| Popis: |
Proteases have evolved in all kingdoms of life with the capability to catalyze irreversible and highly regulated hydrolysis of peptide bonds. Intramembrane proteases share common features as such enzymes are polytopic membrane proteins with their active sites buried several Ångstrom deep within the lipid bilayer. Although the list of physiological substrates is steadily growing, an important remaining question is whether these proteases have a conserved substrate recognition mechanism. In the present thesis, I focused on the human mitochondrial rhomboid protease PARL and its substrate recognition and cleavage mechanism on the example of PGAM5. Dysfunctional mitochondrial quality control disturbs cellular energy metabolism and programmed cell death, triggering disruptive diseases such like neurodegeneration. Genetic deficiency of PGAM5 causes a Parkinson’s-like movement disorder in mice. PARL serves as a safeguard of mitochondrial homeostasis and is processing PGAM5 when the mitochondrial membrane potential is disrupted. Until today, PGAM5 substrate determinants have not been rigorously investigated. Here, I characterize for the first time several cleavage determinants in PGAM5 on basis of mutational studies in human tissue culture, in vitro proteolytic assays with purified recombinant proteins and in a collaborative project using CD spectroscopy and liquid-state NMR. I can show that the N-terminal portion of the PGAM5 TM domain plays a special role and is a critical determinant for PARL-catalyzed processing. Interestingly, besides cleavage resistant forms, I obtained PGAM5 mutants with highly increased cleavage by PARL uncoupling it from its native regulation. NMR analysis revealed that the PGAM5 TM domain harbors two split helices zoned by a hinge-like loop and mutations within the N- or C-terminal helix suggest an altered interaction with PARL or bending into the PARL active site with subsequent modified intramembrane cleavage. Moreover, I found that a balanced net charge in the C-terminal juxtamembrane region prevents premature PGAM5 from PARL-catalyzed cleavage so that cleavage-resistant PGAM5 oligomers can assemble upon mitochondrial import. Under mitochondrial stress after disruption of the membrane potential with CCCP, I propose a model in which PGAM5 oligomers at the inner mitochondrial membrane disassemble into monomers by an unknown mechanism leading to efficient cleavage by PARL in order to trigger PGAM5’s downstream activities. Taken together, my findings indicate that the substrate recognition mechanism of PARL relies on a membrane-potential-dependent oligomeric switch and different substrate features with hierarchical importance. |
