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）：蛋白质原始化是一种至关重要的翻译后修饰，据估计，几百种不同的哺乳动物蛋白是原始化的。尚未确定所有重要的原始化蛋白质及其前化程度的确切身份。现在，这是一个重要的医学重要性问题，这是由于前孕抑制剂在治疗人类癌症中的潜在使用。此外，有迹象表明，汀类药物是使用最广泛的药物类别之一，至少部分通过抑制蛋白质前化来发挥其治疗作用。最近的临床数据表明，他汀类药物是癌症化学预防剂，细胞研究表明，他汀类药物的这种作用是由于它们间接抑制了蛋白质预化。因此，开发用于分析的蛋白质组学方法，尤其是对原蛋白的定量，可能具有重要的医学重要性。该提议的特定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烷基凝聚力化的抑制。这项协作提案汇集了吉布斯实验室在原苯化的化学和生物化学方面的专业知识，以及雷格尼尔实验室的生物分析和蛋白质组学经验，用于共同开发和应用工具，用于分析蛋白质前化的工具。提出了以下特定目的：1）开发和评估新技术，以实现蛋白质的物理分离和后续表征。将研究一种在Regnier实验室，半透明表面（SP）色谱法中开发的新方法，该方法将因其选择性吸附脂质蛋白的能力而进行研究。随后的蛋白质解吸将进行标准蛋白质组学质谱分析。 2）开发和评估新技术的选择性化学修饰，隔离和随后的蛋白质特征。将评估针对前蛋白基部分中存在的独特烯烃单元选择性的几种不同的化学反应，以选择性地衍生前肽的肽的能力进行评估。然后，这些方法的优化版本将应用于生物环境中预元化蛋白的选择性修饰。 3）将开发的技术应用于用他汀类药物治疗的细胞中蛋白质的婚前化状态的表征。这些水平将与胰腺肿瘤细胞的细胞抑制作用，细胞毒素和凋亡反应相关。同时，还将研究经他汀类药物治疗的胰β细胞的原始化状态。开发生物标志物以验证患者对他汀类药物，Farnesylsylansferse酶抑制剂的化学治疗或化学预防反应以及其他可能调节蛋白质前化的药物的化学治疗或化学预防反应非常重要。该过程的蛋白质组学分析将提供这样的生物标志物。