Mechanisms and Function of Intramembrane Proteolysis by the gramme-Secretase homologous Signal Peptide Peptidase-like Proteases (SPPL)

膜内蛋白水解被认为是生物化学上不可能的，因为每种蛋白酶都需要水来发挥其催化活性。然而，在研究阿尔茨海默病相关淀粉样蛋白β-肽的蛋白水解产生时，很明显β-分泌酶确实可以在生理条件下在疏水环境中在膜的中间切割。然而，β分泌酶专门切割1型跨膜结构域蛋白，信号肽肽酶（SPP）仅接受2型定向蛋白作为底物。数据库分析导致SPP样蛋白家族（SPPL 2a、B、c和SPPL 3）的鉴定，其功能迄今为止完全未知。最近，我们能够证明SPPLs，像<$-分泌酶，属于GxGD型-乙酰基蛋白酶，我们最初将其定义为一类新的膜内切割蛋白酶。我们现在有兴趣了解SPPL的功能以及它们能够在膜内切割的机制。我们将鉴定其底物，并随后研究体内和体外底物加工，以确定切割位点、切割位点要求和推定的与β-分泌酶的机制相似性。将通过在重组切割产物表达后在动物模型（斑马鱼）中拯救敲低表型来验证底物。我们的研究结果将有助于理解GxGD蛋白酶的膜内蛋白水解的新方案，这似乎在关键的生理和病理过程中非常重要，如阿尔茨海默病，胆固醇代谢，Notch信号，信号肽的去除，免疫监视和丙型肝炎病毒核心蛋白的加工。

Intramembrane proteolysis was thought to be biochemically impossible, because each protease requires water for its catalytic activity. However, upon investigation of the proteolytic generation of the Alzheimer¿s disease associated Amyloid ß-peptide it became clear that ¿-secretase can indeed cut in the middle of the membrane in a hydrophobic environment under physiological conditions. Whereas ¿-secretase exclusively cuts type 1 trans-membrane domain proteins, signal peptide peptidase (SPP) only accepts type 2 oriented proteins as substrates. Data bank analysis lead to the identification of a family of SPP-like proteins (SPPL2a,b,c and SPPL3), whose function is until now completely unknown. Very recently we were able to demonstrate that SPPLs, like ¿-secretase, belong to the GxGD type-aspartyl proteases, which we originally defined as a new class of intramembrane cleaving proteases. We are now interested to understand the function of the SPPLs and the mechanisms by which they are able to cleave within the membrane. We will identify their substrates and will subsequently investigate substrate processing in vivo and in vitro to determine the cleavage sites, cleavage site requirements and a putative mechanistic analogy to ¿-secretase. Substrates will be verified by rescuing knock down phenotypes in an animal model (zebrafish) upon expression of the recombinant cleavage products. Our findings will contribute to the understanding of the novel scheme of intramembrane proteolysis by GxGD proteases, which appears to be of great importance in pivotal physiological and pathological processes, such as Alzheimer¿s disease, Cholesterol metabolism, Notch-signaling, removal of signal peptides, immune surveillance, and processing of the Hepatitis C viral core protein.