Mutations in vacuolar protein sorting 35 ortholog (VPS35), a gene that encodes a core component of the retromer complex, have recently been shown to cause late-onset, autosomal dominant familial Parkinson's disease (PD). The molecular mechanism(s) by which VPS35 mutations induce progressive neurodegeneration in PD is not yet known. The work described in this dissertation aimed to characterize a novel interaction between VPS35 and the PD-associated protein parkin and to determine the molecular mechanisms of mutant VPS35-induced neurodegeneration by defining and characterizing the differential interactome of mutant and WT VPS35. First, we demonstrate the selective interaction of parkin with VPS35 that facilitates the robust atypical poly-ubiquitination of VPS35 by parkin. Parkin-mediated ubiquitination does not affect the steady-state level or turnover of VPS35 in cell culture models or conditional and germline parkin knockout mice. We show that loss of parkin induces a decrease in the levels of WASH1, FAM21 and ATG9A, as well as disrupting the sorting of ATG9A. We next took an unbiased approach to look at differential protein interactors of VPS35 variants in cellular-based and in vivo-based models. Our cellular-based approaches included both a yeast-two-hybrid (Y2H) and tandem affinity purification screen (TAP). Our Y2H screen identified two novel protein interactors of VPS35, while our TAP screen, even after several optimization attempts, was unable to identify novel or differential interactors of VPS35. Finally, we performed co-immunoprecipitation from the brain of a newly developed D620N VPS35 knockin mouse model to identify TBC1D5 as a differential interacting protein of VPS35 variants, which was validated in mammalian cell culture. Taken together, our data supports a role for the interactions of parkin and TBC1D5 with VPS35 in the endolysosomal system and VPS35-induced neurodegeneration.
|Commitee:||Ma, Jiyan, Sempere, Lorenzo, West, Andrew|
|School:||Van Andel Research Institute|
|School Location:||United States -- Michigan|
|Source:||DAI-B 80/09(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Neurosciences, Cellular biology, Physiology, Epidemiology|
|Keywords:||Differential interacting protein, Parkin-mediated ubiquitination, Progressive neurodegeneration|
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