scholarly journals SPEAR: a proteomics approach for simultaneous protein expression and redox analysis

2021 ◽  
Author(s):  
Shani Doron ◽  
Nardy Lampl ◽  
Alon Savidor ◽  
Corine Katina ◽  
Alexandra Gabashvili ◽  
...  
Keyword(s):  
Protein Expression ◽  
Oxidation State ◽  
Active Sites ◽  
Redox Regulation ◽  
Effective Means ◽  
Stress Conditions ◽  
Cysteine Oxidation ◽  
Redox Proteomics ◽  
Redox Biology ◽  
Proteomics Approach

Oxidation and reduction of protein cysteinyl thiols serve as molecular switches, which is considered the most central mechanism for redox regulation of biological processes, altering protein structure, biochemical activity, subcellular localization, and binding affinity. Redox proteomics allows for the global identification of redox-modified cysteine (Cys) sites and quantification of their oxidation/reduction responses, serving as a hypothesis-generating platform to stimulate redox biology mechanistic research. Here, we developed Simultaneous Protein Expression and Redox (SPEAR) analysis, a new redox-proteomics approach based on differential labeling of oxidized and reduced cysteines with light and heavy isotopic forms of commercially available isotopically-labeled N-ethylmaleimide (NEM). The presented method does not require enrichment for labeled peptides, thus enabling simultaneous quantification of Cys oxidation state and protein abundance. Using SPEAR, we were able to quantify the in-vivo oxidation state of thousands of cysteines across the Arabidopsis proteome under steady-state and oxidative stress conditions. Functional assignment of the identified redox-sensitive proteins demonstrated the widespread effect of oxidative conditions on various cellular functions and highlighted the enrichment of chloroplast-targeted proteins. SPEAR provides a simple, straightforward, and cost-effective means of studying redox proteome dynamics. The presented data provide a global quantitative view of cysteine oxidation of well-known redox-regulated active sites and many novel redox-sensitive sites whose role in plant acclimation to stress conditions remains to be further explored.  

scholarly journals Spatial and temporal alterations in protein structure by EGF regulate cryptic cysteine oxidation

2019 ◽  
Author(s):  
Jessica B Behring ◽  
Sjoerd van der Post ◽  
Arshag D Mooradian ◽  
Matthew J Egan ◽  
Maxwell I Zimmerman ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Redox Regulation ◽  
Receptor Tyrosine Kinases ◽  
Redox Signaling ◽  
Signaling Networks ◽  
Cysteine Oxidation ◽  
Time Resolved ◽  
Cellular Redox ◽  
Redox Biology ◽  
Stimulation Of

AbstractStimulation of receptor tyrosine kinases (RTK) such as EGF locally increase reactive oxygen species (ROS) levels at the plasma membrane that oxidize cysteines in proteins to enhance downstream signaling. Spatial confinement of ROS is an important regulatory mechanism to redox signaling, but it remains unknown why stimulation of different receptor tyrosine kinases (RTKs) at the plasma membrane target distinct sets of downstream proteins. To uncover additional mechanisms specifying which cysteines are redox regulated by EGF stimulation, we performed time-resolved quantification of the oxidation of 4,200 cysteine sites subsequent to EGF stimulation in A431 cells. EGF induces three distinct spatiotemporal patterns of cysteine oxidation in functionally organized protein networks, consistent with the spatial confinement model. Unexpectedly, protein crystal structure analysis and molecular dynamic simulation indicate widespread redox regulation of cryptic cysteines that are only solvent exposed upon changes in protein conformation. Phosphorylation and increased flux of nucleotide substrates serve as two distinct modes by which EGF specifies which cryptic cysteines become solvent exposed and redox regulated. Since proteins structurally regulated by different RTKs or cellular perturbations are largely unique, solvent exposure and redox regulation of cryptic cysteines is an important mechanism contextually delineating redox signaling networks.Significance StatementCellular redox processes are interconnected, but are not in equilibrium. Thus, understanding the redox biology of cells requires a systems-level, rather than reductionist, approach. Factors specifying which cysteines are redox regulated by a stimulus remain poorly characterized but are critical to understanding the fundamental properties of redox signaling networks. Here, we show that EGF stimulation induces oxidation of specific cysteines in 3 distinct spatiotemporal patterns. Redox regulated proteins include many proteins in the EGF pathway as well as many cysteines with known functional importance. Many redox regulated cysteines are cryptic and solvent exposed by changes in protein structure that were induced by EGF treatment. The novel finding that cryptic cysteines are redox regulated has important implications for how redox signaling networks are specified and regulated to minimize crosstalk. In addition, this time-resolved dataset of the redox kinetics of 4,200 cysteine sites is an important resource for others and is an important technological achievement towards systems-level understanding of cellular redox biology.

scholarly journals Is Oxidized Thioredoxin a Major Trigger for Cysteine Oxidation? Clues from a Redox Proteomics Approach

2013 ◽  
Vol 18 (13) ◽  
pp. 1549-1556 ◽  
Author(s):  
Sarela García-Santamarina ◽  
Susanna Boronat ◽  
Isabel A. Calvo ◽  
Miguel Rodríguez-Gabriel ◽  
José Ayté ◽  
...  
Keyword(s):  
Cysteine Oxidation ◽  
Redox Proteomics ◽  
Proteomics Approach

Valence variation of phase-pure M1 MoVNbTe oxide by plasma treatment for improved catalytic performance in oxidative dehydrogenation of ethane

RSC Advances ◽  
2015 ◽  
Vol 5 (111) ◽  
pp. 91295-91301 ◽  
Author(s):  
Xin Chen ◽  
Qianli Yang ◽  
Bozhao Chu ◽  
Hang An ◽  
Yi Cheng
Keyword(s):  
Surface Modification ◽  
Metal Oxide ◽  
Plasma Treatment ◽  
Oxidative Dehydrogenation ◽  
Oxidation State ◽  
Active Sites ◽  
Oxygen Plasma ◽  
Catalyst Surface ◽  
Catalytic Performance ◽  
Phase Pure

This work presents a new method of catalyst surface modification by using oxygen plasma to change the oxidation state of active sites in metal oxide catalysts.

scholarly journals Melatonin protects mesenchymal stem cells from autophagy‐mediated death under ischaemic ER‐stress conditions by increasing prion protein expression

2018 ◽  
Vol 52 (2) ◽  
pp. e12545 ◽  
Author(s):  
Jun Hee Lee ◽  
Yeo Min Yoon ◽  
Yong‐Seok Han ◽  
Seo Kyung Jung ◽  
Sang Hun Lee
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Protein Expression ◽  
Er Stress ◽  
Prion Protein ◽  
Stress Conditions

scholarly journals Proteome-Wide Survey Reveals Norbornene Is Complementary to Dimedone and Related Nucleophilic Probes for Cysteine Sulfenic Acid

2019 ◽  
Author(s):  
Lisa Alcock ◽  
Maike Langini ◽  
Kai Stühler ◽  
Marc Remke ◽  
Michael Perkins ◽  
...  
Keyword(s):  
Redox Regulation ◽  
Chemical Reactivity ◽  
Live Cells ◽  
Chemical Probes ◽  
Cysteine Oxidation ◽  
Sulfenic Acid ◽  
Proteomics Analysis ◽  
New Members ◽  
Specific Chemical ◽  
Future Studies

<p>Detection of cysteine sulfenic acid in live cells is critical in advancing our understanding of cysteine redox chemistry and its biological function. Accordingly, there is a need to develop sulfenic acid-specific chemical probes with distinct reaction mechanisms to facilitate proteome-wide detection of this important posttranslational modification. Herein, we report the first whole-cell proteomics analysis using a norbornene probe to detect cysteine sulfenic acid in live HeLa cells. Comparison of the enriched proteins to those identified using dimedone and other <i>C</i>-nucleophilic probes revealed a complementary reactivity profile. Remarkably, 148 new members of the sulfenome were identified. These discoveries highlight how subtle differences in chemical reactivity of both the probes and cysteine residues influence detection. Overall, this study expands our understanding of protein oxidation at cysteine and reveals new proteins to consider for future studies of cysteine oxidation, redox regulation and signaling, and the biochemistry of oxidative stress. </p>

scholarly journals Proteome-Wide Survey Reveals Norbornene Is Complementary to Dimedone and Related Nucleophilic Probes for Cysteine Sulfenic Acid

2019 ◽  
Author(s):  
Lisa Alcock ◽  
Maike Langini ◽  
Kai Stühler ◽  
Marc Remke ◽  
Michael Perkins ◽  
...  
Keyword(s):  
Redox Regulation ◽  
Chemical Reactivity ◽  
Live Cells ◽  
Chemical Probes ◽  
Cysteine Oxidation ◽  
Sulfenic Acid ◽  
Proteomics Analysis ◽  
New Members ◽  
Specific Chemical ◽  
Future Studies

<p>Detection of cysteine sulfenic acid in live cells is critical in advancing our understanding of cysteine redox chemistry and its biological function. Accordingly, there is a need to develop sulfenic acid-specific chemical probes with distinct reaction mechanisms to facilitate proteome-wide detection of this important posttranslational modification. Herein, we report the first whole-cell proteomics analysis using a norbornene probe to detect cysteine sulfenic acid in live HeLa cells. Comparison of the enriched proteins to those identified using dimedone and other <i>C</i>-nucleophilic probes revealed a complementary reactivity profile. Remarkably, 148 new members of the sulfenome were identified. These discoveries highlight how subtle differences in chemical reactivity of both the probes and cysteine residues influence detection. Overall, this study expands our understanding of protein oxidation at cysteine and reveals new proteins to consider for future studies of cysteine oxidation, redox regulation and signaling, and the biochemistry of oxidative stress. </p>

scholarly journals The effect of calnexin deletion on the expression level of PDI in Saccharomyces cerevisiae under heat stress conditions

2008 ◽  
Vol 13 (1) ◽  
Author(s):  
Huili Zhang ◽  
Jianwei He ◽  
Yanyan Ji ◽  
Akio Kato ◽  
Youtao Song
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Heat Stress ◽  
Protein Expression ◽  
Molecular Chaperone ◽  
Mrna Level ◽  
Stress Conditions ◽  
Mrna Levels ◽  
Wild Type ◽  
Wild Type Yeast

AbstractWe cultured calnexin-disrupted and wild-type Saccharomyces cerevisiae strains under conditions of heat stress. The growth rate of the calnexin-disrupted yeast was almost the same as that of the wild-type yeast under those conditions. However, the induced mRNA level of the molecular chaperone PDI in the ER was clearly higher in calnexin-disrupted S. cerevisiae relative to the wild type at 37°C, despite being almost the same in the two strains under normal conditions. The western blotting analysis for PDI protein expression in the ER yielded results that show a parallel in their mRNA levels in the two strains. We suggest that PDI may interact with calnexin under heat stress conditions, and that the induction of PDI in the ER can recover part of the function of calnexin in calnexin-disrupted yeast, and result in the same growth rate as in wild-type yeast.

Pinpointing cysteine oxidation sites by high-resolution proteomics reveals a mechanism of redox-dependent inhibition of human STING

2021 ◽  
Vol 14 (680) ◽  
pp. eaaw4673
Author(s):  
Natalia Zamorano Cuervo ◽  
Audray Fortin ◽  
Elise Caron ◽  
Stéfany Chartier ◽  
Nathalie Grandvaux
Keyword(s):  
Posttranslational Modifications ◽  
Protein Function ◽  
Disulfide Bonds ◽  
Redox Regulation ◽  
Mass Spectrometry Analysis ◽  
Type I ◽  
Cysteine Oxidation ◽  
Spectrometry Analysis ◽  
Host Interactions ◽  
In Cells

Protein function is regulated by posttranslational modifications (PTMs), among which reversible oxidation of cysteine residues has emerged as a key regulatory mechanism of cellular responses. Given the redox regulation of virus-host interactions, the identification of oxidized cysteine sites in cells is essential to understand the underlying mechanisms involved. Here, we present a proteome-wide identification of reversibly oxidized cysteine sites in oxidant-treated cells using a maleimide-based bioswitch method coupled to mass spectrometry analysis. We identified 2720 unique oxidized cysteine sites within 1473 proteins with distinct abundances, locations, and functions. Oxidized cysteine sites were found in numerous signaling pathways, many relevant to virus-host interactions. We focused on the oxidation of STING, the central adaptor of the innate immune type I interferon pathway, which is stimulated in response to the detection of cytosolic DNA by cGAS. We demonstrated the reversible oxidation of Cys148 and Cys206 of STING in cells. Molecular analyses led us to establish a model in which Cys148 oxidation is constitutive, whereas Cys206 oxidation is inducible by oxidative stress or by the natural ligand of STING, 2′3′-cGAMP. Our data suggest that the oxidation of Cys206 prevented hyperactivation of STING by causing a conformational change associated with the formation of inactive polymers containing intermolecular disulfide bonds. This finding should aid the design of therapies targeting STING that are relevant to autoinflammatory disorders, immunotherapies, and vaccines.

scholarly journals Redox Regulation and Oxidative Stress: The Particular Case of the Stallion Spermatozoa

Antioxidants ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 567 ◽  
Author(s):  
Fernando J. Peña ◽  
Cristian O’Flaherty ◽  
José M. Ortiz Rodríguez ◽  
Francisco E. Martín Cano ◽  
Gemma L. Gaitskell-Phillips ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Redox Regulation ◽  
Redox Homeostasis ◽  
Reactive Oxygen ◽  
Mitochondrial Activity ◽  
Oxygen Species ◽  
Redox Biology ◽  
Major Interest ◽  
And Oxidative Stress

Redox regulation and oxidative stress have become areas of major interest in spermatology. Alteration of redox homeostasis is recognized as a significant cause of male factor infertility and is behind the damage that spermatozoa experience after freezing and thawing or conservation in a liquid state. While for a long time, oxidative stress was just considered an overproduction of reactive oxygen species, nowadays it is considered as a consequence of redox deregulation. Many essential aspects of spermatozoa functionality are redox regulated, with reversible oxidation of thiols in cysteine residues of key proteins acting as an “on–off” switch controlling sperm function. However, if deregulation occurs, these residues may experience irreversible oxidation and oxidative stress, leading to malfunction and ultimately death of the spermatozoa. Stallion spermatozoa are “professional producers” of reactive oxygen species due to their intense mitochondrial activity, and thus sophisticated systems to control redox homeostasis are also characteristic of the spermatozoa in the horse. As a result, and combined with the fact that embryos can easily be collected in this species, horses are a good model for the study of redox biology in the spermatozoa and its impact on the embryo.

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