人视网膜色素上皮细胞中线粒体谷胱甘肽摄取载体蛋白的特性与调控。
Characterization and Regulation of Carrier Proteins of Mitochondrial Glutathione Uptake in Human Retinal Pigment Epithelium Cells.
机构信息
Department of Ophthalmology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
The Stephen J. Ryan Initiative for Macular Research (RIMR), Doheny Eye Institute, Los Angeles, California, United States.
出版信息
Invest Ophthalmol Vis Sci. 2019 Feb 1;60(2):500-516. doi: 10.1167/iovs.18-25686.
PURPOSE
To characterize two mitochondrial membrane transporters 2-oxoglutarate (OGC) and dicarboxylate (DIC) in human RPE (hRPE) and to elucidate their role in the regulation of mitochondrial glutathione (mGSH) uptake and cell death in oxidative stress.
METHODS
The localization of OGC and DIC proteins in confluent hRPE, polarized hRPE monolayers and mouse retina was assessed by immunoblotting and confocal microscopy. Time- and dose-dependent expression of the two carriers were determined after treatment of hRPE with H2O2, phenyl succinate (PS), and butyl malonate (BM), respectively, for 24 hours. The effect of inhibition of OGC and DIC on apoptosis (TUNEL), mGSH, and mtDNA was determined. Silencing of OGC by siRNA knockdown on RPE cell death was studied. Kinetics of caspase 3/7 activation with OGC and DIC inhibitors and effect of cotreatment with glutathione monoethyl ester (GSH-MEE) was determined using the IncuCyte live cell imaging.
RESULTS
OGC and DIC are expressed in hRPE mitochondria and exhibited a time- and dose-dependent decrease with stress. Pharmacologic inhibition caused a decrease in OGC and DIC in mitochondria without changes in mtDNA and resulted in increased apoptosis and mGSH depletion. GSH-MEE prevented apoptosis through restoration of mGSH. OGC siRNA exacerbated apoptotic cell death in stressed RPE which was inhibited by increased mGSH from GSH-MEE cotreatment.
CONCLUSIONS
Characterization and mechanism of action of two carrier proteins of mGSH uptake in RPE are reported. Regulation of OGC and DIC will be of value in devising therapeutic strategies for retinal disorders such as AMD.
目的
研究两种线粒体膜转运蛋白 2-氧戊二酸（OGC）和二羧酸（DIC）在人 RPE（hRPE）中的作用，并阐明它们在调节线粒体谷胱甘肽（mGSH）摄取和氧化应激中的细胞死亡中的作用。
方法
通过免疫印迹和共聚焦显微镜评估 OGC 和 DIC 蛋白在汇合的 hRPE、极化的 hRPE 单层和小鼠视网膜中的定位。用 H2O2、苯丁二酸（PS）和丁基丙二酸（BM）分别处理 hRPE 24 小时后，确定两种载体的时间和剂量依赖性表达。抑制 OGC 和 DIC 对细胞凋亡（TUNEL）、mGSH 和 mtDNA 的影响。用 siRNA 敲低 OGC 研究其对 RPE 细胞死亡的影响。用 IncuCyte 活细胞成像技术测定 OGC 和 DIC 抑制剂对 caspase 3/7 激活的动力学以及与谷胱甘肽单乙酯（GSH-MEE）联合治疗的效果。
结果
OGC 和 DIC 在 hRPE 线粒体中表达，并表现出随应激的时间和剂量依赖性降低。药理抑制导致线粒体中 OGC 和 DIC 减少，而 mtDNA 无变化，导致细胞凋亡增加和 mGSH 耗竭。GSH-MEE 通过恢复 mGSH 来防止凋亡。OGC siRNA 加剧了应激 RPE 中的细胞凋亡，而用 GSH-MEE 联合治疗增加 mGSH 则可抑制其凋亡。
结论
报告了 RPE 中 mGSH 摄取的两种载体蛋白的特性和作用机制。调节 OGC 和 DIC 将对制定 AMD 等视网膜疾病的治疗策略具有重要价值。
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