Probing the role of cysteine sulfenylation in cell signaling

描述（由申请人提供）：蛋白质亚磺酰化，即通过过氧化氢（H2 O2）对半胱氨酸巯基侧链进行基于氧化还原的修饰，是信号转导中的重要机制。失调的蛋白质亚磺酰化有助于一系列人类病理学，包括癌症。然而，努力阐明蛋白质亚磺酰化在生理和疾病中的不同作用，迄今为止，缺乏在天然环境中探测这些修饰的技术。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，直接在细胞中检测蛋白质磺酰化的变化。到目前为止，我们的初步研究已经确定了几个新的细胞内蛋白质的目标过氧化氢在生长因子信号，包括表皮生长因子受体（EGFR）。具体来说，我们已经发现，H2 O2直接修改EGFR的ATP结合位点内的半胱氨酸残基，氧化刺激其酪氨酸激酶活性，虽然这种效果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对氧化还原信号传导、化学生物学和癌症领域具有重要意义的四个主要问题。该提案的目的1将定义EGFR的亚磺酰化调节其激酶活性的分子机制。为了确定H2 O2介导的信号传导中决定选择性的特征，我们将研究 Aim 2中EGFR靶向蛋白酪氨酸磷酸酶（PTP）的亚磺酰化、定位和酶活性。我们将在目标3中使用基于靶点的和化学报告基因/蛋白质组学方法评估响应生长因子产生的细胞内H2 O2的其他靶点。虽然亚磺酰化在细胞中是一种可逆的修饰，但将亚磺酰化蛋白质再循环为还原巯基形式（RSH）的因素在很大程度上是不明确的。我们将在目标4中测试负责可逆亚磺酰化的候选还原酶。我们的化学工具在细胞中的开发和应用为阐明蛋白质亚磺酰化的机制提供了前所未有的机会。鉴于蛋白质的异常亚磺酰化与侵袭性癌症表型有关，并且H2 O2代谢酶中的遗传病变可能导致肿瘤发生，因此定义控制可逆蛋白质亚磺酰化的机制对于理解人类生理学和疾病至关重要。我们预计，这些研究将确定如何亚磺酰化的蛋白质调节信号网络，细胞生长的基础，并确定关键酶，控制亚磺酰化。最终，这将有助于 该研究所致力于确定癌症的新生物标志物和治疗靶点，并在方法学方面取得进展，扩大蛋白质组学技术在生物和生物医学发现中的范围和实用性。 公共卫生相关性：拟议的研究与公共卫生有关，因为发现调节生理蛋白亚磺酰化的细胞机制最终有望增加我们对与氧化应激和异常H2 O2-基信号转导相关的病理生理学的理解，并在疾病诊断，患者分层，以及在氧化还原疗法的新时代监测疗效。因此，拟议的研究与NIH的使命的一部分有关，该使命涉及发展基础知识，这将有助于促进基础发现、创新研究策略及其应用，作为最终保护和改善健康的基础。

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.