Role of Rce1p in the Biogenesis of CaaX Proteins

描述（由申请人提供）：我们的目标是定义Ras转化酶（Rce 1 p）的酶性质，Rce 1 p是CaaX蛋白生物合成所需的蛋白酶。CaaX蛋白是脂化分子，其通常在重要的细胞途径中充当信号分子。Ras和RhoB是关键的例子。由于CaaX蛋白在细胞转化中的作用（例如，Ras的活化形式与30%的癌症相关），调节这些蛋白质的生物合成的策略正被探索作为新的抗癌疗法。rce 1 p是这些策略中的一个新目标。Rce 1 p是一种非典型蛋白酶，具有多个膜跨度，缺乏典型的蛋白酶基序。到目前为止，Rce 1 p的机制仍然不明确。我们假设Rce 1 p活性位点由Rce 1 p直系同源物之间恒定保守的残基子集组成。在我们的初步研究中，利用S。在酿酒酵母系统中，我们1）鉴定了对Rce 1 p功能至关重要的4个残基，2）发现了一种潜在的Rce 1 p特异性和不可逆抑制剂的新化合物，3）利用双重遗传/生化报告基因和缺失分析来评估Rce 1 p C-末端的拓扑结构和重要性。我们已经开发了一套连贯的生化，遗传，化学和分子方法，围绕我们的研究结果，将用于定义活性位点和酶的Rce 1 p的特性。具体而言，我们将使用一种新的定量遗传分析，详细说明电荷和位置的残基被认为是关键的酶活性的重要性，并评估Rce 1 p突变体的底物特异性。我们将使用一种体外试验，监测淬灭的荧光肽底物的裂解，以评估新的抑制剂和突变对Rce 1 p动力学参数的影响。变种人最后，我们将使用双重遗传/生化拓扑报告和删除的方法来确定Rce 1 p的功能结构域。总之，该提案将阐明Rce 1 p的酶性质，Rce 1 p是具有生物医学重要性的新兴多跨膜结合蛋白酶类的成员。

DESCRIPTION (provided by applicant): Our goal is to define the enzymatic properties of the Ras Converting Enzyme (Rce1p), a protease that is required for CaaX protein biosynthesis. CaaX proteins are lipidated molecules that often function as signaling molecules in important cellular pathways. Ras and RhoB are key examples. Because of the role that CaaX proteins have in cellular transformation (e.g., activated forms of Ras are associated with 30% of all cancers), strategies that regulate the biosynthesis of these proteins are being explored as novel anticancer therapies. Rce1p is a new target in these strategies. Rce1p is an atypical protease, having multiple membrane spans and lacking a canonical protease motif. To date, the mechanism of Rce1p remains undefined. We hypothesize that the Rce1p active site is comprised of a subset of residues that are invariably conserved between Rce1p orthologs. In our preliminary studies, which take advantage of the S. cerevisiae system, we have 1) Identified 4 residues that are critically important for Rce1p function, 2) Discovered a novel compound that is potentially a specific and irreversible inhibitor of Rce1p, and 3) Utilized a dual genetic/ biochemical reporter and deletion analysis to assess the topology and importance of the Rce1p C-terminus. We have developed a coherent set of biochemical, genetic, chemical, and molecular approaches around our findings that will be used for defining the active site and enzymatic properties of Rce1p. Specifically, we will use a novel quantitative genetic assay to detail the importance of charge and position for residues deemed critical for enzymatic activity and to evaluate the substrate specificity of Rce1p mutants. We will use an in vitro assay that monitors cleavage of a quenched fluorescent peptide substrate to evaluate the effect of novel inhibitors and mutations on the kinetic parameters of Rce1p. mutants. Finally, we will use a dual genetic/biochemical topology reporter and deletion approaches to identify the functional domains of Rce1p. In sum, this proposal will clarify the enzymatic properties of Rce1p, a member of an emerging class of multi-span membrane-bound proteases having biomedical importance.