Probing the role of cysteine sulfenylation in cell signaling

描述（由申请人提供）：蛋白质亚砜化是过氧化氢（H2O2）对半胱氨酸硫醇侧链的氧化还原修饰，是信号转导的重要机制。失调的蛋白质亚砜化有助于一系列的人类病理，包括癌症。然而，迄今为止，由于缺乏在天然环境中探测这些修饰的技术，阐明蛋白质亚砜化在生理和疾病中的各种作用的努力受到了影响。为了解决这个问题，我们最近引入了一种新的化学蛋白质组学策略来直接检测细胞中蛋白质亚砜化的变化。迄今为止，我们的初步研究已经确定了生长因子信号传导过程中H2O2的几个新的细胞内蛋白靶点，包括表皮生长因子受体（EGFR）。具体来说，我们已经发现H2O2直接修饰EGFR atp结合位点内的半胱氨酸残基，并且氧化刺激其酪氨酸激酶活性，尽管这种作用的生化机制仍有待充分阐明。在本提案中，我们将应用我们的化学探针和分析工具套件来解决氧化还原信号，化学生物学和癌症领域中具有重要意义的四个主要问题。该提案的目的1将定义EGFR的磺化调节其激酶活性的分子机制。为了确定在h2o2介导的信号中决定选择性的特征，我们将检查

DESCRIPTION (provided by applicant): Protein sulfenylation, the redox-based modification of cysteine thiol side chains by hydrogen peroxide (H2O2), is an important mechanism in signal transduction. Dysregulated protein sulfenylation contributes to a range of human pathologies, including cancer. However, efforts to elucidate the diverse roles of protein sulfenylation in physiology and disease have, to date, suffered from a lack of techniques to probe these modifications in native environments. To address this problem, we have recently introduced a new chemical proteomic strategy to detect changes in protein sulfenylation directly in cells. To date, our preliminary studies have identified several novel intracellular protein targets of H2O2 during growth factor signaling, including the epidermal growth factor receptor (EGFR). Specifically, we have discovered that H2O2 directly modifies a cysteine residue within the ATP-binding site of EGFR, and that oxidation stimulates its tyrosine kinase activity, though the biochemical mechanism for this effect remains to be fully elucidated. In this proposal, we will apply our suite of chemical probes and analytical tools to address four major questions of high significance to the fields of redox signaling, chemical biology, and cancer. Aim 1 of the proposal will define the molecular mechanism by which sulfenylation of EGFR regulates its kinase activity. To identify features that dictate selectivity in H2O2-mediated signaling, we will examine sulfenylation, localization, and enzyme activity of EGFR-targeted protein tyrosine phosphatases (PTPs) in Aim 2. We will evaluate additional targets of intracellular H2O2 generated in response to growth factors using target-based and chemical reporter/proteomic methods in Aim 3. While sulfenylation is a reversible modification in cells, the factors that recycle sulfenylated proteinsto their reduced thiol form (RSH) are largely ill defined. We will test candidate reductases responsible for reversible sulfenylation in Aim 4. The development and application of our chemical tools in cells provides an unprecedented opportunity to elucidate mechanisms that govern sulfenylation of proteins. Given that aberrant sulfenylation of proteins has been linked to aggressive cancer phenotypes and that genetic lesions in H2O2-metabolizing enzymes can contribute to tumorigenesis, defining the mechanisms that control reversible protein sulfenylation is vital for understanding human physiology and disease. We anticipate that these studies will define how sulfenylation of proteins regulates signaling networks that underlie cell growth and identify key enzymes that controls desulfenylation. Ultimately, this will facilitate the identification of new biomarkers and therapeutic targets for cancer, as well as produce methodological advances that expand the scope and utility of proteomic technologies for biological and biomedical discoveries. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because the discovery of cellular mechanisms that regulate physiological protein sulfenylation is ultimately expected to increase our understanding of the pathophysiology associated with oxidative stress and abnormal H2O2-based signal transduction, with translational potential for clinical medicine in the key areas of disease diagnosis, patient stratification, and monitoring efficacy in the new era of redox-based therapeutics. Thus, the proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge that will help to foster fundamental discoveries, innovative research strategies, and their applications as a basis for ultimately protecting and improving health.