The rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its cleavage of substrates such as PINK1, PGAM5, and Smac, which have crucial roles in mitochondrial quality control and apoptosis. To gain insight into the catalytic properties of the PARL protease, we expressed human PARL in yeast and used FRET-based kinetic assays to measure proteolytic activity in vitro. We show PARL activity in detergent is enhanced by cardiolipin. Significantly higher turnover rates are observed for PARL reconstituted in proteoliposomes, with Smac being cleaved most rapidly at a rate of 1 min-1. PGAM5 is cleaved with the highest efficiency compared to PINK1 and Smac. In proteoliposomes, a truncated β-cleavage form of PARL is more active than the full-length enzyme for hydrolysis of PINK1, PGAM5 and Smac. Multiplex substrate profiling reveals a substrate preference for PARL with a bulky side chain Phe in P1, which is distinct from small side chain residues typically found with bacterial rhomboid proteases. This study using recombinant PARL provides fundamental insights into its catalytic activity and substrate preferences.

中文翻译：

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洞察线粒体菱形蛋白酶PARL的催化性能

菱形蛋白酶PARL通过裂解诸如PINK1，PGAM5和Smac之类的底物，是线粒体稳态的关键调节剂，而底物在线粒体质量控制和细胞凋亡中具有至关重要的作用。为了深入了解PARL蛋白酶的催化特性，我们在酵母中表达了人PARL，并使用了基于FRET的动力学分析来测量体外蛋白水解活性。我们显示清洁剂中的PARL活性被心磷脂增强。在蛋白脂质体中重组的PARL观察到明显更高的周转率，其中Smac以1 min-1的速度被最快地裂解。与PINK1和Smac相比，PGAM5的切割效率最高。在蛋白脂质体中，PARL的截短β切割形式比全长酶对PINK1，PGAM5和Smac的水解更有活性。多重底物谱分析揭示了底物偏爱P1中具有庞大侧链Phe的PARL，这与细菌菱形蛋白酶通常发现的小侧链残基不同。这项使用重组PARL进行的研究提供了对其催化活性和底物偏好的基本了解。