Doctor of Philosophy
Shaw, Gary S.
The RBR E3 ligase parkin is recruited to the outer mitochondrial membrane (OMM) during oxidative stress where it becomes activated and ubiquitinates numerous proteins. Parkin activation involves binding of a phosphorylated ubiquitin (pUb), followed by phosphorylation of parkin itself, both mediated by the OMM kinase, PINK1. However, targeted mitochondrial proteins have little structural or sequence similarity, with the commonality between substrates being proximity to the OMM. The objective of this thesis was to identify the molecular consequences of parkin phosphorylation, interaction with pUb and how this promotes ubiquitination activity of known Ub-acceptor proteins and parkin itself.
The three-dimensional structure of the isolated pUbl domain revealed structural changes that alter the surfaces charges to disrupt the autoinhibitory interaction. In the presence of pUb, this releases pUbl and promotes the active form of parkin. Parkinson’s disease related substitutions throughout the Ubl domain reduce the rate of phosphorylation by PINK1, destabilize the domain and alter autoubiquitination.
Also shown is a model of activated parkin bound to pUb and an E2-Ub conjugate established from NMR chemical shift perturbations. The RING0/Rcat interface is distant from the E2-Ub binding site but it remodelled to increase exposure to the catalytic cysteine. These experiments provide evidence that parkin phosphorylation and E2 Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the Rcat domain to drive parkin activity.
Finally, parkin was shown to efficiently ubiquitinate acceptor proteins pre-ligated to pUb and phosphorylation of parkin triggers autoubiquitination activity. Parkin phosphorylation is not required for acceptor-pUb ubiquitination. In fact, only phospho-parkin induced self-ubiquitination and deletion of Ubl or mutation at K211N inhibited self-ubiquitination. We propose divergent parkin mechanisms whereby parkin-mediated ubiquitination of acceptor proteins is driven by binding to pre-existing pUb and subsequent parkin phosphorylation triggers autoubiquitination. This finding is critical for understanding parkin’s role in mitochondrial homeostasis and has implications on targets for therapeutics.
Summary for Lay Audience
Parkinson’s disease (PD) is a devastating neurodegenerative disorder that affects more than 6 million people around the world. The typical “shaking disorder” symptoms arise as brain cells that create dopamine, an important signalling molecule for coordination and movement, are damaged and die. In the majority of cases, PD development is sporadic and damage occurs with age-related to environmental factors. In approximately 10% of cases, PD is genetically inherited through mutations in several genes and typically causes much earlier development of symptoms (under 40 years of age). The gene which encodes the enzyme Parkin is mutated in over 50% of familial PD patients.
Parkin exhibits protective properties by tagging proteins on damaged mitochondria with a small molecule called ubiquitin and this event signals for mitochondrial removal. When mutated, parkin does not efficiently transfer ubiquitin for this signal, leading to increased cellular exposure to damaging agents normally contained within mitochondria. Using biophysical techniques and functional assays, this thesis investigates the three-dimensional protein structure of parkin and the molecular details of ubiquitin transfer. Under normal conditions, parkin exists in a state that is inactive. Gradual activation occurs in two stages, both driven by another PD associated gene product, PINK1. In the first stage, parkin is recruited to a protein pre-labeled with a modified ubiquitin molecule and this enables parkin to transfer additional ubiquitins onto that protein. In the second stage, parkin itself becomes modified and further activated. This leads to additional ubiquitin ligation and is the likely signal for initiating mitochondrial degradation. Known PD mutations have various affects in all aspects of parkin function from activation to ubiquitin transfer. Dopamine producing brain cells are particularly sensitive to mitochondrial damage. Investigation into parkin’s native structure and function is important for understanding the defects that lead to PD and for influencing potential therapeutics.
Dunkerley, Karen, "New Perspectives on Phosphorylation State in the Parkin Ubiquitination Cascade" (2021). Electronic Thesis and Dissertation Repository. 7861.
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Available for download on Thursday, June 29, 2023