Insights into the catalytic properties of the mitochondrial rhomboid protease PARL

Abstract

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.

Cleavage of PGAM5 by the intramembrane protease PARL is governed by transmembrane helix dynamics and oligomeric state

“…Consistent with this, we now show that the preference of bulky amino acids in the P1 position (Lysyk et al, 2021) only results in modest effects and cleavage rate may be primary governed by TM helix dynamics. For PINK1 conserved helix-destabilizing glycine residues in the C-terminal portion of the TM domain are invariant for PARL-catalyzed cleavage (Meissner et al, 2011).…”

“…Additional to human tissue culture, we examined wt and mutant PGAM5 TM domains in an in vitro cleavage assay based on detergent-solubilized recombinant human PARL (Fig. 1C) (Lysyk et al, 2021). Since it is not known to what extend the amino acid sequence surrounding the scissile peptide bond influences cleavage specificity, we started analyzing the F23A mutant of PGAM5, which removes the bulky amino acid at P1 that had been shown to be favored in a peptide-based multiplex in vitro assay (Lysyk et al, 2021).…”

“…2C and S2C). We speculate that the effect caused by the TM domain mutations is at least partially dependent on the context of the lipid bilayer and consequently any semi-quantitative detergent-based cleavage assay is only suitable to reveal influence of the primary amino acid sequence surrounding the cleavage site (Lysyk et al, 2021).…”

“…Construction of pcDNA3.1-PARL, pcDNA3.1-PGAM5-FLAG, pcDNA3.1-PGAM5 S24F -FLAG and recombinant pET25b(+)-MBP-PGAM5 expression plasmids have been described previously (Lysyk et al, 2021;Meissner et al, 2011;Sekine et al, 2012). Mutations in the TM domain, WDxxWD motif and juxtamembrane region of PGAM5 were introduced by Quik-Change site-directed mutagenesis (Stratagene).…”

“…PARL belongs to the rhomboid intramembrane proteases and was found to cleave PGAM5 in the second half of the TM domain leading to the release of the C-terminal phosphatase domain into the intermembrane space (IMS). Depending on the assay system, PARL cleavage has been mapped between amino acids F23-S24 (Lysyk et al, 2021) or S24-A25 (Sekine et al, 2012), respectively. Recently, PARL-dependent mitochondrial release of PGAM5 that is thought to occur via proteasome-mediated rupture of the outer mitochondrial membrane through Parkin has been shown to trigger Wnt/β-catenin signaling (Bernkopf et al, 2018;Rauschenberger et al, 2017;Yamaguchi et al, 2019).…”