Regulation of the apoptotic machinery by the E3 ubiquitin ligase Parkin
Document TypePhD thesis
Access StatusOpen Access
© 2018 Dr. Jonathan Bernardini
The cyto-protective molecule Parkin is a criticial player in autosomal-recessive Parkinson’s disease, with approximately half of all patients with this condition carrying a mutant allele of PRKN/PARK2. The E3 ubiquitin ligase Parkin acts in concert with its upstream kinase, PINK1, to survey the mitochondrial network and rapidly induce mitophagy in response to damage. Parkin-mediated ubiquitination of mitochondrial substrates leads to the selective tagging and quarantine of damaged mitochondria. The accumulation of ubiquitinated mitochondrial outer membrane proteins allows autophagy receptors to bind and subsequently recruit and extend the autophagophore. This leads to the specific autolysosomal degradation of damaged mitochondria and the maintainence of mitochondrial homeostasis. The motor-deficit associated with Parkinson’s disease is caused by the selective cell death of dopaminergic neurons in the brain and is associated with homozygous loss of function mutations in either PINK1 or Parkin in early onset-disease. However, the link between PINK1/Parkin-mediated mitophagy and cell death remains unclear. Here I present evidence that Parkin modulates intrinsic apoptosis by slowing the kinetics of both BAK- and BAX-mediated cell death. I have characterised BAK as a novel substrate of Parkin and identified K113 as the key site of ubiquitination (Chapter 3). This modification in the hydrophobic groove slows BAK activation, dimerisation and oligomerisation on model membranes and on mitochondria. I hypothesise that BAK inhibition by Parkin stalls mitochondrial outer membrane permeabilisation, allowing for the effective autophagic clearance of damaged mitochondria to prevent errant apoptosis. I also identified a potential new mechanism by which Parkin signalling can inhibit BAX-mediated apoptosis (Chapter 4). The mechanism by which Parkin inhibits BAX is distinct from that of BAK however, as I observed no direct ubiquitination of BAX. Instead, my data suggests that VDAC2, a key BAX interacting protein and predominant Parkin substrate, is the key determinant of Parkin-mediated inhibition of BAX. Furthermore I confirmed the lysine residues in VDAC2 that are required for its ubiquitination during mitophagy and postulate that abrogating VDAC2 ubiquitination will restore BAX-mediated apoptosis during Parkin signalling. Furthermore, this thesis interrogated the role of PINK1/Parkin signalling following apoptotic damage to mitochondria and discusses implications for inflammatory signalling in the context of Parkinson’s disease (Chapter 5). Here I propose a mechanism for Parkin signalling in silencing inflammation caused by persistent apoptotic mitochondria. This provides a link between defective autophagic clearance of damaged mitochondria and the induction of inflammatory signalling commonly observed in Parkinson’s disease. I postulate that mitochondrial dysfunction and BAK/BAX-dependent release of mitochondrial DNA are the key drivers of neuroinflammation in Parkinson’s disease and characterise murine models that will prove invaluable in interrogating this hypothesis. This thesis provides a comprehensive interrogation of the interplay between PINK1/Parkin signalling and the intrinsic apoptotic machinery. Here I describe a multi-faceted coordination of apoptosis and inflammation by Parkin and provide an in depth model for how Parkin acts as a master regulator in the context of Parkinson’s disease.
KeywordsUbiquitin; Parkin; BAK; BAX; Apoptosis; Mitochondria; Mitophagy
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- Medical Biology - Theses