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Protein Science : a Publication of the... May 2017Human metallothionein 1a, a protein with two cysteine-rich metal-binding domains (α with 11 Cys and β with 9), was analyzed in its metal-free form by selective,...
Human metallothionein 1a, a protein with two cysteine-rich metal-binding domains (α with 11 Cys and β with 9), was analyzed in its metal-free form by selective, covalent Cys modification coupled with ESI-MS. The modification profiles of the isolated β- and α-fragments reacted with p-benzoquinone (Bq), N-ethylmalemide (NEM) and iodoacetamide (IAM) were compared with the full length protein using ESI-mass spectral data to follow the reaction pathway. Under denaturing conditions at low pH, the reaction profile with each modifier followed pathways that resulted in stochastic, Normal distributions of species whose maxima was equal to the mol. eq. of modifier added. Our interpretation of modification at this pH is that reaction with the cysteines is unimpeded when the full protein or those of its isolated domains are denatured. At neutral pH, where the protein is expected to be folded in a more compact structure, there is a difference in the larger Bq and NEM modification, whose reaction profiles indicate a cooperative pattern. The reaction profile with IAM under native conditions follows a similar stochastic distribution as at low pH, suggesting that this modifier is small enough to access the cysteines unimpeded by the compact structure. The data emphasize the utility of residue modification coupled with electrospray ionization mass spectrometry for the study of protein structure.
Topics: Cysteine; Humans; Hydrogen-Ion Concentration; Mass Spectrometry; Metallothionein; Protein Domains; Protein Folding; Protein Processing, Post-Translational
PubMed: 28187517
DOI: 10.1002/pro.3139 -
BMC Bioinformatics Sep 2020S-glutathionylation is the formation of disulfide bonds between the tripeptide glutathione and cysteine residues of the protein, protecting them from irreversible...
BACKGROUND
S-glutathionylation is the formation of disulfide bonds between the tripeptide glutathione and cysteine residues of the protein, protecting them from irreversible oxidation and in some cases causing change in their functions. Regulatory glutathionylation of proteins is a controllable and reversible process associated with cell response to the changing redox status. Prediction of cysteine residues that undergo glutathionylation allows us to find new target proteins, which function can be altered in pathologies associated with impaired redox status. We set out to analyze this issue and create new tool for predicting S-glutathionylated cysteine residues.
RESULTS
One hundred forty proteins with experimentally proven S-glutathionylated cysteine residues were found in the literature and the RedoxDB database. These proteins contain 1018 non-S-glutathionylated cysteines and 235 S-glutathionylated ones. Based on 235 S-glutathionylated cysteines, non-redundant positive dataset of 221 heptapeptide sequences of S-glutathionylated cysteines was made. Based on 221 heptapeptide sequences, a position-specific matrix was created by analyzing the protein sequence near the cysteine residue (three amino acid residues before and three after the cysteine). We propose the method for calculating the glutathionylation propensity score, which utilizes the position-specific matrix and a criterion for predicting glutathionylated peptides.
CONCLUSION
Non-S-glutathionylated sites were enriched by cysteines in - 3 and + 3 positions. The proposed prediction method demonstrates 76.6% of correct predictions of S-glutathionylated cysteines. This method can be used for detecting new glutathionylation sites, especially in proteins with an unknown structure.
Topics: Algorithms; Animals; Computational Biology; Cysteine; Glutathione; Humans; Peptides; Protein Processing, Post-Translational; Proteins
PubMed: 32921310
DOI: 10.1186/s12859-020-03571-w -
Nature Communications Jul 2023Protein-S-glutathionylation is a post-translational modification involving the conjugation of glutathione to protein thiols, which can modulate the activity and...
Protein-S-glutathionylation is a post-translational modification involving the conjugation of glutathione to protein thiols, which can modulate the activity and structure of key cellular proteins. Glutaredoxins (GLRX) are oxidoreductases that regulate this process by performing deglutathionylation. However, GLRX has five cysteines that are potentially vulnerable to oxidative modification, which is associated with GLRX aggregation and loss of activity. To date, GLRX cysteines that are oxidatively modified and their relative susceptibilities remain unknown. We utilized molecular modeling approaches, activity assays using recombinant GLRX, coupled with site-directed mutagenesis of each cysteine both individually and in combination to address the oxidizibility of GLRX cysteines. These approaches reveal that C8 and C83 are targets for S-glutathionylation and oxidation by hydrogen peroxide in vitro. In silico modeling and experimental validation confirm a prominent role of C8 for dimer formation and aggregation. Lastly, combinatorial mutation of C8, C26, and C83 results in increased activity of GLRX and resistance to oxidative inactivation and aggregation. Results from these integrated computational and experimental studies provide insights into the relative oxidizability of GLRX's cysteines and have implications for the use of GLRX as a therapeutic in settings of dysregulated protein glutathionylation.
Topics: Animals; Cysteine; Glutaredoxins; Glutathione; Mammals; Oxidation-Reduction; Proteins
PubMed: 37507364
DOI: 10.1038/s41467-023-39664-2 -
Plant, Cell & Environment Jan 2020After 30 years of intensive work, nitric oxide (NO) has just started to be characterized as a relevant regulatory molecule on plant development and responses to stress.... (Review)
Review
After 30 years of intensive work, nitric oxide (NO) has just started to be characterized as a relevant regulatory molecule on plant development and responses to stress. Its reactivity as a free radical determines its mode of action as an inducer of posttranslational modifications of key target proteins through cysteine S-nitrosylation and tyrosine nitration. Many of the NO-triggered regulatory actions are exerted in tight coordination with phytohormone signaling. This review not only summarizes and updates the information accumulated on how NO is synthesized, sensed, and transduced in plants but also makes emphasis on controversies, deficiencies, and misconceptions that are hampering our present knowledge on the biology of NO in plants. The development of noninvasive accurate tools for the endogenous NO quantitation as well as the implementation of genetic approaches that overcome misleading pharmacological experiments will be critical for getting significant advances in better knowledge of NO homeostasis and regulatory actions in plants.
Topics: Cysteine; Nitric Oxide; Plant Development; Plant Growth Regulators; Plant Proteins; Plants; Protein Processing, Post-Translational; Signal Transduction; Stress, Physiological; Tyrosine
PubMed: 31323702
DOI: 10.1111/pce.13617 -
The Journal of Biological Chemistry 20218-Oxoguanine glycosylase (OGG1) is a base excision repair enzyme responsible for the recognition and removal of 8-oxoguanine, a commonly occurring oxidized DNA...
8-Oxoguanine glycosylase (OGG1) is a base excision repair enzyme responsible for the recognition and removal of 8-oxoguanine, a commonly occurring oxidized DNA modification. OGG1 prevents the accumulation of mutations and regulates the transcription of various oxidative stress-response genes. In addition to targeting DNA, oxidative stress can affect proteins like OGG1 itself, specifically at cysteine residues. Previous work has shown that the function of OGG1 is sensitive to oxidants, with the cysteine residues of OGG1 being the most likely site of oxidation. Due to the integral role of OGG1 in maintaining cellular homeostasis under oxidative stress, it is important to understand the effect of oxidants on OGG1 and the role of cysteines in its structure and function. In this study, we investigate the role of the cysteine residues in the function of OGG1 by mutating and characterizing each cysteine residue. Our results indicate that the cysteines in OGG1 fall into four functional categories: those that are necessary for (1) glycosylase activity (C146 and C255), (2) lyase activity (C140S, C163, C241, and C253), and (3) structural stability (C253) and (4) those with no known function (C28 and C75). These results suggest that under conditions of oxidative stress, cysteine can be targeted for modifications, thus altering the response of OGG1 and affecting its downstream cellular functions.
Topics: Cysteine; DNA Glycosylases; DNA Repair; Electrophoretic Mobility Shift Assay; Oxidation-Reduction; Oxidative Stress
PubMed: 33203705
DOI: 10.1074/jbc.RA120.016126 -
Biomolecules Mar 2021Protein homeostasis is an essential component of proper cellular function; however, sustaining protein health is a challenging task, especially during the aerobic... (Review)
Review
Protein homeostasis is an essential component of proper cellular function; however, sustaining protein health is a challenging task, especially during the aerobic lifestyle. Natural cellular oxidants may be involved in cell signaling and antibacterial defense; however, imbalanced levels can lead to protein misfolding, cell damage, and death. This merges together the processes of protein homeostasis and redox regulation. At the heart of this process are redox-regulated proteins or thiol-based switches, which carefully mediate various steps of protein homeostasis across folding, localization, quality control, and degradation pathways. In this review, we discuss the "redox code" of the proteostasis network, which shapes protein health during cell growth and aging. We describe the sources and types of thiol modifications and elaborate on diverse strategies of evolving antioxidant proteins in proteostasis networks during oxidative stress conditions. We also highlight the involvement of cysteines in protein degradation across varying levels, showcasing the importance of cysteine thiols in proteostasis at large. The individual examples and mechanisms raised open the door for extensive future research exploring the interplay between the redox and protein homeostasis systems. Understanding this interplay will enable us to re-write the redox code of cells and use it for biotechnological and therapeutic purposes.
Topics: Animals; Cysteine; Humans; Oxidation-Reduction; Oxidative Stress; Proteins; Sulfhydryl Compounds
PubMed: 33809923
DOI: 10.3390/biom11030469 -
International Journal of Molecular... Oct 2014In order to use cyanobacteria for the biological production of hydrogen, it is important to thoroughly study the function and the regulation of the hydrogen-production... (Review)
Review
In order to use cyanobacteria for the biological production of hydrogen, it is important to thoroughly study the function and the regulation of the hydrogen-production machine in order to better understand its role in the global cell metabolism and identify bottlenecks limiting H2 production. Most of the recent advances in our understanding of the bidirectional [Ni-Fe] hydrogenase (Hox) came from investigations performed in the widely-used model cyanobacterium Synechocystis PCC6803 where Hox is the sole enzyme capable of combining electrons with protons to produce H2 under specific conditions. Recent findings suggested that the Hox enzyme can receive electrons from not only NAD(P)H as usually shown, but also, or even preferentially, from ferredoxin. Furthermore, plasmid-encoded functions and glutathionylation (the formation of a mixed-disulfide between the cysteines residues of a protein and the cysteine residue of glutathione) are proposed as possible new players in the function and regulation of hydrogen production.
Topics: Bacterial Proteins; Cyanobacteria; Cysteine; Electron Transport; Ferredoxins; Glutathione; Hydrogen; Hydrogenase; Multigene Family; Oxidative Stress
PubMed: 25365180
DOI: 10.3390/ijms151119938 -
STAR Protocols Mar 2024Pinpointing functional, structural, and redox-sensitive cysteines is a central challenge of chemoproteomics. Here, we present a protocol comprising two dual-enrichment...
Pinpointing functional, structural, and redox-sensitive cysteines is a central challenge of chemoproteomics. Here, we present a protocol comprising two dual-enrichment cysteine chemoproteomic techniques that enable capture of cysteines (Cys-LoC) and quantification of cysteine oxidation state (Cys-LOx) in a localization-specific manner. We describe steps for utilizing TurboID-mediated protein biotinylation for enrichment of compartment-specific proteins, followed by click-mediated biotinylation and enrichment of cysteine-containing peptides. Thus, changes to compartment-specific cysteine identification and redox state can be assessed in a variety of contexts. For complete details on the use and execution of this protocol, please refer to Yan et al. (2023)..
Topics: Cysteine; Proteins; Peptides; Organelles; Oxidation-Reduction
PubMed: 38329879
DOI: 10.1016/j.xpro.2024.102865 -
Chimia Nov 2016Our laboratory focuses on chemical proteomics-enabled discovery of new cysteine-reactive small molecules with intriguing biomedical activities as well as identification... (Review)
Review
Our laboratory focuses on chemical proteomics-enabled discovery of new cysteine-reactive small molecules with intriguing biomedical activities as well as identification and detailed characterization of their proteomic targets. In this overview article, we summarize our progress since 2013 in this research field. We have developed a novel mass spectrometry-based chemoproteomic method that allows detection and monitoring of up to ~3000 reactive cysteines in any cellular proteome. This is achieved via strategic use of two clickable, cysteine-reactive chemical probes with complementary substrate selectivity profiles, iodoacetamide and ethynyl benziodoxolone. Using this method, we have been able to identify the direct biological targets of curcumin, a diarylheptanoid natural product with anticancer activity, and deoxyelephantopin, a highly cytotoxic natural sesquiterpene lactone. Furthermore, we have developed chloromethyl triazoles (CMTs) as a novel chemical scaffold for cysteine-reactive inhibitors that can be accessed from commercially available substrates in only two chemical steps. From a small collection of chloromethyl triazoles, we have identified compound AA-CW236 as the first non-pseudosubstrate inhibitor of MGMT, a DNA repair protein that renders several devastating cancer forms resistant to chemotherapy.
Topics: Cysteine; Drug Discovery; Humans; Mass Spectrometry; Models, Molecular; Molecular Structure; Proteomics
PubMed: 28661335
DOI: 10.2533/chimia.2016.764 -
STAR Protocols Jun 2021Differential amino acid reactivity with chemical probes can provide valuable information on the functionality and ligandability of proteins in native biological systems....
Differential amino acid reactivity with chemical probes can provide valuable information on the functionality and ligandability of proteins in native biological systems. Here, we present a quantitative, multiplexed chemical proteomic protocol for in-depth reactivity and ligandability profiling of cysteines in proteins in quiescent and stimulated T cells. This protocol illuminates dynamic immune state-dependent alterations in cysteine reactivity, revealing chemoselective and stereoselective small-molecule interactions with cysteines in structurally and functionally diverse proteins that lack chemical probes. For complete details on the use and execution of this protocol, please refer to Vinogradova et al. (2020).
Topics: Cysteine; Humans; Proteome; Proteomics; T-Lymphocytes
PubMed: 33899026
DOI: 10.1016/j.xpro.2021.100458