Substrate recognition and cleavage by the mitochondrial rhomboid protease PARL

菱形体是普遍保守的丝氨酸蛋白酶，影响多种重要的细胞过程。虽然对细菌菱形体的分析首次揭示了同源底物是如何被选择的，但对于更远的线粒体菱形蛋白酶PARL，我们所知甚少。该项目的中心目标是鉴定新的PARL底物并破译裂解决定因素。这是特别相关的，因为PARL具有与细菌和真核生物分泌途径中的经典菱形蛋白酶相反的活性位点拓扑结构，表明它已经进化出独特的识别和门控机制。先前结合经典遗传学和候选底物测试的工作已经揭示了几种可以被PARL切割的线粒体蛋白，然而，这些报道的相关性目前仍在争论中。我们和其他人最近报道了帕金森病相关蛋白激酶PINK1是一种生理PARL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parl催化的加工。由于其他几种膜内蛋白酶底物也存在相关特征，这表明膜内蛋白水解存在共同的原则。在这里，我们建议系统地定义组织培养细胞中PARL的生理底物谱，并在无细胞系统中解读其特异性。具体目标有：1)生理PARL底物的系统鉴定底物裂解要求的表征PINK1跨膜螺旋构象动力学的研究为此，我们计划开发一种洗涤剂溶解和纯化PARL的体外测定方法，以便对单个底物决定因素的作用进行详细的动力学分析。利用蛋白质组学方法鉴定生理和人工PARL底物的切割位点将为确定其切割位点共识和开发预测算法提供有价值的资源。结合该联盟进行的生物物理分析，这种增强的序列分析可能会提供跨膜螺旋动力学如何影响parl催化裂解的理解。

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.