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，但是这些现象如何影响细胞蛋白的分子细节在很大程度上没有表征。该建议的目的是开发实验和预测方法，以鉴定和分子分析半胱氨酸残基的蛋白质进行氧化修饰，从而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，我们将能够将基于蛋白质组学的新技术应用于首次在多蛋白质，多条纹，全细胞格式中对氧化还原敏感的信号传导蛋白的原位识别。这将允许研究以发现和假设驱动的模式进行，以揭示响应活性氧（ROS）的产生的细胞信号通路的分子细节。 多项研究强调了蛋白质中活化（低PKA）胱烷基残基作为氧化修饰的主要靶标的重要性，并表明过氧化氢是与受体刺激的细胞信号传导有关的最重要的ROS。过氧化物连接的半胱氨酸氧化的直接产物是半胱氨酸硫酸（CYS-SOH）。因此，使用可检测的试剂将Cys-SOH捕获在细胞蛋白中形成，将产生一种敏感而综合的方式，将氧化还原响应性的胱天章基因在细胞信号传导途径中定位。迄今为止，尚无以适合蛋白质组学方法的方式捕获该物种的方法。因此，必须以单独的针对性基础进行含有Cys-SOH蛋白质的识别。因此，我们提出了四个特定的目的，以（i）基于已知的磺胺酸试剂二聚体（ii）使用Dimedone和试剂制定“ Redox-Profighing”方案，创建和验证荧光团和生物素连接的试剂对CYS-SOH的反应性。在AIM 1中创建，以在细胞内形成的蛋白质中捕获Cys-SOH，并检测参与特定信号通路涉及的蛋白质中Cys-SOH形成的程度和位置，（iii）使用生物信息学方法来开发“活跃的位点分析”方法确定可能是氧化还原修饰的靶标的特定的白细胞蛋白酶残基，（iv）验证并应用特定目标2和3的实验和预测方法作为总细胞蛋白/蛋白质组学格式中的工具，以分析癌症在癌症过程中的修饰。相关的细胞信号事件。这些工具将引入氧化还原蛋白质组学的新时代，并能够对氧化应激和抗氧化剂疗法对细胞途径和信号网络的影响进行蛋白质组规模研究。