Role of Rce1p in the Biogenesis of CaaX Proteins

我们的目标是定义RAS转换酶(RCELP)的酶性质，这是一种能够 是CAAX蛋白生物合成所必需的。CAAX蛋白是脂化分子，其功能通常是 重要细胞通路中的信号分子。RAS和RhoB是关键的例子。因为这个角色 CAAX蛋白在细胞转化中的作用(例如，活化形式的RAS与30%的 所有癌症)，调控这些蛋白质的生物合成的策略正在被探索为新的抗肿瘤 癌症疗法。Rcelp是这些战略中的新目标。 Rcelp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，RCELP的作用机制尚不清楚。我们假设Rcelp的活动站点是 由在RCELP同源基因之间总是保守的残基的子集组成。在我们的预赛中 利用酿酒酵母系统的研究，我们1)确定了四种残留物，它们是 对Rcelp功能至关重要，2)发现了一种新的化合物，它可能是一种特定的和 以及3)利用双重遗传/生化报告和缺失分析 评估Rcelp C末端的拓扑和重要性。 我们已经开发了一套连贯的生化、遗传、化学和分子方法 我们的发现将被用来定义RCELP的活性部位和酶性质。具体来说， 我们将使用一种新的定量遗传分析来详细说明残留物的电荷和位置的重要性 被认为对酶活性和评估Rcelp突变体的底物专一性至关重要。我们会 使用体外试验监测猝灭的荧光肽底物的裂解来评估效果 新型抑制剂和突变对Reel P.突变体动力学参数的影响。最后，我们将使用DUAL 利用遗传/生化拓扑报告和缺失方法确定RCELP的功能结构域。 总之，这项提议将澄清Rcelp的酶特性，Rcelp是新兴类别的成员 具有生物医学重要性的多跨膜结合蛋白水解酶。

Our goal is to define the enzymatic properties of the Ras Converting Enzyme (Rcelp), a protease that is required for CaaX protein biosynthesis. CaaX proteins are lipidated molecules that often function as signaling molecules in important cellularpathways. 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 biosynthesisof these proteins are being explored as novel anti- cancer therapies. Rcelp is a new target in these strategies. Rcelp is an atypical protease, having multiple membrane spans and lacking a canonical protease motif. To date, the mechanism of Rcelp remains undefined. We hypothesize that the Rcelp active site is comprised of a subset of residues that are invariably conserved between Rcelp orthologs. In our preliminary studies, which take advantage of the S. cerevisiae system, we have 1) identified four residues that are critically important for Rcelp function, 2) discovered a novel compound that is potentially a specific and irreversible inhibitor of Rcelp, and 3) utilized a dual genetic/ biochemical reporter and deletion analysis to assess the topology and importance of the Rcelp 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 Rcelp. 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 Rcelp 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 Reel p.mutants. Finally, we will use a dual genetic/biochemical topology reporter and deletion approaches to identify the functional domains of Rcelp. In sum, this proposal will clarify the enzymatic properties of Rcelp, a member of an emerging class of multi-span membrane-boundproteases having biomedical importance.