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Role of Rce1p in the Biogenesis of CaaX Proteins

Rce1p 在 CaaX 蛋白生物发生中的作用

基本信息

批准号：
7568648
负责人：
Walter K Schmidt
金额：
$ 0.78万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-07-01 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7568648
关键词：
Active Sites Address Advantage-S Affinity Labels Anabolism Antineoplastic Agents Area Biochemical Genetics Biogenesis Biological Assay Cell membrane Cells Charge Chemicals Chimera organism Class Clinical Collaborations Colorectal Development Endopeptidases Enzymes Fluorescence Future Genetic screening method Goals Human In Vitro Incidence Ketones Kinetics Localized Malignant Neoplasms Membrane Methods Modification Molecular Molecular Target Monitor Mutate Mutation Nature Oncogene Proteins Orthologous Gene Pancreas Peptide Hydrolases Peptides Phenotype Positioning Attribute Property Protein Biosynthesis Proteins Quantitative Genetics Relative (related person)Reporter Role Saccharomyces cerevisiae Signal Transduction Signaling Molecule Site Structure Substrate Specificity Sum System Testing Yeasts affinity labeling base cancer therapy carbobenzoxyphenylalanine deletion analysis drug development drug discovery in vitro Assay in vivo inhibitor/antagonist innovation insight member metaplastic cell transformation mutant novel presenilin response scaffold tool

项目摘要

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.

我们的目标是确定Ras转化酶（Rcelp）的酶学性质，Rcelp是一种蛋白酶