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

先天性肌营养不良是一组以发病年龄较早为特征的退行性肌肉疾病，可根据受影响蛋白的功能和细胞定位进行分类。例如，dystrophin-Glyco-Protein复合体(DGC)亚单位的致病变异已被鉴定，包括Duchenne肌营养不良症和Duchenne肌营养不良症，分别由编码dystrophin和dystro-Glycan的基因突变引起。这两种蛋白都是DGC的核心，DGC是一种重要的细胞黏附受体复合体，通过将肌动蛋白细胞骨架连接到骨骼肌细胞的基底板来维持肌肉的完整性。营养不良多糖是一种蛋白质前体，被切割成外周的高度糖基化的a-和跨膜b-营养不良聚糖。α-营养不良多糖的糖基化对其功能至关重要，这一发现强调了这一发现，即修改α-营养不良糖链的糖基转移酶的变异也会导致肌肉营养不良。此外，影响内质网(ER)到高尔基体膜运输机制组件的变体，如TRAPPC11、GOSR2和BET1，最近已被添加到遗传CMD的名单中。GOSR2和BET1是复杂的伴侣和SNARE(可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体)蛋白，对于内质网、内质网-高尔基体中间体和高尔基体之间囊泡介导的膜转运的对接和融合是必不可少的。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。这项计划中的实验将集中于BET1和GOSR2在DG复合体在肌管和体内膜运输中的作用，以提供对肌肉疾病发病机制的分子见解。特别是，我们将在分子水平上分析在GOSR2中发现的未知意义的新的双等位基因变异，这些变异出现在先天性小头畸形、癫痫和肌病患者中，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内质网到高尔基体陷阱在体内的作用。我们期望这项提案的结果将确定由ER-to Golgi陷阱BET1和GOSR2引起的CMD是属于血糖营养不良症还是属于另一种亚型。

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.