Research

17 pages, 2705 KiB

Open AccessArticle

Searching for the Best Transthyretin Aggregation Protocol to Study Amyloid Fibril Disruption

by Elisabete Ferreira, Zaida L. Almeida, Pedro F. Cruz, Marta Silva e Sousa, Paula Veríssimo and Rui M. M. Brito

Int. J. Mol. Sci. 2022, 23(1), 391; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23010391 - 30 Dec 2021

Cited by 5 | Viewed by 2184

Abstract

Several degenerative amyloid diseases, with no fully effective treatment, affect millions of people worldwide. These pathologies—amyloidoses—are known to be associated with the formation of ordered protein aggregates and highly stable and insoluble amyloid fibrils, which are deposited in multiple tissues and organs. The [...] Read more.

Several degenerative amyloid diseases, with no fully effective treatment, affect millions of people worldwide. These pathologies—amyloidoses—are known to be associated with the formation of ordered protein aggregates and highly stable and insoluble amyloid fibrils, which are deposited in multiple tissues and organs. The disruption of preformed amyloid aggregates and fibrils is one possible therapeutic strategy against amyloidosis; however, only a few compounds have been identified as possible fibril disruptors in vivo to date. To properly identify chemical compounds as potential fibril disruptors, a reliable, fast, and economic screening protocol must be developed. For this purpose, three amyloid fibril formation protocols using transthyretin (TTR), a plasma protein involved in several amyloidoses, were studied using thioflavin-T fluorescence assays, circular dichroism (CD), turbidity, dynamic light scattering (DLS), and transmission electron microscopy (TEM), in order to characterize and select the most appropriate fibril formation protocol. Saturation transfer difference nuclear magnetic resonance spectroscopy (STD NMR) was successfully used to study the interaction of doxycycline, a known amyloid fibril disruptor, with preformed wild-type TTR (TTRwt) aggregates and fibrils. DLS and TEM were also used to characterize the effect of doxycycline on TTRwt amyloid species disaggregation. A comparison of the TTR amyloid morphology formed in different experimental conditions is also presented. Full article

(This article belongs to the Special Issue The Role of Environment in Amyloid Aggregation)

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14 pages, 2417 KiB

Open AccessArticle

Aggregation Condition–Structure Relationship of Mouse Prion Protein Fibrils

by Jēkabs Fridmanis, Zigmantas Toleikis, Tomas Sneideris, Mantas Ziaunys, Raitis Bobrovs, Vytautas Smirnovas and Kristaps Jaudzems

Int. J. Mol. Sci. 2021, 22(17), 9635; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22179635 - 06 Sep 2021

Cited by 4 | Viewed by 2175

Abstract

Prion diseases are associated with conformational conversion of cellular prion protein into a misfolded pathogenic form, which resembles many properties of amyloid fibrils. The same prion protein sequence can misfold into different conformations, which are responsible for variations in prion disease phenotypes (prion [...] Read more.

Prion diseases are associated with conformational conversion of cellular prion protein into a misfolded pathogenic form, which resembles many properties of amyloid fibrils. The same prion protein sequence can misfold into different conformations, which are responsible for variations in prion disease phenotypes (prion strains). In this work, we use atomic force microscopy, FTIR spectroscopy and magic-angle spinning NMR to devise structural models of mouse prion protein fibrils prepared in three different denaturing conditions. We find that the fibril core region as well as the structure of its N- and C-terminal parts is almost identical between the three fibrils. In contrast, the central part differs in length of β-strands and the arrangement of charged residues. We propose that the denaturant ionic strength plays a major role in determining the structure of fibrils obtained in a particular condition by stabilizing fibril core interior-facing glutamic acid residues. Full article

(This article belongs to the Special Issue The Role of Environment in Amyloid Aggregation)

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21 pages, 5043 KiB

Open AccessArticle

Generalizable Compositional Features Influencing the Proteostatic Fates of Polar Low-Complexity Domains

by Sean M. Cascarina, Joshua P. Kaplan, Mikaela R. Elder, Lindsey Brookbank and Eric D. Ross

Int. J. Mol. Sci. 2021, 22(16), 8944; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22168944 - 19 Aug 2021

Cited by 1 | Viewed by 1510

Abstract

Protein aggregation is associated with a growing list of human diseases. A substantial fraction of proteins in eukaryotic proteomes constitutes a proteostasis network—a collection of proteins that work together to maintain properly folded proteins. One of the overarching functions of the proteostasis network [...] Read more.

Protein aggregation is associated with a growing list of human diseases. A substantial fraction of proteins in eukaryotic proteomes constitutes a proteostasis network—a collection of proteins that work together to maintain properly folded proteins. One of the overarching functions of the proteostasis network is the prevention or reversal of protein aggregation. How proteins aggregate in spite of the anti-aggregation activity of the proteostasis machinery is incompletely understood. Exposed hydrophobic patches can trigger degradation by the ubiquitin-proteasome system, a key branch of the proteostasis network. However, in a recent study, we found that model glycine (G)-rich or glutamine/asparagine (Q/N)-rich prion-like domains differ in their susceptibility to detection and degradation by this system. Here, we expand upon this work by examining whether the features controlling the degradation of our model prion-like domains generalize broadly to G-rich and Q/N-rich domains. Experimentally, native yeast G-rich domains in isolation are sensitive to the degradation-promoting effects of hydrophobic residues, whereas native Q/N-rich domains completely resist these effects and tend to aggregate instead. Bioinformatic analyses indicate that native G-rich domains from yeast and humans tend to avoid degradation-promoting features, suggesting that the proteostasis network may act as a form of selection at the molecular level that constrains the sequence space accessible to G-rich domains. However, the sensitivity or resistance of G-rich and Q/N-rich domains, respectively, was not always preserved in their native protein contexts, highlighting that proteins can evolve other sequence features to overcome the intrinsic sensitivity of some LCDs to degradation. Full article

(This article belongs to the Special Issue The Role of Environment in Amyloid Aggregation)

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20 pages, 4215 KiB

Open AccessArticle

Variability of Amyloid Propensity in Imperfect Repeats of CsgA Protein of Salmonella enterica and Escherichia coli

by Natalia Szulc, Marlena Gąsior-Głogowska, Jakub W. Wojciechowski, Monika Szefczyk, Andrzej M. Żak, Michał Burdukiewicz and Malgorzata Kotulska

Int. J. Mol. Sci. 2021, 22(10), 5127; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22105127 - 12 May 2021

Cited by 7 | Viewed by 3184

Abstract

CsgA is an aggregating protein from bacterial biofilms, representing a class of functional amyloids. Its amyloid propensity is defined by five fragments (R1–R5) of the sequence, representing non-perfect repeats. Gate-keeper amino acid residues, specific to each fragment, define the fragment’s propensity for self-aggregation [...] Read more.

CsgA is an aggregating protein from bacterial biofilms, representing a class of functional amyloids. Its amyloid propensity is defined by five fragments (R1–R5) of the sequence, representing non-perfect repeats. Gate-keeper amino acid residues, specific to each fragment, define the fragment’s propensity for self-aggregation and aggregating characteristics of the whole protein. We study the self-aggregation and secondary structures of the repeat fragments of Salmonella enterica and Escherichia coli and comparatively analyze their potential effects on these proteins in a bacterial biofilm. Using bioinformatics predictors, ATR-FTIR and FT-Raman spectroscopy techniques, circular dichroism, and transmission electron microscopy, we confirmed self-aggregation of R1, R3, R5 fragments, as previously reported for Escherichia coli, however, with different temporal characteristics for each species. We also observed aggregation propensities of R4 fragment of Salmonella enterica that is different than that of Escherichia coli. Our studies showed that amyloid structures of CsgA repeats are more easily formed and more durable in Salmonella enterica than those in Escherichia coli. Full article

(This article belongs to the Special Issue The Role of Environment in Amyloid Aggregation)

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16 pages, 4562 KiB

Open AccessArticle

Temperature-Dependent Structural Variability of Prion Protein Amyloid Fibrils

by Mantas Ziaunys, Andrius Sakalauskas, Kamile Mikalauskaite, Ruta Snieckute and Vytautas Smirnovas

Int. J. Mol. Sci. 2021, 22(10), 5075; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22105075 - 11 May 2021

Cited by 15 | Viewed by 2678

Abstract

Prion protein aggregation into amyloid fibrils is associated with the onset and progression of prion diseases—a group of neurodegenerative amyloidoses. The process of such aggregate formation is still not fully understood, especially regarding their polymorphism, an event where the same type of protein [...] Read more.

Prion protein aggregation into amyloid fibrils is associated with the onset and progression of prion diseases—a group of neurodegenerative amyloidoses. The process of such aggregate formation is still not fully understood, especially regarding their polymorphism, an event where the same type of protein forms multiple, conformationally and morphologically distinct structures. Considering that such structural variations can greatly complicate the search for potential antiamyloid compounds, either by having specific propagation properties or stability, it is important to better understand this aggregation event. We have recently reported the ability of prion protein fibrils to obtain at least two distinct conformations under identical conditions, which raised the question if this occurrence is tied to only certain environmental conditions. In this work, we examined a large sample size of prion protein aggregation reactions under a range of temperatures and analyzed the resulting fibril dye-binding, secondary structure and morphological properties. We show that all temperature conditions lead to the formation of more than one fibril type and that this variability may depend on the state of the initial prion protein molecules. Full article

(This article belongs to the Special Issue The Role of Environment in Amyloid Aggregation)

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15 pages, 4302 KiB

Open AccessArticle

Amyloid Structural Changes Studied by Infrared Microspectroscopy in Bigenic Cellular Models of Alzheimer’s Disease

by Agnes Paulus, Anders Engdahl, Yiyi Yang, Antonio Boza-Serrano, Sara Bachiller, Laura Torres-Garcia, Alexander Svanbergsson, Megg G. Garcia, Gunnar K. Gouras, Jia-Yi Li, Tomas Deierborg and Oxana Klementieva

Int. J. Mol. Sci. 2021, 22(7), 3430; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22073430 - 26 Mar 2021

Cited by 4 | Viewed by 2957

Abstract

Alzheimer’s disease affects millions of lives worldwide. This terminal disease is characterized by the formation of amyloid aggregates, so-called amyloid oligomers. These oligomers are composed of β-sheet structures, which are believed to be neurotoxic. However, the actual secondary structure that contributes most to [...] Read more.

Alzheimer’s disease affects millions of lives worldwide. This terminal disease is characterized by the formation of amyloid aggregates, so-called amyloid oligomers. These oligomers are composed of β-sheet structures, which are believed to be neurotoxic. However, the actual secondary structure that contributes most to neurotoxicity remains unknown. This lack of knowledge is due to the challenging nature of characterizing the secondary structure of amyloids in cells. To overcome this and investigate the molecular changes in proteins directly in cells, we used synchrotron-based infrared microspectroscopy, a label-free and non-destructive technique available for in situ molecular imaging, to detect structural changes in proteins and lipids. Specifically, we evaluated the formation of β-sheet structures in different monogenic and bigenic cellular models of Alzheimer’s disease that we generated for this study. We report on the possibility to discern different amyloid signatures directly in cells using infrared microspectroscopy and demonstrate that bigenic (amyloid-β, α-synuclein) and (amyloid-β, Tau) neuron-like cells display changes in β-sheet load. Altogether, our findings support the notion that different molecular mechanisms of amyloid aggregation, as opposed to a common mechanism, are triggered by the specific cellular environment and, therefore, that various mechanisms lead to the development of Alzheimer’s disease. Full article

(This article belongs to the Special Issue The Role of Environment in Amyloid Aggregation)

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9 pages, 6664 KiB

Open AccessArticle

Cold Atmospheric Plasma Modification of Amyloid β

by Maho Yagi-Utsumi, Tomohiro Tanaka, Yoko Otsubo, Akira Yamashita, Shinji Yoshimura, Motohiro Nishida and Koichi Kato

Int. J. Mol. Sci. 2021, 22(6), 3116; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22063116 - 18 Mar 2021

Cited by 3 | Viewed by 2416

Abstract

Cold atmospheric plasma (CAP) has attracted much attention in the fields of biotechnology and medicine owing to its potential utility in clinical applications. Recently accumulating evidence has demonstrated that CAP influences protein structures. However, there remain open questions regarding the molecular mechanisms behind [...] Read more.

Cold atmospheric plasma (CAP) has attracted much attention in the fields of biotechnology and medicine owing to its potential utility in clinical applications. Recently accumulating evidence has demonstrated that CAP influences protein structures. However, there remain open questions regarding the molecular mechanisms behind the CAP-induced structural perturbations of biomacromolecules. Here, we investigated the potential effects of CAP irradiation of amyloid β (Aβ), an amyloidogenic protein associated with Alzheimer’s disease. Using nuclear magnetic resonance spectroscopy, we observed gradual spectral changes in Aβ after a 10 s CAP pretreatment, which also suppressed its fibril formation, as revealed by thioflavin T assay. As per mass spectrometric analyses, these effects were attributed to selective oxidation of the methionine residue (Met) at position 35. Interestingly, this modification occurred when Aβ was dissolved into a pre-irradiated buffer, indicating that some reactive species oxidize the Met residue. Our results strongly suggest that the H₂O₂ generated in the solution by CAP irradiation is responsible for Met oxidation, which inhibits Aβ amyloid formation. The findings of the present study provide fundamental insights into plasma biology, giving clues for developing novel applications of CAP. Full article

(This article belongs to the Special Issue The Role of Environment in Amyloid Aggregation)

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13 pages, 4811 KiB

Open AccessArticle

Effect of Ionic Strength on Thioflavin-T Affinity to Amyloid Fibrils and Its Fluorescence Intensity

by Kamile Mikalauskaite, Mantas Ziaunys, Tomas Sneideris and Vytautas Smirnovas

Int. J. Mol. Sci. 2020, 21(23), 8916; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21238916 - 24 Nov 2020

Cited by 23 | Viewed by 3081

Abstract

The formation of amyloid fibrils is linked to multiple neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease. Despite years of research and countless studies on the topic of such aggregate formation, as well as their resulting structure, the current knowledge is still fairly limited. [...] Read more.

The formation of amyloid fibrils is linked to multiple neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease. Despite years of research and countless studies on the topic of such aggregate formation, as well as their resulting structure, the current knowledge is still fairly limited. One of the main aspects prohibiting effective aggregation tracking is the environment’s effect on amyloid-specific dyes, namely thioflavin-T (ThT). Currently, there are only a few studies hinting at ionic strength being one of the factors that modulate the dye’s binding affinity and fluorescence intensity. In this work we explore this effect under a range of ionic strength conditions, using insulin, lysozyme, mouse prion protein, and α-synuclein fibrils. We show that ionic strength is an extremely important factor affecting both the binding affinity, as well as the fluorescence intensity of ThT. Full article

(This article belongs to the Special Issue The Role of Environment in Amyloid Aggregation)

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