Function and molecular mechanism of signal peptide peptidase (SPP) in regulated membrane protein abundance control

信号肽肽酶（SPP）在调节膜蛋白丰度控制中的功能和分子机制

• 批准号: 407070872
• 负责人: Professor Dr. Marius Lemberg
• 金额: --
• 依托单位: Zentrum für Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/407070872?language=en
• 关键词: Function; molecular; mechanism; signal; peptide

Intramembrane proteases have the unusual property of cleaving peptide bonds within the lipid bilayer of cellular membranes, an environment not obviously suited to a water-requiring hydrolysis reaction. These unusual enzymes include S2P, rhomboids, RCE1, presenilin/γ-secretase and signal peptide peptidase (SPP) and SPP-like proteases. Intramembrane proteolysis was initially identified with its common role in the selective release of bioactive peptides from the membrane, but we hypothesize that these evolutionary widespread proteases are primarily involved in the control of membrane protein homeostasis. As a proof of concept for this idea, we have recently showed that the yeast SPP orthologue Ypf1 regulates protein turnover at the Endoplasmic Reticulum (ER). We have termed this pathway, which controls the levels of nutrient transporters according to cellular needs, ERAD-R (ER Associated Degradation-Regulatory). Consistent with such a function, we and others showed that human SPP functionally interacts with the ERAD factor Derlin1 and the E3 ubiquitin ligase TRC8 forming a 500-kDa complex that cleaves certain tail-anchored and type II membrane proteins: The parallels to Ypf1 suggest an evolutionarily conserved mechanism. However, the molecular function of SPP in ERAD and its physiological role in humans are still ill defined. Our recent unpublished work demonstrates that human SPP controls the abundance of the squalene synthase FDFT1 that serves as a key branching point between cholesterol synthesis and a non-sterol branch of the isoprenoid pathway. Here, we propose a study to systematically define the physiological substrate spectrum of SPP in tissue culture cells by quantitative proteomics and to analyze the molecular mechanism of ERAD-R in regulated protein abundance control. Specific goals are: 1.) Systematic identification of physiological SPP substrates 2.) Studying the function of SPP-mediated ERAD-R 3.) Deciphering regulatory principles and mechanismsTo this end, we aim to study the human SPP in established cell-based assays and to reconstitute the SPP ERAD complex in vitro. Taken together, this project will open a new view on protein homeostasis control in eukaryotic cells, with important implications in a broad variety of essential cellular processes ranging from regulation of lipid biosynthesis to abundance control of polytopic membrane proteins.

膜内蛋白酶具有切割细胞膜脂双层内的肽键的不寻常性质，这种环境显然不适合需要水的水解反应。这些不常见的酶包括S2 P、菱形蛋白酶、RCE 1、早老素/γ-分泌酶和信号肽肽酶（SPP）以及SPP样蛋白酶。膜内蛋白水解最初被确定其共同的作用，从膜上的生物活性肽的选择性释放，但我们假设，这些进化广泛的蛋白酶主要参与膜蛋白稳态的控制。作为这一想法的概念证明，我们最近表明，酵母SPP直向同源物Ypf 1调节内质网（ER）的蛋白质周转。我们将这种根据细胞需要控制营养转运蛋白水平的途径称为ERAD-R（ER相关降解调节）。与这样的功能一致，我们和其他人表明，人类SPP与ERAD因子Derlin 1和E3泛素连接酶TRC 8在功能上相互作用，形成一个500 kDa的复合物，切割某些尾锚定和II型膜蛋白：与Ypf 1的相似之处表明了一种进化上保守的机制。然而，SPP在ERAD中的分子功能及其在人类中的生理作用仍然不清楚。我们最近未发表的工作表明，人SPP控制角鲨烯合酶FDFT 1的丰度，FDFT 1作为胆固醇合成和类异戊二烯途径的非固醇分支之间的关键分支点。在这里，我们提出了一项研究，系统地确定SPP在组织培养细胞中的生理底物谱的定量蛋白质组学和分析ERAD-R在调节蛋白质丰度控制的分子机制。具体目标是：（1）。生理SPP底物的系统鉴定2.）研究SPP介导的ERAD-R3的功能解密调控原理和机制为此，我们的目标是在建立的基于细胞的测定中研究人SPP，并在体外重建SPP ERAD复合物。两者合计，该项目将打开一个新的观点在真核细胞中的蛋白质稳态控制，具有重要的影响，在各种各样的基本细胞过程，从脂质生物合成的调节丰度控制的多位膜蛋白。