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

膜内蛋白酶具有在细胞膜脂质双层内切割肽键的不寻常性质，这种环境显然不适合需要水的水解反应。这些不寻常的酶包括S2P、菱形酶、RCE1、早老素/γ-分泌酶和信号肽肽酶（SPP）及SPP样蛋白酶。膜内蛋白水解最初被确定为其在选择性释放生物活性肽中的共同作用，但我们假设这些进化广泛的蛋白酶主要参与控制膜蛋白稳态。作为这一观点的概念证明，我们最近发现酵母SPP同源物Ypf1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命名为erd - r （ER相关降解调节）。与这种功能一致，我们和其他人表明，人类SPP在功能上与ERAD因子Derlin1和E3泛素连接酶TRC8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Ypf1的相似之处表明了一种进化保守的机制。然而，SPP在ERAD中的分子功能及其在人体中的生理作用仍不明确。我们最近未发表的研究表明，人类SPP控制角鲨烯合成酶FDFT1的丰度，FDFT1是胆固醇合成和类异戊二烯途径非固醇分支之间的关键分支点。本研究拟采用定量蛋白质组学的方法系统定义组织培养细胞中SPP的生理底物谱，并分析erder - r调控蛋白丰度的分子机制。具体目标有：1)生理SPP底物的系统鉴定研究spp介导的erd - r 3的功能。为此，我们的目标是在已建立的基于细胞的分析中研究人类SPP，并在体外重建SPP ERAD复合物。综上所述，该项目将为真核细胞中蛋白质稳态控制开辟一个新的视角，在从脂质生物合成调节到多聚膜蛋白丰度控制的各种基本细胞过程中具有重要意义。

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 Our recent unpublished work demonstrates that human SPP controls the abundance of the squalene synthase FDFT1 that serves as a key branching point between cholesterol synthesis and a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.