Proteomic Analysis of Protein Prenylation

蛋白质异戊二烯化的蛋白质组学分析

• 批准号: 6942399
• 负责人: RICHARD A GIBBS
• 金额: $ 13.44万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-23 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6942399
• 关键词: HMG coA reductases; alkenes; antineoplastics; apoptosis; biomarker; cell growth regulation; cell line; chemical reaction; chromatography; cytotoxicity; enzyme inhibitors; farnesyl compound; mass spectrometry; neoplastic cell; posttranslational modifications; prenylation; protein purification; protein quantitation /detection; protein structure function; proteomics; technology /technique development

DESCRIPTION (provided by applicant): Protein prenylation is a critically important post-translational modification, and it has been estimated that several hundred different mammalian proteins are prenylated. The exact identity of all important prenylated proteins, and the extent of their prenylation, has not been established. This is now an issue of significant medical importance, due the potential use of prenylation inhibitors in treatment of human cancers. Moreover, there are indications that the statins, one of the most widely used drug classes, exert their therapeutic effects at least in part through the inhibition of protein prenylation. Recent clinical data has suggested that the statins serve as cancer chemopreventative agents, and cellular studies have indicated that this effect of the statins is due to their indirect inhibition of protein prenylation. Thus, the development of proteomic methods for the analysis, and particularly the quantitation of prenylated proteins, may be of major medical importance. The specific hypothesis of this proposal is that the cytostatic and apoptotic response of tumor cells to statins is due to the inhibition of the geranylgeranylation of signal transduction proteins in tumor cells. This collaborative proposal brings together the expertise of the Gibbs laboratory in the chemical and biochemical aspects of prenylation, with the bioanalytical and proteomic experience of the Regnier laboratory for the joint development and application of tools for the analysis of protein prenylation. The following specific aims are proposed: 1) To develop and evaluate new techniques for the physical isolation and subsequent characterization of prenylated proteins. A novel method developed in the Regnier laboratory, semipermeable surface (SPS) chromatography, will be investigated for its ability to selectively adsorb lipidated proteins. Subsequent desorption of the proteins will be followed by standard proteomic mass spectrometric analysis. 2) To develop and evaluate new techniques for the selective chemical modification, isolation and subsequent characterization of prenylated proteins. Several different chemical reactions selective for the unique olefin units present in the prenyl moieties will be evaluated for their ability to selectively derivatize prenylated peptides. The optimized versions of these methods will then be applied to the selective modification of prenylated proteins in a biological milieu. 3) To apply the techniques developed to the characterization of the prenylation status of proteins in cells treated with statins. These levels will be correlated with cytostatic, cytotoxJc, and apoptotic responses of pancreatic tumor cells. In parallel, the prenylation status of statin-treated cultured pancreatic beta-cells will also be investigated. It is critically important to develop biomarkers to validate the chemotherapeutic or chemopreventative response of patients to treatment with statins, farnesyltransferase inhibitors, and other drugs that may modulated protein prenylation. The proteomic analysis of this process would provide such a biomarker.

描述（由申请人提供）：蛋白质异戊二烯化是一种极其重要的翻译后修饰，据估计有数百种不同的哺乳动物蛋白质被异戊二烯化。所有重要异戊二烯化蛋白质的确切身份及其异戊二烯化程度尚未确定。由于异戊二烯化抑制剂在治疗人类癌症中的潜在用途，这现在是一个具有重要医学意义的问题。此外，有迹象表明，他汀类药物（最广泛使用的药物类别之一）至少部分通过抑制蛋白质异戊二烯化来发挥其治疗作用。最近的临床数据表明他汀类药物可作为癌症化学预防剂，细胞研究表明他汀类药物的这种作用是由于其间接抑制蛋白质异戊二烯化。因此，开发用于分析的蛋白质组学方法，特别是异戊二烯化蛋白质的定量，可能具有重大的医学重要性。该提案的具体假设是，肿瘤细胞对他汀类药物的细胞抑制和凋亡反应是由于抑制了肿瘤细胞中信号转导蛋白的香叶基香叶基化。该合作提案汇集了吉布斯实验室在异戊二烯化化学和生化方面的专业知识以及雷尼尔实验室的生物分析和蛋白质组学经验，共同开发和应用蛋白质异戊二烯化分析工具。提出以下具体目标： 1) 开发和评估异戊二烯化蛋白质的物理分离和后续表征的新技术。 Regnier 实验室开发的一种新方法，即半透表面 (SPS) 色谱法，将研究其选择性吸附脂化蛋白质的能力。随后蛋白质解吸后将进行标准蛋白质组质谱分析。 2) 开发和评估异戊二烯化蛋白质的选择性化学修饰、分离和后续表征的新技术。将评估对异戊二烯基部分中存在的独特烯烃单元具有选择性的几种不同化学反应选择性衍生异戊二烯化肽的能力。这些方法的优化版本将应用于生物环境中异戊二烯化蛋白质的选择性修饰。 3) 将所开发的技术应用于表征他汀类药物处理的细胞中蛋白质的异戊二烯化状态。这些水平将与胰腺肿瘤细胞的细胞抑制、细胞毒性和凋亡反应相关。同时，还将研究他汀类药物处理的培养胰腺β细胞的异戊二烯化状态。开发生物标志物来验证患者对他汀类药物、法尼基转移酶抑制剂和其他可能调节蛋白质异戊二烯化的药物的化疗或化学预防反应至关重要。这一过程的蛋白质组学分析将提供这样的生物标志物。