Study Rationale:
Recent studies have revealed a role for PINK1 and PARKIN, two genes mutated in early onset Parkinson’s disease, function to promote a cellular process called ubiquitylation of mitochondrial outer membrane proteins. This process promotes natural and healthy degradation of mitochondria. This study will develop a platform for quantification of PINK1-PARKIN activity in cells and in tissues, with the goal of being able to monitor activity in disease tissues.
Hypothesis:
It is possible to use the study of proteins to quantify the activity of the PINK1-PARKIN pathway in tissues
Study Design:
We will develop molecular probes of PARKIN ubiquitylation and will use these for assay development. The assays (experimental set-ups) will be developed using cell lines with activated PARKIN pathways and then extended to tissues.
Impact on Diagnosis/Treatment of Parkinson’s Disease:
Currently, measuring the activity of PINK1-PARKIN pathway on mitochondrial ubiquitylation in tissues is not possible. Thus, our work will provide needed tools to detect the status of the pathway in patient tissues and/or models of Parkinson’s disease.
Next Steps for Development:
It should be possible to develop method protocols that can be extended to other academic research centers.
Final Outcome
Proteins PINK1 and parkin prevent cell death by destroying damaged mitochondria, cell’s energy generators. The goal of this project was to develop a method for quantifying ubiquitylation, the ability of parkin to designate mitochondrial proteins for destruction. To accomplish this goal, we were planning to use proteomics, a set of techniques for a large-scale study of proteins. We created proteomics-based experimental approach that allows us to reliably identify and quantify parkin-dependent ubiquitylation of more than a dozen mitochondrial proteins. Using this system, we closely followed the ubiquitylation process and all its participants simultaneously over time. Other experimental setups we developed allowed us to demonstrate that the entire process is controlled by the PINK1 gene and to better understand the ubiquitylation process in neural cells in vitro.
November 2016
Presentations & Publications
I presented an interim set of data at the Keystone Symposia Ubiquitin Signaling meeting in Whistler British Columbia in March 2016, as well as the Novo Nordisk Cell Signaling meeting in Copenhagen in October 2016 and acknowledged MJFF support.