Objective/Rationale:
The PARK2 (Parkin) gene is frequently mutated in Parkinson’s disease. Several lines of evidence indicate that Parkin functions to catalyze the conjugation of the small protein ubiquitin to cellular proteins. However, endogenous substrates of Parkin and their contribution to Parkinson’s disease is poorly understood. This project seeks to employ newly developed technology to address this important question.
Project Description:
We have developed “diGly capture” methodology, which allows the identification of sites of modification of proteins with ubiquitin using mass spectrometry, and have used this approach to provide a global view of the ubiquitin modified proteome in human cells. The attachment of ubiquitin to proteins can have several outcomes, including turnover by the proteasome and recruitment of ubiquitin-binding co-factors. In the context of Parkin, it is thought that its action leads to ubiquitination of proteins on the surface of mitochondria that facilitate engulfment of damaged mitochondria by autophagosomes. We will use the diGly capture approach to elucidate the proteins that are ubiquitinated in a Parkin-dependent manner upon damage of mitochondria. Target proteins will be assessed for their function in recruitment of mitochondria to autophagosomes.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
The identification of specific Parkin substrates, and more importantly, the sites of ubiquitin modification in these proteins will facilitate the development of novel reagents that can be used to examine the extent of Parkin defects in patient material, and may also suggest approaches to by-passing the need for Parkin to maintain mitochondrial homeostasis.
Anticipated Outcome:
We anticipate identifying new substrates of Parkin and candidates for involvement in mitophagy. In the long term, functional experients may result in the identification of proteins that serve specific signaling functions in terms of recruitment of mitochondria to autophagosomes. We will develop a web-based resource for researchers in the Parkin field to aid in the functional analysis of the Parkin pathway.
Progress Report
Final Outcome
The goal of this work was to identify novel Parkin substrations utilizing a quantitative proteomic methodology. We employed a new quantitative diGLY profiling approach to profile the ubiquitination targets of PARKIN (PARK2), an E3 ubiquitin ligase that is mutated in Parkinson’s Disease. Operationally, we employed mitochondrial depolarization as the trigger to activate PARKIN in a PINK1 dependent manner. Using the approach in two different cell types (HCT116 and SH-SY5Y neuronal cells), we identified several hundred ubiquitination sites whose abundance is increased by at least 2-fold in dozens of proteins, a substantial fraction of which are located on the surface of mitochondria. In additional profiling experiments, we demonstrated the dependence of many of these ubiquitination events on the presence of PINK1 and PARKIN, and that a subset of candidate substrates physically associate with PARKIN. The ubiquitination of many sites increased by 30-60 fold upon mitochondrial depolarization. These studies suggest that activation of PARKIN leads to wide-spread ubiquitination of mitochondria, which promotes ensuing engulfment of damaged mitochondria by autophagosomes. These studies provide a resource for elucidating how PARKIN alters the structure and function of the mitochondrial proteome, thereby controlling mitochondrial homeostasis.
Presentations & Publications
Manuscript in preparation
Sarraf S, Kim W, Sowa M, Gygi SP, and Harper JW (2012) Global PARKIN substrate identification through quantitative diGLY profiling. In preparation
November 2012