Insights into the catalytic properties of the mitochondrial rhomboid protease PARL

Area of Science:

• Mitochondrial biology and biochemistry
• Protease function and regulation
• Lipid-protein interactions

Background:

• The rhomboid protease PARL regulates mitochondrial homeostasis by cleaving key substrates like PINK1, PGAM5, and Smac/Diablo.
• PARL's in vitro catalytic properties and the influence of lipids on its activity remain poorly understood.

Purpose of the Study:

• To characterize the in vitro catalytic properties of human PARL.
• To investigate the effect of lipids, particularly cardiolipin, on PARL activity.
• To determine PARL's substrate specificity and compare the activity of full-length and truncated PARL forms.

Main Methods:

• Isolation of recombinant human PARL expressed in yeast.
• Utilized Förster resonance energy transfer (FRET)-based kinetic assays to measure proteolytic activity.
• Reconstituted PARL in proteoliposomes to mimic the mitochondrial environment.
• Employed multiplex peptide profiling to analyze substrate preferences.

Main Results:

• PARL activity in detergent is enhanced by cardiolipin, a mitochondrial inner membrane lipid.
• PARL reconstituted in proteoliposomes exhibits significantly higher turnover rates, with Smac/Diablo cleaved at 1 min^-1.
• PGAM5 is cleaved with the highest catalytic efficiency (k_cat/K_M) among tested substrates.
• A truncated β-cleavage form of PARL shows increased activity against substrates compared to full-length PARL.
• PARL prefers substrates with a bulky P1 residue (e.g., Phe), differing from bacterial rhomboid proteases.

Conclusions:

• This study provides the first in vitro characterization of PARL's catalytic activity and lipid dependence.
• Findings reveal distinct substrate preferences and highlight the enhanced activity of a truncated PARL form.
• Results offer fundamental insights into PARL's role in mitochondrial function and inform the design of PARL-specific inhibitors.