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Michael A Marletta - One of the best experts on this subject based on the ideXlab platform.
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Comparative and integrative metabolomics reveal that S-Nitrosation inhibits physiologically relevant metabolic enzymes.
Journal of Biological Chemistry, 2018Co-Authors: Joel Bruegger, Brian C. Smith, Sarah L. Wynia-smith, Michael A MarlettaAbstract:Cysteine S-Nitrosation is a reversible post-translational modification mediated by nitric oxide (•NO)-derived agents. S-Nitrosation participates in cellular signaling and is associated with several diseases such as cancer, cardiovascular diseases, and neuronal disorders. Despite the physiological importance of this nonclassical •NO-signaling pathway, little is understood about how much S-Nitrosation affects protein function. Moreover, identifying physiologically relevant targets of S-Nitrosation is difficult because of the dynamics of transNitrosation and a limited understanding of the physiological mechanisms leading to selective protein S-Nitrosation. To identify proteins whose activities are modulated by S-Nitrosation, we performed a metabolomics study comparing WT and endothelial nitric-oxide synthase knockout mice. We integrated our results with those of a previous proteomics study that identified physiologically relevant S-nitrosated cysteines, and we found that the activity of at least 21 metabolic enzymes might be regulated by S-Nitrosation. We cloned, expressed, and purified four of these enzymes and observed that S-Nitrosation inhibits the metabolic enzymes 6-phosphogluconate dehydrogenase, Δ1-pyrroline-5-carboxylate dehydrogenase, catechol-O-methyltransferase, and d-3-phosphoglycerate dehydrogenase. Furthermore, using site-directed mutagenesis, we identified the predominant cysteine residue influencing the observed activity changes in each enzyme. In summary, using an integrated metabolomics approach, we have identified several physiologically relevant S-Nitrosation targets, including metabolic enzymes, which are inhibited by this modification, and we have found the cysteines modified by S-Nitrosation in each enzyme.
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chemoproteomic strategy to quantitatively monitor transNitrosation uncovers functionally relevant s Nitrosation sites on cathepsin d and hadh2
Chemistry & Biology, 2016Co-Authors: Yani Zhou, Michael A Marletta, Brian C. Smith, Kelsey S. Kalous, Sarah L Wyniasmith, Shalise M Couvertier, Eranthie WeerapanaAbstract:S-Nitrosoglutathione (GSNO) is an endogenous transNitrosation donor involved in S-Nitrosation of a variety of cellular proteins, thereby regulating diverse protein functions. Quantitative proteomic methods are necessary to establish which cysteine residues are most sensitive to GSNO-mediated transNitrosation. Here, a competitive cysteine-reactivity profiling strategy was implemented to quantitatively measure the sensitivity of >600 cysteine residues to transNitrosation by GSNO. This platform identified a subset of cysteine residues with a high propensity for GSNO-mediated transNitrosation. Functional characterization of previously unannotated S-Nitrosation sites revealed that S-Nitrosation of a cysteine residue distal to the 3-hydroxyacyl-CoA dehydrogenase type 2 (HADH2) active site impaired catalytic activity. Similarly, S-Nitrosation of a non-catalytic cysteine residue in the lysosomal aspartyl protease cathepsin D (CTSD) inhibited proteolytic activation. Together, these studies revealed two previously uncharacterized cysteine residues that regulate protein function, and established a chemical-proteomic platform with capabilities to determine substrate specificity of other cellular transNitrosation agents.
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Site-specific and redox-controlled S-Nitrosation of thioredoxin.
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Katherine T Barglow, Steven R. Tannenbaum, Charles G Knutson, John S Wishnok, Michael A MarlettaAbstract:Protein S-Nitrosation on cysteine residues has emerged as an important posttranslational modification in mammalian cells. Previous studies have suggested a primary role for thioredoxin (Trx) in controlling protein S-Nitrosation reactions. Human Trx contains five conserved Cys, including two redox-active catalytic Cys (Cys32 and Cys35) and three non-active-site Cys (Cys62, Cys69, and Cys73), all of which have been reported as targets of S-Nitrosation. Prior reports have studied thermodynamic end points of Nitrosation reactions; however, the kinetics of Trx Nitrosation has not previously been investigated. Using the transNitrosation agent, S-nitrosoglutathione, a kinetic analysis of the selectivity and redox dependence of Trx Nitrosation at physiologically relevant concentrations and times was performed, utilizing a mass spectrometry-based method for the direct analysis of the nitrosated Trx. Reduced Trx (rTrx) was nitrosated 2.7-times faster than oxidized Trx (oTrx), and rTrx was nitrosated selectively on Cys62, whereas oTrx was nitrosated only on Cys73. These sites of Nitrosation were confirmed at the peptide level using a novel modification of the biotin-switch technique called the reductive switch. These results suggest separate signaling pathways for Trx-SNO under different cellular redox states.
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thioredoxin catalyzes the s Nitrosation of the caspase 3 active site cysteine
Nature Chemical Biology, 2005Co-Authors: Douglas A Mitchell, Michael A MarlettaAbstract:Nitric oxide (NO) signaling through the formation of cGMP is well established; however, there seems to be an increasing role for cGMP-independent NO signaling. Although key molecular details remain unanswered, S-Nitrosation represents an example of cGMP-independent NO signaling. This modification has garnered recent attention as it has been shown to modulate the function of several important biochemical pathways1,2,3. Although an analogy to O-phosphorylation can be drawn4, little is known about protein nitrosothiol regulation in vivo. In solution, NO readily reacts with oxygen to yield a nitrosating agent, but this process alone provides no specificity for Nitrosation5. This lack of specificity is exemplified by the in vitro poly-S-Nitrosation of caspase-3 (Casp-3, ref. 6) and the ryanodine receptor7. Previous in vivo work with Casp-3 suggests that a protein-assisted process may be responsible for selective S-Nitrosation of the catalytic cysteine (Cys163)8. We demonstrated that a single cysteine in thioredoxin (Trx) is capable of a targeted, reversible transNitrosation reaction with Cys163 of Casp-3. A greater understanding of how S-Nitrosation is mediated has broad implications for cGMP-independent signaling. The example described here also suggests a new role for Trx in the regulation of apoptosis.
Michael P Murphy - One of the best experts on this subject based on the ideXlab platform.
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identification of s nitrosated mitochondrial proteins by s nitrosothiol difference in gel electrophoresis sno dige implications for the regulation of mitochondrial function by reversible s Nitrosation
Biochemical Journal, 2010Co-Authors: Edward T Chouchani, Sergiy M Nadtochiy, Paul S Brookes, Ian M. Fearnley, Kathryn S Lilley, Robin Andrew James Smith, Thomas R Hurd, Michael P MurphyAbstract:The S-Nitrosation of mitochondrial proteins as a consequence of NO metabolism is of physiological and pathological significance. We previously developed a MitoSNO (mitochondria-targeted S-nitrosothiol) that selectively S-nitrosates mitochondrial proteins. To identify these S-nitrosated proteins, here we have developed a selective proteomic methodology, SNO-DIGE (S-nitrosothiol difference in gel electrophoresis). Protein thiols in control and MitoSNO-treated samples were blocked, then incubated with copper(II) and ascorbate to selectively reduce S-nitrosothiols. The samples were then treated with thiol-reactive Cy3 (indocarbocyanine) or Cy5 (indodicarbocyanine) fluorescent tags, mixed together and individual protein spots were resolved by 2D (two-dimensional) gel electrophoresis. Fluorescent scanning of these gels revealed S-nitrosated proteins by an increase in Cy5 red fluorescence, allowing for their identification by MS. Parallel analysis by Redox-DIGE enabled us to distinguish S-nitrosated thiol proteins from those which became oxidized due to NO metabolism. We identified 13 S-nitrosated mitochondrial proteins, and a further four that were oxidized, probably due to evanescent S-Nitrosation relaxing to a reversible thiol modification. We investigated the consequences of S-Nitrosation for three of the enzymes identified using SNO-DIGE (aconitase, mitochondrial aldehyde dehydrogenase and α-ketoglutarate dehydrogenase) and found that their activity was selectively and reversibly inhibited by S-Nitrosation. We conclude that the reversible regulation of enzyme activity by S-Nitrosation modifies enzymes central to mitochondrial metabolism, whereas identification and functional characterization of these novel targets provides mechanistic insight into the potential physiological and pathological roles played by this modification. More generally, the development of SNO-DIGE facilitates robust investigation of protein S-Nitrosation across the proteome.
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Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol Difference In Gel Electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-Nitrosation
Biochemical Journal, 2010Co-Authors: Edward Chouchani, Thomas Hurd, Sergiy M Nadtochiy, Paul S Brookes, Ian M. Fearnley, Kathryn S Lilley, Robin Andrew James Smith, Michael P MurphyAbstract:The S-Nitrosation of mitochondrial proteins as a consequence of nitric oxide (NO) metabolism is of physiological and pathological significance. We previously developed a mitochondria-targeted S-nitrosothiol (MitoSNO) that selectively S-nitrosates mitochondrial proteins. To identify these S-nitrosated proteins, here we have developed a selective proteomic methodology, S-nitrosothiol Difference In Gel Electrophoresis (SNO-DIGE). Protein thiols in control and MitoSNO-treated samples were blocked, then incubated with copper(II) and ascorbate to selectively reduce S-nitrosothiols. The samples were then treated with thiol-reactive Cy3 or Cy5 fluorescent tags, mixed together and individual protein spots were resolved by 2D gel electrophoresis. Fluorescent scanning of these gels revealed S-nitrosated proteins by an increase in Cy5 red fluorescence, allowing for their identification by mass spectrometry. Parallel analysis by Redox-DIGE enabled us to distinguish S-nitrosated thiol proteins from those which became oxidized due to NO metabolism. We identified 13 S-nitrosated mitochondrial proteins, and a further 4 that were oxidized, probably due to evanescent S-Nitrosation relaxing to a reversible thiol modification. We investigated the consequences of S-Nitrosation for three of the enzymes identified using SNO-DIGE (aconitase, aldehyde dehydrogenase and a-ketoglutarate dehydrogenase) and found that their activity was selectively and reversibly inhibited by S-Nitrosation. We conclude that the reversible regulation of enzyme activity by S-Nitrosation modifies enzymes central to mitochondrial metabolism, while identification and functional characterization of these novel targets provides mechanistic insight into the potential physiological and pathological roles played by this modification. More generally, the development of SNO-DIGE facilitates robust investigation of protein S-Nitrosation across the proteome.
Enduo Wang - One of the best experts on this subject based on the ideXlab platform.
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nitrosative stress inhibits aminoacylation and editing activities of mitochondrial threonyl trna synthetase by s Nitrosation
Nucleic Acids Research, 2020Co-Authors: Wenqiang Zheng, Yuying Zhang, Qin Yao, Yuzhe Chen, Xinhua Qiao, Enduo WangAbstract:Structure and/or function of proteins are frequently affected by oxidative/nitrosative stress via posttranslational modifications. Aminoacyl-tRNA synthetases (aaRSs) constitute a class of ubiquitously expressed enzymes that control cellular protein homeostasis. Here, we found the activity of human mitochondrial (mt) threonyl-tRNA synthetase (hmtThrRS) is resistant to oxidative stress (H2O2) but profoundly sensitive to nitrosative stress (S-nitrosoglutathione, GSNO). Further study showed four Cys residues in hmtThrRS were modified by S-Nitrosation upon GSNO treatment, and one residue was one of synthetic active sites. We analyzed the effect of modification at individual Cys residue on aminoacylation and editing activities of hmtThrRS in vitro and found that both activities were decreased. We further confirmed that S-Nitrosation of mtThrRS could be readily detected in vivo in both human cells and various mouse tissues, and we systematically identified dozens of S-Nitrosation-modified sites in most aaRSs, thus establishing both mitochondrial and cytoplasmic aaRS species with S-Nitrosation ex vivo and in vivo, respectively. Interestingly, a decrease in the S-Nitrosation modification level of mtThrRS was observed in a Huntington disease mouse model. Overall, our results establish, for the first time, a comprehensive S-Nitrosation-modified aaRS network and a previously unknown mechanism on the basis of the inhibitory effect of S-Nitrosation on hmtThrRS.
Edward T Chouchani - One of the best experts on this subject based on the ideXlab platform.
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identification and quantification of protein s Nitrosation by nitrite in the mouse heart during ischemia
Journal of Biological Chemistry, 2017Co-Authors: Edward T Chouchani, Andrew M James, Carmen Methner, Victoria R Pell, Tracy A Prime, Brian K Erickson, Marleen Forkink, Gigi Y Lau, Thomas P Bright, Katja E MengerAbstract:Nitrate (NO3-) and nitrite (NO2-) are known to be cardioprotective and to alter energy metabolism in vivo NO3- action results from its conversion to NO2- by salivary bacteria, but the mechanism(s) by which NO2- affects metabolism remains obscure. NO2- may act by S-nitrosating protein thiols, thereby altering protein activity. But how this occurs, and the functional importance of S-Nitrosation sites across the mammalian proteome, remain largely uncharacterized. Here we analyzed protein thiols within mouse hearts in vivo using quantitative proteomics to determine S-Nitrosation site occupancy. We extended the thiol-redox proteomic technique, isotope-coded affinity tag labeling, to quantify the extent of NO2--dependent S-Nitrosation of proteins thiols in vivo Using this approach, called SNOxICAT (S-nitrosothiol redox isotope-coded affinity tag), we found that exposure to NO2- under normoxic conditions or exposure to ischemia alone results in minimal S-Nitrosation of protein thiols. However, exposure to NO2- in conjunction with ischemia led to extensive S-Nitrosation of protein thiols across all cellular compartments. Several mitochondrial protein thiols exposed to the mitochondrial matrix were selectively S-nitrosated under these conditions, potentially contributing to the beneficial effects of NO2- on mitochondrial metabolism. The permeability of the mitochondrial inner membrane to HNO2, but not to NO2-, combined with the lack of S-Nitrosation during anoxia alone or by NO2- during normoxia places constraints on how S-Nitrosation occurs in vivo and on its mechanisms of cardioprotection and modulation of energy metabolism. Quantifying S-nitrosated protein thiols now allows determination of modified cysteines across the proteome and identification of those most likely responsible for the functional consequences of NO2- exposure.
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identification of s nitrosated mitochondrial proteins by s nitrosothiol difference in gel electrophoresis sno dige implications for the regulation of mitochondrial function by reversible s Nitrosation
Biochemical Journal, 2010Co-Authors: Edward T Chouchani, Sergiy M Nadtochiy, Paul S Brookes, Ian M. Fearnley, Kathryn S Lilley, Robin Andrew James Smith, Thomas R Hurd, Michael P MurphyAbstract:The S-Nitrosation of mitochondrial proteins as a consequence of NO metabolism is of physiological and pathological significance. We previously developed a MitoSNO (mitochondria-targeted S-nitrosothiol) that selectively S-nitrosates mitochondrial proteins. To identify these S-nitrosated proteins, here we have developed a selective proteomic methodology, SNO-DIGE (S-nitrosothiol difference in gel electrophoresis). Protein thiols in control and MitoSNO-treated samples were blocked, then incubated with copper(II) and ascorbate to selectively reduce S-nitrosothiols. The samples were then treated with thiol-reactive Cy3 (indocarbocyanine) or Cy5 (indodicarbocyanine) fluorescent tags, mixed together and individual protein spots were resolved by 2D (two-dimensional) gel electrophoresis. Fluorescent scanning of these gels revealed S-nitrosated proteins by an increase in Cy5 red fluorescence, allowing for their identification by MS. Parallel analysis by Redox-DIGE enabled us to distinguish S-nitrosated thiol proteins from those which became oxidized due to NO metabolism. We identified 13 S-nitrosated mitochondrial proteins, and a further four that were oxidized, probably due to evanescent S-Nitrosation relaxing to a reversible thiol modification. We investigated the consequences of S-Nitrosation for three of the enzymes identified using SNO-DIGE (aconitase, mitochondrial aldehyde dehydrogenase and α-ketoglutarate dehydrogenase) and found that their activity was selectively and reversibly inhibited by S-Nitrosation. We conclude that the reversible regulation of enzyme activity by S-Nitrosation modifies enzymes central to mitochondrial metabolism, whereas identification and functional characterization of these novel targets provides mechanistic insight into the potential physiological and pathological roles played by this modification. More generally, the development of SNO-DIGE facilitates robust investigation of protein S-Nitrosation across the proteome.
Wenqiang Zheng - One of the best experts on this subject based on the ideXlab platform.
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nitrosative stress inhibits aminoacylation and editing activities of mitochondrial threonyl trna synthetase by s Nitrosation
Nucleic Acids Research, 2020Co-Authors: Wenqiang Zheng, Yuying Zhang, Qin Yao, Yuzhe Chen, Xinhua Qiao, Enduo WangAbstract:Structure and/or function of proteins are frequently affected by oxidative/nitrosative stress via posttranslational modifications. Aminoacyl-tRNA synthetases (aaRSs) constitute a class of ubiquitously expressed enzymes that control cellular protein homeostasis. Here, we found the activity of human mitochondrial (mt) threonyl-tRNA synthetase (hmtThrRS) is resistant to oxidative stress (H2O2) but profoundly sensitive to nitrosative stress (S-nitrosoglutathione, GSNO). Further study showed four Cys residues in hmtThrRS were modified by S-Nitrosation upon GSNO treatment, and one residue was one of synthetic active sites. We analyzed the effect of modification at individual Cys residue on aminoacylation and editing activities of hmtThrRS in vitro and found that both activities were decreased. We further confirmed that S-Nitrosation of mtThrRS could be readily detected in vivo in both human cells and various mouse tissues, and we systematically identified dozens of S-Nitrosation-modified sites in most aaRSs, thus establishing both mitochondrial and cytoplasmic aaRS species with S-Nitrosation ex vivo and in vivo, respectively. Interestingly, a decrease in the S-Nitrosation modification level of mtThrRS was observed in a Huntington disease mouse model. Overall, our results establish, for the first time, a comprehensive S-Nitrosation-modified aaRS network and a previously unknown mechanism on the basis of the inhibitory effect of S-Nitrosation on hmtThrRS.
