Kresge Eye Institute, Wayne State University, 4717 St. Antoine, Detroit, MI 48201, USA.
Kresge Eye Institute, Wayne State University, 4717 St. Antoine, Detroit, MI 48201, USA.
Exp Eye Res. 2014 Apr;121:168-77. doi: 10.1016/j.exer.2014.02.010. Epub 2014 Mar 4.
Mitochondrial transcription factor A (TFAM) is one of the key regulators of the transcription of mtDNA. In diabetes, despite increase in gene transcripts of TFAM, its protein levels in the mitochondria are decreased and mitochondria copy numbers become subnormal. The aim of this study is to investigate the mechanism(s) responsible for decreased mitochondrial TFAM in diabetes. Using retinal endothelial cells, we have investigated the effect of overexpression of cytosolic chaperone, Hsp70, and TFAM on glucose-induced decrease in mitochondrial TFAM levels, and the transcription of mtDNA-encoded genes, NADH dehydrogenase subunit 6 (ND6) and cytochrome b (Cytb). To investigate the role of posttranslational modifications in subnormal mitochondrial TFAM, ubiquitination of TFAM was assessed, and the results were confirmed in the retina from streptozotocin-induced diabetic rats. While overexpression of Hsp70 failed to prevent glucose-induced decrease in mitochondrial TFAM and transcripts of ND6 and Cytb, overexpression of TFAM ameliorated decrease in its mitochondrial protein levels and transcriptional activity. TFAM was ubiquitinated by high glucose, and PYR-41, an inhibitor of ubiquitination, prevented TFAM ubiquitination and restored the transcriptional activity. Similarly, TFAM was ubiquitinated in the retina from diabetic rats, and it continued to be modified after reinstitution of normal glycemia. Our results clearly imply that the ubiquitination of TFAM impedes its transport to the mitochondria resulting in subnormal mtDNA transcription and mitochondria dysfunction, and inhibition of ubiquitination restores mitochondrial homeostasis. Reversal of hyperglycemia does not provide any benefit to TFAM ubiquitination. Thus, strategies targeting posttranslational modification could provide an avenue to preserve mitochondrial homeostasis, and inhibit the development/progression of diabetic retinopathy.
线粒体转录因子 A（TFAM）是调节线粒体 DNA 转录的关键调节因子之一。在糖尿病中，尽管 TFAM 的基因转录物增加，但线粒体中的其蛋白水平降低，线粒体拷贝数变得异常。本研究旨在探讨糖尿病中线粒体 TFAM 减少的机制。我们使用视网膜内皮细胞研究了胞质伴侣 Hsp70 和 TFAM 的过表达对葡萄糖诱导的线粒体 TFAM 水平降低以及 mtDNA 编码基因 NADH 脱氢酶亚单位 6（ND6）和细胞色素 b（Cytb）转录的影响。为了研究翻译后修饰在异常线粒体 TFAM 中的作用，评估了 TFAM 的泛素化，并且在链脲佐菌素诱导的糖尿病大鼠的视网膜中证实了该结果。虽然 Hsp70 的过表达未能防止葡萄糖诱导的线粒体 TFAM 和 ND6 和 Cytb 转录物的减少，但 TFAM 的过表达改善了其线粒体蛋白水平和转录活性的减少。TFAM 被高葡萄糖泛素化，并且泛素化抑制剂 PYR-41 可防止 TFAM 泛素化并恢复转录活性。同样，糖尿病大鼠的视网膜中 TFAM 被泛素化，并且在重新建立正常血糖水平后它仍在继续修饰。我们的结果清楚地表明，TFAM 的泛素化阻碍了其向线粒体的转运，导致异常的 mtDNA 转录和线粒体功能障碍，并且抑制泛素化可恢复线粒体的动态平衡。逆转高血糖症对 TFAM 泛素化没有任何益处。因此，针对翻译后修饰的策略可能提供一种保留线粒体动态平衡并抑制糖尿病性视网膜病变发展/进展的途径。
