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%的蛋白质通过异戊烯化修饰。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;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。该加工途径的组成反应是目前使用的和研究的抗癌药物的靶点。此外，这种修饰的一类靶蛋白，即异源三聚体G蛋白的3个亚基，是约一半临床有用药物的介体的关键成分。因此，该处理途径的生理和病理功能与从参与心血管疾病治疗的那些到参与癌症化疗的那些的广泛治疗剂的作用密切相关。异戊烯化涉及多个酶促步骤。在异戊烯化之后，通常对蛋白质进行蛋白水解处理以去除最后3个氨基酸，并且对新的C-末端进行羧甲基化。我们已经表征了异三聚体G蛋白3亚基的异戊烯化的异质性，并且在该途径的所有三种酶促反应中发现了生物学上显著的变化。为了表征这种变异的功能意义，我们集中在一个特定的亚组的异戊二烯化处理的蛋白质（例如在人类中的G35），但不是由随后的反应，在这个复杂的途径。我们之所以关注这个变体，是因为它的加工模式依赖于序列。约10%的异戊二烯化蛋白质具有相似的序列决定簇。使用G35作为模型蛋白，我们将通过检验G35介导细胞中独特信号事件的假设来阐明这种可变加工模式的功能意义（目的1），通过检验异戊烯化G3亚基的差异蛋白水解决定其细胞内运输和作用的功能位点（Aim 2）的假设，并通过鉴定和验证蛋白质-与G35在细胞中的独特功能相关的蛋白质相互作用（Aim 3）。最后，我们将评估变异异戊二烯基加工信号在人类基因组其他预测蛋白质中的一般作用（目的4）。这些研究将扩展已知的异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异戊二烯基加工在细胞中的作用、异戊二烯基加工的变化如何与蛋白质功能相关、通过异源三聚体G蛋白进行信号传导的一般机制、通过异源三聚体G蛋白发挥其作用的大量治疗剂的作用以及机制具有重要意义。特异性或巧合地靶向蛋白质异戊二烯化反应的抗癌药物的后果和效用。 公共卫生相关性：该项目研究了一种特殊的脂质分子，称为异戊二烯基，当附着在细胞中的蛋白质上时，如何改变它们的结构和功能。将异戊烯基添加到蛋白质上的酶和修饰的蛋白质本身是许多不同种类药物的靶点，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工作的理解，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.