Profiling of Redox-Sensitive Signaling Proteins

描述(申请人提供)：氧化损伤和氧化还原信号是致癌细胞转化的重要组成部分，然而这些现象如何影响细胞蛋白质的分子细节在很大程度上仍未确定。这项提议的目标是开发实验和预测方法来鉴定和分子分析半胱氨酸残基上经历氧化修饰的蛋白质，这些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，我们将能够首次将我们基于蛋白质组学的新技术应用于多蛋白质、多途径、全细胞形式的氧化还原敏感信号蛋白的原位鉴定。这将使研究能够在发现驱动和假设驱动的两种模式下进行，以揭开细胞信号通路对活性氧物种(ROS)产生的反应的分子细节。许多研究强调了蛋白质中活化的(低pKA)半胱氨酸残基作为氧化修饰的主要目标的重要性，并表明过氧化氢是参与受体刺激的细胞信号转导的最重要的ROS。过氧半胱氨酸氧化的直接产物是半胱氨酸磺酸(Cys-SOH)。因此，使用可检测的试剂在细胞蛋白中形成Cys-SOH时捕获Cys-SOH将产生一种灵敏和全面的方法来定位细胞信号通路中氧化还原反应的半胱氨基残基。到目前为止，还不存在以蛋白质组学类型的方法捕获该物种的方法；因此，含有Cys-SOH的蛋白质的鉴定必须在个别的、有针对性的基础上进行。因此，我们提出了四个具体目标：(I)基于已知的磺酸试剂dimedone，创建和验证针对Cys-SOH的荧光团和生物素连接试剂；(Ii)开发“氧化还原图谱”方案，使用在Aim 1中创建的试剂，捕获Cys-SOH在细胞内形成的蛋白质中，并检测特定信号通路中涉及的蛋白质中Cys-SOH的形成程度和位置；(Iii)利用生物信息学方法开发“活性部位图谱”方法，以确定可能成为氧化还原修饰目标的特定半胱氨基残基，以及(Iv)验证和应用特定AIMS 2和3的实验和预测方法，作为总细胞蛋白/蛋白质组学形式的工具，以分析与癌症相关的细胞信号事件过程中的蛋白质修饰。这些工具将开启氧化还原蛋白质组学的新纪元，并使氧化应激和抗氧化治疗对细胞通路和信号网络的影响进行蛋白质组规模的研究。

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.