Study Rationale:
Failure in the quality control of mitochondria (power houses of the cell) in neurons may contribute to Parkinson's disease (PD). Parkin is an enzyme that tags proteins from damaged mitochondria with ubiquitin (a small protein) to promote their degradation (break down). Mutations in Parkin contribute to some forms of PD. Parkin adopts an inactive conformation (structure) in the context of healthy mitochondria. Many studies have now revealed that, in the presence of damaged mitochondria, Parkin becomes activated by binding phosphorylated ubiquitin and by phosphorylation (addition of molecules to a protein) of the kinase PINK1. Activation of Parkin ubiquitin ligase activity results from a three-dimensional (3D) rearrangement within the Parkin protein. The Parkin conformation necessary to label dysfunctional mitochondria is currently unknown.
Hypothesis:
This project aims to determine, at the atomic level, 3D conformations of activated Parkin to gain insight into the mechanism of ubiquitin transfer.
Study Design:
We plan to generate crystals of different activated forms of Parkin and use X-ray crystallography (a technique used to study the atomic and molecular structure of crystals) to determine their atomic structure. We also aim to determine the structure of the first step of Parkin ubiquitination activity (e.g., the structure of phosphorylated Parkin binding to the ubiquitin-conjugating enzyme E2). To maximize the presence of the two enzymes in the crystal, we will generate fusions of Parkin and E2 proteins. We also will determine the structure of phosphorylated Parkin with an ubiquitin molecule attached to its active cysteine (a building block of proteins), ready for the ubiquitin transfer from Parkin to its substrate. To enhance crystal production, we will also carry out crystallization experiments in the presence of small antibody (an immune system protein) fragments that specifically bind and stabilize activated forms of Parkin. The production of these specific antibodies will be done in collaboration with Dr. Gräslund in Sweden.
Impact on Diagnosis/Treatment of Parkinson's disease:
The structures of different forms of activated Parkin may pinpoint regions or residues of the protein as potential drug targets. The structures could be used to design drugs to interact with Parkin to boost its quality control activity for the treatment of sporadic PD and to overcome Parkin mutations in genetic forms.
Next Steps for Development:
A successful outcome of this project would lead to strategies for drug design and drug validation experiments, both in vitro and in vivo. Ultimately, this study will aid the screening of lead compounds for future testing of drugs for PD.