The Study Of Interaction Of Parkin And OGCP And The Mutation Detection Of OGCP Gene

PART I: The Study of Interaction of Parkin and OGCPParkin gene is one of the virulence genes for Parkinson's disease. As one of the E3 enzymes of ubiquitin-proteasome pathway (UPP), Parkin mediates many substrate proteins' ubiquitination which has close relationship with the nosogenesis of Parkinson's disease. 2-Oxoglutarate carrier protein(OGCP) is one of the proteins in mitochondrion. We studied the interaction of Parkin and OGCP in this research.The full-length CDS of Parkin and OGCP were amplificationed from the human fetus brain cDNA library and were cloned to the pCMV-Tag4 vector and pcDNA3.1-myc-his(-)B vector differentialy to make the eukaryotic expression vector pCMV-Parkin and pcDNA3.1-myc-OGCP. After validation of vector construction by sequencing, we transfectioned those vectors to the HEK293 cells. By using the immunoblotting and immunofluorescence and confocal microscope, we discovered that the pCMV-Parkin and pcDNA3.1-myc-0GCP expressed Parkin-Flag and OGCP-myc correctly. Parkin-Flag was expressed in both the kytoplasm and nuclei and OGCP-myc localized in the mitochondrion which showed the merge to Flag and myc didn't change the subcellular localization of Parkin and OGCP. We also successfully established the cell line which expressed OGCP-myc stably.Using the immunofluorescence and confocal microscope , we found that in the HEK293 cell which was cotransfectioned pCMV-Parkin and pcDNA3.1-myc-0GCP, Parkin-Flag and OGCP-myc can be co-localized, co-immunoprecipitation experiment validated the interaction of Parkin-Flag and OGCP-myc.In Vitro Ubiquitination Assays showed that in the presence of El enzyme, UbcH7 and ubiqutin, Parkin-Flag can promote the ubiquitination of OGCP-myc. In vivo Ubiquitination Assays supported this result, that showed OGCP is one of the substrate proteins of Parkin and Parkin mediates OGCP's ubiquitination.By interfering OGCP-myc/HEK293 cells with proteasome-inhibitor...