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Biological Systems at the Protein Level

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: closed (31 October 2019) | Viewed by 21119

Special Issue Editor

College of Arts and Science, University of Missouri, 125 Chemistry Bldg., Columbia, MO 65211, USA
Interests: biological mass spectrometry; separation of complex mixtures; quantitative proteomics; directed metabolite analysis
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Biological systems can be defined from cells through plants and animals. These systems can respond to a variety of stimuli or environmental conditions. The protein and molecular responses can be measured by a variety of methods, often using mass spectrometry. Of particular interest are quantitative expression profiling (proteins or metabolites), response-associated post-translational modifications, and pathway analysis.

Dr. C. Michael Greenlief
Guest Editor

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Keywords

  • post-translational modifications
  • pathway analysis
  • metabolomics
  • proteomics
  • structural biology
  • quantitative expression profiling

Published Papers (5 papers)

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Research

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15 pages, 2761 KiB  
Article
Screening and Identification of Novel cGAS Homologues Using a Combination of in Vitro and In Vivo Protein Synthesis
by Jascha Rolf, Regine Siedentop, Stephan Lütz and Katrin Rosenthal
Int. J. Mol. Sci. 2020, 21(1), 105; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21010105 - 22 Dec 2019
Cited by 17 | Viewed by 4761
Abstract
The cyclic GMP-AMP synthase (cGAS) catalyzes the synthesis of the multifunctional second messenger, cGAMP, in metazoans. Although numerous cGAS homologues are predicted in protein databases, the catalytic activity towards cGAMP synthesis has been proven for only four of them. Therefore, we selected five [...] Read more.
The cyclic GMP-AMP synthase (cGAS) catalyzes the synthesis of the multifunctional second messenger, cGAMP, in metazoans. Although numerous cGAS homologues are predicted in protein databases, the catalytic activity towards cGAMP synthesis has been proven for only four of them. Therefore, we selected five novel and yet uncharacterized cGAS homologues, which cover a broad range in the field of vertebrates. Cell-free protein synthesis (CFPS) was used for a pre-screening to investigate if the cGAS genes originating from higher organisms can be efficiently expressed in a bacterial expression system. As all tested cGAS variants were expressible, enzymes were synthesized in vivo to supply higher amounts for a subsequent in vitro activity assay. The assays were carried out with purified enzymes and revealed vast differences in the activity of the homologues. For the first time, the cGAS homologues from the Przewalski’s horse, naked mole-rat, bald eagle, and zebrafish were proven to catalyze the synthesis of cGAMP. The extension of the list of described cGAS variants enables the acquisition of further knowledge about the structural and molecular mechanism of cGAS, potentially leading to functional improvement of the enzyme. Full article
(This article belongs to the Special Issue Biological Systems at the Protein Level)
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19 pages, 4621 KiB  
Article
Novel Peptidomic Approach for Identification of Low and High Molecular Weight Tauopathy Peptides Following Calpain Digestion, and Primary Culture Neurotoxic Challenges
by Hamad Yadikar, Connor Johnson, Niko Pafundi, Edwin Mouhawasse, Lynn Nguyen, Isabel Torres, Milin Kurup, Zhihui Yang, Firas Kobeissy, Richard Yost and Kevin K. Wang
Int. J. Mol. Sci. 2019, 20(20), 5213; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20205213 - 21 Oct 2019
Cited by 7 | Viewed by 3731
Abstract
Tauopathy is a class of a neurodegenerative disorder linked with tau hyperphosphorylation, proteolysis, and aggregation. Tau can be subjected to proteolysis upon calpain activation in Alzheimer disease (AD), and traumatic brain injury (TBI). We and others have extensively researched calpain-mediated tau breakdown products [...] Read more.
Tauopathy is a class of a neurodegenerative disorder linked with tau hyperphosphorylation, proteolysis, and aggregation. Tau can be subjected to proteolysis upon calpain activation in Alzheimer disease (AD), and traumatic brain injury (TBI). We and others have extensively researched calpain-mediated tau breakdown products (Tau-BDP; 45K, 35K, and 17K). Tau proteolysis might also generate low molecular weight (LMW ≤10K) proteolytic peptides after neurodegenerative damage. In this study, we have subjected purified tau protein (phospho and non-phospho) and mouse brain lysate to calpain-1 digestion to characterize the LMW generated by nano-liquid chromatography coupled to electrospray ionization to tandem mass spectrometry (nano-LC-ESI-MS/MS). We have also challenged differentiated primary cerebrocortical neuronal cultures (CTX) with neurotoxic agents (calcium ionophore calcimycin (A23187), staurosporine (STS), N-methyl-D-aspartate (NMDA), and Maitotoxin (MTX)) that mimic neurodegeneration to investigate the peptidome released into the conditioned cell media. We used a simple workflow in which we fractionate LMW calpain-mediated tau peptides by ultrafiltration (molecular weight cut-off value (MWCO) of 10K) and subject filtrate fractions to nano-LC-MS/MS analysis. The high molecular weight (HMW) peptides and intact proteins retained on the filter were analyzed separately by western blotting using total and phospho-specific tau antibodies. We have identified several novel proteolytic tau peptides (phosphorylated and non-phosphorylated) that are only present in samples treated with calpain or cell-based calpain activation model (particularly N- and C-terminal peptides). Our findings can help in developing future research strategies emphasizing on the suppression of tau proteolysis as a target. Full article
(This article belongs to the Special Issue Biological Systems at the Protein Level)
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16 pages, 2241 KiB  
Article
The G9a Histone Methyltransferase Inhibitor BIX-01294 Modulates Gene Expression during Plasmodium falciparum Gametocyte Development and Transmission
by Che Julius Ngwa, Meike Jutta Kiesow, Lindsey Marie Orchard, Afia Farrukh, Manuel Llinás and Gabriele Pradel
Int. J. Mol. Sci. 2019, 20(20), 5087; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20205087 - 14 Oct 2019
Cited by 17 | Viewed by 3441
Abstract
Transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is initiated by specialized sexual cells, the gametocytes. In the human, gametocytes are formed in response to stress signals and following uptake by a blood-feeding Anopheles mosquito initiate sexual reproduction. [...] Read more.
Transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is initiated by specialized sexual cells, the gametocytes. In the human, gametocytes are formed in response to stress signals and following uptake by a blood-feeding Anopheles mosquito initiate sexual reproduction. Gametocytes need to fine-tune their gene expression in order to develop inside the mosquito to continue life-cycle progression. Previously, we showed that post-translational histone acetylation controls gene expression during gametocyte development and transmission. However, the role of histone methylation remains poorly understood. We here use the histone G9a methyltransferase inhibitor BIX-01294 to investigate the role of histone methylation in regulating gene expression in gametocytes. In vitro assays demonstrated that BIX-01294 inhibits intraerythrocytic replication with a half maximal inhibitory concentration (IC50) of 13.0 nM. Furthermore, BIX-01294 significantly impairs gametocyte maturation and reduces the formation of gametes and zygotes. Comparative transcriptomics between BIX-01294-treated and untreated immature, mature and activated gametocytes demonstrated greater than 1.5-fold deregulation of approximately 359 genes. The majority of these genes are transcriptionally downregulated in the activated gametocytes and could be assigned to transcription, translation, and signaling, indicating a contribution of histone methylations in mediating gametogenesis. Our combined data show that inhibitors of histone methylation may serve as a multi-stage antimalarial. Full article
(This article belongs to the Special Issue Biological Systems at the Protein Level)
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16 pages, 2980 KiB  
Article
Extra Purified Exosomes from Human Placenta Contain an Unpredictable Small Number of Different Major Proteins
by Evgeniya E. Burkova, Alina E. Grigor’eva, Dmitrii V. Bulgakov, Pavel S. Dmitrenok, Valentin V. Vlassov, Elena I. Ryabchikova, Sergey E. Sedykh and Georgy A. Nevinsky
Int. J. Mol. Sci. 2019, 20(10), 2434; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20102434 - 16 May 2019
Cited by 32 | Viewed by 4665
Abstract
Exosomes are nanovesicles (30–100 nm) containing various RNAs and different proteins. Exosomes are important in intracellular communication, immune function, etc. Exosomes from different sources including placenta were mainly obtained by different types of centrifugation and ultracentrifugations and were reported to contain from a [...] Read more.
Exosomes are nanovesicles (30–100 nm) containing various RNAs and different proteins. Exosomes are important in intracellular communication, immune function, etc. Exosomes from different sources including placenta were mainly obtained by different types of centrifugation and ultracentrifugations and were reported to contain from a few dozen to thousands of different proteins. First crude exosome preparations from four placentas (normal pregnancy) were obtained here using several standard centrifugations but then were additionally purified by gel filtration on Sepharose 4B. Individual preparations demonstrated different gel filtration profiles showing good or bad separation of exosome peaks from two peaks of impurity proteins and their complexes. According to electron microscopy, exosomes before gel filtration contain vesicles of different size, ring-shaped structures forming by ferritin and clusters of aggregated proteins and their complexes. After filtration through 220 nm filters and gel filtration exosomes display typically for exosome morphology and size (30–100 nm) and do not contain visible protein admixtures. Identification of exosome proteins was carried out by MS and MS/MS MALDI mass spectrometry of proteins’ tryptic hydrolyzates after their SDS-PAGE and 2D electrophoresis. We have obtained unexpected results. Good, purified exosomes contained only 11–13 different proteins: CD9, CD81, CD-63, hemoglobin subunits, interleukin-1 receptor, annexin A1, annexin A2, annexin A5, cytoplasmic actin, alkaline phosphatase, serotransferin, and probably human serum albumin and immunoglobulins. We assume that a possible number of exosome proteins found previously using crude preparations may be very much overestimated. Our data may be important for study of biological functions of pure exosomes. Full article
(This article belongs to the Special Issue Biological Systems at the Protein Level)
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Review

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16 pages, 2426 KiB  
Review
Homologous Recombination under the Single-Molecule Fluorescence Microscope
by Dalton R. Gibbs and Soma Dhakal
Int. J. Mol. Sci. 2019, 20(23), 6102; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20236102 - 03 Dec 2019
Cited by 9 | Viewed by 4109
Abstract
Homologous recombination (HR) is a complex biological process and is central to meiosis and for repair of DNA double-strand breaks. Although the HR process has been the subject of intensive study for more than three decades, the complex protein–protein and protein–DNA interactions during [...] Read more.
Homologous recombination (HR) is a complex biological process and is central to meiosis and for repair of DNA double-strand breaks. Although the HR process has been the subject of intensive study for more than three decades, the complex protein–protein and protein–DNA interactions during HR present a significant challenge for determining the molecular mechanism(s) of the process. This knowledge gap is largely because of the dynamic interactions between HR proteins and DNA which is difficult to capture by routine biochemical or structural biology methods. In recent years, single-molecule fluorescence microscopy has been a popular method in the field of HR to visualize these complex and dynamic interactions at high spatiotemporal resolution, revealing mechanistic insights of the process. In this review, we describe recent efforts that employ single-molecule fluorescence microscopy to investigate protein–protein and protein–DNA interactions operating on three key DNA-substrates: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and four-way DNA called Holliday junction (HJ). We also outline the technological advances and several key insights revealed by these studies in terms of protein assembly on these DNA substrates and highlight the foreseeable promise of single-molecule fluorescence microscopy in advancing our understanding of homologous recombination. Full article
(This article belongs to the Special Issue Biological Systems at the Protein Level)
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