Mechanistic characterization of substrate recognition by gamma-secretase

该项目测试了膜内蛋白酶γ -分泌酶存在一类新的非常规底物的假设。这些新的底物将用于定义尚未理解的分子特征，使膜蛋白成为分泌酶的底物。识别这些特征是研究小组的核心相关性，将使我们能够了解膜内蛋白酶不同家族之间在底物识别和切割方面的相似性，以及差异。我们发现了γ -分泌酶的一个意想不到的新功能，它与菱形膜内蛋白酶家族共享，即全长膜蛋白的直接脱落，而不需要通过脱落预先截断底物的外畴。我们已经确定了B细胞成熟抗原（BCMA）作为一种新的底物。分泌酶的裂解导致BCMA外结构域的分泌和BCMA受体功能的调节。与已知的γ -分泌酶底物相比，BCMA具有短的外畴，但我们的数据表明，BCMA序列中必须存在额外的基序，这使得BCMA可以被γ -分泌酶直接切割。从生物信息学的角度来看，我们已经鉴定出100种具有短外结构域的膜蛋白，这表明它们中的一些与BCMA一起形成了一种新的、非常规的γ -分泌酶底物，而其他的则是非底物，不会被γ -分泌酶切割。鉴定这两组蛋白（底物和非底物）对于理解除外结构域长度外哪些特征将膜蛋白定义为γ -分泌酶底物至关重要。这个项目有三个部分。1. 首先，我们将使用无偏蛋白质组学方法和候选方法系统地确定100种具有短外结构域的膜蛋白中哪些是新的底物，哪些是γ -分泌酶的非底物。2. 其次，我们将通过结合细胞测定和体外γ分泌酶测定来验证选定的γ分泌酶底物和非底物。3. 第三，我们将使用结构域交换实验和机制分析-分泌酶底物和非底物的分子结构。该项目采用了最先进的技术，并与该财团的其他合作伙伴密切合作。作为附加价值，该项目对更全面地破译γ -分泌酶的生理功能具有直接意义，这对开发针对癌症和神经退行性疾病的γ -分泌酶的药物也很重要。

This project tests the hypothesis that a new class of unconventional substrates exists for the intramembrane protease gamma-secretase. These new substrates will be used to define the as yet non-understood molecular features, which make a membrane protein a substrate for gamma-secretase. Identifying these features is of central relevance for the research group and will allow us to understand the similarities, but also the differences in substrate recognition and cleavage between the different families of intramembrane proteases. We have discovered an unexpected, novel function for gamma-secretase which is shared with the rhomboid intramembrane protease family, namely the direct shedding of a full-length membrane protein without a prior truncation of the substrates' ectodomain by shedding. We have identified the B cell maturation antigen (BCMA) as a novel substrate. gamma-secretase cleavage results in secretion of the BCMA ectodomain and modulation of BCMA receptor function. In contrast to known gamma-secretase substrates BCMA has a short ectodomain, but our data suggest that additional motifs must exist in the BCMA sequence, which allow BCMA to be directly cleaved by gamma-secretase. Bioinformatically, we have identified 100 additional membrane proteins with a short ectodomain, suggesting that several of them - together with BCMA - form a new, unconventional class of gamma-secretase substrates, while others will be non-substrates and not be cleaved by gamma-secretase. Identification of both sets of proteins (substrates and non-substrates) is essential to understand which features besides the ectodomain length define a membrane protein as a gamma-secretase substrate. The project has three parts. 1. First, we will use an unbiased proteomic approach and a candidate approach to systematically determine which of the 100 membrane proteins with short ectodomains are new substrates, and which are non-substrates for gamma-secretase. 2. Second, we will validate selected gamma-secretase substrates and non-substrates by combining cellular assays and in vitro gamma-secretase assays. 3. Third, we will use domain swap experiments and mechanistically analyze the molecular architecture of gamma-secretase substrates and non-substrates. The project uses state-of-the-art technologies and a close collaboration with other partners in this consortium. As an added value this project has direct implications for deciphering more comprehensively the physiological functions of gamma-secretase, which is also important for drug development targeting gamma-secretase in cancer and neurodegeneration.