Global redox proteome and phosphoproteome analysis reveals redox switch in Akt

AbstractProtein oxidation sits at the intersection of multiple signalling pathways, yet the magnitude and extent of crosstalk between oxidation and other post-translational modifications remains unclear. Here, we delineate global changes in adipocyte signalling networks following acute oxidative stress and reveal considerable crosstalk between cysteine oxidation and phosphorylation-based signalling. Oxidation of key regulatory kinases, including Akt, mTOR and AMPK influences the fidelity rather than their absolute activation state, highlighting an unappreciated interplay between these modifications. Mechanistic analysis of the redox regulation of Akt identified two cysteine residues in the pleckstrin homology domain (C60 and C77) to be reversibly oxidized. Oxidation at these sites affected Akt recruitment to the plasma membrane by stabilizing the PIP3 binding pocket. Our data provide insights into the interplay between oxidative stress-derived redox signalling and protein phosphorylation networks and serve as a resource for understanding the contribution of cellular oxidation to a range of diseases.

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Effects of heterologous expression of human cyclic nucleotide phosphodiesterase 3A (hPDE3A) on redox regulation in yeast

Biochemical Journal ◽

10.1042/bcj20160572 ◽

2016 ◽

Vol 473 (22) ◽

pp. 4205-4225 ◽

Cited By ~ 1

Author(s):

Dong Keun Rhee ◽

Jung Chae Lim ◽

Steven C. Hockman ◽

Faiyaz Ahmad ◽

Dong Ho Woo ◽

...

Keyword(s):

Oxidative Stress ◽

Redox Regulation ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Negative Regulator ◽

Antioxidant Genes ◽

Post Translational Modifications ◽

Thioredoxin System ◽

Antioxidant Gene Expression ◽

Camp Levels

Oxidative stress plays a pivotal role in pathogenesis of cardiovascular diseases and diabetes; however, the roles of protein kinase A (PKA) and human phosphodiesterase 3A (hPDE3A) remain unknown. Here, we show that yeast expressing wild-type (WT) hPDE3A or K13R hPDE3A (putative ubiquitinylation site mutant) exhibited resistance or sensitivity to exogenous hydrogen peroxide (H2O2), respectively. H2O2-stimulated ROS production was markedly increased in yeast expressing K13R hPDE3A (Oxidative stress Sensitive 1, OxiS1), compared with yeast expressing WT hPDE3A (Oxidative stress Resistant 1, OxiR1). In OxiR1, YAP1 and YAP1-dependent antioxidant genes were up-regulated, accompanied by a reduction in thioredoxin peroxidase. In OxiS1, expression of YAP1 and YAP1-dependent genes was impaired, and the thioredoxin system malfunctioned. H2O2 increased cyclic adenosine monophosphate (cAMP)-hydrolyzing activity of WT hPDE3A, but not K13R hPDE3A, through PKA-dependent phosphorylation of hPDE3A, which was correlated with its ubiquitinylation. The changes in antioxidant gene expression did not directly correlate with differences in cAMP–PKA signaling. Despite differences in their capacities to hydrolyze cAMP, total cAMP levels among OxiR1, OxiS1, and mock were similar; PKA activity, however, was lower in OxiS1 than in OxiR1 or mock. During exposure to H2O2, however, Sch9p activity, a target of Rapamycin complex 1-regulated Rps6 kinase and negative-regulator of PKA, was rapidly reduced in OxiR1, and Tpk1p, a PKA catalytic subunit, was diffusely spread throughout the cytosol, with PKA activation. In OxiS1, Sch9p activity was unchanged during exposure to H2O2, consistent with reduced activation of PKA. These results suggest that, during oxidative stress, TOR-Sch9 signaling might regulate PKA activity, and that post-translational modifications of hPDE3A are critical in its regulation of cellular recovery from oxidative stress.

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A molecular switch for Cdc48 activity and localization during oxidative stress and aging

10.1101/733709 ◽

2019 ◽

Cited By ~ 1

Author(s):

Meytal Radzinski ◽

Ohad Yogev ◽

Yarden Yesharim ◽

Esther S. Brielle ◽

Ran Israeli ◽

...

Keyword(s):

Oxidative Stress ◽

Protein Degradation ◽

Polypeptide Chain ◽

Thiol Group ◽

Oxidative Stress Response ◽

Mitochondrial Fragmentation ◽

Chronological Aging ◽

Sensitive Site ◽

Redox Switch ◽

Cellular Oxidation

SummaryControl over a healthy proteome begins with the birth of the polypeptide chain and ends with coordinated protein degradation. One of the major players in eukaryotic protein degradation is the essential and highly conserved ATPase, Cdc48 (p97/VCP in mammals). Cdc48 mediates clearance of misfolded proteins from the nucleus, cytosol, ER, mitochondria, and more. Here we dissect the crosstalk between cellular oxidation and Cdc48 activity by identification of a redox-sensitive site, Cys115. By integrating proteomics, biochemistry, microscopy, and bioinformatics, we show that removal of Cys115’s redox-sensitive thiol group leads to accumulation of Cdc48 in the nucleus and consequently, results in severe defects in the oxidative stress response, mitochondrial fragmentation, and a decrease in ERAD and sterol biogenesis. We have thus identified a unique redox switch in Cdc48, which may provide a clearer picture of the importance of Cdc48’s localization in maintaining a “healthy” proteome during oxidative stress and chronological aging in yeast.

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Potential Modulation of Sirtuins by Oxidative Stress

Oxidative Medicine and Cellular Longevity ◽

10.1155/2016/9831825 ◽

2016 ◽

Vol 2016 ◽

pp. 1-12 ◽

Cited By ~ 33

Author(s):

Leonardo Santos ◽

Carlos Escande ◽

Ana Denicola

Keyword(s):

Oxidative Stress ◽

Posttranslational Modifications ◽

Histone Deacetylases ◽

Redox Regulation ◽

Molecular Mechanisms ◽

Compensatory Mechanism ◽

Cysteine Oxidation ◽

Catalytic Core

Sirtuins are a conserved family of NAD-dependent protein deacylases. Initially proposed as histone deacetylases, it is now known that they act on a variety of proteins including transcription factors and metabolic enzymes, having a key role in the regulation of cellular homeostasis. Seven isoforms are identified in mammals (SIRT1–7), all of them sharing a conserved catalytic core and showing differential subcellular localization and activities. Oxidative stress can affect the activity of sirtuins at different levels: expression, posttranslational modifications, protein-protein interactions, and NAD levels. Mild oxidative stress induces the expression of sirtuins as a compensatory mechanism, while harsh or prolonged oxidant conditions result in dysfunctional modified sirtuins more prone to degradation by the proteasome. Oxidative posttranslational modifications have been identifiedin vitroandin vivo, in particular cysteine oxidation and tyrosine nitration. In addition, oxidative stress can alter the interaction with other proteins, like SIRT1 with its protein inhibitor DBC1 resulting in a net increase of deacetylase activity. In the same way, manipulation of cellular NAD levels by pharmacological inhibition of other NAD-consuming enzymes results in activation of SIRT1 and protection against obesity-related pathologies. Nevertheless, further research is needed to establish the molecular mechanisms of redox regulation of sirtuins to further design adequate pharmacological interventions.

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How N-Acetylcysteine Supplementation Affects Redox Regulation, Especially at Mitohormesis and Sarcohormesis Level: Current Perspective

Antioxidants ◽

10.3390/antiox10020153 ◽

2021 ◽

Vol 10 (2) ◽

pp. 153

Author(s):

Aslı Devrim-Lanpir ◽

Lee Hill ◽

Beat Knechtle

Keyword(s):

Oxidative Stress ◽

Oxidative Metabolism ◽

Adaptive Response ◽

Redox Regulation ◽

Therapeutic Agent ◽

Metabolic Effects ◽

Metabolic Adaptations ◽

Current Perspective ◽

Exercise Induced ◽

Response To Exercise

Exercise frequently alters the metabolic processes of oxidative metabolism in athletes, including exposure to extreme reactive oxygen species impairing exercise performance. Therefore, both researchers and athletes have been consistently investigating the possible strategies to improve metabolic adaptations to exercise-induced oxidative stress. N-acetylcysteine (NAC) has been applied as a therapeutic agent in treating many diseases in humans due to its precursory role in the production of hepatic glutathione, a natural antioxidant. Several studies have investigated NAC’s possible therapeutic role in oxidative metabolism and adaptive response to exercise in the athletic population. However, still conflicting questions regarding NAC supplementation need to be clarified. This narrative review aims to re-evaluate the metabolic effects of NAC on exercise-induced oxidative stress and adaptive response developed by athletes against the exercise, especially mitohormetic and sarcohormetic response.

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The Role of HO-1 and Its Crosstalk with Oxidative Stress in Cancer Cell Survival

Cells ◽

10.3390/cells10092401 ◽

2021 ◽

Vol 10 (9) ◽

pp. 2401

Author(s):

Shih-Kai Chiang ◽

Shuen-Ei Chen ◽

Ling-Chu Chang

Keyword(s):

Oxidative Stress ◽

Redox Regulation ◽

Oxidative Status ◽

Multiple Roles ◽

Mitochondrial Enzymes ◽

Heme Oxygenases ◽

Downstream Effects ◽

Cancer Cell Survival ◽

And Oxidative Stress

Heme oxygenases (HOs) act on heme degradation to produce carbon monoxide (CO), free iron, ferritin, and biliverdin. Upregulation of cellular HO-1 levels is signature of oxidative stress for its downstream effects particularly under pro-oxidative status. Subcellular traffics of HO-1 to different organelles constitute a network of interactions compromising a variety of effectors such as pro-oxidants, ROS, mitochondrial enzymes, and nucleic transcription factors. Some of the compartmentalized HO-1 have been demonstrated as functioning in the progression of cancer. Emerging data show the multiple roles of HO-1 in tumorigenesis from pathogenesis to the progression to malignancy, metastasis, and even resistance to therapy. However, the role of HO-1 in tumorigenesis has not been systematically addressed. This review describes the crosstalk between HO-1 and oxidative stress, and following redox regulation in the tumorigenesis. HO-1-regulated signaling pathways are also summarized. This review aims to integrate basic information and current progress of HO-1 in cancer research in order to enhance the understandings and facilitate following studies.

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Comprehensive analyses of the cysteine thiol oxidation of PKM2 reveal the effects of multiple oxidation on cellular oxidative stress response

Biochemical Journal ◽

10.1042/bcj20200897 ◽

2021 ◽

Author(s):

Hayato Irokawa ◽

Satoshi Numasaki ◽

Shin Kato ◽

Kenta Iwai ◽

Atsushi Inose-Maruyama ◽

...

Keyword(s):

Oxidative Stress ◽

Pyruvate Kinase ◽

Redox Regulation ◽

Cysteine Residue ◽

Pentose Phosphate ◽

Signal Pathways ◽

Heterologous Proteins ◽

Cancer Cell Growth ◽

Anti Cancer ◽

Cellular Signal

Redox regulation of proteins via cysteine residue oxidation is involved in the control of various cellular signal pathways. Pyruvate kinase M2 (PKM2), a rate-limiting enzyme in glycolysis, is critical for the metabolic shift from glycolysis to the pentose phosphate pathway under oxidative stress in cancer cell growth. The PKM2 tetramer is required for optimal pyruvate kinase (PK) activity, whereas the inhibition of inter-subunit interaction of PKM2 induced by Cys358 oxidation has reduced PK activity. In the present study, we identified three oxidation-sensitive cysteine residues (Cys358, Cys423 and Cys424) responsible for four oxidation forms via the thiol oxidant diamide and/or hydrogen peroxide (H2O2). Possibly due to obstruction of the dimer-dimer interface, H2O2-induced sulfenylation (-SOH) and diamide-induced modification at Cys424 inhibited tetramer formation and PK activity. Cys423 is responsible for intermolecular disulphide bonds with heterologous proteins via diamide. Additionally, intramolecular polysulphide linkage (–Sn–, n≧3) between Cys358 and an unidentified PKM2 Cys could be induced by diamide. We observed that cells expressing the oxidation-resistant PKM2 (PKM2C358,424A) produced more intracellular reactive oxygen species (ROS) and exhibited greater sensitivity to ROS-generating reagents and ROS-inducible anti-cancer drugs compared to cells expressing wildtype PKM2. These results highlight the possibility that PKM2 inhibition via Cys358 and Cys424 oxidation contributes to eliminating excess ROS and oxidative stress.

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

10.26434/chemrxiv.8874077 ◽

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>

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