Profiling of Redox-Sensitive Signaling Proteins

氧化还原敏感信号蛋白的分析

• 批准号: 6861333
• 负责人: LESLIE B POOLE
• 金额: $ 15.44万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6861333
• 关键词: analytical chemistry; bioinformatics; biological signal transduction; biotechnology; biotin; cell line; cellular oncology; chemical synthesis; cyclohexanones; cysteine; fluorescent dye /probe; ionophores; method development; molecular oncology; oxidative stress; proteins; proteomics; reagent /indicator

DESCRIPTION (provided by applicant): Oxidative damage and redox signaling are important components of oncogenic cell transformation, yet the molecular details of how these phenomena impact cellular proteins remain largely uncharacterized. The goal of this proposal is to develop experimental and predictive methods for the identification and molecular analysis of proteins undergoing oxidative modifications at cysteine residues, impacting cancer-related redox signaling pathways in cells. With these efforts, we will be able to apply our new proteomics-based technology to the in situ identification of redox-sensitive signaling proteins in a multi-protein, multi-pathway, whole cell format for the first time. This will allow for investigations to proceed in both discovery- and hypothesis-driven modes to unravel the molecular details of cell signaling pathways responsive to the production of reactive oxygen species (ROS). Multiple studies have highlighted the importance of activated (low pKa) cysteinyl residues in proteins as the primary targets of oxidative modifications, and have shown that hydrogen peroxide is the most important ROS involved in receptor-stimulated cell signaling. The immediate product of peroxide-linked cysteine oxidation is cysteine sulfenic acid (Cys-SOH). Therefore, trapping of Cys-SOH upon its formation in cellular proteins using a detectable reagent will yield a sensitive and comprehensive way of locating redox-responsive cysteinyl residues in cell signaling pathways. To date, no methods exist to trap this species in a manner that is amenable to proteomics type approaches; thus, identification of Cys-SOH containing proteins must be done on an individual, targeted basis. Therefore, we propose four specific aims to (i) create and validate fluorophore- and biotin-linked reagents reactive toward Cys-SOH based on the known sulfenic acid reagent dimedone, (ii) develop "redox-profiling" protocols, using dimedone and reagents created in Aim 1, to trap Cys-SOH in proteins as formed within cells and detect the extent and location of Cys-SOH formation in proteins involved in a particular signaling pathway, (iii) develop "active site profiling" methods using bioinformatics approaches to identify particular cysteinyl residues likely to be targets of redox modifications, and (iv) validate and apply the experimental and predictive methodologies of Specific Aims 2 and 3 as tools in a total cell protein/proteomics format to analyze protein modification during the course of cancer-relevant cell signaling events. These tools will usher in a new era of redox proteomics and enable proteome-scale studies of effects of oxidative stress and antioxidant therapies on cell pathways and signaling networks.

描述（由申请人提供）：氧化损伤和氧化还原信号传导是致癌细胞转化的重要组成部分，但这些现象如何影响细胞蛋白的分子细节在很大程度上仍未表征。该提案的目标是开发实验和预测方法，用于识别和分子分析在半胱氨酸残基处进行氧化修饰的蛋白质，影响细胞中与癌症相关的氧化还原信号通路。通过这些努力，我们将能够将我们新的基于蛋白质组学的技术首次应用于多蛋白质、多途径、全细胞形式的氧化还原敏感信号蛋白的原位鉴定。这将使调查在发现和假设驱动的模式下进行，以解开细胞信号通路对活性氧（ROS）产生的反应的分子细节。 多项研究强调了蛋白质中活化的（低pKa）半胱氨酰残基作为氧化修饰的主要靶点的重要性，并表明过氧化氢是参与受体刺激的细胞信号传导的最重要的ROS。过氧化物连接的半胱氨酸氧化的直接产物是半胱氨酸次磺酸（Cys-SOH）。因此，使用可检测试剂在Cys-SOH在细胞蛋白中形成时捕获Cys-SOH将产生在细胞信号传导途径中定位氧化还原响应性半胱氨酰残基的灵敏且全面的方法。迄今为止，没有方法存在的方式，是服从蛋白质组学类型的方法来捕获这个物种，因此，鉴定Cys-SOH含有蛋白质必须在一个单独的，有针对性的基础上进行。因此，我们提出了四个具体目标：（i）基于已知的次磺酸试剂二甲酮，创建并验证对Cys-SOH具有反应性的荧光团和生物素连接的试剂，（ii）使用二甲酮和目标1中创建的试剂，开发“氧化还原谱”方案，以捕获细胞内形成的蛋白质中的Cys-SOH，并检测Cys-SOH的程度和位置，参与特定信号传导途径的蛋白质中的SOH形成，（iii）使用生物信息学方法开发“活性位点分析”方法以鉴定可能是氧化还原修饰的靶的特定半胱氨酰残基，和（iv）验证和应用特定目的2和3的实验和预测方法学作为全细胞蛋白质/蛋白质组学形式的工具，以分析癌症相关细胞信号传导事件过程中的蛋白质修饰。这些工具将开创氧化还原蛋白质组学的新时代，并实现氧化应激和抗氧化疗法对细胞途径和信号网络影响的蛋白质组规模研究。