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(申请人提供)：蛋白质预烯基化是一种至关重要的翻译后修饰，据估计，数百种不同的哺乳动物蛋白质被预烯基化。所有重要的前烯基化蛋白质的确切身份以及它们的前烯基化程度尚未确定。这现在是一个具有重大医学意义的问题，因为预烯基化抑制剂在治疗人类癌症方面的潜在用途。此外，有迹象表明，作为最广泛使用的药物类别之一，他汀类药物至少在一定程度上是通过抑制蛋白质预烯基化来发挥治疗作用的。最近的临床数据表明，他汀类药物可以作为癌症的化学预防药物，细胞研究表明，他汀类药物的这种作用是由于它们间接地抑制了蛋白质的前烯基化。因此，蛋白质组学分析方法的发展，特别是对预烯基化蛋白质的定量，可能具有重大的医学意义。这一建议的具体假设是，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老年化。这项合作建议将Gibbs实验室在预烯基化的化学和生化方面的专业知识与Regnier实验室的生物分析和蛋白质组学经验结合在一起，共同开发和应用蛋白质预烯基化分析工具。提出了以下具体目标：1)开发和评估用于物理分离和随后表征预烯基化蛋白质的新技术。在Regnier实验室开发的一种新方法，半渗透表面(SPS)层析，将因其选择性吸附脂化蛋白质的能力而被研究。随后将对蛋白质进行解吸，然后进行标准蛋白质组质谱分析。2)开发和评价用于选择性化学修饰、分离和随后表征预烯基化蛋白质的新技术。将评估几种不同的化学反应对戊烯基部分中存在的独特的烯烃单元的选择性衍生化异戊烯基多肽的能力。然后，这些方法的优化版本将被应用于生物环境中的戊烯基化蛋白质的选择性修饰。3)将开发的技术应用于他汀类药物处理的细胞中蛋白质的预烯基化状态的表征。这些水平将与胰腺肿瘤细胞的细胞抑制、细胞毒性Jc和凋亡反应相关。同时，他汀类药物处理的培养的胰岛β细胞的苯丙基化状态也将被研究。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预苯化的药物的化疗或化学预防反应至关重要。对这一过程的蛋白质组学分析将提供这样的生物标记物。