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Rationalizing PINK1 transmembrane mutations in Parkinson's disease

  • Author / Creator
    Takyi, Emmanuella A. F.
  • PINK1 (Phosphatase-Tensin homologue (PTEN)-induced putative kinase 1) is a protein kinase heavily involved in mitochondrial health and integrity. It has multiple domains: an N-terminal mitochondrial targeting sequence, a transmembrane domain, and a kinase domain. Some mutations, located throughout the entire length of the protein, have been identified in persons diagnosed with Parkinson’s disease (PD). The focus of this thesis is the transmembrane domain of PINK1, particularly PD-linked mutations in this region that have been reported to influence PARL-mediated cleavage. PARL (Presenilin-associated rhomboid-like) protease is responsible for the constitutive proteolysis of PINK1 in the inner mitochondrial membrane in healthy mitochondria. This cleavage event is key for PINK1 signalling pathways in the mitochondrion.Cellular studies have shown defects in proteolysis of PINK1 with mutations in its TM region. Furthermore, cellular studies demonstrate redundancy in the cleavage of PINK1 by various mitochondrial proteases. A gap in knowledge exists to precisely define the effect of these mutations by in vitro analysis. The primary goal of this thesis is to develop a protocol for the overexpression of human PINK1, and to assess whether PD-linked mutations in PINK1 affect PARL protease-mediated cleavage.A protocol for the expression and purification of a truncated human PINK1 (residues 70- 134) was developed. PINK1 PD-linked variants were cloned and recombinantly expressed in Escherichia coli. Recombinant human PINK1 was purified from cell lysates via immobilized metal affinity chromatography and size exclusion chromatography. Additionally, human PARL protease was recombinantly expressed in Pichia pastoris and purified resulting in a sufficient yield for downstream applications. Using a robust Fluorescence Resonance Energy Transfer (FRET)-based kinetic assay adapted in the Lemieux lab, we assessed the kinetic activity of PARL protease towards recombinantly expressed PD-linked variants of human PINK1. This work provides insight into the molecular etiology of PINK1 variants associated with PD.

  • Subjects / Keywords
  • Graduation date
    Spring 2019
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/r3-cxa0-ex18
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.