Proteomic Analysis of Protein Prenylation

描述（由申请人提供）：蛋白质异戊二烯化是一种非常重要的翻译后修饰，据估计有数百种不同的哺乳动物蛋白质被异戊二烯化。所有重要的异戊烯化蛋白质的确切身份及其异戊烯化程度尚未确定。由于异戊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）至少部分地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，他汀类药物作为癌症的化学预防剂，细胞研究表明，他汀类药物的这种作用是由于他们的蛋白质异戊二烯化的间接抑制。因此，用于分析的蛋白质组学方法的开发，特别是异戊烯化蛋白质的定量，可能具有重大的医学重要性。该建议的具体假设是，肿瘤细胞对他汀类药物的细胞生长抑制和凋亡反应是由于抑制肿瘤细胞中信号转导蛋白的香叶基香叶基化。这项合作提案汇集了吉布斯实验室在异戊二烯化的化学和生物化学方面的专业知识，以及Regnier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。提出了以下具体目标：1）开发和评估用于异戊二烯化蛋白质的物理分离和随后表征的新技术。在Regnier实验室开发的一种新方法，半渗透表面（SPS）色谱法，将研究其选择性吸附脂化蛋白的能力。随后对蛋白质进行解吸，然后进行标准蛋白质组质谱分析。2)开发和评估用于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。几种不同的化学反应的独特的烯烃单元中存在的异戊二烯部分的选择性将被评估的能力，选择性地衍生异戊二烯化的肽。这些方法的优化版本，然后将被应用到选择性修饰的异戊二烯化蛋白质在生物环境中。3)将开发的技术应用于他汀类药物处理细胞中蛋白质异戊二烯化状态的表征。这些水平将与胰腺肿瘤细胞的细胞生长抑制、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治疗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。

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.