Substrate recognition and cleavage by the mitochondrial rhomboid protease PARL

• Applicant: Professor Dr. Marius Lemberg
• Subject Area: Biochemistry
• Term: from 2015 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 263531414

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.

• DFG Programme: Research Units
• Subproject of: FOR 2290: Understanding Intramembrane Proteolysis