Role of the ER-to-Golgi SNAREs BET1 and GOSR2 in muscular dystrophies

先天性肌营养不良症（CMDs）是一组退行性肌肉疾病，其特点是发病年龄早，可根据受影响蛋白的功能和细胞定位进行分类。例如，已经确定了肌营养不良蛋白-糖蛋白复合物（DGC）亚基的致病变异，包括杜氏肌营养不良症和糖营养不良症，它们分别由编码肌营养不良蛋白和糖营养不良蛋白的基因突变引起。DGC是一种重要的细胞粘附受体复合物，通过将肌动蛋白细胞骨架连接到骨骼肌细胞的基板来维持肌肉的完整性。糖醛酸是一种蛋白质前体，被分裂成外周高度糖基化的a-糖醛酸和跨膜的b-糖醛酸。糖基化对其功能至关重要，研究结果也强调了糖基转移酶的变异会导致肌营养不良。此外，影响内质网（ER）到高尔基膜运输机制成分的变异，如TRAPPC11、GOSR2和BET1，最近被添加到遗传性cd列表中。GOSR2和BET1是复杂的伴侣蛋白和SNARE（可溶性n -乙基酰亚胺敏感因子附着蛋白受体）蛋白，它们对于内质网、ER-高尔基体中间组分（ERGIC）和高尔基体之间囊泡介导的膜运输的对接和融合至关重要。我们和其他人已经证明BET1和GOSR2的双等位基因变异是功能丧失突变，导致CMD/癫痫综合征。然而，疾病进展的分子机制仍然难以捉摸。本课题的实验将重点关注BET1和GOSR2在肌管和体内DG复合物的膜运输中的作用，为肌肉疾病的发病机制提供分子见解。特别是，我们将在分子水平上分析在先天性小头畸形、癫痫和肌病患者中发现的具有未知意义的GOSR2新双等位基因变异，这些患者的肌酸激酶和乳酸水平升高。我们将对GOSR2和BET1缺陷的成肌细胞和肌管进行比较分析，以确定常见的疾病途径。最后，肌肉特异性GOSR2缺陷小鼠的表型特征将使我们能够分析ER-to-Golgi SNAREs在体内的作用。我们期望这项建议的结果将确定ER-to高尔基SNAREs BET1和GOSR2引起的CMDs是属于糖代谢异常症还是属于另一亚型。

Congenital muscular dystrophies (CMDs) are a group of degenerative muscle disorders, characterized by early age of onset and can be classified by function and cellular localization of affected proteins. For example, pathogenic variants in subunits of the dystrophin-glyco-protein complex (DGC) have been identified, including Duchenne muscular dystrophy and dystroglycanopathies, caused by mutations in the genes encoding dystrophin and dystro-glycan, respectively. Both proteins are central for the DGC, which is an important cell adhesion receptor complex maintaining muscle integrity by linking the actin cytoskeleton to the basal lamina in skeletal muscle cells. Dystroglycan is a protein precursor and is cleaved into peripheral, highly glycosylated a- and a transmembrane b-dystroglycan. Glycosylation of a-dystroglycan is vital for its function, underscored by the findings that also variants in glycosyl¬transferases that modify a-dystroglycan cause muscular dystrophy. Furthermore, variants affecting components of the Endoplasmic Reticulum (ER)-to-Golgi membrane trafficking machinery such as TRAPPC11, GOSR2 and BET1 have been added recently to the list of inherited CMDs. GOSR2 and BET1 are complex partners and SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins that are essential for docking and fusion of vesicle-mediated membrane trafficking between the ER, the ER-Golgi intermediate com-part¬ment (ERGIC) and the Golgi. We and others have shown that biallelic variants in BET1 and GOSR2 are loss-of-function mutations, causing CMD/epilepsy syndromes. However, the molecular mechanism of disease progression remained elusive. The experiments in this proposal will focus on the role of BET1 and GOSR2 in membrane trafficking of the DG complex in myotubes and in vivo to provide molecular insights into the pathogenesis of muscular disorders. In particular, we will analyze on a molecular level novel biallelic variants of unknown significance identified in GOSR2 in patients presented with congenital microcephaly, epilepsy and myopathy with elevated creatine kinase and lactate levels. We will perform a comparative analysis of GOSR2 and BET1 deficient myoblasts and myotubes to identify common disease pathways. Finally, the characterization of the phenotype of muscle specific GOSR2 deficient mice will allow us to analyze the role of ER-to-Golgi SNAREs in vivo. We expect that the outcome of this proposal will establish if the CMDs caused by the ER-to Golgi SNAREs BET1 and GOSR2 are belonging to the dystroglycanopathies or to another subtype.