| Abstract No.: | B-C2090 |
| Country: | Canada |
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| Title: | CHARACTERIZATION OF THE NOVEL INTERACTION BETWEEN PARKIN AND ENDOPHILIN: IMPLICATIONS FOR PAKINSON’S DISEASE |
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| Authors/Affiliations: | 2 Carol Chen*; 2 karl Grenier; 1 Jean-Francois Trempe; 1 Kalle Gehring; 2 Edward Fon;
1 Department of Biochemistry; 2 Montreal Neurological Institute, QC, Canada
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| Content: | Background. Mutations in the parkin gene result in an autosomal recessive juvenile-onset form of Parkinson’s disease (PD). Parkin is an E3 ubiquitin-ligase encoding two ring domains at its C-terminus, which mediates the attachment of ubiquitin onto specific substrate, an IBR (in-between ring motif) and a Ubl (ubiquitin-like) domain at the N-terminus. The inability of parkin to ubiquitinate its substrates is therefore believed to lead to neurodegeneration in PD. Accordingly, we performed a pull down with parkin’s UbL domain in order to identify new binding partners of parkin. Interestingly, a number of these proteins have turned out to contain SH3 (Src homology domain) domains, including endophilin, a protein involved in endocytosis. This was surprising, as SH3 domains were known to bind proline-rich sequences but not ubiquitin or Ubl domains. However, recent studies showed that ubiquitin binds to a subset of SH3 domains. Strikingly, endophilin was found not to bind ubiquitin, contrasting with our finding that endophilin binds the parkin Ubl. Our hypothesis is that endophilin interacts with parkin's Ubl via its SH3 domain, and that PD related parkin mutations in the Ubl domain alter this interaction and thus promote neurodegeneration by affecting endophilin's normal function in endocytosis.
Objectives. The objectives of the present study are to first characterize the structural basis of the Ubl-SH3 interaction between parkin and endophilin. Next, we will determine whether endophilin is a substrate of parkin-mediated ubiquitination. Finally we will elucidate the functional consequence of parkin-dependent ubiquitination of endophilin.
Materials and methods. The interaction between parkin and endophilin has been validated by pull-down and co-immunoprecipitation assays. NMR structural studies were performed to identify critical residues involved in the Ubl-SH3 interaction. Mutagenesis was then performed to confirm the importance of these residues in the Ubl-SH3 interaction. Ubiquitination assays were used to determine whether parkin ubiquitinates endophilin.
Results. We found that the interaction between parkin and endophilin is mediated by the Ubl-SH3 interaction. Deletion of either domain effectively abolished the interaction. NMR studies identified N8 and K48 residues of parkin’s Ubl as critical residues for binding to endophilin’s SH3 domain. N8A and K48A mutagenesis effectively abolished binding to endophilin thus confirming the importance of these residues. We also found that parkin is able to mono-ubiquitinate rather than poly-ubiquitinate endophilin. Consistent with this finding, endophilin steady state levels are similar in parkin wild-type and knockout mouse brain, indicating the lack of parkin-dependent proteasome-mediated degradation of endophilin.
Conclusion. We have identified a novel interaction between endophilin and parkin. Moreover, we have shown that this interaction is mediated by the parkin Ubl domain and the endophilin SH3 domain. The N8 and K48 residues within the Ubl are critical for the interaction. Finally, we have demonstrated that endophilin is mono-ubiquitinated by parkin, and therefore that PD related parkin mutations might promote neurodegeneration by affecting the regulation of endophilin’s normal function in endocytosis.
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