Profiling of Redox-Sensitive Signaling Proteins

氧化还原敏感信号蛋白的分析

• 批准号: 7060447
• 负责人: LESLIE B POOLE
• 金额: $ 14.02万
• 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 2008-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7060447
• 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然未知。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半胱氨酸残基发生氧化修饰的蛋白质，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，我们将能够首次将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。这将使研究能够以发现驱动和假设驱动的模式进行，以揭示响应活性氧（ROS）产生的细胞信号传导途径的分子细节。 多项研究强调了蛋白质中活化（低 pKa）半胱氨酰残基作为氧化修饰主要目标的重要性，并表明过氧化氢是参与受体刺激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，使用可检测试剂捕获细胞蛋白中形成的 Cys-SOH 将产生一种灵敏且全面的方法来定位细胞信号传导途径中的氧化还原响应性半胱氨酰残基。迄今为止，尚不存在以适合蛋白质组学类型方法的方式捕获该物种的方法。因此，含有 Cys-SOH 的蛋白质的鉴定必须在个体、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双甲酮，创建并验证对 Cys-SOH 具有反应性的荧光团和生物素连接试剂；(ii) 使用双甲酮和试剂开发“氧化还原分析”方案在目标 1 中创建，捕获细胞内形成的蛋白质中的 Cys-SOH，并检测参与特定信号传导途径的蛋白质中 Cys-SOH 形成的程度和位置，(iii) 开发“活性位点”使用生物信息学方法来识别可能成为氧化还原修饰目标的特定半胱氨酰残基的“分析”方法，以及（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质/蛋白质组学格式的工具来分析蛋白质癌症相关细胞信号传导事件过程中的修饰。这些工具将开创氧化还原蛋白质组学的新时代，并使蛋白质组规模的研究成为可能，研究氧化应激和抗氧化疗法对细胞途径和信号网络的影响。

项目成果

The requirement of reversible cysteine sulfenic acid formation for T cell activation and function.

T 细胞激活和功能需要可逆的半胱氨酸磺酸形成。

• 发表时间: 2007-11-15
• 作者: Michalek, Ryan D;Nelson, Kimberly J;Holbrook, Beth C;Yi, John S;Stridiron, Daya;Daniel, Larry W;Fetrow, Jacquelyn S;King, S Bruce;Poole, Leslie B;Grayson, Jason M
• 通讯作者: Grayson, Jason M

Functional site profiling and electrostatic analysis of cysteines modifiable to cysteine sulfenic acid.

可修饰为半胱氨酸磺酸的半胱氨酸的功能位点分析和静电分析。

• 发表时间: 2008-02
• 作者: Salsbury Jr, Freddie R;Knutson, Stacy T;Poole, Leslie B;Fetrow, Jacquelyn S
• 通讯作者: Fetrow, Jacquelyn S

Measurement of protein sulfenic acid content.

蛋白质次磺酸含量的测定。

• 发表时间: 2008-11
• 作者: Poole; Leslie B
• 通讯作者: Leslie B

Synthesis of chemical probes to map sulfenic acid modifications on proteins.

合成化学探针以绘制蛋白质上的次磺酸修饰图。

• DOI: 10.1021/bc050257s
• 发表时间: 2005-11-01
• 期刊: Bioconjugate chemistry
• 影响因子: 4.7
• 作者: L. Poole;Bu‐Bing Zeng;S. Knaggs;Mamudu Yakubu;S. King
• 通讯作者: S. King

Use of dimedone-based chemical probes for sulfenic acid detection evaluation of conditions affecting probe incorporation into redox-sensitive proteins.

使用基于双甲酮的化学探针进行磺酸检测评估影响探针掺入氧化还原敏感蛋白的条件。

• DOI: 10.1016/s0076-6879(10)73003-2
• 发表时间: 2010
• 期刊: Methods in enzymology
• 作者: Klomsiri, Chananat;Nelson, Kimberly J.;Bechtold, Erika;Soito, Laura;Johnson, Lynnette C.;Lowther, W. Todd;Ryu, Seong-Eon;King, S. Bruce;Furdui, Cristina M.;Poole, Leslie B.
• 通讯作者: Poole, Leslie B.