Generation and Function of Variable Prenyl Protein Processing

描述(由申请人提供)：人类基因组中大约2%的蛋白质预计会被预烯基化修饰。它们包括广泛的底物，但其中最突出的是许多参与细胞信号、生长调节、细胞进展和分化的关键蛋白质；例如，这些蛋白质包括ras原癌基因产物和异源三聚体G蛋白的3个亚基。这一加工途径的组成反应是目前使用和研究中的抗癌药物的靶点。此外，这种修饰的一类靶蛋白，异三聚体G蛋白的3个亚基，是大约一半临床有用药物的介体的关键成分。因此，这一过程的生理和病理功能与多种治疗药物的作用密切相关，从心血管疾病的治疗到癌症化疗。预酯化涉及多个酶促步骤。在预烯基化之后，蛋白质通常被蛋白质水解性加工以去除最后3个氨基酸，并且新的C末端被羧甲基化。我们已经表征了异三聚体G蛋白3亚单位预烯基化的异质性，并在这一途径的所有三种酶反应中发现了生物学上的显著差异。为了表征这种变异的功能意义，我们集中在可变加工蛋白质的特定子集(以人类的G35为例)，这些蛋白质是通过预烯基化加工的，而不是通过这一复杂途径中的后续反应加工的。我们之所以关注这个变体，是因为它的处理模式依赖于序列。大约10%的预烯基化蛋白质具有相似的序列决定因素。我们将以G35作为模型蛋白，通过检验G35介导细胞内独特信号事件的假说(目标1)，检验预烯基化G3亚单位的差异蛋白分解决定其细胞内运输和作用功能位点的假说(目标2)，以及通过鉴定和验证与G35在细胞中的独特功能相关的蛋白质-蛋白质相互作用(目标3)，阐明这种可变处理模式的功能意义。最后，我们将评估变异的戊烯基加工信号在人类基因组中其他预测蛋白质中的一般作用(目标4)。这些研究将扩大已知的戊烯加工反应的多样性，并确定这种变异的功能意义。这项工作的结果将对细胞中异戊烯加工的作用，异戊烯加工的变异如何与蛋白质功能相关，通过异三聚体G蛋白传递信号的一般机制，大量通过异三聚体G蛋白发挥作用的治疗药物的作用，以及特异性或巧合靶向蛋白质预烯基化反应的抗癌药物的机制、后果和用途具有重要意义。 与公共健康相关：拟议的项目研究一种特殊的脂类分子，当它与细胞中的蛋白质结合时，是如何改变它们的结构和功能的。将戊烯基团添加到蛋白质中的酶和修饰的蛋白质本身是许多不同类型药物的靶标，包括用于治疗心脏病和癌症的药物。拟议的工作将增加我们对这些药物如何发挥作用的了解，并将帮助我们开发更有效的药物来治疗这些常见疾病。

DESCRIPTION (provided by applicant): About 2% of proteins in the human genome are predicted to be modified by prenylation. These include a broad range of substrates, but prominent among them are many key proteins involved in cell signaling, growth regulation, cell progression and differentiation; for example these include the ras proto-oncogene products and the 3 subunits of the heterotrimeric G proteins. The constituent reactions of this processing pathway are the targets of both currently used and investigational anticancer drugs. In addition, one class of target proteins for this modification, the 3 subunits of the heterotrimeric G proteins, are key constituents of the mediators of about half of all clinically useful drugs. Thus the physiological and pathological function of this processing pathway is closely related to the actions of a wide range of therapeutics from those involved in treatment of cardiovascular diseases to those involved in cancer chemotherapy. Prenylation involves multiple enzymatic steps. Following prenylation, the protein is typically proteolytically processed to remove the last 3 amino acids and the new C- terminus is carboxymethylated. We have characterized the heterogeneity of the prenylation of the heterotrimeric G protein 3 subunits and have found biologically significant variation in all three enzymatic reactions of this pathway. To characterize the functional significance of this variation we are concentrating on a specific subset of variably processed proteins (exemplified by G35 in humans) that are processed by prenylation but not by the subsequent reactions in this complex pathway. We are concentrating on this variant because its processing pattern is sequence dependent. About 10% of prenylated proteins have similar sequence determinants. Using G35 as a model protein we will elucidate the functional significance of this variable processing pattern by testing the hypothesis that G35 mediates unique signaling events in cells (Aim 1), by testing the hypothesis that differential proteolysis of prenylated G3 subunits determines their intracellular trafficking and functional site of action (Aim 2) and by identifying and validating protein-protein interactions related to the unique functions of G35 in cells (Aim 3). Finally, we will evaluate the general role of the variant prenyl processing signal in other predicted proteins from the human genome (Aim 4). These studies will extend the known diversity of the prenyl processing reactions and define the functional significance of this variation. The results of this work will have significance for the role of prenyl processing in cells, for how variation of prenyl processing is related to protein function, for the general mechanisms for signaling through heterotrimeric G proteins, for the actions of the large number of therapeutics that exert their effects through heterotrimeric G proteins, and for the mechanism, consequences and utility of anti-cancer drugs that specifically or coincidently target the protein prenylation reactions. PUBLIC HEALTH RELEVANCE: The proposed project studies how a particular kind of lipid molecule referred to as a prenyl group when attached to proteins in cells alters their structure and function. The enzymes that add prenyl groups to proteins and the modified proteins themselves are targets for many different kinds of drugs including those used to treat heart diseases and cancer. The proposed work will increase our understanding of how these drugs work and will help us develop more effective drugs to treat these common illnesses.