Structural and mechanistic basis of Parl activity and regulation.

The mitochondrial rhomboid protease Parl governs apoptosis, morphology, metabolism and might be implicated in Parkinson's disease, but the structural basis of its activity and complex regulation remain unknown. We report the discovery of γ-cleavage, a proteolytic event on the loop connecting the first transmembrane helix (TMH) of Parl to the 6-TMH catalytic rhomboid domain of the protease. This cleavage disrupts the '1+6' structure that defines every mitochondrial rhomboid and generates a new form of Parl, PROD (Parl-rhomboid-domain). Structure-function analysis of Parl suggests that γ-cleavage could be implicated in eliminating Parl proteolytic activity, and structural modeling of PROD reveals structural conservation with the bacterial rhomboid GlpG. However, unlike bacterial rhomboids, which employ a diad-based mechanism of catalysis, Parl appears to use a conserved mitochondrial rhomboid-specific Asp residue on TMH-5 in a triad-based mechanism of catalysis. This work provides unexpected insights into the structural determinants regulating Parl stability and activity in vivo, and reveals a complex cascade of proteolytic events controlling the function of the protease in the mitochondrion.

Hax1 lacks BH modules and is peripherally associated to heavy membranes: implications for Omi/HtrA2 and PARL activity in the regulation of mitochondrial stress and apoptosis.

Hax1 has an important role in immunodeficiency syndromes and apoptosis. A recent report (Chao et al., Nature, 2008) proposed that the Bcl-2-family-related protein, Hax1, suppresses apoptosis in lymphocytes and neurons through a mechanism that involves its association to the inner mitochondrial membrane rhomboid protease PARL, to proteolytically activate the serine protease Omi/HtrA2 and eliminate active Bax. This model implies that the control of cell-type sensitivity to pro-apoptotic stimuli is governed by the PARL/Hax1 complex in the mitochondria intermembrane space and, more generally, that Bcl-2-family-related proteins can control mitochondrial outer-membrane permeabilization from inside the mitochondrion. Further, it defines a novel, anti-apoptotic Opa1-independent pathway for PARL. In this study, we present evidence that, in vivo, the activity of Hax1 cannot be mechanistically coupled to PARL because the two proteins are confined in distinct cellular compartments and their interaction in vitro is an artifact. We also show by sequence analysis and secondary structure prediction that Hax1 is extremely unlikely to be a Bcl-2-family-related protein because it lacks Bcl-2 homology modules. These results indicate a different function and mechanism of Hax1 in apoptosis and re-opens the question of whether mammalian PARL, in addition to apoptosis, regulates mitochondrial stress response through Omi/HtrA2 processing.