Proteomic Profiling of Cancer-Related Redox Signaling Pathways

癌症相关氧化还原信号通路的蛋白质组学分析

基本信息

批准号：
7790611
负责人：
LESLIE B POOLE
金额：
$ 26.05万
依托单位：
WAKE FOREST UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-05-01 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7790611
关键词：
Address Affect Amino Acids Antineoplastic Agents Apoptosis Avidin Biological Biotin Biotinylation Cancer Biology Cell Culture Techniques Cell Cycle Arrest Cell Line Cell Proliferation Regulation Cell division Cells Chemistry Cisplatin Color Communities Computing Methodologies Detection Development Digestion Drug resistance Evaluation Event Gel Goals Grant Hydrogen Peroxide Individual Investigation Ionizing radiation Label Malignant Neoplasms Malignant neoplasm of ovary Mass Spectrum Analysis Mediating Methods Modification Molecular Molecular Analysis Molecular Profiling Molecular Target Monitor Nitrogen Oxidants Oxidation-Reduction Oxygen PTEN gene Pathway interactions Patients Peptides Peroxonitrite Pharmaceutical Preparations Phosphoric Monoester Hydrolases Play Post-Translational Protein Processing Proteins Proteomics Reaction Reactive Oxygen Species Reagent Regulation Relative (related person)Research Role Running Sampling Signal Pathway Signal Transduction Signaling Molecule Site Stable Isotope Labeling Staging Stimulus System TNF gene Target Populations Taxane Compound Technology Testing Tumor Promoters Work base cancer cell cancer initiation cancer therapy cell killing cysteinesulfenic acid cytokine disulfide bond docetaxel improved in vivo innovative technologies new technology novel oxidation polyacrylamide gels preference protein function research study response taxane tool two-dimensional

项目摘要

DESCRIPTION (provided by applicant): It is widely appreciated that reactive oxygen species (ROS) play a major role in the initiation of cancer, and they are also implicated in many cancer therapies, such as ionizing radiation, cisplatin and taxanes. More recently, it has been discovered that cancer cells produce ROS as signaling molecules that promote proliferation. Unfortunately, the molecular details of how redox regulation affects cell signaling events are far from clear. New experimental and computational technologies that we have developed are uniquely suited to identifying the molecular targets that are modified by ROS, either as a result of ROS damage or ROS signaling. With the reagents and methods that we have recently developed, we can now evaluate the "redox profile" of cell populations by targeting uniquely reactive cysteine sulfenic acid (Cys-SOH) groups, the initial intermediates generated following reaction of activated protein thiolate groups with hydrogen peroxide and peroxynitrite (and perhaps other ROS). In this R33 application, our labeling technology will be further developed for quantification and multiplex analysis, so that it will have broad applicability in: 1) the investigation of basic mechanisms of ROS damage and ROS signaling; 2) molecular profiling to stratify patients with cancers that are sensitive to ROS-generating therapies; and 3) the development of novel cancer therapies based on the inhibition of ROS-dependent proliferative signaling. The following Specific Aims are proposed: 1) to develop reagents and methods of use for additional new, multicolor fluorescently-labeled Cys-SOH reagents for multiplex analysis of samples; 2) to develop quantitative mass spectrometry methods, which have some major advantages over gel-based methods (including direct readout of protein identity and numerous posttranslational modifications); and 3) to use the new quantitative methods to detect and identify Cys-SOH modified proteins generated during ROS-dependent signaling in HEK-293 cells and ovarian cancer cells. Taken together, the approaches developed in Specific Aims 1 and 2 will provide new tools for the research community to use to study the mechanisms of redox regulation and signaling. In Specific Aim 3, these tools will be used to determine the targets of ROS in the regulation of cell proliferation and apoptosis. First we will continue our study of NF-?B regulation in HEK-293 cells in response to cytokine (TNF-?) and tumor promoter (TPA) stimulation. Second, we will use ovarian cancer cells treated with cisplatin or taxane to determine which protein oxidations are critical to regulating survival and apoptosis. Besides providing specific information about the mechanism of redox regulation and signaling, these biological experiments will allow us to further refine our reagents and methods to make them most useful to the cancer biology community. These approaches to detecting functional oxidative modifications to cellular proteins hold promise in identifying specific protein targets that mediate the actions of anticancer drugs, e.g. through their effects on cell cycle arrest, cell division or apoptosis. An outgrowth of these studies could also be the development of new anticancer drugs and the ability to predict efficacy of a given drug in the treatment of individual patients.

描述（由申请人提供）：人们普遍认为，活性氧（ROS）在癌症的发生中发挥着重要作用，并且它们还涉及许多癌症治疗，例如电离辐射、顺铂和紫杉烷。最近，人们发现癌细胞产生ROS作为促进增殖的信号分子。不幸的是，氧化还原调节如何影响细胞信号事件的分子细节尚不清楚。我们开发的新实验和计算技术特别适合识别 ROS 修饰的分子靶标，无论是由于 ROS 损伤还是 ROS 信号传导。利用我们最近开发的试剂和方法，我们现在可以通过靶向独特的反应性半胱氨酸磺酸 (Cys-SOH) 基团（活化的蛋白质硫醇盐基团与氢反应后产生的初始中间体）来评估细胞群的“氧化还原谱”过氧化物和过氧亚硝酸盐（也许还有其他活性氧）。在本次R33应用中，我们的标记技术将进一步发展为定量和多重分析，从而在以下方面具有广泛的应用性：1）ROS损伤和ROS信号传导基本机制的研究； 2) 通过分子分析对对 ROS 生成疗法敏感的癌症患者进行分层； 3）基于抑制ROS依赖性增殖信号传导的新型癌症疗法的开发。提出以下具体目标： 1) 开发用于样品多重分析的其他新型多色荧光标记 Cys-SOH 试剂的试剂和使用方法； 2) 开发定量质谱方法，该方法比基于凝胶的方法具有一些主要优势（包括直接读出蛋白质特性和大量翻译后修饰）； 3) 使用新的定量方法检测和鉴定 HEK-293 细胞和卵巢癌细胞中 ROS 依赖性信号传导过程中产生的 Cys-SOH 修饰蛋白。总而言之，具体目标 1 和 2 中开发的方法将为研究界提供新的工具，用于研究氧化还原调节和信号传导机制。在Specific Aim 3中，这些工具将用于确定ROS在细胞增殖和凋亡调节中的靶标。首先，我们将继续研究 HEK-293 细胞中 NF-κB 对细胞因子 (TNF-α) 和肿瘤促进剂 (TPA) 刺激的调节反应。其次，我们将使用顺铂或紫杉烷处理的卵巢癌细胞来确定哪些蛋白质氧化对于调节存活和细胞凋亡至关重要。除了提供有关氧化还原调节和信号传导机制的具体信息外，这些生物学实验将使我们能够进一步完善我们的试剂和方法，使其对癌症生物学界最有用。这些检测细胞蛋白质功能性氧化修饰的方法有望识别介导抗癌药物作用的特定蛋白质靶标，例如抗癌药物。通过它们对细胞周期停滞、细胞分裂或细胞凋亡的影响。这些研究的成果还可能是新抗癌药物的开发以及预测特定药物在个体患者治疗中的疗效的能力。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Simple synthesis of 1,3-cyclopentanedione derived probes for labeling sulfenic acid proteins.

用于标记次磺酸蛋白的 1,3-环戊二酮衍生探针的简单合成。

DOI：
10.1039/c1cc12127h
发表时间：
2011-08-28
期刊：
Chemical communications
影响因子：
4.9
作者：
Qian J;Klomsiri C;Wright MW;King SB;Tsang AW;Poole LB;Furdui CM
通讯作者：
Furdui CM

Isoform-specific regulation of Akt by PDGF-induced reactive oxygen species.

PDGF 诱导的活性氧对 Akt 的亚型特异性调节。

DOI：
发表时间：
2011-06-28
期刊：
影响因子：
11.1
作者：
Wani, Revati;Qian, Jiang;Yin, Leimiao;Bechtold, Erika;King, S Bruce;Poole, Leslie B;Paek, Eunok;Tsang, Allen W;Furdui, Cristina M
通讯作者：
Furdui, Cristina M

A simple and effective strategy for labeling cysteine sulfenic acid in proteins by utilization of β-ketoesters as cleavable probes.

利用β-酮酯作为可裂解探针来标记蛋白质中的半胱氨酸磺酸的一种简单而有效的策略。