核黄素调节多种酰基辅酶A脱氢缺陷症ETF-QO突变蛋白稳定性的机制研究

多种酰基辅酶A脱氢缺陷症（MADD）是常见的脂质沉积性肌病，临床异质性明显，主要致病基因为ETFDH，分为核黄素反应型（RR-MADD）和非核黄素反应型，后者缺乏有效治疗手段。我们前期报道了中国ETFDH的主要突变热点，发现突变蛋白稳定性下降，更易被E3连接酶CHIP介导的泛素-蛋白酶体途径(UPP)降解，核黄素可能增加突变蛋白稳定性而恢复其表达，但机制不明。结合本病表型异质性现象，我们推测不同亚型突变蛋白间可能存在稳定性差异，研究核黄素稳定突变蛋白的内在途径有助阐明疗效差异的本质。本项目以患者成纤维细胞、已构建质粒转染293T细胞及MADD鼠为模型，利用分子动力学模拟、RNAi、泛素化修饰分析等手段，从突变蛋白空间结构及稳定性、蛋白与核黄素互作、CHIP介导的UPP调控等方面，探讨突变对蛋白稳定性的影响、核黄素稳定突变蛋白的机制、及非RR-MADD干预靶点，为治疗代谢性肌病提供依据。

Multiple acyl-CoA dehydrogenation deficiency (MADD) is a common lipid storage myopathy with obvious clinical heterogeneity. The main pathogenic gene is ETFDH, which is divided into riboflavin-responsive type (RR-MADD) and non-nuclear yellow Primitive response type, the latter lacks effective treatment. We previously reported the main mutation hotspots of ETFDH in China, and found that the stability of the mutant protein decreased and was more easily degraded by the ubiquitin-proteasome pathway (UPP) mediated by the E3 ligase CHIP. Riboflavin may increase the stability of the mutant protein and restore it. Expressed, but the mechanism is unknown. Combining the phenomenon of differences in riboflavin reactivity, we speculate that there may be differences in stability between mutant proteins of different subtypes. Studying the internal pathways of riboflavin stabilizing mutant proteins can help clarify the nature of the difference in efficacy. This project uses patient fibroblasts, constructed plasmid transfected 293T cells and MADD mice as models, using molecular dynamics simulation, RNAi, ubiquitination modification analysis and other methods, from the spatial structure and stability of the mutant protein, protein and riboflavin In terms of protein interaction, CHIP-mediated UPP regulation, etc., explore the effect of mutation on protein stability, the mechanism of riboflavin stabilizing the mutant protein, and non-RR-MADD intervention targets to provide a basis for the treatment of metabolic myopathy.