Analysis of intramembrane-cleaving proteases and of MEM-superfamily proteases in prokaryotes with Bacillus subtilis as model organism

越来越多的证据表明，跨膜调节蛋白通过膜内切割蛋白酶的蛋白水解切割参与细菌中多种重要的跨膜信号传导过程。这种所谓的“调节性膜内蛋白水解”（RIP）使细菌能够对细胞外信号和压力做出反应，例如致病性。在高等生物中，RIP在基因调控、通讯和分化的细胞过程中起着核心作用。对于这两者，调节RIP中蛋白酶活性的分子机制知之甚少。在我们的工作中，我们将详细研究细菌中跨膜调节蛋白的RIP，以革兰氏阳性模式细菌枯草芽孢杆菌为例。先前，我们已经鉴定并分析了催化特定B RIP的第一（“位点-1”）和第二（“位点-2”）蛋白酶。枯草杆菌转录因子在这里，我们将集中在迄今未知的机制，调节这两种酶的蛋白水解活性的调查。我们的目标是确定激活位点1蛋白酶的分子信号和假定因子。此外，将分析防止通过位点2蛋白酶过早切割的机制。此外，膜内蛋白水解在调节额外的B。将检测枯草杆菌转录因子。最后，我们打算分配功能的其他膜内蛋白酶。总之，我们的工作将有助于揭示RIP中蛋白酶活性调节的一般机制。

There is growing evidence that proteolytic cleavage of membrane spanning regulatory proteins through intramembrane cleaving proteases is involved in a variety of important transmembrane signal-ing processes in bacteria. This so called ‘regulated intramembrane proteolysis’ (RIP) enables bacteria to respond to extracellular signals and stresses, e.g. in pathogenicity. In higher organisms, RIP holds a central role in cellular processes in gene regulation, communication and differentiation. For both, molecular mechanisms that regulate protease activity in RIP are less understood. In our work, we will examine RIP of membrane-spanning regulatory proteins in bacteria at the example of the Gram-positive model bacterium Bacillus subtilis in detail. Previously, we have identified and analyzed the first (‘site-1’) and second (‘site-2’) protease catalyzing RIP of a specific B. subtilis transcription factor. Here, we will focus on the investigation of the so far unknown mechanisms that modulate proteolytic activity of both of these enzymes. We aim to identify the molecular signal(s) and the putative factor(s) that activate the site-1 protease. Also, the mechanism that prevents premature cleavage through the site-2 protease will be analyzed. Furthermore, the role of intramembrane proteolysis in the regulation of additional B. subtilis transcription factors will be examined. Finally, we intend to assign function to other intramembrane proteases. In summary, our work will significantly contribute to unravel the gen-eral mechanisms that regulate activity of proteases involved in RIP.