Data from Inducible Nitric Oxide Synthase Drives mTOR Pathway Activation and Proliferation of Human Melanoma by Reversible Nitrosylation of TSC2
Esther López-RiveraPadmini JayaramanFalguni ParikhMichael A. DaviesSühendan EkmekçiogluSudeh IzadmehrDenái R. MiltonJerry E. ChipukElizabeth A. GrimmYeriel EstradaJulio A. Aguirre‐GhisoAndrew G. Sikora
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<div>Abstract<p>Melanoma is one of the cancers of fastest-rising incidence in the world. Inducible nitric oxide synthase (iNOS) is overexpressed in melanoma and other cancers, and previous data suggest that iNOS and nitric oxide (NO) drive survival and proliferation of human melanoma cells. However, specific mechanisms through which this occurs are poorly defined. One candidate is the PI3K–AKT–mTOR pathway, which plays a major role in proliferation, angiogenesis, and metastasis of melanoma and other cancers. We used the chick embryo chorioallantoic membrane (CAM) assay to test the hypothesis that melanoma growth is regulated by iNOS-dependent mTOR pathway activation. Both pharmacologic inhibition and siRNA-mediated gene silencing of iNOS suppressed melanoma proliferation and <i>in vivo</i> growth on the CAM in human melanoma models. This was associated with strong downregulation of mTOR pathway activation by Western blot analysis of p-mTOR, p70 ribosomal S6 kinase (p-P70S6K), p-S6RP, and p-4EBP1. iNOS expression and NO were associated with reversible nitrosylation of tuberous sclerosis complex (TSC) 2, and inhibited dimerization of TSC2 with its inhibitory partner TSC1, enhancing GTPase activity of its target Ras homolog enriched in brain (Rheb), a critical activator of mTOR signaling. Immunohistochemical analysis of tumor specimens from stage III melanoma patients showed a significant correlation between iNOS expression levels and expression of the mTOR pathway members. Exogenously supplied NO was also sufficient to reverse the mTOR pathway inhibition by the B-Raf inhibitor vemurafenib. In summary, covalent modification of TSC2 by iNOS-derived NO is associated with impaired TSC2/TSC1 dimerization, mTOR pathway activation, and proliferation of human melanoma. This model is consistent with the known association of iNOS overexpression and poor prognosis in melanoma and other cancers. <i>Cancer Res; 74(4); 1067–78. ©2014 AACR</i>.</p></div>Keywords:
S-Nitrosylation
Nitrosylation
S-Nitrosylation, the covalent addition of a nitrogen monoxide group to a cysteine thiol, has been shown to modify the function of a broad spectrum of mammalian, plant, and microbial proteins and thereby to convey the ubiquitous influence of nitric oxide on cellular signal transduction and host defense. Accumulating evidence indicates that dysregulated, diminished, or excessive S-nitrosylation may be implicated in a wide range of pathophysiological conditions. A recent study establishes a functional relationship between inhibitory S-nitrosylation of the redox enzyme protein disulfide isomerase (PDI), defects in regulation of protein folding within the endoplasmic reticulum (ER), and neurodegeneration. Further, an examination of human brains afflicted with Parkinson's or Alzheimer's disease supports a causal role for the S-nitrosylation of PDI and consequent ER stress in these prevalent neurodegenerative disorders.
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The biological effects of nitric oxide (NO) are in significant part mediated through S-nitrosylation of cysteine thiol. Work on model thiol substrates has raised the idea that molecular oxygen (O2) is required for S-nitrosylation by NO; however, the relevance of this mechanism at the low physiological pO2 of tissues is unclear. Here we have used a proteomic approach to study S-nitrosylation reactions in situ. We identify endogenously S-nitrosylated proteins in subcellular organelles, including dihydrolipoamide dehydrogenase and catalase, and show that these, as well as hydroxymethylglutaryl-CoA synthase and sarcosine dehydrogenase (SarDH), are S-nitrosylated by NO under strictly anaerobic conditions. S-Nitrosylation of SarDH by NO is best rationalized by a novel mechanism involving the covalently bound flavin of the enzyme. We also identify a set of mitochondrial proteins that can be S-nitrosylated through multiple reaction channels, including anaerobic/oxidative, NO/O2, and GSNO-mediated transnitrosation. Finally, we demonstrate that steady state levels of S-nitrosylation are higher in mitochondrial extracts than the intact organelles, suggesting the importance of denitrosylation reactions. Collectively, our results provide new insight into the determinants of S-nitrosothiol levels in subcellular compartments. The biological effects of nitric oxide (NO) are in significant part mediated through S-nitrosylation of cysteine thiol. Work on model thiol substrates has raised the idea that molecular oxygen (O2) is required for S-nitrosylation by NO; however, the relevance of this mechanism at the low physiological pO2 of tissues is unclear. Here we have used a proteomic approach to study S-nitrosylation reactions in situ. We identify endogenously S-nitrosylated proteins in subcellular organelles, including dihydrolipoamide dehydrogenase and catalase, and show that these, as well as hydroxymethylglutaryl-CoA synthase and sarcosine dehydrogenase (SarDH), are S-nitrosylated by NO under strictly anaerobic conditions. S-Nitrosylation of SarDH by NO is best rationalized by a novel mechanism involving the covalently bound flavin of the enzyme. We also identify a set of mitochondrial proteins that can be S-nitrosylated through multiple reaction channels, including anaerobic/oxidative, NO/O2, and GSNO-mediated transnitrosation. Finally, we demonstrate that steady state levels of S-nitrosylation are higher in mitochondrial extracts than the intact organelles, suggesting the importance of denitrosylation reactions. Collectively, our results provide new insight into the determinants of S-nitrosothiol levels in subcellular compartments.
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Nitric oxide (NO) is a reactive free radical with pleiotropic functions that participates in diverse biological processes in plants, such as germination, root development, stomatal closing, abiotic stress and defense responses. It acts mainly through redox-based modification of cysteine residue(s) of target proteins, called protein S-nitrosylation. In this way NO regulates numerous cellular functions and signaling events in plants. Identification of S-nitrosylated substrates and their exact target cysteine residue(s) is very important to reveal the molecular mechanisms and regulatory roles of S-nitrosylation. In addition to the necessity of protein-protein interaction for trans-nitrosylation and denitrosylation reactions, the cellular redox environment and cysteine thiol micro-environment have been proposed important factors for the specificity of protein S-nitrosylation. Several methods have recently been developed for the proteomic identification of target proteins. However, the specificity of NO-based cysteine modification is still less defined. In this review, we discuss formation and specificity of S-nitrosylation. Special focus will be on potential S-nitrosylation motifs, site-specific proteomic analyses, computational predictions using different algorithms and on structural analysis of cysteine S-nitrosylation. In this review, we discuss formation and specificity of S-nitrosylation. Special focus will be on potential S-nitrosylation motifs, site-specific proteomic analyses, computational predictions using different algorithm and on structural analysis of cysteine S-nitrosylation.
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Plants are exposed to various environmental stresses that affect crop growth and production. During stress, various physiological and biochemical changes including the production of nitric oxide (NO), take place. It is clear that NO could work through either transcriptional or post-translational level. The redox-based post-translational modification S-nitrosylation – the covalent attachment of an NO moiety to a reactive cysteine thiol of a protein to form an S-nitrosothiol (SNO) – has attracted increasing attention in the regulation of abiotic stress signalling. So far, the relevance of S-nitrosylation of certain proteins has been investigated under abiotic stress. In this work, we focus on the current state of knowledge regarding S-nitrosylation in plants under abiotic stress, and provide a better understanding of the relevance of S-nitrosylation in plant response to abiotic stress.
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S-nitrosylation, the covalent attachment of a nitric oxide (NO) moiety to a cysteine thiol, is a prototypically redox-based post-translational modification established initially in animals. The S-nitrosylated protein (SNO) can serve to stabilize and diversify NO-related signals, which is also controlled through the regulation of NO biosynthesis adversely. S-nitrosylation occurs with exquisite specificity affected by the primary or higher-order structures of proteins as well as some physiological factors. The approach termed SNOSID (SNO site identification) provides an effective tool for identifying S-nitrosylated cysteine residues. The recently obtained evidences suggest that S-nitrosylation may also have a central function in plant biology,especially in plant resistance to diseases.
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