Generation and Function of Variable Prenyl Protein Processing

描述（由申请人提供）：预计人类基因组中约2%的蛋白质可被戊酰化修饰。这些包括广泛的底物，但其中突出的是参与细胞信号传导、生长调节、细胞进展和分化的许多关键蛋白质；例如，这些包括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的和正在研究的抗癌药物的靶点。此外，这种修饰的一类靶蛋白，异源三聚体G蛋白的3个亚基，是大约一半临床有用药物的介质的关键成分。因此，这一加工途径的生理和病理功能与广泛的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的治疗。戊烯酰化涉及多个酶促步骤。在戊酰化之后，蛋白质通常进行蛋白水解处理以去除最后3个氨基酸，新的C端被羧甲基化。我们已经表征了异三聚体G蛋白3亚基的戊酰化的异质性，并发现了该途径的所有三种酶促反应的生物学显著差异。为了描述这种变异的功能意义，我们将重点放在可变加工蛋白质的一个特定子集上（例如人类中的G35），这些蛋白质被前置酰化处理，但不被这一复杂途径中的后续反应处理。我们之所以关注这种变体，是因为它的处理模式依赖于序列。大约10%的戊酰化蛋白具有类似的序列决定因子。使用G35作为模型蛋白，我们将通过验证G35介导细胞内独特信号事件的假设（目标1），通过验证G3亚基的差异蛋白水解决定其细胞内运输和功能作用位点的假设（目标2），以及通过识别和验证与G35在细胞中独特功能相关的蛋白-蛋白相互作用（目标3），阐明这种可变加工模式的功能意义。最后，我们将评估变异戊烯基加工信号在人类基因组其他预测蛋白中的一般作用（目的4）。这些研究将扩展已知的戊烯基加工反应的多样性，并确定这种变化的功能意义。这项工作的结果将对细胞中戊烯基加工的作用，戊烯基加工的变化与蛋白质功能的关系，通过异三聚体G蛋白信号传导的一般机制，通过异三聚体G蛋白发挥作用的大量治疗方法的作用，以及特异性或偶然靶向蛋白质戊烯基化反应的抗癌药物的机制，后果和效用具有重要意义。

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.