Regarding nNA mechanisms: (i) nanoencapsulation provides a slow-prolonged release to promote antimicrobial action over time, and (ii) prevents interactions with food constituents that in turn impair antimicrobial action. Besides in vitro antifungal and antibacterial, nNA also demonstrated antioxidant activity—potential to shelf life extension in food. However, of the studies involving nanoencapsulated natural antimicrobials used in this review, little attention was placed on proximate composition, sensory, and rheological evaluation. We encourage further in situ studies once data differ from in vitro assay, suggesting food matrix greatly influences NA mechanisms. Full article

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9 pages, 1422 KiB

Open AccessCommunication

Low-Coverage Whole Genome Sequencing Using Laser Capture Microscopy with Combined Digital Droplet PCR: An Effective Tool to Study Copy Number and Kras Mutations in Early Lung Adenocarcinoma Development

by Elizabeth A. Mickler, Huaxin Zhou, Tzu L. Phang, Mark W. Geraci, Robert S. Stearman and Catherine R. Sears

Int. J. Mol. Sci. 2021, 22(21), 12034; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222112034 - 06 Nov 2021

Cited by 1 | Viewed by 1823

Defining detailed genomic characterization of early tumor progression is critical to identifying key regulators and pathways in carcinogenesis as potentially druggable targets. In human lung cancer, work to characterize early cancer development has mainly focused on squamous cancer, as the earliest lesions are [...] Read more.

Defining detailed genomic characterization of early tumor progression is critical to identifying key regulators and pathways in carcinogenesis as potentially druggable targets. In human lung cancer, work to characterize early cancer development has mainly focused on squamous cancer, as the earliest lesions are more proximal in the airways and often accessible by repeated bronchoscopy. Adenocarcinomas are typically located distally in the lung, limiting accessibility for biopsy of pre-malignant and early stages. Mouse lung cancer models recapitulate many human genomic features and provide a model for tumorigenesis with pre-malignant atypical adenomatous hyperplasia and in situ adenocarcinomas often developing contemporaneously within the same animal. Here, we combined tissue characterization and collection by laser capture microscopy (LCM) with digital droplet PCR (ddPCR) and low-coverage whole genome sequencing (LC-WGS). ddPCR can be used to identify specific missense mutations in Kras (Kirsten rat sarcoma viral oncogene homolog, here focused on Kras Q61) and estimate the percentage of mutation predominance. LC-WGS is a cost-effective method to infer localized copy number alterations (CNAs) across the genome using low-input DNA. Combining these methods, the histological stage of lung cancer can be correlated with appearance of Kras mutations and CNAs. The utility of this approach is adaptable to other mouse models of human cancer. Full article

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16 pages, 2801 KiB

Open AccessArticle

Exploring the Assembly of Resorc[4]arenes for the Construction of Supramolecular Nano-Aggregates

by Fabio Buonsenso, Francesca Ghirga, Isabella Romeo, Gabriella Siani, Serena Pilato, Deborah Quaglio, Marco Pierini, Bruno Botta and Andrea Calcaterra

Int. J. Mol. Sci. 2021, 22(21), 11785; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222111785 - 29 Oct 2021

Cited by 4 | Viewed by 2098

Many biologically active compounds feature low solubility in aqueous media and, thus, poor bioavailability. The formation of the host-guest complex by using calixarene-based macrocycles (i.e., resorcinol-derived cyclic oligomers) with a good solubility profile can improve solubilization of hydrophobic drugs. Herein, we explore the [...] Read more.

Many biologically active compounds feature low solubility in aqueous media and, thus, poor bioavailability. The formation of the host-guest complex by using calixarene-based macrocycles (i.e., resorcinol-derived cyclic oligomers) with a good solubility profile can improve solubilization of hydrophobic drugs. Herein, we explore the ability of resorc[4]arenes to self-assemble in polar solutions, to form supramolecular aggregates, and to promote water-solubility of an isoflavone endowed with anti-cancer activity, namely Glabrescione B (GlaB). Accordingly, we synthesized several architectures featuring a different pattern of substitution on the upper rim including functional groups able to undergo acid dissociation (i.e., carboxyl and hydroxyl groups). The aggregation phenomenon of the amphiphilic resorc[4]arenes has been investigated in a THF/water solution by UV–visible spectroscopy, at different pH values. Based on their ionization properties, we demonstrated that the supramolecular assembly of resorc[4]arene-based systems can be modulated at given pH values, and thus promoting the solubility of GlaB. Full article

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26 pages, 3945 KiB

Open AccessArticle

Investigating Pathogenetic Mechanisms of Alzheimer’s Disease by Systems Biology Approaches for Drug Discovery

by Shan-Ju Yeh, Ming-Hsun Chung and Bor-Sen Chen

Int. J. Mol. Sci. 2021, 22(20), 11280; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222011280 - 19 Oct 2021

Cited by 3 | Viewed by 4615

Alzheimer’s disease (AD) is the most common cause of dementia, characterized by progressive cognitive decline and neurodegenerative disorder. Abnormal aggregations of intracellular neurofibrillary tangles (NFTs) and unusual accumulations of extracellular amyloid-β (Aβ) peptides are two important pathological features in AD brains. However, in [...] Read more.

Alzheimer’s disease (AD) is the most common cause of dementia, characterized by progressive cognitive decline and neurodegenerative disorder. Abnormal aggregations of intracellular neurofibrillary tangles (NFTs) and unusual accumulations of extracellular amyloid-β (Aβ) peptides are two important pathological features in AD brains. However, in spite of large-scale clinical studies and computational simulations, the molecular mechanisms of AD development and progression are still unclear. In this study, we divided all of the samples into two groups: early stage (Braak score I–III) and later stage (Braak score IV–VI). By big database mining, the candidate genetic and epigenetic networks (GEN) have been constructed. In order to find out the real GENs for two stages of AD, we performed systems identification and system order detection scheme to prune false positives with the help of corresponding microarray data. Applying the principal network projection (PNP) method, core GENs were extracted from real GENs based on the projection values. By the annotation of KEGG pathway, we could obtain core pathways from core GENs and investigate pathogenetic mechanisms for the early and later stage of AD, respectively. Consequently, according to pathogenetic mechanisms, several potential biomarkers are identified as drug targets for multiple-molecule drug design in the treatment of AD. Full article

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21 pages, 4074 KiB

Open AccessArticle

Photophysical Properties of BADAN Revealed in the Study of GGBP Structural Transitions

by Alexander V. Fonin, Sergey A. Silonov, Iuliia A. Antifeeva, Olga V. Stepanenko, Olesya V. Stepanenko, Anna S. Fefilova, Olga I. Povarova, Anastasia A. Gavrilova, Irina M. Kuznetsova and Konstantin K. Turoverov

Int. J. Mol. Sci. 2021, 22(20), 11113; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222011113 - 15 Oct 2021

Cited by 2 | Viewed by 2100

The fluorescent dye BADAN (6-bromoacetyl-2-dimetylaminonaphtalene) is widely used in various fields of life sciences, however, the photophysical properties of BADAN are not fully understood. The study of the spectral properties of BADAN attached to a number of mutant forms of GGBP, as well [...] Read more.

The fluorescent dye BADAN (6-bromoacetyl-2-dimetylaminonaphtalene) is widely used in various fields of life sciences, however, the photophysical properties of BADAN are not fully understood. The study of the spectral properties of BADAN attached to a number of mutant forms of GGBP, as well as changes in its spectral characteristics during structural changes in proteins, allowed to shed light on the photophysical properties of BADAN. It was shown that spectral properties of BADAN are determined by at least one non-fluorescent and two fluorescent isomers with overlapping absorbing bands. It was found that BADAN fluorescence is determined by the unsolvated “PICT” (planar intramolecular charge transfer state) and solvated “TICT” (twisted intramolecular charge transfer state) excited states. While “TICT” state can be formed both as a result of the “PICT” state solvation and as a result of light absorption by the solvated ground state of the dye. BADAN fluorescence linked to GGBP/H152C apoform is quenched by Trp 183, but this effect is inhibited by glucose intercalation. New details of the changes in the spectral characteristics of BADAN during the unfolding of the protein apo and holoforms have been obtained. Full article

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13 pages, 2976 KiB

Open AccessArticle

Label-Free, Real-Time Measurement of Metabolism of Adherent and Suspended Single Cells by In-Cell Fourier Transform Infrared Microspectroscopy

by Tommaso Vannocci, Luca Quaroni, Antonio de Riso, Giulia Milordini, Magda Wolna, Gianfelice Cinque and Annalisa Pastore

Int. J. Mol. Sci. 2021, 22(19), 10742; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms221910742 - 04 Oct 2021

Cited by 2 | Viewed by 1826

We used infrared (IR) microscopy to monitor in real-time the metabolic turnover of individual mammalian cells in morphologically different states. By relying on the intrinsic absorption of mid-IR light by molecular components, we could discriminate the metabolism of adherent cells as compared to [...] Read more.

We used infrared (IR) microscopy to monitor in real-time the metabolic turnover of individual mammalian cells in morphologically different states. By relying on the intrinsic absorption of mid-IR light by molecular components, we could discriminate the metabolism of adherent cells as compared to suspended cells. We identified major biochemical differences between the two cellular states, whereby only adherent cells appeared to rely heavily on glycolytic turnover and lactic fermentation. We also report spectroscopic variations that appear as spectral oscillations in the IR domain, observed only when using synchrotron infrared radiation. We propose that this effect could be used as a reporter of the cellular conditions. Our results are instrumental in establishing IR microscopy as a label-free method for real-time metabolic studies of individual cells in different morphological states, and in more complex cellular ensembles. Full article

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17 pages, 2316 KiB

Open AccessArticle

Reducing Myosin II and ATP-Dependent Mechanical Activity Increases Order and Stability of Intracellular Organelles

by Ishay Wohl and Eilon Sherman

Int. J. Mol. Sci. 2021, 22(19), 10369; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms221910369 - 26 Sep 2021

Cited by 3 | Viewed by 1854

Organization of intracellular content is affected by multiple simultaneous processes, including diffusion in a viscoelastic and structured environment, intracellular mechanical work and vibrations. The combined effects of these processes on intracellular organization are complex and remain poorly understood. Here, we studied the organization [...] Read more.

Organization of intracellular content is affected by multiple simultaneous processes, including diffusion in a viscoelastic and structured environment, intracellular mechanical work and vibrations. The combined effects of these processes on intracellular organization are complex and remain poorly understood. Here, we studied the organization and dynamics of a free Ca⁺⁺ probe as a small and mobile tracer in live T cells. Ca⁺⁺, highlighted by Fluo-4, is localized in intracellular organelles. Inhibiting intracellular mechanical work by myosin II through blebbistatin treatment increased cellular dis-homogeneity of Ca⁺⁺-rich features in length scale < 1.1 μm. We detected a similar effect in cells imaged by label-free bright-field (BF) microscopy, in mitochondria-highlighted cells and in ATP-depleted cells. Blebbistatin treatment also reduced the dynamics of the Ca⁺⁺-rich features and generated prominent negative temporal correlations in their signals. Following Guggenberger et al. and numerical simulations, we suggest that diffusion in the viscoelastic and confined medium of intracellular organelles may promote spatial dis-homogeneity and stability of their content. This may be revealed only after inhibiting intracellular mechanical work and related cell vibrations. Our described mechanisms may allow the cell to control its organization via balancing its viscoelasticity and mechanical activity, with implications to cell physiology in health and disease. Full article

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17 pages, 2993 KiB

Open AccessReview

Lipid Self-Assemblies under the Atomic Force Microscope

by Aritz B. García-Arribas, Félix M. Goñi and Alicia Alonso

Int. J. Mol. Sci. 2021, 22(18), 10085; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms221810085 - 18 Sep 2021

Cited by 5 | Viewed by 2954

Lipid model membranes are important tools in the study of biophysical processes such as lipid self-assembly and lipid–lipid interactions in cell membranes. The use of model systems to adequate and modulate complexity helps in the understanding of many events that occur in cellular [...] Read more.

Lipid model membranes are important tools in the study of biophysical processes such as lipid self-assembly and lipid–lipid interactions in cell membranes. The use of model systems to adequate and modulate complexity helps in the understanding of many events that occur in cellular membranes, that exhibit a wide variety of components, including lipids of different subfamilies (e.g., phospholipids, sphingolipids, sterols…), in addition to proteins and sugars. The capacity of lipids to segregate by themselves into different phases at the nanoscale (nanodomains) is an intriguing feature that is yet to be fully characterized in vivo due to the proposed transient nature of these domains in living systems. Model lipid membranes, instead, have the advantage of (usually) greater phase stability, together with the possibility of fully controlling the system lipid composition. Atomic force microscopy (AFM) is a powerful tool to detect the presence of meso- and nanodomains in a lipid membrane. It also allows the direct quantification of nanomechanical resistance in each phase present. In this review, we explore the main kinds of lipid assemblies used as model membranes and describe AFM experiments on model membranes. In addition, we discuss how these assemblies have extended our knowledge of membrane biophysics over the last two decades, particularly in issues related to the variability of different model membranes and the impact of supports/cytoskeleton on lipid behavior, such as segregated domain size or bilayer leaflet uncoupling. Full article

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15 pages, 2593 KiB

Open AccessArticle

Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure

by Jorge Barroca-Ferreira, Pedro Cruz-Vicente, Marino F. A. Santos, Sandra M. Rocha, Teresa Santos-Silva, Cláudio J. Maia and Luís A. Passarinha

Int. J. Mol. Sci. 2021, 22(18), 10012; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms221810012 - 16 Sep 2021

Cited by 5 | Viewed by 2717

Background: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. Methods: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was [...] Read more.

Background: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. Methods: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was evaluated by manipulating the ionic strength of binding and elution phases, followed by a Co-Immunoprecipitation (Co-IP) polishing. Several potential stabilizing additives were assessed, and the melting temperature (Tm) values ranked the best/worst compounds. The secondary structure of STEAP1 was identified by circular dichroism. Results: The STEAP1 was not fully captured with 1.375 M (Butyl), in contrast with interfering heterologous proteins, which were strongly retained and mostly eluted with water. This single step demonstrated higher selectivity of Butyl-Sepharose for host impurities removal from injected crude samples. Co-IP allowed recovering a purified fraction of STEAP1 and contributed to unveil potential physiologically interacting counterparts with the target. A Tm of ~55 °C was determined, confirming STEAP1 stability in the purification buffer. A predominant α-helical structure was identified, ensuring the protein’s structural stability. Conclusions: A method for successfully isolating human STEAP1 from LNCaP cells was provided, avoiding the use of detergents to achieve stability, even outside a membrane-mimicking environment. Full article

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16 pages, 2717 KiB

Open AccessReview

An Overview of Near Infrared Spectroscopy and Its Applications in the Detection of Genetically Modified Organisms

by Soo-In Sohn, Subramani Pandian, Young-Ju Oh, John-Lewis Zinia Zaukuu, Hyeon-Jung Kang, Tae-Hun Ryu, Woo-Suk Cho, Youn-Sung Cho, Eun-Kyoung Shin and Byoung-Kwan Cho

Int. J. Mol. Sci. 2021, 22(18), 9940; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22189940 - 14 Sep 2021

Cited by 15 | Viewed by 4442

Near-infrared spectroscopy (NIRS) has become a more popular approach for quantitative and qualitative analysis of feeds, foods and medicine in conjunction with an arsenal of chemometric tools. This was the foundation for the increased importance of NIRS in other fields, like genetics and [...] Read more.

Near-infrared spectroscopy (NIRS) has become a more popular approach for quantitative and qualitative analysis of feeds, foods and medicine in conjunction with an arsenal of chemometric tools. This was the foundation for the increased importance of NIRS in other fields, like genetics and transgenic monitoring. A considerable number of studies have utilized NIRS for the effective identification and discrimination of plants and foods, especially for the identification of genetically modified crops. Few previous reviews have elaborated on the applications of NIRS in agriculture and food, but there is no comprehensive review that compares the use of NIRS in the detection of genetically modified organisms (GMOs). This is particularly important because, in comparison to previous technologies such as PCR and ELISA, NIRS offers several advantages, such as speed (eliminating time-consuming procedures), non-destructive/non-invasive analysis, and is inexpensive in terms of cost and maintenance. More importantly, this technique has the potential to measure multiple quality components in GMOs with reliable accuracy. In this review, we brief about the fundamentals and versatile applications of NIRS for the effective identification of GMOs in the agricultural and food systems. Full article

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13 pages, 1988 KiB

Open AccessArticle

Muscimol Directly Activates the TREK-2 Channel Expressed in GABAergic Neurons through Its N-Terminus

by Eun-Jin Kim, Oh-Sang Kwon, Chang-Gi Hur, Marie Merci Nyiramana, Dong-Kun Lee, Seong-Geun Hong, Jaehee Han and Dawon Kang

Int. J. Mol. Sci. 2021, 22(17), 9320; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22179320 - 27 Aug 2021

Cited by 1 | Viewed by 2821

The two-pore domain K⁺ (K_2P) channel, which is involved in setting the resting membrane potential in neurons, is an essential target for receptor agonists. Activation of the γ-aminobutyric acid (GABA) receptors (GABA_AR and GABA_BR) reduces cellular [...] Read more.

The two-pore domain K⁺ (K_2P) channel, which is involved in setting the resting membrane potential in neurons, is an essential target for receptor agonists. Activation of the γ-aminobutyric acid (GABA) receptors (GABA_AR and GABA_BR) reduces cellular excitability through Cl^- influx and K⁺ efflux in neurons. Relatively little is known about the link between GABA_AR and the K⁺ channel. The present study was performed to identify the effect of GABAR agonists on K_2P channel expression and activity in the neuroblastic B35 cells that maintain glutamic acid decarboxylase (GAD) activity and express GABA. TASK and TREK/TRAAK mRNA were expressed in B35 cells with a high level of TREK-2 and TRAAK. In addition, TREK/TRAAK proteins were detected in the GABAergic neurons obtained from GABA transgenic mice. Furthermore, TREK-2 mRNA and protein expression levels were markedly upregulated in B35 cells by GABA_AR and GABA_BR agonists. In particular, muscimol, a GABA_AR agonist, significantly increased TREK-2 expression and activity, but the effect was reduced in the presence of the GABA_AR antagonist bicuculine or TREK-2 inhibitor norfluoxetine. In the whole-cell and single-channel patch configurations, muscimol increased TREK-2 activity, but the muscimol effect disappeared in the N-terminal deletion mutant. These results indicate that muscimol directly induces TREK-2 activation through the N-terminus and suggest that muscimol can reduce cellular excitability by activating the TREK-2 channel and by inducing Cl^- influx in GABAergic neurons. Full article

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16 pages, 5035 KiB

Open AccessArticle

Decreased Interactions between Calmodulin and a Mutant Huntingtin Model Might Reduce the Cytotoxic Level of Intracellular Ca²⁺: A Molecular Dynamics Study

by Sanda Nastasia Moldovean and Vasile Chiş

Int. J. Mol. Sci. 2021, 22(16), 9025; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22169025 - 21 Aug 2021

Cited by 2 | Viewed by 2788

Mutant huntingtin (m-HTT) proteins and calmodulin (CaM) co-localize in the cerebral cortex with significant effects on the intracellular calcium levels by altering the specific calcium-mediated signals. Furthermore, the mutant huntingtin proteins show great affinity for CaM that can lead to a further stabilization [...] Read more.

Mutant huntingtin (m-HTT) proteins and calmodulin (CaM) co-localize in the cerebral cortex with significant effects on the intracellular calcium levels by altering the specific calcium-mediated signals. Furthermore, the mutant huntingtin proteins show great affinity for CaM that can lead to a further stabilization of the mutant huntingtin aggregates. In this context, the present study focuses on describing the interactions between CaM and two huntingtin mutants from a biophysical point of view, by using classical Molecular Dynamics techniques. The huntingtin models consist of a wild-type structure, one mutant with 45 glutamine residues and the second mutant with nine additional key-point mutations from glutamine residues into proline residues (9P(EM) model). Our docking scores and binding free energy calculations show higher binding affinities of all HTT models for the C-lobe end of the CaM protein. In terms of dynamic evolution, the 9P(EM) model triggered great structural changes into the CaM protein’s structure and shows the highest fluctuation rates due to its structural transitions at the helical level from α-helices to turns and random coils. Moreover, our proposed 9P(EM) model suggests much lower interaction energies when compared to the 45Qs-HTT mutant model, this finding being in good agreement with the 9P(EM)’s antagonistic effect hypothesis on highly toxic protein–protein interactions. Full article

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18 pages, 4927 KiB

Open AccessArticle

BRAF Modulates Stretch-Induced Intercellular Gap Formation through Localized Actin Reorganization

by Anna Hollósi, Katalin Pászty, Miklós Kellermayer, Guillaume Charras and Andrea Varga

Int. J. Mol. Sci. 2021, 22(16), 8989; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22168989 - 20 Aug 2021

Cited by 1 | Viewed by 2053

Mechanical forces acting on cell–cell adhesion modulate the barrier function of endothelial cells. The actively remodeled actin cytoskeleton impinges on cell–cell adhesion to counteract external forces. We applied stress on endothelial monolayers by mechanical stretch to uncover the role of BRAF in the [...] Read more.

Mechanical forces acting on cell–cell adhesion modulate the barrier function of endothelial cells. The actively remodeled actin cytoskeleton impinges on cell–cell adhesion to counteract external forces. We applied stress on endothelial monolayers by mechanical stretch to uncover the role of BRAF in the stress-induced response. Control cells responded to external forces by organizing and stabilizing actin cables in the stretched cell junctions. This was accompanied by an increase in intercellular gap formation, which was prevented in BRAF knockdown monolayers. In the absence of BRAF, there was excess stress fiber formation due to the enhanced reorganization of actin fibers. Our findings suggest that stretch-induced intercellular gap formation, leading to a decrease in barrier function of blood vessels, can be reverted by BRAF RNAi. This is important when the endothelium experiences changes in external stresses caused by high blood pressure, leading to edema, or by immune or cancer cells in inflammation or metastasis. Full article

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13 pages, 3281 KiB

Open AccessReview

Application of Monolayer Graphene and Its Derivative in Cryo-EM Sample Preparation

by Ke Wu, Di Wu, Li Zhu and Yi Wu

Int. J. Mol. Sci. 2021, 22(16), 8940; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22168940 - 19 Aug 2021

Cited by 1 | Viewed by 5875

Cryo-electron microscopy (Cryo-EM) has become a routine technology for resolving the structure of biological macromolecules due to the resolution revolution in recent years. The specimens are typically prepared in a very thin layer of vitrified ice suspending in the holes of the perforated [...] Read more.

Cryo-electron microscopy (Cryo-EM) has become a routine technology for resolving the structure of biological macromolecules due to the resolution revolution in recent years. The specimens are typically prepared in a very thin layer of vitrified ice suspending in the holes of the perforated amorphous carbon film. However, the samples prepared by directly applying to the conventional support membranes may suffer from partial or complete denaturation caused by sticking to the air–water interface (AWI). With the application in materials, graphene has also been used recently to improve frozen sample preparation instead of a suspended conventional amorphous thin carbon. It has been proven that graphene or graphene oxide and various chemical modifications on its surface can effectively prevent particles from adsorbing to the AWI, which improves the dispersion, adsorbed number, and orientation preference of frozen particles in the ice layer. Their excellent properties and thinner thickness can significantly reduce the background noise, allowing high-resolution three-dimensional reconstructions using a minimum data set. Full article

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13 pages, 2651 KiB

Open AccessArticle

High Power Electromagnetic Waves Exposure of Healthy and Tumor Bearing Mice: Assessment of Effects on Mice Growth, Behavior, Tumor Growth, and Vessel Permeabilization

by Jelena Kolosnjaj-Tabi, Muriel Golzio, Elisabeth Bellard, Alexandre Catrain, Thomas Chretiennot, Quentin Saurin, Jacques Tarayre, René Vezinet and Marie-Pierre Rols

Int. J. Mol. Sci. 2021, 22(16), 8516; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22168516 - 07 Aug 2021

Cited by 2 | Viewed by 1678

High power radiofrequencies may transiently or permanently disrupt the functioning of electronic devices, but their effect on living systems remains unknown. With the aim to evaluate the safety and biological effects of narrow-band and wide-band high-power electromagnetic (HPEM) waves, we studied their effects [...] Read more.

High power radiofrequencies may transiently or permanently disrupt the functioning of electronic devices, but their effect on living systems remains unknown. With the aim to evaluate the safety and biological effects of narrow-band and wide-band high-power electromagnetic (HPEM) waves, we studied their effects upon exposure of healthy and tumor-bearing mice. In field experiments, the exposure to 1.5 GHz narrow-band electromagnetic fields with the incident amplitude peak value level in the range of 40 kV/m and 150 MHz wide-band electric fields with the amplitude peak value in the range of 200 kV/m, did not alter healthy and tumor-bearing animals’ growth, nor it had any impact on cutaneous murine tumors’ growth. While we did not observe any noticeable behavioral changes in mice during the exposure to narrow-band signals when wide-band HPEM signals were applied, mice could behave in a similar way as they respond to loud noise signals: namely, if a mouse was exploring the cage prior to signal application, it returned to companion mates when wide-band HPEM signals were applied. Moreover, the effect of wide-band signals was assessed on normal blood vessels permeability in real-time in dorsal-chamber-bearing mice exposed in a pilot study using wide-band signal applicators. Our pilot study conducted within the applicator and performed at the laboratory scale suggests that the exposure to wide-band signals with the amplitude of 47.5 kV/m does not result in increased vessel permeability. Full article

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16 pages, 4122 KiB

Open AccessArticle

Effects of Visfatin on Intracellular Mechanics and Catabolism in Human Primary Chondrocytes through Glycogen Synthase Kinase 3β Inactivation

by Shun-Fu Chang, Kuo-Chin Huang, Kuan-Han Lee, Yao-Chang Chiang, Wei-Ru Lee, Rong-Ze Hsieh, Yu-Ping Su and Shun-Chi Wu

Int. J. Mol. Sci. 2021, 22(15), 8107; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22158107 - 28 Jul 2021

Cited by 5 | Viewed by 1928

Osteoarthritis (OA) is still a recalcitrant musculoskeletal disease on account of its complex biochemistry and mechanical stimulations. Apart from stimulation by external mechanical forces, the regulation of intracellular mechanics in chondrocytes has also been linked to OA development. Recently, visfatin has received significant [...] Read more.

Osteoarthritis (OA) is still a recalcitrant musculoskeletal disease on account of its complex biochemistry and mechanical stimulations. Apart from stimulation by external mechanical forces, the regulation of intracellular mechanics in chondrocytes has also been linked to OA development. Recently, visfatin has received significant attention because of the clinical finding of the positive correlation between its serum/synovial level and OA progression. However, the precise mechanism involved is still unclear. This study determined the effect of visfatin on intracellular mechanics and catabolism in human primary chondrocytes isolated from patients. The intracellular stiffness of chondrocytes was analyzed by the particle-tracking microrheology method. It was shown that visfatin damages the microtubule and microfilament networks to influence intracellular mechanics to decrease the intracellular elasticity and viscosity via glycogen synthase kinase 3β (GSK3β) inactivation induced by p38 signaling. Further, microtubule network destruction in human primary chondrocytes is predominantly responsible for the catabolic effect of visfatin on the cyclooxygenase 2 upregulation. The present study shows a more comprehensive interpretation of OA development induced by visfatin through biochemical and biophysical perspectives. Finally, the role of GSK3β inactivation, and subsequent regulation of intracellular mechanics, might be considered as theranostic targets for future drug development for OA. Full article

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19 pages, 4312 KiB

Open AccessArticle

Effect of Dimer Structure and Inhomogeneous Broadening of Energy Levels on the Action of Flavomononucleotide in Rigid Polyvinyl Alcohol Films

by Hanna Grajek, Jacek Kubicki, Ignacy Gryczyński, Jerzy Karolczak, Grażyna Żurkowska, Agnieszka I. Piotrowicz-Cieślak and Piotr Bojarski

Int. J. Mol. Sci. 2021, 22(14), 7759; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22147759 - 20 Jul 2021

Cited by 1 | Viewed by 2283

The results of time-resolved fluorescence measurements of flavin mononucleotide (FMN) in rigid polyvinyl alcohol films (PVA) demonstrate that fluorescence intensity decays are strongly accelerated in the presence of fluorescent dimers and nonradiative energy transfer processes. The fluorescence decay originating both from H and [...] Read more.

The results of time-resolved fluorescence measurements of flavin mononucleotide (FMN) in rigid polyvinyl alcohol films (PVA) demonstrate that fluorescence intensity decays are strongly accelerated in the presence of fluorescent dimers and nonradiative energy transfer processes. The fluorescence decay originating both from H and J dimer states of FMN was experimentally observed for the first time. The mean fluorescence lifetimes for FMN dimers were obtained:

{⟨τ⟩}_{f l}

= 2.66 ns (at λ_exc = 445 nm) and

{⟨τ⟩}_{f l}

= 2.02 (at λ_exc = 487 nm) at λ_obs = 600 nm and T = 253 K from H and J state of dimers, respectively. We show that inhomogeneous orientational broadening of energy levels (IOBEL) affects the shape of the fluorescence decay and leads to the dependence of the average monomer fluorescence lifetime on excitation wavelength. IOBEL affected the nonradiative energy transfer and indicated that different flavin positioning in the protein pocket could (1) change the spectroscopic properties of flavins due to the existence of “blue” and “red” fluorescence centers, and (2) diminish the effectiveness of energy transfer between FMN molecules. Full article

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21 pages, 3458 KiB

Open AccessArticle

The Mode of SN38 Derivatives Interacting with Nicked DNA Mimics Biological Targeting of Topo I Poisons

by Wojciech Bocian, Beata Naumczuk, Magdalena Urbanowicz, Jerzy Sitkowski, Anna Bierczyńska-Krzysik, Elżbieta Bednarek, Katarzyna Wiktorska, Małgorzata Milczarek and Lech Kozerski

Int. J. Mol. Sci. 2021, 22(14), 7471; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22147471 - 12 Jul 2021

Cited by 3 | Viewed by 2249

The compounds 7-ethyl-9-(N-methylamino)methyl-10-hydroxycamptothecin (2) and 7-ethyl-9-(N-morpholino)methyl-10-hydroxycamptothecin (3) are potential topoisomerase I poisons. Moreover, they were shown to have favorable anti-neoplastic effects on several tumor cell lines. Due to these properties, the compounds are being considered [...] Read more.

The compounds 7-ethyl-9-(N-methylamino)methyl-10-hydroxycamptothecin (2) and 7-ethyl-9-(N-morpholino)methyl-10-hydroxycamptothecin (3) are potential topoisomerase I poisons. Moreover, they were shown to have favorable anti-neoplastic effects on several tumor cell lines. Due to these properties, the compounds are being considered for advancement to the preclinical development stage. To gain better insights into the molecular mechanism with the biological target, here, we conducted an investigation into their interactions with model nicked DNA (1) using different techniques. In this work, we observed the complexity of the mechanism of action of the compounds 2 and 3, in addition to their decomposition products: compound 4 and SN38. Using DOSY experiments, evidence of the formation of strongly bonded molecular complexes of SN38 derivatives with DNA duplexes was provided. The molecular modeling based on cross-peaks from the NOESY spectrum also allowed us to assign the geometry of a molecular complex of DNA with compound 2. Confirmation of the alkylation reaction of both compounds was obtained using MALDI–MS. Additionally, in the case of 3, alkylation was confirmed in the recording of cross-peaks in the ¹H/¹³C HSQC spectrum of ¹³C-enriched compound 3. In this work, we showed that the studied compounds—parent compounds 2 and 3, and their potential metabolite 4 and SN38—interact inside the nick of 1, either forming the molecular complex or alkylating the DNA nitrogen bases. In order to confirm the influence of the studied compounds on the topoisomerase I relaxation activity of supercoiled DNA, the test was performed based upon the measurement of the fluorescence of DNA stain which can differentiate between supercoiled and relaxed DNA. The presented results confirmed that studied SN38 derivatives effectively block DNA relaxation mediated by Topo I, which means that they stop the machinery of Topo I activity. Full article

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20 pages, 1413 KiB

Open AccessReview

Human Radiosensitivity and Radiosusceptibility: What Are the Differences?

by Laura El-Nachef, Joelle Al-Choboq, Juliette Restier-Verlet, Adeline Granzotto, Elise Berthel, Laurène Sonzogni, Mélanie L. Ferlazzo, Audrey Bouchet, Pierre Leblond, Patrick Combemale, Stéphane Pinson, Michel Bourguignon and Nicolas Foray

Int. J. Mol. Sci. 2021, 22(13), 7158; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22137158 - 02 Jul 2021

Cited by 30 | Viewed by 4055

The individual response to ionizing radiation (IR) raises a number of medical, scientific, and societal issues. While the term “radiosensitivity” was used by the pioneers at the beginning of the 20st century to describe only the radiation-induced adverse tissue reactions related to cell [...] Read more.

The individual response to ionizing radiation (IR) raises a number of medical, scientific, and societal issues. While the term “radiosensitivity” was used by the pioneers at the beginning of the 20st century to describe only the radiation-induced adverse tissue reactions related to cell death, a confusion emerged in the literature from the 1930s, as “radiosensitivity” was indifferently used to describe the toxic, cancerous, or aging effect of IR. In parallel, the predisposition to radiation-induced adverse tissue reactions (radiosensitivity), notably observed after radiotherapy appears to be caused by different mechanisms than those linked to predisposition to radiation-induced cancer (radiosusceptibility). This review aims to document these differences in order to better estimate the different radiation-induced risks. It reveals that there are very few syndromes associated with the loss of biological functions involved directly in DNA damage recognition and repair as their role is absolutely necessary for cell viability. By contrast, some cytoplasmic proteins whose functions are independent of genome surveillance may also act as phosphorylation substrates of the ATM protein to regulate the molecular response to IR. The role of the ATM protein may help classify the genetic syndromes associated with radiosensitivity and/or radiosusceptibility. Full article

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16 pages, 2737 KiB

Open AccessArticle

Engineering of Janus-Like Dendrimers with Peptides Derived from Glycoproteins of Herpes Simplex Virus Type 1: Toward a Versatile and Novel Antiviral Platform

by Annarita Falanga, Valentina Del Genio, Elizabeth A. Kaufman, Carla Zannella, Gianluigi Franci, Marcus Weck and Stefania Galdiero

Int. J. Mol. Sci. 2021, 22(12), 6488; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22126488 - 17 Jun 2021

Cited by 16 | Viewed by 2111

Novel antiviral nanotherapeutics, which may inactivate the virus and block it from entering host cells, represent an important challenge to face viral global health emergencies around the world. Using a combination of bioorthogonal copper-catalyzed 1,3-dipolar alkyne/azide cycloaddition (CuAAC) and photoinitiated thiol–ene coupling, monofunctional [...] Read more.

Novel antiviral nanotherapeutics, which may inactivate the virus and block it from entering host cells, represent an important challenge to face viral global health emergencies around the world. Using a combination of bioorthogonal copper-catalyzed 1,3-dipolar alkyne/azide cycloaddition (CuAAC) and photoinitiated thiol–ene coupling, monofunctional and bifunctional peptidodendrimer conjugates were obtained. The conjugates are biocompatible and demonstrate no toxicity to cells at biologically relevant concentrations. Furthermore, the orthogonal addition of multiple copies of two different antiviral peptides on the surface of a single dendrimer allowed the resulting bioconjugates to inhibit Herpes simplex virus type 1 at both the early and the late stages of the infection process. The presented work builds on further improving this attractive design to obtain a new class of therapeutics. Full article

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11 pages, 2358 KiB

Open AccessArticle

A Glimpse into the Structural Properties of the Intermediate and Transition State in the Folding of Bromodomain 2 Domain 2 by Φ Value Analysis

by Leonore Novak, Maria Petrosino, Daniele Santorelli, Roberta Chiaraluce, Valerio Consalvi, Alessandra Pasquo and Carlo Travaglini-Allocatelli

Int. J. Mol. Sci. 2021, 22(11), 5953; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22115953 - 31 May 2021

Cited by 1 | Viewed by 1921

Bromodomains (BRDs) are small protein interaction modules of about 110 amino acids that selectively recognize acetylated lysine in histones and other proteins. These domains have been identified in a variety of multi-domain proteins involved in transcriptional regulation or chromatin remodeling in eukaryotic cells. [...] Read more.

Bromodomains (BRDs) are small protein interaction modules of about 110 amino acids that selectively recognize acetylated lysine in histones and other proteins. These domains have been identified in a variety of multi-domain proteins involved in transcriptional regulation or chromatin remodeling in eukaryotic cells. BRD inhibition is considered an attractive therapeutic approach in epigenetic disorders, particularly in oncology. Here, we present a Φ value analysis to investigate the folding pathway of the second domain of BRD2 (BRD2(2)). Using an extensive mutational analysis based on 25 site-directed mutants, we provide structural information on both the intermediate and late transition state of BRD2(2). The data reveal that the C-terminal region represents part of the initial folding nucleus, while the N-terminal region of the domain consolidates its structure only later in the folding process. Furthermore, only a small number of native-like interactions have been identified, suggesting the presence of a non-compact, partially folded state with scarce native-like characteristics. Taken together, these results indicate that, in BRD2(2), a hierarchical mechanism of protein folding can be described with non-native interactions that play a significant role in folding. Full article

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14 pages, 3014 KiB

Open AccessArticle

Serotonin Promotes Serum Albumin Interaction with the Monomeric Amyloid β Peptide

by Ekaterina A. Litus, Alexey S. Kazakov, Evgenia I. Deryusheva, Ekaterina L. Nemashkalova, Marina P. Shevelyova, Aliya A. Nazipova, Maria E. Permyakova, Elena V. Raznikova, Vladimir N. Uversky and Sergei E. Permyakov

Int. J. Mol. Sci. 2021, 22(11), 5896; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22115896 - 31 May 2021

Cited by 11 | Viewed by 2669

Prevention of amyloid β peptide (Aβ) deposition via facilitation of Aβ binding to its natural depot, human serum albumin (HSA), is a promising approach to preclude Alzheimer’s disease (AD) onset and progression. Previously, we demonstrated the ability of natural HSA ligands, fatty acids, [...] Read more.

Prevention of amyloid β peptide (Aβ) deposition via facilitation of Aβ binding to its natural depot, human serum albumin (HSA), is a promising approach to preclude Alzheimer’s disease (AD) onset and progression. Previously, we demonstrated the ability of natural HSA ligands, fatty acids, to improve the affinity of this protein to monomeric Aβ by a factor of 3 (BBRC, 510(2), 248–253). Using plasmon resonance spectroscopy, we show here that another HSA ligand related to AD pathogenesis, serotonin (SRO), increases the affinity of the Aβ monomer to HSA by a factor of 7/17 for Aβ₄₀/Aβ₄₂, respectively. Meanwhile, the structurally homologous SRO precursor, tryptophan (TRP), does not affect HSA’s affinity to monomeric Aβ, despite slowdown of the association and dissociation processes. Crosslinking with glutaraldehyde and dynamic light scattering experiments reveal that, compared with the TRP-induced effects, SRO binding causes more marked changes in the quaternary structure of HSA. Furthermore, molecular docking reveals distinct structural differences between SRO/TRP complexes with HSA. The disintegration of the serotonergic system during AD pathogenesis may contribute to Aβ release from HSA in the central nervous system due to impairment of the SRO-mediated Aβ trapping by HSA. Full article

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20 pages, 7535 KiB

Open AccessArticle

KEAP1 Cancer Mutants: A Large-Scale Molecular Dynamics Study of Protein Stability

by Carter J. Wilson, Megan Chang, Mikko Karttunen and Wing-Yiu Choy

Int. J. Mol. Sci. 2021, 22(10), 5408; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22105408 - 20 May 2021

Cited by 4 | Viewed by 3642

We have performed 280

μ

s of unbiased molecular dynamics (MD) simulations to investigate the effects of 12 different cancer mutations on Kelch-like ECH-associated protein 1 (KEAP1) (G333C, G350S, G364C, G379D, R413L, R415G, A427V, G430C, R470C, R470H, R470S and G476R), one of the [...] Read more.

We have performed 280

μ

s of unbiased molecular dynamics (MD) simulations to investigate the effects of 12 different cancer mutations on Kelch-like ECH-associated protein 1 (KEAP1) (G333C, G350S, G364C, G379D, R413L, R415G, A427V, G430C, R470C, R470H, R470S and G476R), one of the frequently mutated proteins in lung cancer. The aim was to provide structural insight into the effects of these mutants, including a new class of ANCHOR (additionally NRF2-complexed hypomorph) mutant variants. Our work provides additional insight into the structural dynamics of mutants that could not be analyzed experimentally, painting a more complete picture of their mutagenic effects. Notably, blade-wise analysis of the Kelch domain points to stability as a possible target of cancer in KEAP1. Interestingly, structural analysis of the R470C ANCHOR mutant, the most prevalent missense mutation in KEAP1, revealed no significant change in structural stability or NRF2 binding site dynamics, possibly indicating an covalent modification as this mutant’s mode of action. Full article

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27 pages, 27333 KiB

Open AccessArticle

The Study on Molecular Profile Changes of Pathogens via Zinc Nanocomposites Immobilization Approach

by Agnieszka Rogowska, Viorica Railean-Plugaru, Paweł Pomastowski, Justyna Walczak-Skierska, Anna Król-Górniak, Adrian Gołębiowski and Bogusław Buszewski

Int. J. Mol. Sci. 2021, 22(10), 5395; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22105395 - 20 May 2021

Cited by 6 | Viewed by 2097

The most critical group of all includes multidrug resistant bacteria that pose a particular threat in hospitals, as they can cause severe and often deadly infections. Modern medicine still faces the difficult task of developing new agents for the effective control of bacterial-based [...] Read more.

The most critical group of all includes multidrug resistant bacteria that pose a particular threat in hospitals, as they can cause severe and often deadly infections. Modern medicine still faces the difficult task of developing new agents for the effective control of bacterial-based diseases. The targeted administration of nanoparticles can enhance the efficiency of conventional pharmaceutical agents. However, the interpretation of interfaces’ interactions between nanoparticles and biological systems still remains a challenge for researchers. In fact, the current research presents a strategy for using ZnO NPs immobilization with ampicillin and tetracycline. Firstly, the study provides the mechanism of the ampicillin and tetracycline binding on the surface of ZnO NPs. Secondly, it examines the effect of non-immobilized ZnO NPs, immobilized with ampicillin (ZnONPs/AMP) and tetracycline (ZnONPs/TET), on the cells’ metabolism and morphology, based on the protein and lipid profiles. A sorption kinetics study showed that the antibiotics binding on the surface of ZnONPs depend on their structure. The efficiency of the process was definitely higher in the case of ampicillin. In addition, flow cytometry results showed that immobilized nanoparticles present a different mechanism of action. Moreover, according to the MALDI approach, the antibacterial activity mechanism of the investigated ZnO complexes is mainly based on the destruction of cell membrane integrity by lipids and proteins, which is necessary for proper cell function. Additionally, it was noticed that some of the identified changes indicate the activation of defense mechanisms by cells, leading to a decrease in the permeability of a cell’s external barriers or the synthesis of repair proteins. Full article

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16 pages, 2055 KiB

Open AccessArticle

Some Dietary Phenolic Compounds Can Activate Thyroid Peroxidase and Inhibit Lipoxygenase-Preliminary Study in the Model Systems

by Ewa Habza-Kowalska, Agnieszka A. Kaczor, Damian Bartuzi, Jacek Piłat and Urszula Gawlik-Dziki

Int. J. Mol. Sci. 2021, 22(10), 5108; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22105108 - 12 May 2021

Cited by 7 | Viewed by 2086

The presented research concerns the triple activity of trans-cinnamic (tCA), ferulic (FA) and syringic acids (SA). They act as thyroid peroxidase (TPO) activators, lipoxygenase (LOX) inhibitors and show antiradical activity. All compounds showed a dose-dependent TPO activatory effect, thus the AC₅₀ [...] Read more.

The presented research concerns the triple activity of trans-cinnamic (tCA), ferulic (FA) and syringic acids (SA). They act as thyroid peroxidase (TPO) activators, lipoxygenase (LOX) inhibitors and show antiradical activity. All compounds showed a dose-dependent TPO activatory effect, thus the AC₅₀ value (the concentration resulting in 50% activation) was determined. The tested compounds can be ranked as follows: tCA > FA > SA with AC₅₀ = 0.10, 0.39, 0.69 mM, respectively. Strong synergism was found between FA and SA. The activatory effects of all tested compounds may result from interaction with the TPO allosteric site. It was proposed that conformational change resulting from activator binding to TPO allosteric pocket results from the flexibility of a nearby loop formed by residues Val352-Tyr363. All compounds act as uncompetitive LOX inhibitors. The most effective were tCA and SA, whereas the weakest was FA (IC₅₀ = 0.009 mM and IC₅₀ 0.027 mM, respectively). In all cases, an interaction between the inhibitors carboxylic groups and side-chain atoms of Arg102 and Arg139 in an allosteric pocket of LOX was suggested. FA/tCA and FA/SA acted synergistically, whereas tCA/SA demonstrated antagonism. The highest antiradical activity was found in the case of SA (IC₅₀ = 0.22 mM). FA/tCA and tCA/SA acted synergistically, whereas antagonism was found for the SA/FA mixture. Full article

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29 pages, 4923 KiB

Open AccessArticle

Isolation and Characterization of Human Colon Adenocarcinoma Stem-Like Cells Based on the Endogenous Expression of the Stem Markers

by Sergei A. Koshkin, Olga V. Anatskaya, Alexander E. Vinogradov, Vladimir N. Uversky, Guy W. Dayhoff II, Margarita A. Bystriakova, Valery A. Pospelov and Elena N. Tolkunova

Int. J. Mol. Sci. 2021, 22(9), 4682; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22094682 - 28 Apr 2021

Cited by 6 | Viewed by 2754

Background: Cancer stem cells’ (CSCs) self-maintenance is regulated via the pluripotency pathways promoting the most aggressive tumor phenotype. This study aimed to use the activity of these pathways for the CSCs’ subpopulation enrichment and separating cells characterized by the OCT4 and SOX2 expression. [...] Read more.

Background: Cancer stem cells’ (CSCs) self-maintenance is regulated via the pluripotency pathways promoting the most aggressive tumor phenotype. This study aimed to use the activity of these pathways for the CSCs’ subpopulation enrichment and separating cells characterized by the OCT4 and SOX2 expression. Methods: To select and analyze CSCs, we used the SORE6x lentiviral reporter plasmid for viral transduction of colon adenocarcinoma cells. Additionally, we assessed cell chemoresistance, clonogenic, invasive and migratory activity and the data of mRNA-seq and intrinsic disorder predisposition protein analysis (IDPPA). Results: We obtained the line of CSC-like cells selected on the basis of the expression of the OCT4 and SOX2 stem cell factors. The enriched CSC-like subpopulation had increased chemoresistance as well as clonogenic and migration activities. The bioinformatic analysis of mRNA seq data identified the up-regulation of pluripotency, development, drug resistance and phototransduction pathways, and the downregulation of pathways related to proliferation, cell cycle, aging, and differentiation. IDPPA indicated that CSC-like cells are predisposed to increased intrinsic protein disorder. Conclusion: The use of the SORE6x reporter construct for CSCs enrichment allows us to obtain CSC-like population that can be used as a model to search for the new prognostic factors and potential therapeutic targets for colon cancer treatment. Full article

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21 pages, 5386 KiB

Open AccessArticle

Red-Edge Excitation Shift Spectroscopy (REES): Application to Hidden Bound States of Ligands in Protein–Ligand Complexes

by Md Lutful Kabir, Feng Wang and Andrew H. A. Clayton

Int. J. Mol. Sci. 2021, 22(5), 2582; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22052582 - 04 Mar 2021

Cited by 6 | Viewed by 2676

Ligand-protein binding is responsible for the vast majority of bio-molecular functions. Most experimental techniques examine the most populated ligand-bound state. The determination of less populated, intermediate, and transient bound states is experimentally challenging. However, hidden bound states are also important because these can [...] Read more.

Ligand-protein binding is responsible for the vast majority of bio-molecular functions. Most experimental techniques examine the most populated ligand-bound state. The determination of less populated, intermediate, and transient bound states is experimentally challenging. However, hidden bound states are also important because these can strongly influence ligand binding and unbinding processes. Here, we explored the use of a classical optical spectroscopic technique, red-edge excitation shift spectroscopy (REES) to determine the number, population, and energetics associated with ligand-bound states in protein–ligand complexes. We describe a statistical mechanical model of a two-level fluorescent ligand located amongst a finite number of discrete protein microstates. We relate the progressive emission red shift with red-edge excitation to thermodynamic parameters underlying the protein–ligand free energy landscape and to photo-physical parameters relating to the fluorescent ligand. We applied the theoretical model to published red-edge excitation shift data from small molecule inhibitor–kinase complexes. The derived thermodynamic parameters allowed dissection of the energetic contribution of intermediate bound states to inhibitor–kinase interactions. Full article

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11 pages, 31615 KiB

Open AccessArticle

25-Hydroxycholesterol Effect on Membrane Structure and Mechanical Properties

by Marco M. Domingues, Bárbara Gomes, Axel Hollmann and Nuno C. Santos

Int. J. Mol. Sci. 2021, 22(5), 2574; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22052574 - 04 Mar 2021

Cited by 12 | Viewed by 2724

Cholesterol is responsible for the plasticity of plasma membranes and is involved in physiological and pathophysiological responses. Cholesterol homeostasis is regulated by oxysterols, such as 25-hydroxycholesterol. The presence of 25-hydroxycholesterol at the membrane level has been shown to interfere with several viruses’ entry [...] Read more.

Cholesterol is responsible for the plasticity of plasma membranes and is involved in physiological and pathophysiological responses. Cholesterol homeostasis is regulated by oxysterols, such as 25-hydroxycholesterol. The presence of 25-hydroxycholesterol at the membrane level has been shown to interfere with several viruses’ entry into their target cells. We used atomic force microscopy to assess the effect of 25-hydroxycholesterol on different properties of supported lipid bilayers with controlled lipid compositions. In particular, we showed that 25-hydroxycholesterol inhibits the lipid-condensing effects of cholesterol, rendering the bilayers less rigid. This study indicates that the inclusion of 25-hydroxycholesterol in plasma membranes or the conversion of part of their cholesterol content into 25-hydroxycholesterol leads to morphological alterations of the sphingomyelin (SM)-enriched domains and promotes lipid packing inhomogeneities. These changes culminate in membrane stiffness variations. Full article

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11 pages, 888 KiB

Open AccessArticle

SAAFEC-SEQ: A Sequence-Based Method for Predicting the Effect of Single Point Mutations on Protein Thermodynamic Stability

by Gen Li, Shailesh Kumar Panday and Emil Alexov

Int. J. Mol. Sci. 2021, 22(2), 606; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22020606 - 09 Jan 2021

Cited by 57 | Viewed by 4245

Modeling the effect of mutations on protein thermodynamics stability is useful for protein engineering and understanding molecular mechanisms of disease-causing variants. Here, we report a new development of the SAAFEC method, the SAAFEC-SEQ, which is a gradient boosting decision tree machine learning method [...] Read more.

Modeling the effect of mutations on protein thermodynamics stability is useful for protein engineering and understanding molecular mechanisms of disease-causing variants. Here, we report a new development of the SAAFEC method, the SAAFEC-SEQ, which is a gradient boosting decision tree machine learning method to predict the change of the folding free energy caused by amino acid substitutions. The method does not require the 3D structure of the corresponding protein, but only its sequence and, thus, can be applied on genome-scale investigations where structural information is very sparse. SAAFEC-SEQ uses physicochemical properties, sequence features, and evolutionary information features to make the predictions. It is shown to consistently outperform all existing state-of-the-art sequence-based methods in both the Pearson correlation coefficient and root-mean-squared-error parameters as benchmarked on several independent datasets. The SAAFEC-SEQ has been implemented into a web server and is available as stand-alone code that can be downloaded and embedded into other researchers’ code. Full article

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2020

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10 pages, 1940 KiB

Open AccessArticle

BION-2: Predicting Positions of Non-Specifically Bound Ions on Protein Surface by a Gaussian-Based Treatment of Electrostatics

by H. B. Mihiri Shashikala, Arghya Chakravorty, Shailesh Kumar Panday and Emil Alexov

Int. J. Mol. Sci. 2021, 22(1), 272; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010272 - 29 Dec 2020

Cited by 3 | Viewed by 2076

Ions play significant roles in biological processes—they may specifically bind to a protein site or bind non-specifically on its surface. Although the role of specifically bound ions ranges from actively providing structural compactness via coordination of charge–charge interactions to numerous enzymatic activities, non-specifically [...] Read more.

Ions play significant roles in biological processes—they may specifically bind to a protein site or bind non-specifically on its surface. Although the role of specifically bound ions ranges from actively providing structural compactness via coordination of charge–charge interactions to numerous enzymatic activities, non-specifically surface-bound ions are also crucial to maintaining a protein’s stability, responding to pH and ion concentration changes, and contributing to other biological processes. However, the experimental determination of the positions of non-specifically bound ions is not trivial, since they may have a low residential time and experience significant thermal fluctuation of their positions. Here, we report a new release of a computational method, the BION-2 method, that predicts the positions of non-specifically surface-bound ions. The BION-2 utilizes the Gaussian-based treatment of ions within the framework of the modified Poisson–Boltzmann equation, which does not require a sharp boundary between the protein and water phase. Thus, the predictions are done by the balance of the energy of interaction between the protein charges and the corresponding ions and the de-solvation penalty of the ions as they approach the protein. The BION-2 is tested against experimentally determined ion’s positions and it is demonstrated that it outperforms the old BION and other available tools. Full article

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21 pages, 6537 KiB

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Polar Lipid Fraction E from Sulfolobus acidocaldarius and Dipalmitoylphosphatidylcholine Can Form Stable yet Thermo-Sensitive Tetraether/Diester Hybrid Archaeosomes with Controlled Release Capability

by Umme Ayesa and Parkson Lee-Gau Chong

Int. J. Mol. Sci. 2020, 21(21), 8388; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21218388 - 09 Nov 2020

Cited by 11 | Viewed by 2972

Archaeosomes have drawn increasing attention in recent years as novel nano-carriers for therapeutics. The main obstacle of using archaeosomes for therapeutics delivery has been the lack of an efficient method to trigger the release of entrapped content from the otherwise extremely stable structure. [...] Read more.

Archaeosomes have drawn increasing attention in recent years as novel nano-carriers for therapeutics. The main obstacle of using archaeosomes for therapeutics delivery has been the lack of an efficient method to trigger the release of entrapped content from the otherwise extremely stable structure. Our present study tackles this long-standing problem. We made hybrid archaeosomes composed of tetraether lipids, called the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius, and the synthetic diester lipid dipalmitoylphosphatidylcholine (DPPC). Differential polarized phase-modulation and steady-state fluorometry, confocal fluorescence microscopy, zeta potential (ZP) measurements, and biochemical assays were employed to characterize the physical properties and drug behaviors in PLFE/DPPC hybrid archaeosomes in the presence and absence of live cells. We found that PLFE lipids have an ordering effect on fluid DPPC liposomal membranes, which can slow down the release of entrapped drugs, while PLFE provides high negative charges on the outer surface of liposomes, which can increase vesicle stability against coalescence among liposomes or with cells. Furthermore, we found that the zeta potential in hybrid archaeosomes with 30 mol% PLFE and 70 mol% DPPC (designated as PLFE/DPPC(3:7) archaeosomes) undergoes an abrupt increase from −48 mV at 37 °C to −16 mV at 44 °C (termed the ZP transition), which we hypothesize results from DPPC domain melting and PLFE lipid ‘flip-flop’. The anticancer drug doxorubicin (DXO) can be readily incorporated into PLFE/DPPC(3:7) archaeosomes. The rate constant of DXO release from PLFE/DPPC(3:7) archaeosomes into Tris buffer exhibited a sharp increase (~2.5 times), when the temperature was raised from 37 to 42 °C, which is believed to result from the liposomal structural changes associated with the ZP transition. This thermo-induced sharp increase in drug release was not affected by serum proteins as a similar temperature dependence of drug release kinetics was observed in human blood serum. A 15-min pre-incubation of PLFE/DPPC(3:7) archaeosomal DXO with MCF-7 breast cancer cells at 42 °C caused a significant increase in the amount of DXO entering into the nuclei and a considerable increase in the cell’s cytotoxicity under the 37 °C growth temperature. Taken together, our data suggests that PLFE/DPPC(3:7) archaeosomes are stable yet potentially useful thermo-sensitive liposomes wherein the temperature range (from 37 to 42–44 °C) clinically used for mild hyperthermia treatment of tumors can be used to trigger drug release for medical interventions. Full article

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31 pages, 9702 KiB

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Coevolution, Dynamics and Allostery Conspire in Shaping Cooperative Binding and Signal Transmission of the SARS-CoV-2 Spike Protein with Human Angiotensin-Converting Enzyme 2

by Gennady Verkhivker

Int. J. Mol. Sci. 2020, 21(21), 8268; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21218268 - 04 Nov 2020

Cited by 23 | Viewed by 3567

Binding to the host receptor is a critical initial step for the coronavirus SARS-CoV-2 spike protein to enter into target cells and trigger virus transmission. A detailed dynamic and energetic view of the binding mechanisms underlying virus entry is not fully understood and [...] Read more.

Binding to the host receptor is a critical initial step for the coronavirus SARS-CoV-2 spike protein to enter into target cells and trigger virus transmission. A detailed dynamic and energetic view of the binding mechanisms underlying virus entry is not fully understood and the consensus around the molecular origins behind binding preferences of SARS-CoV-2 for binding with the angiotensin-converting enzyme 2 (ACE2) host receptor is yet to be established. In this work, we performed a comprehensive computational investigation in which sequence analysis and modeling of coevolutionary networks are combined with atomistic molecular simulations and comparative binding free energy analysis of the SARS-CoV and SARS-CoV-2 spike protein receptor binding domains with the ACE2 host receptor. Different from other computational studies, we systematically examine the molecular and energetic determinants of the binding mechanisms between SARS-CoV-2 and ACE2 proteins through the lens of coevolution, conformational dynamics, and allosteric interactions that conspire to drive binding interactions and signal transmission. Conformational dynamics analysis revealed the important differences in mobility of the binding interfaces for the SARS-CoV-2 spike protein that are not confined to several binding hotspots, but instead are broadly distributed across many interface residues. Through coevolutionary network analysis and dynamics-based alanine scanning, we established linkages between the binding energy hotspots and potential regulators and carriers of signal communication in the virus–host receptor complexes. The results of this study detailed a binding mechanism in which the energetics of the SARS-CoV-2 association with ACE2 may be determined by cumulative changes of a number of residues distributed across the entire binding interface. The central findings of this study are consistent with structural and biochemical data and highlight drug discovery challenges of inhibiting large and adaptive protein–protein interfaces responsible for virus entry and infection transmission. Full article

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12 pages, 5050 KiB

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Super-Resolution Live Cell Microscopy of Membrane-Proximal Fluorophores

by Verena Richter, Peter Lanzerstorfer, Julian Weghuber and Herbert Schneckenburger

Int. J. Mol. Sci. 2020, 21(19), 7099; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21197099 - 26 Sep 2020

Cited by 9 | Viewed by 2562

Here, we present a simple and robust experimental setup for the super-resolution live cell microscopy of membrane-proximal fluorophores, which is comparably easy to perform and to implement. The method is based on Structured Illumination Microscopy (SIM) with a switchable spatial light modulator (SLM) [...] Read more.

Here, we present a simple and robust experimental setup for the super-resolution live cell microscopy of membrane-proximal fluorophores, which is comparably easy to perform and to implement. The method is based on Structured Illumination Microscopy (SIM) with a switchable spatial light modulator (SLM) and exchangeable objective lenses for epi-illumination and total internal reflection fluorescence (TIRF) microscopy. While, in the case of SIM (upon epi-illumination), cell layers of about 1–2 µm in close proximity to the plasma membrane can be selected by software, layers in the 100 nm range are assessed experimentally by TIRF-SIM. To show the applicability of this approach, both methods are used to measure the translocation of the glucose transporter 4 (GLUT4) from intracellular vesicles to the plasma membrane upon stimulation by insulin or insulin-mimetic compounds, with a lateral resolution of around 100 nm and an axial resolution of around 200 nm. While SIM is an appropriate method to visualize the intracellular localization of GLUT4 fused with a green fluorescent protein, TIRF-SIM permits the quantitative evaluation of its fluorescence in the plasma membrane. These imaging methods are discussed in the context of fluorescence lifetime kinetics, providing additional data for the molecular microenvironment. Full article

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30 pages, 8493 KiB

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Zooming into the Dark Side of Human Annexin-S100 Complexes: Dynamic Alliance of Flexible Partners

by Judith Weisz and Vladimir N. Uversky

Int. J. Mol. Sci. 2020, 21(16), 5879; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21165879 - 16 Aug 2020

Cited by 20 | Viewed by 3291

Annexins and S100 proteins form two large families of Ca²⁺-binding proteins. They are quite different both structurally and functionally, with S100 proteins being small (10–12 kDa) acidic regulatory proteins from the EF-hand superfamily of Ca²⁺-binding proteins, and with annexins [...] Read more.

Annexins and S100 proteins form two large families of Ca²⁺-binding proteins. They are quite different both structurally and functionally, with S100 proteins being small (10–12 kDa) acidic regulatory proteins from the EF-hand superfamily of Ca²⁺-binding proteins, and with annexins being at least three-fold larger (329 ± 12 versus 98 ± 7 residues) and using non-EF-hand-based mechanism for calcium binding. Members of both families have multiple biological roles, being able to bind to a large cohort of partners and possessing a multitude of functions. Furthermore, annexins and S100 proteins can interact with each other in either a Ca²⁺-dependent or Ca²⁺-independent manner, forming functional annexin-S100 complexes. Such functional polymorphism and binding indiscrimination are rather unexpected, since structural information is available for many annexins and S100 proteins, which therefore are considered as ordered proteins that should follow the classical “one protein–one structure–one function” model. On the other hand, the ability to be engaged in a wide range of interactions with multiple, often unrelated, binding partners and possess multiple functions represent characteristic features of intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs); i.e., functional proteins or protein regions lacking unique tertiary structures. The aim of this paper is to provide an overview of the functional roles of human annexins and S100 proteins, and to use the protein intrinsic disorder perspective to explain their exceptional multifunctionality and binding promiscuity. Full article

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15 pages, 5762 KiB

Open AccessArticle

Insights into the Effect of Curcumin and (–)-Epigallocatechin-3-Gallate on the Aggregation of Aβ(1–40) Monomers by Means of Molecular Dynamics

by Francesco Tavanti, Alfonso Pedone and Maria Cristina Menziani

Int. J. Mol. Sci. 2020, 21(15), 5462; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21155462 - 30 Jul 2020

Cited by 19 | Viewed by 2165

In this study, we compared the effects of two well-known natural compounds on the early step of the fibrillation process of amyloid-β (1–40), responsible for the formation of plaques in the brains of patients affected by Alzheimer’s disease (AD). The use of extensive [...] Read more.

In this study, we compared the effects of two well-known natural compounds on the early step of the fibrillation process of amyloid-β (1–40), responsible for the formation of plaques in the brains of patients affected by Alzheimer’s disease (AD). The use of extensive replica exchange simulations up to the µs scale allowed us to characterize the inhibition activity of (–)-epigallocatechin-3-gallate (EGCG) and curcumin (CUR) on unfolded amyloid fibrils. A reduced number of β-strands, characteristic of amyloid fibrils, and an increased distance between the amino acids that are responsible for the intra- and interprotein aggregations are observed. The central core region of the amyloid-β (Aβ(1–40)) fibril is found to have a high affinity to EGCG and CUR due to the presence of hydrophobic residues. Lastly, the free binding energy computed using the Poisson Boltzmann Surface Ares suggests that EGCG is more likely to bind to unfolded Aβ(1–40) fibrils and that this molecule can be a good candidate to develop new and more effective congeners to treat AD. Full article

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18 pages, 2727 KiB

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Low-Dose Ionizing Radiation Modulates Microglia Phenotypes in the Models of Alzheimer’s Disease

by Sujin Kim, Hyunju Chung, Han Ngoc Mai, Yunkwon Nam, Soo Jung Shin, Yong Ho Park, Mi Joo Chung, Jong Kil Lee, Hak Young Rhee, Geon-Ho Jahng, Youngkyong Kim, Yu Jin Lim, Moonkyoo Kong, Minho Moon and Weon Kuu Chung

Int. J. Mol. Sci. 2020, 21(12), 4532; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21124532 - 25 Jun 2020

Cited by 39 | Viewed by 5241

Alzheimer’s disease (AD) is the most common type of dementia. AD involves major pathologies such as amyloid-β (Aβ) plaques and neurofibrillary tangles in the brain. During the progression of AD, microglia can be polarized from anti-inflammatory M2 to pro-inflammatory M1 phenotype. The activation [...] Read more.

Alzheimer’s disease (AD) is the most common type of dementia. AD involves major pathologies such as amyloid-β (Aβ) plaques and neurofibrillary tangles in the brain. During the progression of AD, microglia can be polarized from anti-inflammatory M2 to pro-inflammatory M1 phenotype. The activation of triggering receptor expressed on myeloid cells 2 (TREM2) may result in microglia phenotype switching from M1 to M2, which finally attenuated Aβ deposition and memory loss in AD. Low-dose ionizing radiation (LDIR) is known to ameliorate Aβ pathology and cognitive deficits in AD; however, the therapeutic mechanisms of LDIR against AD-related pathology have been little studied. First, we reconfirm that LDIR (two Gy per fraction for five times)-treated six-month 5XFAD mice exhibited (1) the reduction of Aβ deposition, as reflected by thioflavins S staining, and (2) the improvement of cognitive deficits, as revealed by Morris water maze test, compared to sham-exposed 5XFAD mice. To elucidate the mechanisms of LDIR-induced inhibition of Aβ accumulation and memory loss in AD, we examined whether LDIR regulates the microglial phenotype through the examination of levels of M1 and M2 cytokines in 5XFAD mice. In addition, we investigated the direct effects of LDIR on lipopolysaccharide (LPS)-induced production and secretion of M1/M2 cytokines in the BV-2 microglial cells. In the LPS- and LDIR-treated BV-2 cells, the M2 phenotypic marker CD206 was significantly increased, compared with LPS- and sham-treated BV-2 cells. Finally, the effect of LDIR on M2 polarization was confirmed by detection of increased expression of TREM2 in LPS-induced BV2 cells. These results suggest that LDIR directly induced phenotype switching from M1 to M2 in the brain with AD. Taken together, our results indicated that LDIR modulates LPS- and Aβ-induced neuroinflammation by promoting M2 polarization via TREM2 expression, and has beneficial effects in the AD-related pathology such as Aβ deposition and memory loss. Full article

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19 pages, 2818 KiB

Open AccessArticle

Mapping of CaM, S100A1 and PIP2-Binding Epitopes in the Intracellular N- and C-Termini of TRPM4

by Kristyna Bousova, Ivan Barvik, Petr Herman, Kateřina Hofbauerová, Lenka Monincova, Pavel Majer, Monika Zouharova, Veronika Vetyskova, Klara Postulkova and Jiri Vondrasek

Int. J. Mol. Sci. 2020, 21(12), 4323; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21124323 - 17 Jun 2020

Cited by 6 | Viewed by 3183

Molecular determinants of the binding of various endogenous modulators to transient receptor potential (TRP) channels are crucial for the understanding of necessary cellular pathways, as well as new paths for rational drug designs. The aim of this study was to characterise interactions between [...] Read more.

Molecular determinants of the binding of various endogenous modulators to transient receptor potential (TRP) channels are crucial for the understanding of necessary cellular pathways, as well as new paths for rational drug designs. The aim of this study was to characterise interactions between the TRP cation channel subfamily melastatin member 4 (TRPM4) and endogenous intracellular modulators—calcium-binding proteins (calmodulin (CaM) and S100A1) and phosphatidylinositol 4, 5-bisphosphate (PIP₂). We have found binding epitopes at the N- and C-termini of TRPM4 shared by CaM, S100A1 and PIP₂. The binding affinities of short peptides representing the binding epitopes of N- and C-termini were measured by means of fluorescence anisotropy (FA). The importance of representative basic amino acids and their combinations from both peptides for the binding of endogenous TRPM4 modulators was proved using point alanine-scanning mutagenesis. In silico protein–protein docking of both peptides to CaM and S100A1 and extensive molecular dynamics (MD) simulations enabled the description of key stabilising interactions at the atomic level. Recently solved cryo-Electron Microscopy (EM) structures made it possible to put our findings into the context of the entire TRPM4 channel and to deduce how the binding of these endogenous modulators could allosterically affect the gating of TRPM4. Moreover, both identified binding epitopes seem to be ideally positioned to mediate the involvement of TRPM4 in higher-order hetero-multimeric complexes with important physiological functions. Full article

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15 pages, 4109 KiB

Open AccessArticle

Hydrophobic Amino Acids as Universal Elements of Protein-Induced DNA Structure Deformation

by Kateřina Faltejsková, David Jakubec and Jiří Vondrášek

Int. J. Mol. Sci. 2020, 21(11), 3986; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21113986 - 02 Jun 2020

Cited by 5 | Viewed by 3446

Interaction with the DNA minor groove is a significant contributor to specific sequence recognition in selected families of DNA-binding proteins. Based on a statistical analysis of 3D structures of protein–DNA complexes, we propose that distortion of the DNA minor groove resulting from interactions [...] Read more.

Interaction with the DNA minor groove is a significant contributor to specific sequence recognition in selected families of DNA-binding proteins. Based on a statistical analysis of 3D structures of protein–DNA complexes, we propose that distortion of the DNA minor groove resulting from interactions with hydrophobic amino acid residues is a universal element of protein–DNA recognition. We provide evidence to support this by associating each DNA minor groove-binding amino acid residue with the local dimensions of the DNA double helix using a novel algorithm. The widened DNA minor grooves are associated with high GC content. However, some AT-rich sequences contacted by hydrophobic amino acids (e.g., phenylalanine) display extreme values of minor groove width as well. For a number of hydrophobic amino acids, distinct secondary structure preferences could be identified for residues interacting with the widened DNA minor groove. These results hold even after discarding the most populous families of minor groove-binding proteins. Full article

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35 pages, 12204 KiB

Open AccessArticle

Intrinsic Disorder in Tetratricopeptide Repeat Proteins

by Nathan W. Van Bibber, Cornelia Haerle, Roy Khalife, Bin Xue and Vladimir N. Uversky

Int. J. Mol. Sci. 2020, 21(10), 3709; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21103709 - 25 May 2020

Cited by 8 | Viewed by 3572

Among the realm of repeat containing proteins that commonly serve as “scaffolds” promoting protein-protein interactions, there is a family of proteins containing between 2 and 20 tetratricopeptide repeats (TPRs), which are functional motifs consisting of 34 amino acids. The most distinguishing feature of [...] Read more.

Among the realm of repeat containing proteins that commonly serve as “scaffolds” promoting protein-protein interactions, there is a family of proteins containing between 2 and 20 tetratricopeptide repeats (TPRs), which are functional motifs consisting of 34 amino acids. The most distinguishing feature of TPR domains is their ability to stack continuously one upon the other, with these stacked repeats being able to affect interaction with binding partners either sequentially or in combination. It is known that many repeat-containing proteins are characterized by high levels of intrinsic disorder, and that many protein tandem repeats can be intrinsically disordered. Furthermore, it seems that TPR-containing proteins share many characteristics with hybrid proteins containing ordered domains and intrinsically disordered protein regions. However, there has not been a systematic analysis of the intrinsic disorder status of TPR proteins. To fill this gap, we analyzed 166 human TPR proteins to determine the degree to which proteins containing TPR motifs are affected by intrinsic disorder. Our analysis revealed that these proteins are characterized by different levels of intrinsic disorder and contain functional disordered regions that are utilized for protein-protein interactions and often serve as targets of various posttranslational modifications. Full article

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15 pages, 3163 KiB

Open AccessArticle

Characteristic Analysis of Homo- and Heterodimeric Complexes of Human Mitochondrial Pyruvate Carrier Related to Metabolic Diseases

by Jinyi Lee, Zeyu Jin, Donghan Lee, Ji-Hye Yun and Weontae Lee

Int. J. Mol. Sci. 2020, 21(9), 3403; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21093403 - 11 May 2020

Cited by 13 | Viewed by 4307

Human mitochondrial pyruvate carriers (hMPCs), which are required for the uptake of pyruvate into mitochondria, are associated with several metabolic diseases, including type 2 diabetes and various cancers. Yeast MPC was recently demonstrated to form a functional unit of heterodimers. However, human MPC-1 [...] Read more.

Human mitochondrial pyruvate carriers (hMPCs), which are required for the uptake of pyruvate into mitochondria, are associated with several metabolic diseases, including type 2 diabetes and various cancers. Yeast MPC was recently demonstrated to form a functional unit of heterodimers. However, human MPC-1 (hMPC-1) and MPC-2 (hMPC-2) have not yet been individually isolated for their detailed characterization, in particular in terms of their structural and functional properties, namely, whether they exist as homo- or heterodimers. In this study, hMPC-1 and hMPC-2 were successfully isolated in micelles and they formed stable homodimers. However, the heterodimer state was found to be dominant when both hMPC-1 and hMPC-2 were present. In addition, as heterodimers, the molecules exhibited a higher binding capacity to both substrates and inhibitors, together with a larger structural stability than when they existed as homodimers. Taken together, our results demonstrated that the hetero-dimerization of hMPCs is the main functional unit of the pyruvate metabolism, providing a structural insight into the transport mechanisms of hMPCs. Full article

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15 pages, 1579 KiB

Open AccessArticle

Data-Driven Modeling Identifies TIRAP-Independent MyD88 Activation Complex and Myddosome Assembly Strategy in LPS/TLR4 Signaling

by Xiang Li, Chuan-Qi Zhong, Zhiyong Yin, Hong Qi, Fei Xu, Qingzu He and Jianwei Shuai

Int. J. Mol. Sci. 2020, 21(9), 3061; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21093061 - 26 Apr 2020

Cited by 22 | Viewed by 3582

TLR4 complexes are essential for the initiation of the LPS-induced innate immune response. The Myddosome, which mainly contains TLR4, TIRAP, MyD88, IRAK1/4 and TRAF6 proteins, is regarded as a major complex of TLR4. Although the Myddosome has been well studied, a quantitative description [...] Read more.

TLR4 complexes are essential for the initiation of the LPS-induced innate immune response. The Myddosome, which mainly contains TLR4, TIRAP, MyD88, IRAK1/4 and TRAF6 proteins, is regarded as a major complex of TLR4. Although the Myddosome has been well studied, a quantitative description of the Myddosome assembly dynamics is still lacking. Furthermore, whether some unknown TLR4 complexes exist remains unclear. In this study, we constructed a SWATH-MS data-based mathematical model that describes the component assembly dynamics of TLR4 complexes. In addition to Myddosome, we suggest that a TIRAP-independent MyD88 activation complex is formed upon LPS stimulation, in which TRAF6 is not included. Furthermore, quantitative analysis reveals that the distribution of components in TIRAP-dependent and -independent MyD88 activation complexes are LPS stimulation-dependent. The two complexes compete for recruiting IRAK1/4 proteins. MyD88 forms higher-order assembly in the Myddosome and we show that the strategy to form higher-order assembly is also LPS stimulation-dependent. MyD88 forms a long chain upon weak stimulation, but forms a short chain upon strong stimulation. Higher-order assembly of MyD88 is directly determined by the level of TIRAP in the Myddosome, providing a formation mechanism for efficient signaling transduction. Taken together, our study provides an enhanced understanding of component assembly dynamics and strategies in TLR4 complexes. Full article

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16 pages, 1631 KiB

Open AccessArticle

SAAMBE-3D: Predicting Effect of Mutations on Protein–Protein Interactions

by Swagata Pahari, Gen Li, Adithya Krishna Murthy, Siqi Liang, Robert Fragoza, Haiyuan Yu and Emil Alexov

Int. J. Mol. Sci. 2020, 21(7), 2563; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21072563 - 07 Apr 2020

Cited by 55 | Viewed by 6117

Maintaining wild type protein–protein interactions is essential for the normal function of cell and any mutation that alter their characteristics can cause disease. Therefore, the ability to correctly and quickly predict the effect of amino acid mutations is crucial for understanding disease effects [...] Read more.

Maintaining wild type protein–protein interactions is essential for the normal function of cell and any mutation that alter their characteristics can cause disease. Therefore, the ability to correctly and quickly predict the effect of amino acid mutations is crucial for understanding disease effects and to be able to carry out genome-wide studies. Here, we report a new development of the SAAMBE method, SAAMBE-3D, which is a machine learning-based approach, resulting in accurate predictions and is extremely fast. It achieves the Pearson correlation coefficient ranging from 0.78 to 0.82 depending on the training protocol in benchmarking five-fold validation test against the SKEMPI v2.0 database and outperforms currently existing algorithms on various blind-tests. Furthermore, optimized and tested via five-fold cross-validation on the Cornell University dataset, the SAAMBE-3D achieves AUC of 1.0 and 0.96 on a homo and hereto-dimer test datasets. Another important feature of SAAMBE-3D is that it is very fast, it takes less than a fraction of a second to complete a prediction. SAAMBE-3D is available as a web server and as well as a stand-alone code, the last one being another important feature allowing other researchers to directly download the code and run it on their local computer. Combined all together, SAAMBE-3D is an accurate and fast software applicable for genome-wide studies to assess the effect of amino acid mutations on protein–protein interactions. The webserver and the stand-alone codes (SAAMBE-3D for predicting the change of binding free energy and SAAMBE-3D-DN for predicting if the mutation is disruptive or non-disruptive) are available. Full article

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11 pages, 1870 KiB

Open AccessArticle

Distal Unfolding of Ferricytochrome c Induced by the F82K Mutation

by Daniela Lalli, Camilla Rosa, Marco Allegrozzi and Paola Turano

Int. J. Mol. Sci. 2020, 21(6), 2134; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21062134 - 20 Mar 2020

Cited by 6 | Viewed by 2335

It is well known that axial coordination of heme iron in mitochondrial cytochrome c has redox-dependent stability. The Met80 heme iron axial ligand in the ferric form of the protein is relatively labile and can be easily replaced by alternative amino acid side [...] Read more.

It is well known that axial coordination of heme iron in mitochondrial cytochrome c has redox-dependent stability. The Met80 heme iron axial ligand in the ferric form of the protein is relatively labile and can be easily replaced by alternative amino acid side chains under non-native conditions induced by alkaline pH, high temperature, or denaturing agents. Here, we showed a redox-dependent destabilization induced in human cytochrome c by substituting Phe82—conserved amino acid and a key actor in cytochrome c intermolecular interactions—with a Lys residue. Introducing a positive charge at position 82 did not significantly affect the structure of ferrous cytochrome c but caused localized unfolding of the distal site in the ferric state. As revealed by ¹H NMR fingerprint, the ferric form of the F82K variant had axial coordination resembling the renowned alkaline species, where the detachment of the native Met80 ligand favored the formation of multiple conformations involving distal Lys residues binding to iron, but with more limited overall structural destabilization. Full article

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14 pages, 4478 KiB

Open AccessArticle

In Silico Insights into Protein–Protein Interaction Disruptive Mutations in the PCSK9-LDLR Complex

by William R. Martin, Felice C. Lightstone and Feixiong Cheng

Int. J. Mol. Sci. 2020, 21(5), 1550; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21051550 - 25 Feb 2020

Cited by 13 | Viewed by 5739

Gain-of-function mutations in PCSK9 (proprotein convertase subtilisin/kexin type 9) lead to reduced uptake of LDL (low density lipoprotein) cholesterol and, therefore, increased plasma LDL levels. However, the mechanism by which these mutants reduce LDL reuptake is not fully understood. Here, we have used [...] Read more.

Gain-of-function mutations in PCSK9 (proprotein convertase subtilisin/kexin type 9) lead to reduced uptake of LDL (low density lipoprotein) cholesterol and, therefore, increased plasma LDL levels. However, the mechanism by which these mutants reduce LDL reuptake is not fully understood. Here, we have used molecular dynamics simulations, MM/PBSA (Molecular Mechanics/Poisson–Boltzmann Surface Area) binding affinity calculations, and residue interaction networks, to investigate the protein–protein interaction (PPI) disruptive effects of two of PCSK9′s gain-of-function mutations, Ser127Arg and Asp374Tyr on the PCSK9 and LDL receptor complex. In addition to these PPI disruptive mutants, a third, non-interface mutation (Arg496Trp) is included as a positive control. Our results indicate that Ser127Arg and Asp374Tyr confer significantly improved binding affinity, as well as different binding modes, when compared to the wild-type. These PPI disruptive mutations lie between the EGF(A) (epidermal growth factor precursor homology domain A) of the LDL receptor and the catalytic domain of PCSK9 (Asp374Tyr) and between the prodomain of PCSK9 and the β-propeller of the LDL receptor (Ser127Arg). The interactions involved in these two interfaces result in an LDL receptor that is sterically inhibited from entering its closed conformation. This could potentially implicate the prodomain as a target for small molecule inhibitors. Full article

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13 pages, 3771 KiB

Open AccessArticle

Mathematical Model of ATM Activation and Chromatin Relaxation by Ionizing Radiation

by Yongfeng Li and Francis A. Cucinotta

Int. J. Mol. Sci. 2020, 21(4), 1214; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21041214 - 12 Feb 2020

Cited by 4 | Viewed by 3286

We propose a comprehensive mathematical model to study the dynamics of ionizing radiation induced Ataxia-telangiectasia mutated (ATM) activation that consists of ATM activation through dual mechanisms: the initiative activation pathway triggered by the DNA damage-induced local chromatin relaxation and the primary activation pathway [...] Read more.

We propose a comprehensive mathematical model to study the dynamics of ionizing radiation induced Ataxia-telangiectasia mutated (ATM) activation that consists of ATM activation through dual mechanisms: the initiative activation pathway triggered by the DNA damage-induced local chromatin relaxation and the primary activation pathway consisting of a self-activation loop by interplay with chromatin relaxation. The model is expressed as a series of biochemical reactions, governed by a system of differential equations and analyzed by dynamical systems techniques. Radiation induced double strand breaks (DSBs) cause rapid local chromatin relaxation, which is independent of ATM but initiates ATM activation at damage sites. Key to the model description is how chromatin relaxation follows when active ATM phosphorylates KAP-1, which subsequently spreads throughout the chromatin and induces global chromatin relaxation. Additionally, the model describes how oxidative stress activation of ATM triggers a self-activation loop in which PP2A and ATF2 are released so that ATM can undergo autophosphorylation and acetylation for full activation in relaxed chromatin. In contrast, oxidative stress alone can partially activate ATM because phosphorylated ATM remains as a dimer. The model leads to predictions on ATM mediated responses to DSBs, oxidative stress, or both that can be tested by experiments. Full article

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17 pages, 2163 KiB

Open AccessArticle

Using the Gibbs Function as a Measure of Human Brain Development Trends from Fetal Stage to Advanced Age

by Edward A. Rietman, Sophie Taylor, Hava T. Siegelmann, Marco A. Deriu, Marco Cavaglia and Jack A. Tuszynski

Int. J. Mol. Sci. 2020, 21(3), 1116; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21031116 - 07 Feb 2020

Cited by 5 | Viewed by 2950

We propose to use a Gibbs free energy function as a measure of the human brain development. We adopt this approach to the development of the human brain over the human lifespan: from a prenatal stage to advanced age. We used proteomic expression [...] Read more.

We propose to use a Gibbs free energy function as a measure of the human brain development. We adopt this approach to the development of the human brain over the human lifespan: from a prenatal stage to advanced age. We used proteomic expression data with the Gibbs free energy to quantify human brain’s protein–protein interaction networks. The data, obtained from BioGRID, comprised tissue samples from the 16 main brain areas, at different ages, of 57 post-mortem human brains. We found a consistent functional dependence of the Gibbs free energies on age for most of the areas and both sexes. A significant upward trend in the Gibbs function was found during the fetal stages, which is followed by a sharp drop at birth with a subsequent period of relative stability and a final upward trend toward advanced age. We interpret these data in terms of structure formation followed by its stabilization and eventual deterioration. Furthermore, gender data analysis has uncovered the existence of functional differences, showing male Gibbs function values lower than female at prenatal and neonatal ages, which become higher at ages 8 to 40 and finally converging at late adulthood with the corresponding female Gibbs functions. Full article

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12 pages, 2455 KiB

Open AccessArticle

C-Terminal Domain of the Human Zinc Transporter hZnT8 Is Structurally Indistinguishable from Its Disease Risk Variant (R325W)

by Raheem Ullah, Aamir Shehzad, Majid Ali Shah, Matteo De March, Fouzia Ismat, Mazhar Iqbal, Silvia Onesti, Moazur Rahman and Michael J. McPherson

Int. J. Mol. Sci. 2020, 21(3), 926; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21030926 - 31 Jan 2020

Cited by 6 | Viewed by 2905

The human zinc transporter 8 (hZnT8) plays important roles in the storage of insulin in the secretory vesicles of pancreatic β cells. hZnT8 consists of a transmembrane domain, with its N- and C-termini protruding into the cytoplasm. Interestingly, the exchange of arginine to [...] Read more.

The human zinc transporter 8 (hZnT8) plays important roles in the storage of insulin in the secretory vesicles of pancreatic β cells. hZnT8 consists of a transmembrane domain, with its N- and C-termini protruding into the cytoplasm. Interestingly, the exchange of arginine to tryptophan at position 325 in the C-terminal domain (CTD) increases the risk of developing type 2 diabetes mellitus (T2D). In the present study, the CTDs of hZnT8 (the wild-type (WT) and its disease risk variant (R325W)) were expressed, purified, and characterized in their native forms by biophysical techniques. The data reveal that the CTDs form tetramers which are stabilized by zinc binding, and exhibit negligible differences in their secondary structure content and zinc-binding affinities in solution. These findings provide the basis for conducting further structural studies aimed at unravelling the molecular mechanism underlying the increased susceptibility to develop T2D, which is modulated by the disease risk variant. Full article

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39 pages, 9779 KiB

Open AccessReview

Integrated Computational Approaches and Tools for Allosteric Drug Discovery

by Olivier Sheik Amamuddy, Wayde Veldman, Colleen Manyumwa, Afrah Khairallah, Steve Agajanian, Odeyemi Oluyemi, Gennady M. Verkhivker and Özlem Tastan Bishop

Int. J. Mol. Sci. 2020, 21(3), 847; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21030847 - 28 Jan 2020

Cited by 60 | Viewed by 9768

Understanding molecular mechanisms underlying the complexity of allosteric regulation in proteins has attracted considerable attention in drug discovery due to the benefits and versatility of allosteric modulators in providing desirable selectivity against protein targets while minimizing toxicity and other side effects. The proliferation [...] Read more.

Understanding molecular mechanisms underlying the complexity of allosteric regulation in proteins has attracted considerable attention in drug discovery due to the benefits and versatility of allosteric modulators in providing desirable selectivity against protein targets while minimizing toxicity and other side effects. The proliferation of novel computational approaches for predicting ligand–protein interactions and binding using dynamic and network-centric perspectives has led to new insights into allosteric mechanisms and facilitated computer-based discovery of allosteric drugs. Although no absolute method of experimental and in silico allosteric drug/site discovery exists, current methods are still being improved. As such, the critical analysis and integration of established approaches into robust, reproducible, and customizable computational pipelines with experimental feedback could make allosteric drug discovery more efficient and reliable. In this article, we review computational approaches for allosteric drug discovery and discuss how these tools can be utilized to develop consensus workflows for in silico identification of allosteric sites and modulators with some applications to pathogen resistance and precision medicine. The emerging realization that allosteric modulators can exploit distinct regulatory mechanisms and can provide access to targeted modulation of protein activities could open opportunities for probing biological processes and in silico design of drug combinations with improved therapeutic indices and a broad range of activities. Full article

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7 pages, 1728 KiB

Open AccessCommunication

Unveiling the Molecular Basis of the Noonan Syndrome-Causing Mutation T42A of SHP2

by Angelo Toto, Francesca Malagrinò, Lorenzo Visconti, Francesca Troilo and Stefano Gianni

Int. J. Mol. Sci. 2020, 21(2), 461; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21020461 - 10 Jan 2020

Cited by 8 | Viewed by 2741

Noonan syndrome (NS) is a genetic disorder caused by the hyperactivation of the RAS-MAPK molecular pathway. About 50% of NS cases are caused by mutations affecting the SHP2 protein, a multi-domain phosphatase with a fundamental role in the regulation of the RAS-MAPK pathway. [...] Read more.

Noonan syndrome (NS) is a genetic disorder caused by the hyperactivation of the RAS-MAPK molecular pathway. About 50% of NS cases are caused by mutations affecting the SHP2 protein, a multi-domain phosphatase with a fundamental role in the regulation of the RAS-MAPK pathway. Most NS-causing mutations influence the stability of the inactive form of SHP2. However, one NS-causing mutation, namely T42A, occurs in the binding pocket of the N-SH2 domain of the protein. Here, we present a quantitative characterization of the effect of the T42A mutation on the binding of the N-terminal SH2 domain of SHP2 with a peptide mimicking Gab2, a fundamental interaction that triggers the activation of the phosphatase in the cellular environment. Our results show that whilst the T42A mutation does not affect the association rate constant with the ligand, it causes a dramatic increase of the affinity for Gab2. This effect is due to a remarkable decrease of the microscopic dissociation rate constant of over two orders of magnitudes. In an effort to investigate the molecular basis of the T42A mutation in causing Noonan syndrome, we also compare the experimental results with a more conservative variant, T42S. Our findings are discussed in the context of the structural data available on SHP2. Full article

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13 pages, 4003 KiB

Open AccessArticle

Conformational Plasticity of the Active Site Entrance in E. coli Aspartate Transcarbamoylase and Its Implication in Feedback Regulation

by Zhen Lei, Nan Wang, Hongwei Tan, Jimin Zheng and Zongchao Jia

Int. J. Mol. Sci. 2020, 21(1), 320; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21010320 - 03 Jan 2020

Cited by 3 | Viewed by 2530

Aspartate transcarbamoylase (ATCase) has been studied for decades and Escherichia coli ATCase is referred as a “textbook example” for both feedback regulation and cooperativity. However, several critical questions about the catalytic and regulatory mechanisms of E. coli ATCase remain unanswered, especially about its [...] Read more.

Aspartate transcarbamoylase (ATCase) has been studied for decades and Escherichia coli ATCase is referred as a “textbook example” for both feedback regulation and cooperativity. However, several critical questions about the catalytic and regulatory mechanisms of E. coli ATCase remain unanswered, especially about its remote feedback regulation. Herein, we determined a structure of E. coli ATCase in which a key residue located (Arg167) at the entrance of the active site adopted an uncommon open conformation, representing the first wild-type apo-form E. coli ATCase holoenzyme that features this state. Based on the structure and our results of enzymatic characterization, as well as molecular dynamic simulations, we provide new insights into the feedback regulation of E. coli ATCase. We speculate that the binding of pyrimidines or purines would affect the hydrogen bond network at the interface of the catalytic and regulatory subunit, which would further influence the stability of the open conformation of Arg167 and the enzymatic activity of ATCase. Our results not only revealed the importance of the previously unappreciated open conformation of Arg167 in the active site, but also helped to provide rationalization for the mechanism of the remote feedback regulation of ATCase. Full article

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14 pages, 2080 KiB

Open AccessArticle

In Situ Proteolysis Condition-Induced Crystallization of the XcpVWX Complex in Different Lattices

by Yichen Zhang, Shu Wang and Zongchao Jia

Int. J. Mol. Sci. 2020, 21(1), 308; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21010308 - 02 Jan 2020

Cited by 1 | Viewed by 3147

Although prevalent in the determination of protein structures; crystallography always has the bottleneck of obtaining high-quality protein crystals for characterizing a wide range of proteins; especially large protein complexes. Stable fragments or domains of proteins are more readily to crystallize; which prompts the [...] Read more.

Although prevalent in the determination of protein structures; crystallography always has the bottleneck of obtaining high-quality protein crystals for characterizing a wide range of proteins; especially large protein complexes. Stable fragments or domains of proteins are more readily to crystallize; which prompts the use of in situ proteolysis to remove flexible or unstable structures for improving crystallization and crystal quality. In this work; we investigated the effects of in situ proteolysis by chymotrypsin on the crystallization of the XcpVWX complex from the Type II secretion system of Pseudomonas aeruginosa. Different proteolysis conditions were found to result in two distinct lattices in the same crystallization solution. With a shorter chymotrypsin digestion at a lower concentration; the crystals exhibited a P3 hexagonal lattice that accommodates three complex molecules in one asymmetric unit. By contrast; a longer digestion with chymotrypsin of a 10-fold higher concentration facilitated the formation of a compact P2₁2₁2₁ orthorhombic lattice with only one complex molecule in each asymmetric unit. The molecules in the hexagonal lattice have shown high atomic displacement parameter values compared with the ones in the orthorhombic lattice. Taken together; our results clearly demonstrate that different proteolysis conditions can result in the generation of distinct lattices in the same crystallization solution; which can be exploited in order to obtain different crystal forms of a better quality Full article

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2019

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21 pages, 3571 KiB

Open AccessReview

Strategies for Molecular Imprinting and the Evolution of MIP Nanoparticles as Plastic Antibodies—Synthesis and Applications

by Doaa Refaat, Mohamed G. Aggour, Ahmed A. Farghali, Rashmi Mahajan, Jesper G. Wiklander, Ian A. Nicholls and Sergey A. Piletsky

Int. J. Mol. Sci. 2019, 20(24), 6304; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20246304 - 13 Dec 2019

Cited by 108 | Viewed by 7834

Materials that can mimic the molecular recognition-based functions found in biology are a significant goal for science and technology. Molecular imprinting is a technology that addresses this challenge by providing polymeric materials with antibody-like recognition characteristics. Recently, significant progress has been achieved in [...] Read more.

Materials that can mimic the molecular recognition-based functions found in biology are a significant goal for science and technology. Molecular imprinting is a technology that addresses this challenge by providing polymeric materials with antibody-like recognition characteristics. Recently, significant progress has been achieved in solving many of the practical problems traditionally associated with molecularly imprinted polymers (MIPs), such as difficulties with imprinting of proteins, poor compatibility with aqueous environments, template leakage, and the presence of heterogeneous populations of binding sites in the polymers that contribute to high levels of non-specific binding. This success is closely related to the technology-driven shift in MIP research from traditional bulk polymer formats into the nanomaterial domain. The aim of this article is to throw light on recent developments in this field and to present a critical discussion of the current state of molecular imprinting and its potential in real world applications. Full article

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17 pages, 2786 KiB

Open AccessArticle

Near-Infrared Markers based on Bacterial Phytochromes with Phycocyanobilin as a Chromophore

by Olesya V. Stepanenko, Olga V. Stepanenko, Olesya G. Shpironok, Alexander V. Fonin, Irina M. Kuznetsova and Konstantin K. Turoverov

Int. J. Mol. Sci. 2019, 20(23), 6067; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20236067 - 02 Dec 2019

Cited by 10 | Viewed by 3726

Biomarkers engineered on the basis of bacterial phytochromes with biliverdin IXα (BV) cofactor as a chromophore are increasingly used in cell biology and biomedicine, since their absorption and fluorescence spectra lie within the so-called optical “transparency window” of biological tissues. However, the quantum [...] Read more.

Biomarkers engineered on the basis of bacterial phytochromes with biliverdin IXα (BV) cofactor as a chromophore are increasingly used in cell biology and biomedicine, since their absorption and fluorescence spectra lie within the so-called optical “transparency window” of biological tissues. However, the quantum yield of BV fluorescence in these biomarkers does not exceed 0.145. The task of generating biomarkers with a higher fluorescence quantum yield remains relevant. To address the problem, we proposed the use of phycocyanobilin (PCB) as a chromophore of biomarkers derived from bacterial phytochromes. In this work, we characterized the complexes of iRFP713 evolved from RpBphP2 and its mutant variants with different location of cysteine residues capable of covalent tetrapyrrole attachment with the PCB cofactor. All analyzed proteins assembled with PCB were shown to have a higher fluorescence quantum yield than the proteins assembled with BV. The iRFP713/V256C and iRFP713/C15S/V256C assembled with PCB have a particularly high quantum yield of 0.5 and 0.45, which exceeds the quantum yield of all currently available near-infrared biomarkers. Moreover, PCB has 4 times greater affinity for iRFP713/V256C and iRFP713/C15S/V256C proteins compared to BV. These data establish iRFP713/V256C and iRFP713/C15S/V256C assembled with the PCB chromophore as promising biomarkers for application in vivo. The analysis of the spectral properties of the tested biomarkers allowed for suggesting that the high-fluorescence quantum yield of the PCB chromophore can be attributed to the lower mobility of the D-ring of PCB compared to BV. Full article

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59 pages, 14401 KiB

Open AccessArticle

Intrinsic Disorder of the BAF Complex: Roles in Chromatin Remodeling and Disease Development

by Nashwa El Hadidy and Vladimir N. Uversky

Int. J. Mol. Sci. 2019, 20(21), 5260; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20215260 - 23 Oct 2019

Cited by 19 | Viewed by 6271

The two-meter-long DNA is compressed into chromatin in the nucleus of every cell, which serves as a significant barrier to transcription. Therefore, for processes such as replication and transcription to occur, the highly compacted chromatin must be relaxed, and the processes required for [...] Read more.

The two-meter-long DNA is compressed into chromatin in the nucleus of every cell, which serves as a significant barrier to transcription. Therefore, for processes such as replication and transcription to occur, the highly compacted chromatin must be relaxed, and the processes required for chromatin reorganization for the aim of replication or transcription are controlled by ATP-dependent nucleosome remodelers. One of the most highly studied remodelers of this kind is the BRG1- or BRM-associated factor complex (BAF complex, also known as SWItch/sucrose non-fermentable (SWI/SNF) complex), which is crucial for the regulation of gene expression and differentiation in eukaryotes. Chromatin remodeling complex BAF is characterized by a highly polymorphic structure, containing from four to 17 subunits encoded by 29 genes. The aim of this paper is to provide an overview of the role of BAF complex in chromatin remodeling and also to use literature mining and a set of computational and bioinformatics tools to analyze structural properties, intrinsic disorder predisposition, and functionalities of its subunits, along with the description of the relations of different BAF complex subunits to the pathogenesis of various human diseases. Full article

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9 pages, 1469 KiB

Open AccessCommunication

Multiplexed Competitive Screening of One-Bead-One-Component Combinatorial Libraries Using a ClonePix 2 Colony Sorter

by R. Ashton Lavoie, Alice di Fazio, Ruben G. Carbonell and Stefano Menegatti

Int. J. Mol. Sci. 2019, 20(20), 5119; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20205119 - 16 Oct 2019

Cited by 10 | Viewed by 3372

Screening solid-phase combinatorial libraries of bioactive compounds against fluorescently labeled target biomolecules is an established technology in ligand and drug discovery. Rarely, however, do screening methods include comprehensive strategies—beyond mere library blocking and competitive screening—to ensure binding selectivity of selected leads. This work [...] Read more.

Screening solid-phase combinatorial libraries of bioactive compounds against fluorescently labeled target biomolecules is an established technology in ligand and drug discovery. Rarely, however, do screening methods include comprehensive strategies—beyond mere library blocking and competitive screening—to ensure binding selectivity of selected leads. This work presents a method for multiplexed solid-phase peptide library screening using a ClonePix 2 Colony Picker that integrates (i) orthogonal fluorescent labeling for positive selection against a target protein and negative selection against competitor species with (ii) semi-quantitative tracking of target vs. competitor binding for every library bead. The ClonePix 2 technology enables global at-a-glance evaluation and customization of the parameters for bead selection to ensure high affinity and selectivity of the isolated leads. A case study is presented by screening a peptide library against green-labeled human immunoglobulin G (IgG) and red-labeled host cell proteins (HCPs) using ClonePix 2 to select HCP-binding ligands for flow-through chromatography applications. Using this approach, 79 peptide ligand candidates (6.6% of the total number of ligands screened) were identified as potential HCP-selective ligands, enabling a potential rate of >3,000 library beads screened per hour. Full article

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17 pages, 1908 KiB

Open AccessArticle

Novel Genetic Markers for Early Detection of Elevated Breast Cancer Risk in Women

by Bohua Wu, Yunhui Peng, Julia Eggert and Emil Alexov

Int. J. Mol. Sci. 2019, 20(19), 4828; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20194828 - 28 Sep 2019

Cited by 3 | Viewed by 3686

This study suggests that two newly discovered variants in the MSH2 gene, which codes for a DNA mismatch repair (MMR) protein, can be associated with a high risk of breast cancer. While variants in the MSH2 gene are known to be linked with [...] Read more.

This study suggests that two newly discovered variants in the MSH2 gene, which codes for a DNA mismatch repair (MMR) protein, can be associated with a high risk of breast cancer. While variants in the MSH2 gene are known to be linked with an elevated cancer risk, the MSH2 gene is not a part of the standard kit for testing patients for elevated breast cancer risk. Here we used the results of genetic testing of women diagnosed with breast cancer, but who did not have variants in BRCA1 and BRCA2 genes. Instead, the test identified four variants with unknown significance (VUS) in the MSH2 gene. Here, we carried in silico analysis to develop a classifier that can distinguish pathogenic from benign mutations in MSH2 genes taken from ClinVar. The classifier was then used to classify VUS in MSH2 genes, and two of them, p.Ala272Val and p.Met592Val, were predicted to be pathogenic mutations. These two mutations were found in women with breast cancer who did not have mutations in BRCA1 and BRCA2 genes, and thus they are suggested to be considered as new bio-markers for the early detection of elevated breast cancer risk. However, before this is done, an in vitro validation of mutation pathogenicity is needed and, moreover, the presence of these mutations should be demonstrated in a higher number of patients or in families with breast cancer history. Full article

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15 pages, 4223 KiB

Open AccessArticle

Low Energy Shock Wave Therapy Inhibits Inflammatory Molecules and Suppresses Prostatic Pain and Hypersensitivity in a Capsaicin Induced Prostatitis Model in Rats

by Hung-Jen Wang, Pradeep Tyagi, Yu-Ming Chen, Michael B. Chancellor and Yao-Chi Chuang

Int. J. Mol. Sci. 2019, 20(19), 4777; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20194777 - 26 Sep 2019

Cited by 19 | Viewed by 3676

The effect of low energy shock wave (LESW) therapy on the changes of inflammatory molecules and pain reaction was studied in a capsaicin (10 mM, 0.1 cc) induced prostatitis model in rats. Intraprostatic capsaicin injection induced a pain reaction, including closing of the [...] Read more.

The effect of low energy shock wave (LESW) therapy on the changes of inflammatory molecules and pain reaction was studied in a capsaicin (10 mM, 0.1 cc) induced prostatitis model in rats. Intraprostatic capsaicin injection induced a pain reaction, including closing of the eyes, hypolocomotion, and tactile allodynia, which effects were ameliorated by LESW treatment. LESW therapy (2Hz, energy flux density of 0.12 mJ/mm²) at 200 and 300 shocks significantly decreased capsaicin-induced inflammatory reactions, reflected by a reduction of tissue edema and inflammatory cells, COX-2 and TNF-α stained positive cells, however, the therapeutic effects were not observed at 100 shocks treated group. Capsaicin-induced IL-1β, COX-2, IL-6, caspase-1, and NGF upregulation on day 3 and 7, while NALP1 and TNF-α upregulation was observed on day 7. LESW significantly suppressed the expression of IL-1β, COX-2, caspase-1, NGF on day 3 and IL-1β, TNF-α, COX-2, NALP1, caspase-1, NGF expression on day 7 in a dose-dependent fashion. LESW has no significant effect on IL-6 expression. Intraprostatic capsaicin injection activates inflammatory molecules and induces prostatic pain and hypersensitivity, which effects were suppressed by LESW. These findings might be the potential mechanisms of LESW therapy for nonbacterial prostatitis in humans. Full article

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9 pages, 248 KiB

Open AccessArticle

Robust Sampling of Defective Pathways in Multiple Myeloma

by Juan Luis Fernández-Martínez, Enrique J. de Andrés-Galiana, Francisco Javier Fernández-Ovies, Ana Cernea and Andrzej Kloczkowski

Int. J. Mol. Sci. 2019, 20(19), 4681; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20194681 - 21 Sep 2019

Cited by 4 | Viewed by 2087

We present the analysis of defective pathways in multiple myeloma (MM) using two recently developed sampling algorithms of the biological pathways: The Fisher’s ratio sampler, and the holdout sampler. We performed the retrospective analyses of different gene expression datasets concerning different aspects of [...] Read more.

We present the analysis of defective pathways in multiple myeloma (MM) using two recently developed sampling algorithms of the biological pathways: The Fisher’s ratio sampler, and the holdout sampler. We performed the retrospective analyses of different gene expression datasets concerning different aspects of the disease, such as the existing difference between bone marrow stromal cells in MM and healthy controls (HC), the gene expression profiling of CD34+ cells in MM and HC, the difference between hyperdiploid and non-hyperdiploid myelomas, and the prediction of the chromosome 13 deletion, to provide a deeper insight into the molecular mechanisms involved in the disease. Our analysis has shown the importance of different altered pathways related to glycosylation, infectious disease, immune system response, different aspects of metabolism, DNA repair, protein recycling and regulation of the transcription of genes involved in the differentiation of myeloid cells. The main difference in genetic pathways between hyperdiploid and non-hyperdiploid myelomas are related to infectious disease, immune system response and protein recycling. Our work provides new insights on the genetic pathways involved in this complex disease and proposes novel targets for future therapies. Full article

10 pages, 2622 KiB

Open AccessArticle

Cooperative Binding of KaiB to the KaiC Hexamer Ensures Accurate Circadian Clock Oscillation in Cyanobacteria

by Reiko Murakami, Yasuhiro Yunoki, Kentaro Ishii, Kazuki Terauchi, Susumu Uchiyama, Hirokazu Yagi and Koichi Kato

Int. J. Mol. Sci. 2019, 20(18), 4550; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20184550 - 13 Sep 2019

Cited by 12 | Viewed by 4356

The central oscillator generating cyanobacterial circadian rhythms comprises KaiA, KaiB, and KaiC proteins. Their interactions cause KaiC phosphorylation and dephosphorylation cycles over approximately 24 h. KaiB interacts with phosphorylated KaiC in competition with SasA, an output protein harboring a KaiB-homologous domain. Structural data [...] Read more.

The central oscillator generating cyanobacterial circadian rhythms comprises KaiA, KaiB, and KaiC proteins. Their interactions cause KaiC phosphorylation and dephosphorylation cycles over approximately 24 h. KaiB interacts with phosphorylated KaiC in competition with SasA, an output protein harboring a KaiB-homologous domain. Structural data have identified KaiB–KaiC interaction sites; however, KaiB mutations distal from the binding surfaces can impair KaiB–KaiC interaction and the circadian rhythm. Reportedly, KaiB and KaiC exclusively form a complex in a 6:6 stoichiometry, indicating that KaiB–KaiC hexamer binding shows strong positive cooperativity. Here, mutational analysis was used to investigate the functional significance of this cooperative interaction. Results demonstrate that electrostatic complementarity between KaiB protomers promotes their cooperative assembly, which is indispensable for accurate rhythm generation. SasA does not exhibit such electrostatic complementarity and noncooperatively binds to KaiC. Thus, the findings explain KaiB distal mutation effects, providing mechanistic insights into clock protein interplay. Full article

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16 pages, 1918 KiB

Open AccessArticle

Cooperativity and Steep Voltage Dependence in a Bacterial Channel

by Shang H. Lin, Kai-Ti Chang, Nuval Cherian, Benjamin Wu, Hyo Phee, Christy Cho and Marco Colombini

Int. J. Mol. Sci. 2019, 20(18), 4501; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20184501 - 11 Sep 2019

Cited by 2 | Viewed by 2422

This paper reports on the discovery of a novel three-membrane channel unit exhibiting very steep voltage dependence and strong cooperative behavior. It was reconstituted into planar phospholipid membranes formed by the monolayer method and studied under voltage-clamp conditions. The behavior of the novel [...] Read more.

This paper reports on the discovery of a novel three-membrane channel unit exhibiting very steep voltage dependence and strong cooperative behavior. It was reconstituted into planar phospholipid membranes formed by the monolayer method and studied under voltage-clamp conditions. The behavior of the novel channel-former, isolated from Escherichia coli, is consistent with a linearly organized three-channel unit displaying steep voltage-gating (a minimum of 14 charges in the voltage sensor) that rivals that of channels in mammalian excitable membranes. The channels also display strong cooperativity in that closure of the first channel permits the second to close and closure of the second channel permits closure of the third. All three have virtually the same conductance and selectivity, and yet the first and third close at positive potentials whereas the second closes at negative potentials. Thus, is it likely that the second channel-former is oriented in the membrane in a direction opposite to that of the other two. This novel structure is named “triplin.” The extraordinary behavior of triplin indicates that it must have important and as yet undefined physiological roles. Full article

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15 pages, 2217 KiB

Open AccessArticle

TRPM6 N-Terminal CaM- and S100A1-Binding Domains

by Monika Zouharova, Petr Herman, Kateřina Hofbauerová, Jiri Vondrasek and Kristyna Bousova

Int. J. Mol. Sci. 2019, 20(18), 4430; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20184430 - 09 Sep 2019

Cited by 9 | Viewed by 3283

Transient receptor potential (TRPs) channels are crucial downstream targets of calcium signalling cascades. They can be modulated either by calcium itself and/or by calcium-binding proteins (CBPs). Intracellular messengers usually interact with binding domains present at the most variable TRP regions—N- and C-cytoplasmic termini. [...] Read more.

Transient receptor potential (TRPs) channels are crucial downstream targets of calcium signalling cascades. They can be modulated either by calcium itself and/or by calcium-binding proteins (CBPs). Intracellular messengers usually interact with binding domains present at the most variable TRP regions—N- and C-cytoplasmic termini. Calmodulin (CaM) is a calcium-dependent cytosolic protein serving as a modulator of most transmembrane receptors. Although CaM-binding domains are widespread within intracellular parts of TRPs, no such binding domain has been characterised at the TRP melastatin member—the transient receptor potential melastatin 6 (TRPM6) channel. Another CBP, the S100 calcium-binding protein A1 (S100A1), is also known for its modulatory activities towards receptors. S100A1 commonly shares a CaM-binding domain. Here, we present the first identified CaM and S100A1 binding sites at the N-terminal of TRPM6. We have confirmed the L520-R535 N-terminal TRPM6 domain as a shared binding site for CaM and S100A1 using biophysical and molecular modelling methods. A specific domain of basic amino acid residues (R526/R531/K532/R535) present at this TRPM6 domain has been identified as crucial to maintain non-covalent interactions with the ligands. Our data unambiguously confirm that CaM and S100A1 share the same binding domain at the TRPM6 N-terminus although the ligand-binding mechanism is different. Full article

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20 pages, 1909 KiB

Open AccessArticle

Identifying Methylation Pattern and Genes Associated with Breast Cancer Subtypes

by Lei Chen, Tao Zeng, Xiaoyong Pan, Yu-Hang Zhang, Tao Huang and Yu-Dong Cai

Int. J. Mol. Sci. 2019, 20(17), 4269; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20174269 - 31 Aug 2019

Cited by 35 | Viewed by 5024

Breast cancer is regarded worldwide as a severe human disease. Various genetic variations, including hereditary and somatic mutations, contribute to the initiation and progression of this disease. The diagnostic parameters of breast cancer are not limited to the conventional protein content and can [...] Read more.

Breast cancer is regarded worldwide as a severe human disease. Various genetic variations, including hereditary and somatic mutations, contribute to the initiation and progression of this disease. The diagnostic parameters of breast cancer are not limited to the conventional protein content and can include newly discovered genetic variants and even genetic modification patterns such as methylation and microRNA. In addition, breast cancer detection extends to detailed breast cancer stratifications to provide subtype-specific indications for further personalized treatment. One genome-wide expression–methylation quantitative trait loci analysis confirmed that different breast cancer subtypes have various methylation patterns. However, recognizing clinically applied (methylation) biomarkers is difficult due to the large number of differentially methylated genes. In this study, we attempted to re-screen a small group of functional biomarkers for the identification and distinction of different breast cancer subtypes with advanced machine learning methods. The findings may contribute to biomarker identification for different breast cancer subtypes and provide a new perspective for differential pathogenesis in breast cancer subtypes. Full article

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21 pages, 4872 KiB

Open AccessReview

Structure Determination by Single-Particle Cryo-Electron Microscopy: Only the Sky (and Intrinsic Disorder) is the Limit

by Emeka Nwanochie and Vladimir N. Uversky

Int. J. Mol. Sci. 2019, 20(17), 4186; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20174186 - 27 Aug 2019

Cited by 45 | Viewed by 7501

Traditionally, X-ray crystallography and NMR spectroscopy represent major workhorses of structural biologists, with the lion share of protein structures reported in protein data bank (PDB) being generated by these powerful techniques. Despite their wide utilization in protein structure determination, these two techniques have [...] Read more.

Traditionally, X-ray crystallography and NMR spectroscopy represent major workhorses of structural biologists, with the lion share of protein structures reported in protein data bank (PDB) being generated by these powerful techniques. Despite their wide utilization in protein structure determination, these two techniques have logical limitations, with X-ray crystallography being unsuitable for the analysis of highly dynamic structures and with NMR spectroscopy being restricted to the analysis of relatively small proteins. In recent years, we have witnessed an explosive development of the techniques based on Cryo-electron microscopy (Cryo-EM) for structural characterization of biological molecules. In fact, single-particle Cryo-EM is a special niche as it is a technique of choice for the structural analysis of large, structurally heterogeneous, and dynamic complexes. Here, sub-nanometer atomic resolution can be achieved (i.e., resolution below 10 Å) via single-particle imaging of non-crystalline specimens, with accurate 3D reconstruction being generated based on the computational averaging of multiple 2D projection images of the same particle that was frozen rapidly in solution. We provide here a brief overview of single-particle Cryo-EM and show how Cryo-EM has revolutionized structural investigations of membrane proteins. We also show that the presence of intrinsically disordered or flexible regions in a target protein represents one of the major limitations of this promising technique. Full article

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18 pages, 3054 KiB

Open AccessArticle

A New Approach for Spontaneous Silver Ions Immobilization onto Casein

by Oleksandra Pryshchepa, Gulyaim N. Sagandykova, Paweł Pomastowski, Viorica Railean-Plugaru, Anna Król, Agnieszka Rogowska, Agnieszka Rodzik, Myroslav Sprynskyy and Bogusław Buszewski

Int. J. Mol. Sci. 2019, 20(16), 3864; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20163864 - 08 Aug 2019

Cited by 19 | Viewed by 3514

The work presents the kinetic and isotherm studies of silver binding on casein, which was carried out using batch sorption technique. Moreover, the influence of light irradiation on the process was shown. In order to investigate the mechanism of metal ions sorption by [...] Read more.

The work presents the kinetic and isotherm studies of silver binding on casein, which was carried out using batch sorption technique. Moreover, the influence of light irradiation on the process was shown. In order to investigate the mechanism of metal ions sorption by casein the zero, pseudo-first order kinetics and Weber-Morris intra-particle diffusion as well as Langmuir and Freundlich isotherm models were used. Furthermore, to specify more precisely, the possible binding mechanism, the spectroscopic (FT-IR—Fourier Transform Infrared Spectroscopy, Raman), spectrometric (MALDI-TOF MS—Matrix-Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry), microscopic (SEM—Scanning Electron Microscope, TEM/EDX—Transmission Electron Microscopy/Energy Dispersive X-ray detector) and thermal (TGA—Thermogravimetric Analysis, DTG—Derivative Thermogravimetry) analysis were performed. Kinetic study indicates that silver binding onto casein is a heterogeneous process with two main stages: initial rapid stage related to surface adsorption onto casein with immediate creation of silver nanoparticles and slower second stage of intraglobular diffusion with silver binding in chelated form (metalloproteins) or ion-exchange form. Spectroscopic techniques confirmed the binding process and MALDI-TOF MS analysis show the dominant contribution of the α-casein in the process. Moreover, the treatment of silver-casein complex by artificial physiological fluids was performed. Full article

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11 pages, 2312 KiB

Open AccessCommunication

Non-Ionic Deep Eutectic Liquids: Acetamide–Urea Derived Room Temperature Solvents

by Subramanian Suriyanarayanan, Gustaf D. Olsson, Subban Kathiravan, Natacha Ndizeye and Ian A. Nicholls

Int. J. Mol. Sci. 2019, 20(12), 2857; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20122857 - 12 Jun 2019

Cited by 16 | Viewed by 4246

A family of non-ionic deep eutectic liquids has been developed based upon mixtures of solid N-alkyl derivatives of urea and acetamide that in some cases have melting points below room temperature. The eutectic behaviour and physical characteristics of a series of eleven [...] Read more.

A family of non-ionic deep eutectic liquids has been developed based upon mixtures of solid N-alkyl derivatives of urea and acetamide that in some cases have melting points below room temperature. The eutectic behaviour and physical characteristics of a series of eleven eutectic mixtures are presented, along with a molecular dynamics study-supported hypothesis for the origin of the non-ideal mixing of these substances. Their use as solvents in applications ranging from natural product extraction to organic and polymer synthesis are demonstrated. Full article

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16 pages, 6500 KiB

Open AccessArticle

CUBAN, a Case Study of Selective Binding: Structural Details of the Discrimination between Ubiquitin and NEDD8

by Elena Santonico, Ridvan Nepravishta, Walter Mandaliti, Luisa Castagnoli, Gianni Cesareni and Maurizio Paci

Int. J. Mol. Sci. 2019, 20(5), 1185; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20051185 - 08 Mar 2019

Cited by 4 | Viewed by 2760

The newly identified CUBAN (Cullin binding domain associating with NEDD8) domain recognizes both ubiquitin and the ubiquitin-like NEDD8. Despite the high similarity between the two molecules, CUBAN shows a clear preference for NEDD8, free and conjugated to cullins. We previously characterized the domain [...] Read more.

The newly identified CUBAN (Cullin binding domain associating with NEDD8) domain recognizes both ubiquitin and the ubiquitin-like NEDD8. Despite the high similarity between the two molecules, CUBAN shows a clear preference for NEDD8, free and conjugated to cullins. We previously characterized the domain structure, both alone and in complex with NEDD8. The results here reported are addressed to investigate the determinants that drive the selective binding of CUBAN towards NEDD8 and ubiquitin. The ¹⁵N HSQC NMR perturbation pattern of the labeled CUBAN domain, when combined with either NEDD8 or ubiquitin, shows a clear involvement of hydrophobic residues that characterize the early stages of these interactions. After a slow conformational selection step, hydrophobic and then neutral and polar interactions take place, which drive the correct orientation of the CUBAN domain, leading to differences in the recognition scheme of NEDD8 and ubiquitin. As a result, a cascade of induced fit steps seems to determine the structural preference shown for NEDD8 and therefore the basis of the selectivity of the CUBAN domain. Finally, molecular dynamics analysis was performed to determine by fluctuations the internal flexibility of the CUBAN/NEDD8 complex. We consider that our results, based on a structural investigation mainly focused on the early stages of the recognition, provide a fruitful opportunity to report the different behavior of the same protein with two highly similar binding partners. Full article

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Planned Papers

The below list represents only planned manuscripts. Some of these manuscripts have not been received by the Editorial Office yet. Papers submitted to MDPI journals are subject to peer-review.

Title: Order, Disorder, BAF Complex, and Chromatin Remodeling
Authors: Nashwa El Hadidy and Vladimir N. Uversky

Title: In situ proteolysis condition-induced crystallization of the XcpVWX complex in different crystal lattice
Authors: Jerry Y. Zhang and Zongchao Jia
Affiliation: Department of Biomedical and Molecular Sciences, Queen's University

Title: Structural prerequisites for antimicrobial peptides to induce keratinocyte cell migration
Authors: G. Weindl, et al.

Title: Biophyiscal investigations into the interaction of synthetic anti-LPS peptides with the bacterial phospholipid membrane
Authors: P. Garidel, et al.

Title: Antiinflammatory mode of action of Pep19-2.5
Authors: Klaus Brandenburg, et al.

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