Substrate recognition and cleavage by the mitochondrial rhomboid protease PARL

菱形蛋白是一种普遍保守的丝氨酸蛋白酶，可影响多种重要的细胞过程。虽然对细菌菱形的分析提供了关于同源底物是如何选择的第一次洞察，但对于更远的线粒体菱形蛋白酶parl仍然知之甚少。该项目的中心目标是确定新的PAL底物并破译切割决定因素。这一点特别重要，因为parl具有与细菌中经典的菱形蛋白酶和真核分泌途径相反的活性位点拓扑，表明它已经进化出一种独特的识别和门控机制。先前结合经典遗传学和底物候选测试的工作已经发现了几种可以被PARL切割的线粒体蛋白质，然而，其中一些报告的相关性目前仍在争论中。我们和其他人最近报道，帕金森病相关蛋白激酶PINK1是一种生理性的PAIL底物。有趣的是，我们观察到疾病相关的突变和保守的螺旋不稳定残基的替换干扰了PAL催化的加工。由于相关的特征存在于其他几种膜内蛋白酶底物中，这表明膜内的蛋白分解有共同的原理。在这里，我们建议系统地定义组织培养细胞中PAL的生理底物谱，并在无细胞系统中破译其特异性。具体目标是：1.)生理性PAL底物的系统鉴定2)底物切割要求的表征3。)为了研究PINK1跨膜螺旋的构象动力学，我们计划建立一种洗涤剂增溶和纯化的PARL的体外检测方法，从而能够进行详细的动力学分析，分析单个底物决定因素的作用。用蛋白质组学方法鉴定生理和人工PARL底物的裂解位点，将为确定其裂解位点的一致性和开发预测算法提供有价值的资源。结合在该联盟内进行的生物物理分析，这种增强的序列分析可能会提供对跨膜螺旋动力学如何影响parl催化的切割的理解。

Rhomboids are universally conserved serine proteases that impact on a variety of important cellular processes. While analysis of bacterial rhomboids has provided first insights of how cognate substrates are selected, for the more distant mitochondrial rhomboid protease PARL still only very little is known. Central aims of this project are to identify new PARL substrates and to decipher cleavage determinants. This is of particular relevance since PARL has an active site topology opposite to classical rhomboid proteases in bacteria and the eukaryotic secretory pathway, indicating that it has evolved a unique recognition and gating mechanism. Previous work combining classical genetics and substrate candidate testing has revealed several mitochondrial proteins that can be cleaved by PARL, however, the relevance of a number of these reports are currently still under debate. We and others recently reported that the Parkinsons disease-associated protein kinase PINK1 is a physiological PARL substrat. Intriguingly, we observed that disease-associated mutations and replacement of conserved helix-destabilizing residues interfere with PARL-catalyzed processing. Since related features exist in several other intramembrane protease substrates, this suggests that there are common principles for proteolysis within the membrane. Here we propose to systematically define the physiological substrate spectrum of PARL in tissue culture cells and to decipher its specificity in a cell-free system. Specific goals are: 1.) Systematic identification of physiological PARL substrates 2.) Characterization of requirements of substrate cleavage 3.) Investigation of the conformational dynamics of the PINK1 transmembrane helix To this end, we plan to develop an in vitro assay for detergent-solubilized and purified PARL enabling detailed kinetic analysis analyzing the role of individual substrate determinants. Identification of cleavage sites of physiological as well as artificial PARL substrates by proteomic methods will provide a valuable resource to determine its cleavage site consensus and to develop a prediction algorithm. Combined with biophysical analysis performed within this consortium, this enhanced sequence analysis likely will provide an understanding of how transmembrane helix dynamics impact on PARL-catalyzed cleavage.