Probing the role of cysteine sulfenylation in cell signaling

描述(申请人提供)：蛋白质硫基化，是过氧化氢(H_2O_2)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。调节失调的蛋白质硫基化导致包括癌症在内的一系列人类疾病。然而，到目前为止，由于缺乏在自然环境中探索这些修饰的技术，阐明蛋白质磺化在生理和疾病中的不同作用的努力受到了阻碍。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在细胞中检测蛋白质磺化的变化。到目前为止，我们的初步研究已经确定了几个新的过氧化氢在生长因子信号转导过程中的细胞内蛋白质靶标，包括表皮生长因子受体(EGFR)。具体地说，我们发现过氧化氢直接修饰EGFR ATP结合部位的半胱氨酸残基，氧化刺激其酪氨酸激酶活性，尽管这种作用的生化机制仍未完全阐明。在这项提案中，我们将应用我们的一套化学探针和分析工具来解决氧化还原信号、化学生物学和癌症领域具有高度重要性的四个主要问题。该提案的目标1将定义EGFR的亚磺化调节其激酶活性的分子机制。为了确定在过氧化氢介导的信号传递中决定选择性的特征，我们将研究 在AIM 2中，EGFR靶向蛋白酪氨酸磷酸酶(PTPs)的磺化、定位和酶活性。我们将在AIM 3中使用基于靶标和化学报告/蛋白质组学的方法来评估细胞内因生长因子而产生的额外的H_2O_2靶标。虽然磺化是细胞中的可逆修饰，但将硫基化的蛋白循环为其还原的硫醇形式(RSH)的因子在很大程度上是不明确的。我们将在AIM 4中测试负责可逆亚磺化的候选还原酶。我们的化学工具的开发和在细胞中的应用为阐明控制蛋白质亚磺化的机制提供了前所未有的机会。鉴于蛋白质的异常磺化与侵袭性癌症表型有关，以及过氧化氢代谢酶的遗传损伤可能有助于肿瘤的发生，因此确定控制可逆蛋白质磺化的机制对于了解人类生理和疾病至关重要。我们预计，这些研究将确定蛋白质的亚磺化如何调节支撑细胞生长的信号网络，并确定控制去苯化的关键酶。最终，这将促进 确定新的癌症生物标记物和治疗靶点，以及产生方法学进展，扩大蛋白质组技术的范围和用途，用于生物和生物医学发现。 公共卫生相关性：拟议的研究与公共健康相关，因为调节生理蛋白质磺化的细胞机制的发现最终有望增加我们对与氧化应激和异常的基于过氧化氢的信号转导相关的病理生理学的理解，在基于氧化还原的治疗的新时代，在疾病诊断、患者分层和监测疗效的关键领域，临床医学具有翻译潜力。因此，拟议的研究与NIH使命的一部分相关，该使命涉及发展基础知识，这些基础知识将有助于促进基础发现、创新研究战略及其应用，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.