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

点击翻译按钮获取中文摘要

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.