Analysis of the substrate specificity and activity regulation of rhomboid proteases

膜内蛋白水解酶是一种特殊的酶，能在膜的疏水环境中降解底物。通常，底物以休眠的形式结合到膜上，在分裂时会导致特定的生物反应。菱形体形成膜内丝氨酸蛋白酶家族。在结构上，它们是最具特性的膜内蛋白水解酶，因此它们形成了膜内蛋白分解的范例。有趣的是，编码菱形的基因几乎在所有已测序的生物体中都被发现，但它们中的大多数的功能作用仍然不清楚。从已知的例子来看，菱形生物的生物学功能似乎相当多样化，从细胞通讯到某些寄生虫对宿主细胞的入侵。在菱形研究中有几个关键问题仍然没有答案：菱形如何选择其底物？底物是如何进入活性中心的？菱形可以用作药物靶点吗？可以设计选择性抑制剂吗？菱形活动是如何受到监管的？本提案中描述的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相互矛盾的线索是从对特定蛋白质底物的研究中获得的。我们将通过一种基于蛋白质组学的无偏见的方法来揭示菱形细胞的底物特异性。利用蛋白质组衍生的多肽文库，我们将通过基于质谱学的方法来鉴定不同菱形的剪切键。将对所产生的切割位点进行分析，以便为每个菱形推导出一致的序列。所得到的信息将被用于设计新的报告底物和新型的基于多肽的抑制剂，将分析它们的选择性和它们与菱形结构相互作用的位置。虽然大多数蛋白酶是以非活性酵素的形式合成的，但菱形是以其活性形式转化而来的。有一些迹象表明，膜环境影响底物切割。为了更深入地了解这一点，我们将从不同的原核和真核物种中表达和纯化菱形蛋白，并将它们重组到具有不同脂组成的脂质体中。使用我们最近开发的菱形ABP，我们手头有理想的工具来测量菱形在这些不同环境中的活动状态。总体而言，这项建议将产生关于菱形药物活性调节和底物特异性的有价值的信息，并可能为未来的菱形药物开发奠定基础。

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.