| Abstract No.: | B-C2120 |
| Country: | Canada |
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| Title: | GAINING INSIGHT INTO PARKIN’S STRUCTURE AND UBIQUITIN LIGASE ACTIVITY |
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| Authors/Affiliations: | 1 Ventzi Hristova*; 1 Gary Shaw; 1 Rebecca Jane Rylett;
1 University of Western Ontario, London, ON, Canada
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| Content: | Objectives: Autosomal Recessive Juvenile Parkinson’s disease (ARJP) is a neurodegenerative disorder that affects individuals at 40 years of age or younger. Onset of ARJP is associated with symptoms such as dystonia, bradykinesia, and body tremors. Degeneration of dopaminergic neurons of the substantia nigra is the hallmark of ARJP, however the etiology of these changes remains unknown. Individuals diagnosed with ARJP have mutations in the park2 gene, which encodes the parkin protein.
This study focuses on the structure of parkin and the protein-interactions parkin is involved in, specifically parkin-UbcH8 binding. The goal of this research is to express and purify sufficient amounts of parkin to determine the protein’s structural conformation and domain boundaries. This project also aims to identify what regions of parkin are responsible for the specificity of parkin-UbcH8 interactions.
Materials and Methods: Rat parkin is expressed in BL-21 Codon Plus cells and purified through a series of chromatographic steps conducted under strict conditions to prevent protein degradation. Parkin’s three-dimensional structure is analyzed by nuclear magnetic resonance (NMR) and circular dichroism (CD) techniques, as well as a limited proteolysis approach which utilizes the trypsin and V8 protease enzymes. Liquid chromatography coupled to mass spectrometry (LC-MS) is used for identification of the proteolytic fragments of parkin. Protein-interaction experiments are done in HEK Flp-In cells that are stably over-expressing FLAG-parkin and UbcH8. The proteins are shown to interact by immunoprecipitation methods followed by western blotting. This interaction is also studied in vitro with bacterially purified parkin and UbcH8. The cross-linking reagents DSP and BS3 will be used to covalently attach lysine residues present at the binding surfaces of both proteins.
Results: Parkin was successfully purified and analyzed by both CD and NMR, which showed that the protein contained structured regions with both alpha-helical and beta-sheet components. Preliminary limited proteolysis results indicated the presence of well defined domain boundaries in parkin, which are tightly held together and resistant to protease cleavage. Currently we are investigating the number of zinc ions coordinated by the parkin metalloprotein, and conducting EDTA titration experiments to determine how the gradual removal of zinc ions affects parkin’s folding. Parkin-UbH8 binding studies done in situ and in vitro show a direct interaction between the two proteins. UbcH8 co-immunoprecipitates with parkin in both HEK Flp-In cells and SH-SY5Y neuroblastoma cells. In vitro binding experiments indicate that parkin can bind UbcH8 in a cell free environment in the absence of other proteins.
Conclusion: NMR and CD analysis of parkin indicate that the metalloprotein remains folded following the purification process. The limited proteolysis results identified the most stable, well structured regions of parkin that remained intact following treatment with either V8 or trypsin. These results are key for determining domain boundaries within the parkin protein. Parkin’s ability to bind UbcH8 in situ indicates that this interaction is responsible for the ubiquitination of substrate proteins in HEK Flp-In cells. It also |
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