泛素-蛋白酶体途径在Dysferlin突变蛋白降解中的机制研究

Dysferlinopathy is a group of autosomal recessive inherited muscular disorders. Two major phenotypes are Limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM). It has been identified that dysferlin deficiency caused by DYSF gene mutation, which contribute to defective plasma membrane repair, degeneration and necrosis of muscle fibers. Although it has proposed that interference with the degradation of mutated protein reversed plasma membrane resealing defects, the degradation mechanism of mutant protein has not been clearly understood. We previously observed that c.1667 T>C is a common mutation of DYSF gene identified in 25% dysferlinopathy families. We demonstrated that a marked reduction of dysferlin protein expression in patients, but the level of mRNA expression did not decline. And proteasome inhibitors were capable of increasing dysferlin in cultured, which is suggestive that the degradation of mutant dysferlin protein was regulated by the Ubiquitin-proteasome Pathway (UPP). In the present work, by using the mass spectrometry（MS）analysis, we will systematically screen and prove the special E3 ligase for ubiquitination of dysferlin, identify the ubiquitinated sites and then explore the neworks and “communities”of the interaction between UPP and dysferlin protein, reveal the molecular mechanism of disfuction of the mutant protein. Furthermore, we will interefere specfic targets for the neworks of UPP and dysferlin protein, observe that the change of the physiological function of myoblast, finally provide with the advanced theory basis of treatment for dysferlinopathy.

Dysferlin肌病是临床较常见的一组常染色体隐性遗传性肌病，由DYSF基因突变引起肌膜蛋白dysferlin表达减少或缺失，导致肌膜自我修复障碍和肌纤维变性、坏死。既往研究证实抑制dysferlin突变蛋白的降解，可以部分恢复肌膜的修复功能，但突变蛋白降解的调控机制不明确。我们前期发现c.1667T>C是常见的突变位点，占25%；证实患者骨骼肌dysferlin蛋白显著下调，而mRNA水平无明显改变。利用蛋白酶体抑制剂干预患者来源的肌细胞，发现突变蛋白显著上调，提示DYSF突变后的蛋白降解受泛素-蛋白酶体途径（UPP）调控。在此基础之上，本项目拟用质谱分析等技术，筛选dysferlin蛋白的特异性泛素连接酶E3及泛素化位点，绘制dysferlin蛋白-UPP的互作网络，寻找及调控关键靶点，观察突变蛋白及肌细胞各生理功能的修复状况，揭示突变蛋白功能障碍的分子机制，为治疗提供新思路。

Dysferlin肌病（Dysferlinopathy）是肌膜蛋白dysferlin 缺陷引起的一组常染色体隐性遗传性肌病，根据首先受累肌群，主要分为2型：肢带型肌营养不良2B型(Limb-girdle muscular dystrophy type 2B，LGMD2B)，Miyoshi远端型肌营养不良(Miyoshi Myopathy，MM)。DYSF 基因是本病的致病基因，基因突变后dysferlin蛋白表达下调或缺失，肌细胞的自身修复功能障碍，导致肌细胞变性、坏死，继发炎性反应。我们前期确定了患者骨骼肌dysferlin蛋白显著下调，而mRNA水平无明显改变，是突变蛋白降解加速等分子病理机制。本项目围绕疾病的临床异质性分析、突变体的蛋白泛素化修饰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-分子病理诊断体系的完善，规范化数据库的建设，DYSF基因突变后蛋白降解调控通路的研究，建立了规范化的诊治流程，完善了临床样品数据库，发现了高频突变点和较多新发的突变位点，扩展了DYSF基因的突变谱，获得大数据量的基因型-临床表型分析数据，发现同一基因型可以导致明显的临床异质性以及家系内基因异质性，评估及量化分析dysferlinopathy疾病不同时期下肢肌肉磁共振。在致病机理方面，在外源性和内源性细胞水平上，进一步证实了dysferlin突变蛋白降解受到泛素蛋白酶体途径（UPP）的调控。通过肌肉活检骨骼肌病理组织的高通量泛素化修饰组学分析，证实了泛素化修饰蛋白参与dysferlinopathy疾病一般代谢，分子转运和调控、细胞合成代谢和钙信号传导等生物学过程。鉴定到了与突变dysferlin蛋白降解三种E3连接酶（HSP90AB1、HSPA8和TRIM32），并进一步在体外细胞实验证实HSPA8与dysferlin蛋白存在体内的相互作用，说明dsyferlinopathy发病机制的重要环节是HSPA8介导降解突变型dysferlin蛋白，引起细胞内dysferlin蛋白功能不足，以上结果为后续体内功能干预及修复机制研究提供了理论和实践基础。