Role of Rce1p in the Biogenesis of CaaX Proteins

描述(由申请人提供)：我们的目标是确定RAS转换酶(Rce1p)的酶性质，Rce1p是CAAX蛋白质生物合成所需的一种蛋白酶。CAAX蛋白是脂化分子，通常在重要的细胞通路中起信号分子的作用。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，活化形式的RAS与30%的癌症有关)，调控这些蛋白的生物合成的策略正在被探索为新的抗癌疗法。Rce1p是这些策略中的一个新靶点。Rce1p是一种非典型的蛋白水解酶，具有多个膜跨度，缺乏一个典型的蛋白水解酶基序。到目前为止，Rce1p的作用机制尚不清楚。我们假设Rce1p活性位点由Rce1p同源基因之间总是保守的残基子集组成。在我们的初步研究中，利用酿酒酵母系统，我们1)鉴定了4个对Rce1p功能至关重要的残基，2)发现了一种新的化合物，它可能是Rce1p的特异性和不可逆的抑制剂，以及3)利用双重遗传/生化报告和缺失分析来评估Rce1p C-末端的拓扑结构和重要性。我们围绕我们的发现开发了一套连贯的生化、遗传、化学和分子方法，将用于定义Rce1p的活性部位和酶性质。具体地说，我们将使用一种新的定量遗传分析来详细说明被认为对酶活性至关重要的残基的电荷和位置的重要性，并评估Rce1p突变体的底物特异性。我们将使用一种监测猝灭的荧光肽底物切割的体外实验来评估新的抑制剂和突变对Rce1p动力学参数的影响。变种人。最后，我们将使用遗传/生化双重拓扑报告和缺失的方法来确定Rce1p的功能结构域。总之，这项提议将阐明Rce1p的酶学性质，Rce1p是一类新兴的具有生物医学重要性的多跨膜结合蛋白酶的成员。

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.