Physiological role of the intramembrane proteases SPPL2a/b in the homeostasis of tail-anchored proteins

尾锚定(Tail-Anchored，TA)蛋白是一类拓扑学定义的膜蛋白，包括参与囊泡运输的SNARE蛋白、共翻译易位蛋白亚基和泛素蛋白酶体系统的组成部分。虽然已经确定了几种介导TA蛋白生物发生和膜插入的途径，但控制其降解的机制目前尚不清楚。我们推测，这两种膜内蛋白水解酶信号肽2a和b(SPPL2a/b)有助于TA蛋白在分泌晚期和内体/溶酶体途径中的动态平衡。由于TA蛋白的跨膜区呈II型拓扑结构，且C末端较短，因此TA蛋白满足作为这些酶底物的所有基本要求。SPPL2a和SPPL2b是天冬氨酸基膜内酶，与早老素具有同源性，分别存在于溶酶体/晚内体和质膜上。在小型中试筛选中，我们已经获得了初步数据，SNARE蛋白VAMP-2是SPPL2a的底物，并在体内被该酶在内源条件下加工。利用转基因小鼠模型，我们可以证明VAMP-2在心肌细胞中的动态平衡严重依赖于SPPL2a活性。事实上，与野生型小鼠相比，缺乏或过度表达SPPL2a的小鼠表现出高或极低的VAMP-2水平。在这个设计的项目中，我们计划在细胞水平和体内彻底表征SPPL2a介导的VAMP-2的切割。我们的目标是提供洞察力，这种蛋白分解事件发生在哪个细胞室，以及它是如何被调控的。特别是，我们将分析在我们的SPPL2a/b单、双缺陷和SPPL2a过表达的小鼠模型中，除了心肌细胞外，细胞类型和组织利用膜内蛋白分解来维持VAMP-2的动态平衡。此外，我们想要阐明VAMP-2的蛋白降解控制如何影响这种SNARE蛋白调节的下游过程，如胰岛素调节的葡萄糖转运体GLUT4的运输和心肌细胞中心钠素的分泌。由于这两个过程都具有关键的(病理)生理相关性，因此更好地理解它们的调节将是翻译上的兴趣。我们认为，将VAMP-2确定为SPPL2a底物可以证明SPPL2膜内酶可以调节不同的TA蛋白。为了证实这一点，我们计划在SPPL2a和/或SPPL2b基于候选的方法中筛选分泌和内吞晚期途径的广泛的TA蛋白。这将有助于确定这两种蛋白酶对目前知之甚少的细胞生物学问题的贡献，如何在晚期分泌和内吞途径中控制TA蛋白的水平，以及这些蛋白是如何被翻转的。

Tail-anchored (TA) proteins are a topologically defined class of membrane proteins that include SNARE proteins involved in vesicular traffic, subunits of the co-translational translocon and components of the ubiquitin proteasome system. Whereas several pathways mediating biogenesis and membrane insertion of TA proteins have been identified, mechanisms controlling their degradation are currently poorly understood. We hypothesise that the two intramembrane proteases Signal Peptide Peptidase-like 2a and b (SPPL2a/b) contribute to the homeostasis of TA proteins in the late secretory and endosomal/lysosomal pathway. As their transmembrane segment displays type II topology and their C-terminus is short, TA proteins fulfill all basic requirements to be substrates of these proteases. SPPL2a and SPPL2b are aspartyl intramembrane proteases with homology to presenilins that primarily reside in lysosomes/late endosomes and at the plasma membrane, respectively. In a small pilot screen, we have obtained preliminary data that the SNARE protein VAMP-2 is a substrate of SPPL2a and is processed by this protease under endogenous conditions in vivo. Using transgenic mouse models, we could show that the VAMP-2 homeostasis in cardiomyocytes critically depends on SPPL2a activity. In fact, mice that lack or overexpress SPPL2a showed high or very low VAMP-2 levels compared to wild type mice. In the devised project, we plan to thoroughly characterize SPPL2a-mediated cleavage of VAMP-2 at the cellular level and in vivo. We aim to provide insight in which cellular compartment this proteolytic event occurs and how it is regulated. In particular, we will analyse in our SPPL2a/b single- and double-deficient and SPPL2a overexpressing mouse models which cell types and tissues apart from cardiomyocytes utilise intramembrane proteolysis to maintain VAMP-2 homeostasis. Furthermore, we want to elucidate how the proteolytic control of VAMP-2 impacts on downstream processes which are regulated by this SNARE protein like trafficking of the insulin-regulated glucose transporter GLUT4 and the secretion of the Atrial Natriuretic Peptide in cardiomyocytes. Since both processes are of critical (patho-)physiological relevance, a better understanding of their regulation will be of translational interest. We consider the identification of VAMP-2 as SPPL2a substrate as a proof-of-principle that SPPL2 intramembrane proteases can regulate distinct TA proteins. To substantiate this, we plan to screen a broad panel of TA proteins of the late secretory and endocytic pathway in a candidate-based approach for proteolysis by SPPL2a and/or SPPL2b. This will help to define the contribution of these two proteases to the currently poorly understood cell-biological problem how the levels of TA proteins are controlled in the late secretory and endocytic pathway and how these proteins are turned over.