Retinitis Pigmentosa Associated Rhodopsin Mutant T17M Induces Cell Death

Chapter1:The sub-cellular localization of rhodopsin mutant T17MAim:To investigate the sub-cellular localization of rhodopsin mutant T17M.Methods:pCDNA-3.1-T17M rhodopsin-myc and pCDNA-3.1-WT rhodopsin-myc plasmids were constructed and identified by EcoR I and BamH I double enzyme digestion and sequencing. The overexpression of rhodopsin mutant T17M and WT were detected by Western blot. The sub-cellular localization of rhodopsin mutant T17M and WT were visualized using a confocal microscope.Resluts:1Kbp site band was detected after PCR and double enzyme digestion from pCDNA-3.1-T17M rhodopsin-myc and pCDNA-3.1-WT rhodopsin-myc. DNA sequencing showed a C-T mutation in site50which resulted in T-M amino acid change in rhodopsin protein. About40KD band was detected by Western blot after transfection of rhodopsin T17M plasmid and WT, suggesting the protein successfully expressed in mammalian cell lines. Rhodopsin mutant T17M abnormally localized at endoplasmic reticulum (ER), while rhodopsin WT dominantly localized at plasma membrane. Rhodopsin T17M didn’t colocalize with Golgi apparatus. Conclusion:Rhodopsin T17M mislocates at ER and does not colocalize with Golgi apparatus. Rhodopsin WT locates at plasma membrane Chapter2:The degradation pathway of rhodopsin mutant T17MAIM:To demonstrate the degradation pathway of Rhodopsin mutant T17M.Methods:Protein degradation rate of rhodopsin mutant T17M and WT were detected by MTT method. The effect of lysosomal inhibitors and protease inhibitors on rhodopsin mutant T17M and WT were analyzed by Western blot separately. With siRNA technology to knockdown ERAD-related protein degradation pathways, the degradation pathway of rhodopsin T17M and WT was demonstrated.Results:The relative protein value of rhodopsin mutant T17M and WT in HEK293cells after6h of CHX treatment were0.219±0.032and0.635±0.072(P<0.01) in HEK293cells, and0.302±0.041and0.531±0.052in ARPE-19cells. The relative protein value of rhodopsin mutant T17M and WT in HEK293cells after12h of treatment with lysosomal inhibitor CQ increased from1to1.023±0.265and1.433±0.159(P <0.05), separately. The relative protein value of rhodopsin mutant T17M and WT in HEK293cells after6h of treatment with proteasome inhibitor MG132increased from1to7.213±2.108(P<0.01) and2.011±0.221(P <0.05), separately. After immunoprecipitation, ubiquitination of rhodopsin mutant T17M increased from1to2.200±0.361(P<0.01), while that of WT protein relative value was increased from1to1.160±0.162. After overexpressing p97/VCP-QQ in ARPE-19cells, the relative protein value of contrast and rhodopsin mutant T17M was0.159±0.052and0.558±0.095(P<0.01). The relative protein value of contrast and rhodopsin mutant T17M after Erasin siRNA treatmen was0.230±0.059and0.602±0.064(P<0.01), while the WT one did not change significantly.Conclusion:Rhodopsin T17M was more rapidly degraded than wild type. Rhodopsin WT is degradated by both lysosome and proteasome pathway. But rhodopsin mutant T17M is degradated only by proteasome pathway. The degradation of rhodopsin mutant T17M is realated to ubiquitinized ERAD, which could be inhibited by overexpression of p97/VCP-QQ and earsin knockdown. Chapter3:The mechanism of cell death induced by rhodopsin mutant T17MAIM:To invesitage the mechanism of cell death induced by rhodopsin mutant T17M.Method:pEGFP-CL1stable ARPE-19cell line was established. The activity of the proteasome indicated by level of pEGFP-CL1was detected by Western blot. Rhodopsin protein was overpressed to induce endoplasmic reticulum stress response, and the expression of endoplasmic reticulum-associated protein BIP, CHOP, GRP94, peIF-2a, eIF-2a, active ATF-6a were detected. The effect of PBA was detected by Western blot. The death of ARPE-19cells after tunicamycin treatment was detected by flow cytometry. The ROS level and the effect of ROS scavenging agent NAC and BHA were detected by flow cytometry.Results:pEGFP-CL1ARPE-19cells could be stably expressed uGFP. There was no significant different between the uGFP level of rhodopsin T17M and WT. Mutation T17M upregulated the expression of endoplasmic reticulum stress protein BIP, CHOP, GRP94, peIF-2a, eIF-2a, active ATF-6a. Compared with rhodopsin WT, the expression level of BIP, GRP94, CHOP, peIF-2a/eIF-2a, active ATF-6a was increased from1to2.439±0.363(P＜0.01),2.433±0.802(P<0.01),1.600±0.212(P<0.05),1.567±0.153(P<0.05),2.167±0.306(P<0.01), respectively. The relative protein level of GRP94, CHOP, peIF-2a/eIF-2a, active ATF-6a before and after PBA treatment was2.600±0.854and1.467±0.306(p<0.05),1.921±0.557and1.203±0.239(p<0.05),1.733±0.154and1.167±0.252(p<0.05),2.564±0.406and1.349±0.529(p<0.05), respectively. The death rate of vector, rhodopsin mutant T17M and WT before and after tunicamycin treatment was4.156%±0.501%and4.814%±0.531%,3.879%±0.413%and5.712%±0.574%,7.021%±0.612%and16.213%±3.419%, respectively. The ROS level of rhodopsin WT and T17M was1.136±0.055and1.935±0.088, respectively (P<0.01). The cell death rate of vector group, DMSO group, NAC group and BHA group was3.716%±0.523%,7.322%±1.924%, 4.857%±1.369%(compare to DMSO, P＜0.05),4.271%±0.988%, respectively (compare to DMSO, P<0.01).Conclusion:Overexpression of T17M rhodopsin mutations does not affect the activity of the proteasome. Endoplasmic reticulum stress could be induced by mutant T17M. Overexpression T17M rhodopsin induces cell death and increases cell endoplasmic reticulum stress leading to cell death cytotoxicity. Chemical chaperone PBA reduces endoplasmic reticulum stress induced by rhodopsin mutant T17M. Tunicamycin could strengthen ER stress induced by T17M. T17M rhodopsin mutations increases intracellular levels of ROS. ROS scavengers rescue cell death induced by rhodopsin T17M.