Hydrogen peroxide (H2O2) is an important messenger molecule for diverse cellular processes. H2O2 oxidizes proteinaceous cysteinyl thiols to sulfenic acid, also known as S-sulfenylation, thereby affecting the protein conformation and functionality. Although many proteins have been identified as S-sulfenylation targets in plants, site-specific mapping and quantification remain largely unexplored. By means of a peptide-centric chemoproteomics approach, we mapped 1,537 S-sulfenylated sites on more than 1,000 proteins in Arabidopsis thaliana cells. Proteins involved in RNA homeostasis and metabolism were identified as hotspots for S-sulfenylation. Moreover, S-sulfenylation frequently occurred on cysteines located at catalytic sites of enzymes or on cysteines involved in metal binding, hinting at a direct mode of action for redox regulation. Comparison of human and Arabidopsis S-sulfenylation datasets provided 155 conserved S-sulfenylated cysteines, including Cys181 of the Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE4 (AtMAPK4) that corresponds to Cys161 in the human MAPK1, which has been identified previously as being S-sulfenylated. We show that, by replacing Cys181 of recombinant AtMAPK4 by a redox-insensitive serine residue, the kinase activity decreased, indicating the importance of this noncatalytic cysteine for the kinase mechanism. Altogether, we quantitatively mapped the S-sulfenylated cysteines in Arabidopsis cells under H2O2 stress and thereby generated a comprehensive view on the S-sulfenylation landscape that will facilitate downstream plant redox studies.
EGFR, HER2, and HER3 contribute to the initiation and progression of human cancers, and are therapeutic targets for monoclonal antibodies and tyrosine kinase inhibitors. An important source of resistance to these agents arises from functional redundancy among EGFR, HER2, and HER3. EGFR family members contain conserved extracellular structures that are stabilized by disulfide bonds. Compounds that disrupt extracellular disulfide bonds could inactivate EGFR, HER2, and HER3 in unison. Here we describe the identification of compounds that kill breast cancer cells that overexpress EGFR or HER2. Cell death parallels downregulation of EGFR, HER2, and HER3. These compounds disrupt disulfide bonds and are termed Disulfide Bond Disrupting Agents (DDAs). DDA RBF3 exhibits anticancer efficacy in vivo at 40 mg/kg without evidence of toxicity. DDAs may complement existing EGFR-, HER2-, and HER3-targeted agents that function through alternate mechanisms of action, and combination regimens with these existing drugs may overcome therapeutic resistance.
Post-translational changes in the redox state of cysteine residues can rapidly and reversibly alter protein functions, thereby modulating biological processes. The nematode C. elegans is an ideal model organism for studying cysteine-mediated redox signaling at a network level. Here we present a comprehensive, quantitative, and site-specific profile of the intrinsic reactivity of the cysteinome in wild-type C. elegans. We also describe a global characterization of the C. elegans redoxome in which we measured changes in three major cysteine redox forms after H2O2 treatment. Our data revealed redox-sensitive events in translation, growth signaling, and stress response pathways, and identified redox-regulated cysteines that are important for signaling through the p38 MAP kinase (MAPK) pathway. Our in-depth proteomic dataset provides a molecular basis for understanding redox signaling in vivo, and will serve as a valuable and rich resource for the field of redox biology.
Oxidation of a protein cysteinyl thiol (Cys‐SH) to S‐sulfenic acid (Cys‐SOH) by a reactive oxygen species (e.g., hydrogen peroxide), which is termed protein S‐sulfenylation, is a reversible post‐translational modification that plays a crucial role in redox regulation of protein function in various biological processes. Due to its intrinsically labile nature, protein S‐sulfenylation cannot be directly detected or analyzed. Chemoselective probing has been the method of choice for analyzing S‐sulfenylated proteins either in vitro or in situ, as it allows stabilization and direct detection of this transient oxidative intermediate. However, it remains challenging to globally pinpoint the specific S‐sulfenylated cysteine sites on complex proteomes and to quantify their dynamic changes upon oxidative stress. This unit describes how a benzothiazine‐based chemoselective probe called BTD and mass spectrometry based chemoproteomics can be used to globally and site‐specifically identify and quantify protein S‐sulfenylation. © 2018 by John Wiley & Sons, Inc.
Mirabegron is the first β 3 -adrenoceptor agonist approved for treatment of overactive bladder syndrome. This study aimed to investigate the effects of β 3 -adrenoceptor agonist mirabegron in mouse urethra. The possibility that mirabegron also exerts α 1 -adrenoceptor antagonism was also tested in rat smooth muscle preparations presenting α 1A -(vas deferens and prostate), α 1D -(aorta) and α 1B -adrenoceptors (spleen). EXPERIMENTAL APPROACHFunctional assays were carried out in mouse and rat isolated tissues. Competition assays for the specific binding of [ 3 H]prazosin to membrane preparations of HEK-293 cells expressing each of the human α 1 -adrenoceptors, as well as β-adrenoceptor mRNA expression and cyclic AMP measurements in mouse urethra, were performed. KEY RESULTSMirabegron produced concentration-dependent urethral relaxations that were shifted to the right by the selective β 3 -adrenoceptor antagonist L-748,337 but unaffected by β 1 -and β 2 -adrenoceptor antagonists (atenolol and ICI-118,551 respectively). Mirabegron-induced relaxations were enhanced by the PDE4 inhibitor rolipram, and the agonist stimulated cAMP synthesis. Mirabegron also produced rightward shifts in urethral contractions induced by the α 1 -adrenoceptor agonist phenylephrine. Schild regression analysis revealed that mirabegron behaves as a competitive antagonist of α 1 -adrenoceptors in urethra, vas deferens and prostate (α 1A -adrenoceptor, pA 2 ≅ 5.6) and aorta (α 1D -adrenoceptor, pA 2 ≅ 5.4) but not in spleen (α 1B -adrenoceptor). The affinities estimated for mirabegron in functional assays were consistent with those estimated in radioligand binding with human recombinant α 1A -and α 1D -adrenoceptors (pK i ≅ 6.0). CONCLUSION AND IMPLICATIONSThe effects of mirabegron in urethral smooth muscle are the result of β 3 -adrenoceptor agonism together with α 1A and α 1D -adrenoceptor antagonism. Abbreviations
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