Influence of Familial Parkinson's Disease Mutations on Mitochondrial Localization and Secondary Structure of PINK1

Abstract

Mutations in the PARK6 gene, encoding for the protein PINK1 (Phosphatase-Tensin homologue (PTEN)-induced protein kinase), are associated with early-onset familial Parkinson's disease (PD). PINK1 is found in multiple cellular locations, including the cytosol and both mitochondrial membranes (outer mitochondrial membrane, OMM; and inner mitochondrial membrane, IMM). PINK1 must enter into the IMM in order to be cleaved by PARL (Presenilin-associated rhomboid-like), an IMM protease. Cleavage of PINK1 by PARL activates the degradation of PINK1, indicating a healthy, polarized mitochondrion. Improper processing of PINK1 PD mutants leads to unprocessed PINK1 accumulating on the OMM which induces mitophagy, or the selective autophagy of mitochondria. We hypothesize that accumulation of unprocessed PINK1 on the OMM is either due to improper localization of PINK1 or due to the inability of PARL to cleave the mutated protein. Many traditional protein localization techniques including confocal microscopy cannot distinguish between the inner and outer mitochondrial membrane. Super-resolution microscopy will be used to determine the precise localization patterns to the appropriate mitochondrial membrane of both wildtype PINK1 and PINK1 with point mutations associated to PD. Preliminary confocal microscopy determined that PINK1 constructs are capable of being translated in vivo in HeLa cells. These preliminary results have proved to be promising and further experiments will be conducted using super-resolution microscopy and structured illumination microscopy to determine precise mitochondrial localization patterns in order to determine if PD mutations lead to improper localization of PINK1. Molecular dynamics simulations on PINK1 transmembrane segments harbouring familial Parkinson's disease mutations will be conducted to determine if localization defects are due to structural perturbations. This work sets the stage for an understanding for the role of PINK1 mutations in Parkinson's disease at a molecular level.