脂质沉积性肌病ETFDH基因突变后蛋白调控异常的机制研究

Lipid-storage myopathy (LSM) is pathologically characterized by prominent lipid accumulation in muscle fibers. It is a common inherited muscular disorder, categorized under the broad category of defects in long-chain fatty acid β-oxidation (FAO) metabolism. Some patients' symptoms strikingly ameliorate by ribo?avin treatment (presenting ribo?avin-responsive). Recently, studies have confirmed that late-onset MADD（MADD III）is the most common form of LSM in Chinese population. Their major genetic causes have been identified in mutations of electron transfer flavoprotein dehydrogenase (ETFDH) gene, especially existing some hotspots. And the mutational spectrum may be heterogeneous in various ethnic groups between the southern and northern Chinese population. However, the dis-function of mutational protein and the mechanism of ribo?avin-responsive are unclear. In our previous research , ETFDH-c.250G>A（p.A84T）has been proved to be the most common mutation, representing a high allelic frequency of 83.3% in southern China. We have further confirmed that mutations of ETFDH gene cause declination of protein stability, acceleration of degradation, and eventually leads to ETFDH reduction.It might be related with the cross talk of protein ubiquitin-proteasome system (UPS). In the advance,we will focus on protein posttranslational modification (PTM), by the way of mass spectrometry （MS）analysis , systematically screen and prove the special ligase for ubiquitination of ETFDH,identify the ubiquitinated sites.Then, the cell and animal models will be constructed to explore the regulation and intervention of interaction between ligase and substrate, reveal the mechanism in the regulation abnormality of mutant protein, meanwhile seek out potential target point of riboflavin , finally provide with the advanced theory basis of treatment.

脂质沉积性肌病（LSM）由长链脂肪酸β-氧化代谢障碍导致，为较常见的遗传代谢性肌病，部分患者对核黄素治疗有效。我们及国内其他小组的研究证实晚发型多种酰基辅酶A脱氢缺陷症（MADD）是我国LSM的主要类型，多由电子转移黄素蛋白脱氢酶（ETFDH）基因突变引起，存在突变热点并有南北方人群差异，但突变后蛋白功能改变及核黄素治疗有效的机制不明确。我们发现ETFDH-c.250G>A突变是南方LSM的高频突变热点，并证实了ETFDH突变可引起ETFDH蛋白的稳定性下降，降解加速，这可能与蛋白质泛素化修饰有关。本项目将基于上述研究结果，采用质谱分析等技术，筛选出特异性修饰ETFDH蛋白的泛素连接酶及其他调控蛋白，确定泛素化修饰位点；建立细胞模型和转基因动物模型，探讨ETFDH蛋白泛素化修饰的调控过程，揭示突变后蛋白调控异常的机制，并寻找核黄素的可能作用靶点，为治疗提供理论依据。

脂质沉积性肌病（LSM）是体内长链脂肪酸β氧化（FAO）缺陷导致脂质在肌纤维内大量沉积而引起的一类代谢性肌病，属于临床病理综合征，是神经科较常见的常染色体隐性遗传病。我国LSM主要类型是晚发型多种酰基辅酶A脱氢缺陷症（MADD），主要致病基因为电子转移黄素蛋白脱氢酶（ETFDH）基因，基因突变后导致血清脂酰肉碱谱异常和尿有机酸升高。我们前期确定了中国南方LSM患者的分子遗传学特征及突变蛋白降解加速等分子病理变化规律，本项目围绕疾病的临床异质性分析、突变体的蛋白翻译后修饰（PTM）及其致病机理为关键科学问题，进行临床与基础结合的研究，完成了临床评估体系的完善，规范化数据库的建设，药物疗效临床对比评价，ETFDH基因突变后蛋白降解调控通路及其与线粒体功能障碍的关系，核黄素治疗机理，MADD小鼠模型构建及表型鉴定等多领域的综合研究，建立了规范化的诊治流程，完善了临床样品数据库，扩展了ETFDH基因的突变谱，获得大数据量的临床表型-基因型分析数据，发现相近的基因型可以导致明显的临床异质性，评估和强调了肌肉MRI的诊断价值，客观评价了核黄素的治疗时效性。特别是发病机理方面，在外源性和内源性细胞水平上，证实了ETFDH突变蛋白降解受到泛素蛋白酶体途径（UPP）的精确控制，发现了突变型ETFDH的特异性泛素E3连接酶CHIP及ETFDH-A84T蛋白的2个特异性CHIP泛素化位点（K），并在MADD患者的活体肌组织上，证实CHIP与ETFDH蛋白存在体内的相互作用，证实热休克蛋白70（Hsp70）参与了ETFDH蛋白的降解过程，说明MADD发病机制的重要环节是CHIP介导降解突变型ETFDH，引起细胞内ETFDH蛋白功能不足，进而导致MADD线粒体功能障碍及细胞凋亡。核黄素活性形式FAD干预后稳定了ETFDH突变蛋白的构象，使突变蛋白表达量提高而取得临床效果，此作用并非通过抑制泛素化途径。最后，通过同源重组法构建EtfdhA83T/A83T突变敲入小鼠模型，表型鉴定符合LSM的肌肉病理及生化改变特征，但致死性强，本项目延续性工作采用新型基因编辑技术改进动物模型，以上结果为后续体内功能研究提供了理论和实践基础。