The Effect Of The Interaction Between Mutant Parkin And OGCP On The Apoptosis Of HEK293 Cells And The Mutation Analysis Of HtrA2/Omi Gene In Patients With Parkinson's Disease

PART I : The Effect of the Interaction between Parkin Mutants and OGCP on the Apoptosis of HEK293 CellsParkin Gene is one of the Parkinson's disease-related genes. As a ligase E3 of ubiquitin-proteasome pathway(UPP ), Parkin Protein mediates the ubiquitin process of substrate proteins. The N-terminal of Parkin Gene contains a ubiquitin-like(Ubl) domain and its C-terminal contains a Ring-IBR-Ring (RIR) domain, which is formed with two ring fingers, Ring1 (R1) and Ring2 (R2) and the IBR between them. The RIR fingers in Parkin have been shown to mediate protein-protein interactions with the E2 conjugating proteins that are required for ubiquitination. The amino-terminal ubiquitin-like (Ubl) domain contributes to the recognition of target proteins. The single mutation causing an Arg to Pro substitution at amino-acid position 42 of the Ubl domain and another single mutation causing an Thr to Arg substitution at amino-acid position 240 of the R1 have been identified in AR-JP patients. 2-oxoglutarate carrier protein(OGCP) is one of the proteins in mitochondrion which transports reduced glutathione (GSH) from cytoplasmin to mitochondrialmatrix and eliminates endogenic oxygen free radical and improves the cellular antioxidation capacity. In the early stage work, the interaction between Parkin and OGCP had been confirmed. OGCP was ubiquitinated substrate protein of Parkin. In this paper, we investigated the interaction between the mutation Parkin (R42P or T240R) and OGCP and the effect of the interaction on the HEK293 cells.We transfectioned the constructed pcDNA3.1-myc-OGCP eukaryotic expression vector to the HEK293, and successfully established the cell line which expressed OGCP-myc stably that validated by western blot and RT-PCR. Moreover, through Western blot, we found that in HEK293 cells, the expression of OGCP-myc which was co-transfected with wild-type Parkin was markedly decreased. There was no significant difference among the other three group OGCP-myc expression. That indicates that wild-type Parkin can promote the degradation of OGCP. And mutant Parkin(R42P and T240R) has no obvious effect on the metabolism of OGCP.Through immunofluorescence and laser Confocal microscope, we found that in HEK293 cells, there exited co-orientation between fusion protein Parkin(WT)-vsvg,Parkin(R42P)-vsvg Parkin(T240R)-vsvg and fusion protein OGCP-myc, respectively. Co-immunoprecipitation confirmed the interaction between fusion protein Parkin(WT)-vsvg and fusion protein OGCP-myc, while there was no interaction between fusion protein Parkin(R42P)-vsvg, OGCP-myc, Parkin(T240R)-vsvg and fusion protein OGCP-myc.By Flow Cytometry Methods (FCM) , we examined HEK293 cellS apoptosis using fluorescence FITC as probe, tested the mitochondrion membrane potential(△ψ_M) using Rhodamine 123 as probe and detected the cellular reactive oxygen species(ROS) level using DCFH-DA as probe. The results showed that the cell activity and△ψ_M of all the cell groups induced by 100nmol/l rotenone were significantly lower than those not induced by rotenone. However, the cellular ROS level and the cell apoptosis rate were much higher than those not induced by rotenone. We also found that Parkin could raise the cell activity and△ψ_M of HEK293 cells treated by 100nmol/l rotenone and lowered the cellular ROS level so that it lowered the the cell apoptosis rate induced by 100nmol/l rotenone. And Parkin mutant (R42P and T240R) lowered the cell activity and△ψ_M of HEK293 cells especially the HEK293 cells induced by rotenone, raised the cellular ROS level and improved cell apoptosis.In addition, we also found that OGCP could raise the cell activity and△ψ_M of HEK293 cells induced by 100nmol/l rotenone, lower the cellular ROS level so that it inhibited cell apoptosis induced by rotenone. Furthermore, OGCP could inhibit the decrease of the cell activity and△ψ_M and the increase of the cellular ROS level resulting from Parkin mutant (R42P and T240R) so that it reduced the cell apoptosis resulting from Parkin mutant (R42P and T240R) .PART II: The mutation analysis of HtrA2/Omi gene in patients with Parkinson's diseasePathogenic substitutions in the HtrA serine peptidase 2 (HtrA2), G399S and A141S, are a cause of sporadic Parkinson's disease in the German population. In this study we examined the frequency of these two substitutions in 236 sporadic Parkinson's disease (PD) patients who was older than 50 years and had no family history by Restriction Fragment Length Polymorphism(RFLP). Both substitutions were not observed. At the same time, we performed a mutation screening of the HtrA2/Omi gene in these 236 patients and 200 normal controls with the denaturing high performance liquid chromatogram(DHPLC). Only in one patient, we identified a known polymorphism(rs2231249). These results suggested HtrA2 may play an unimportant role in Chinese Parkinson's disease who was older than 50 years and had no family history.