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Analysis of the substrate specificity and activity regulation of rhomboid proteases

菱形蛋白酶的底物特异性及活性调控分析

基本信息

批准号：
262132838
负责人：
Professor Dr. Steven Verhelst
金额：
--
依托单位：
Dept. of Cellular and Molecular Medicine
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2014
资助国家：
德国
起止时间：
2013-12-31 至 2017-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/262132838?language=en
关键词：
Analysis substrate specificity activity regulation

项目摘要

Intramembrane proteases are unusual enzymes that hydrolyze their substrates in the hydrophobic environment of the membrane. Generally, the substrates are bound to the membrane in a dormant form, which upon cleavage lead to a specific biological response. The rhomboids form the family of intramembrane serine proteases. Structurally, they are the best characterized intramembrane proteases and they therefore form a paradigm for intramembrane proteolysis. Interestingly, genes coding for rhomboids have been found in virtually all sequenced organisms, but the functional role of most of them remains unelucidated. From the examples that are known, it appears that the biological function of rhomboids is quite diverse, ranging from cellular communication to host cell invasion by certain parasites. There are several key questions in rhomboid research that remain unanswered: How does rhomboid choose its substrates? How does the substrate enter the active site? Can rhomboids be used as drug targets? Can selective inhibitors be designed? And how is rhomboid activity regulated? The specific aims of the project described in this proposal deal with several of these questions. Previous contradicting clues about the substrate specificity were obtained from the study of specific protein substrates. We will reveal the substrate specificity of rhomboids by an unbiased, proteomics-based approach. Making use of proteome-derived peptide libraries, we will identify scissile bonds of different rhomboids by mass spectrometry-based methods. The resulting cleavage sites will be analyzed in order to derive a consensus sequence for each individual rhomboid. The resulting information will them be used to design new reporter substrates and novel peptide-based inhibitors, which will be analyzed for their selectivity and their site of interaction with the rhomboid structure. Although most proteases are synthesized as inactive zymogens, rhomboids are translated in their active from. There are some indications that the membrane environment influences substrate cleavage. To gain more insight into this, we will express and purify rhomboids from different prokaryotic and eukaryotic species and reconstitute them in liposomes with different lipid compositions. Using our recently developed rhomboid ABPs, we have the ideal tools in hand to measure the activity state of the rhomboids in these different environments. Overall, this proposal will yield valuable information about the activity regulation and the substrate specificity of rhomboids, and may form the foundation for future rhomboid drug development.

膜内蛋白酶是在膜的疏水环境中水解其底物的不常见的酶。通常，底物以休眠形式与膜结合，其在裂解时导致特异性生物反应。菱形形成膜内丝氨酸蛋白酶家族。在结构上，它们是最具特征的膜内蛋白酶，因此它们形成膜内蛋白水解的范例。有趣的是，几乎在所有已测序的生物中都发现了编码菱形的基因，但其中大多数的功能作用仍未阐明。从已知的例子来看，菱形体的生物学功能似乎相当多样化，从细胞通讯到某些寄生虫入侵宿主细胞。菱形体研究中有几个关键问题尚未得到解答：菱形体如何选择其基底？底物如何进入活性位点？菱形体可以作为药物靶点吗？可以设计选择性抑制剂吗？菱形细胞的活动是如何调节的？本提案所述项目的具体目标涉及其中几个问题。以前关于底物特异性的矛盾线索是从特异性蛋白质底物的研究中获得的。我们将揭示底物特异性的菱形通过一个公正的，基于蛋白质组学的方法。利用蛋白质组衍生的肽库，我们将通过基于质谱的方法鉴定不同菱形的易断裂键。将分析所得切割位点，以获得每个菱形的共有序列。由此产生的信息将用于设计新的报告底物和新的基于肽的抑制剂，将分析它们的选择性和它们与菱形结构的相互作用位点。虽然大多数蛋白酶是作为无活性的酶原合成的，但菱形蛋白酶以其活性形式翻译。有一些迹象表明，膜环境影响底物裂解。为了更深入地了解这一点，我们将表达和纯化来自不同的原核和真核物种的菱形，并将其重组在具有不同脂质成分的脂质体中。使用我们最近开发的菱形ABP，我们拥有理想的工具来测量这些不同环境中菱形的活动状态。总之，这一建议将产生有价值的信息的活动调节和底物特异性的菱形，并可能形成未来的菱形药物开发的基础。