| Abstract No.: | B-C2104 |
| Country: | Canada |
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| Title: | THE E3 LIGASE PARKIN INTERACTS WITH THE PROTEASOMAL SUBUNIT S5A AND THE ENDOCYTIC PROTEIN EPS15. |
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| Authors/Affiliations: | 1 Susan Safadi*; 1 Gary Shaw;
1 University of Western Ontario, ON, Canada
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| Content: | Objectives: Autosomal Recessive Juvenile Parkinsonism (AR-JP) is an early-onset form of Parkinson’s disease (PD) that is clinically indistinguishable from the sporadic form of PD. A major cause of AR-JP results from mutations in the protein Parkin, an E3 ubiquitin ligase, responsible for the tagging of specific proteins mediating cellular trafficking and protein degradation. Parkin has a multidomain architecture that includes an N-terminal Ubiquitin-like domain (Ubld). Mutations in AR-JP have been mapped throughout the Parkin gene, and specifically within its Ubld. The Ubld is essential for the proper function of Parkin and has been shown to interact with proteins containing Ubiquitin Interacting Motifs (UIMs). In this work we present a detailed examination of the interaction of the Parkin Ubld with two UIM containing proteins, the proteasomal subunit S5a and the endocytic protein Eps15. To determine the effects of ARJP causative mutations in the Ubld, we have created two forms of the protein each carrying a disease-state mutation (R42P and K48A).
Materials and Methods: All proteins were expressed and purified from bacteria. Initial His-pull down assays were carried out to determine if the Parkin Ubld and its mutants containing disease state mutations interacted with the S5a subunit. Following confirmation of the interaction more detailed studies were carried out using Nuclear Magnetic Resonance (NMR) Spectroscopy to determine the exact binding surfaces at the amino acid level on the Parkin Ubld and the S5a subunit. The dissociation constant for this interaction was also determined by NMR as well as Isothermal Titration Calorimetry (ITC). The interaction of the Parkin Ubld with Eps15 was carried out in a similar manner. Pull down assays had already confirmed this interaction (Fallon et al. Nat. Cell Biol., 2006). The interaction of Eps15 with Ubiquitin was also characterized by the methods described above for comparison with the Parkin Ubld.
Results: We have shown that the Parkin Ubld binds the S5a subunit of the 26S proteasome and that two ARJP causative mutations (R42P and K48A) disrupt this interaction. We have identified the exact binding surfaces on the Parkin Ubld and the S5a used in this interaction. We have also provided a similar characterization for the interaction of the Parkin Ubld and Ubiquitin with Eps15. The affinities of these interactions have also been determined.
Conclusions: These results provide information into the interactions involved in the normal function of the Parkin Ubld within the cell. The abolished interactions caused by the disease state mutations gives evidence for the dysfunction of the Parkin Ubld in AR-JP.
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