Insights into the catalytic properties of the mitochondrial rhomboid protease PARL

J Biol Chem. Jan-Jun 2021;296:100383. doi: 10.1016/j.jbc.2021.100383. Epub 2021 Feb 6.

Abstract

The rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its cleavage of substrates such as PINK1, PGAM5, and Smac/Diablo, which have crucial roles in mitochondrial quality control and apoptosis. However, the catalytic properties of PARL, including the effect of lipids on the protease, have never been characterized in vitro. To address this, we isolated human PARL expressed in yeast and used FRET-based kinetic assays to measure proteolytic activity in vitro. We show that PARL activity in detergent is enhanced by cardiolipin, a lipid enriched in the mitochondrial inner membrane. Significantly higher turnover rates were observed for PARL reconstituted in proteoliposomes, with Smac/Diablo being cleaved most rapidly at a rate of 1 min-1. In contrast, PGAM5 is cleaved with the highest efficiency (kcat/KM) compared with PINK1 and Smac/Diablo. In proteoliposomes, a truncated β-cleavage form of PARL, a physiological form known to affect mitochondrial fragmentation, is more active than the full-length enzyme for hydrolysis of PINK1, PGAM5, and Smac/Diablo. Multiplex profiling of 228 peptides reveals that PARL prefers substrates with a bulky side chain such as Phe in P1, which is distinct from the preference for 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 that enhance our understanding of its role in mitochondrial function and has implications for specific inhibitor design.

Keywords: GlpG; PGAM5; PINK1; Smac/Diablo; intramembrane proteolysis; membrane protease; mitochondria; rhomboid protease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis Regulatory Proteins / metabolism
  • Catalytic Domain
  • Endopeptidases / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Metalloproteases / genetics
  • Metalloproteases / metabolism*
  • Metalloproteases / physiology*
  • Mitochondria / metabolism
  • Mitochondrial Membranes / metabolism
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism*
  • Mitochondrial Proteins / physiology*
  • Peptide Hydrolases / metabolism
  • Protein Kinases / genetics
  • Protein Kinases / metabolism
  • Proteolysis

Substances

  • Apoptosis Regulatory Proteins
  • Mitochondrial Proteins
  • Protein Kinases
  • PTEN-induced putative kinase
  • Endopeptidases
  • Metalloproteases
  • Peptide Hydrolases
  • PARL protein, human