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

先天性肌营养不良症（CMD）是一组退行性肌肉疾病，其特征在于发病年龄较早，并可根据受影响蛋白质的功能和细胞定位进行分类。例如，已经鉴定了肌营养不良蛋白-糖蛋白复合物（DGC）的亚基中的致病性变体，包括杜氏肌营养不良和肌营养不良聚糖病，其分别由编码肌营养不良蛋白和肌营养不良聚糖的基因中的突变引起。这两种蛋白质都是DGC的核心，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中的基底层来维持肌肉完整性。 肌营养不良聚糖是一种蛋白质前体，并被切割成外周的高度糖基化的a-和跨膜b-肌营养不良聚糖。α-肌营养不良蛋白聚糖的糖基化对其功能至关重要，这一发现强调了修饰α-肌营养不良蛋白聚糖的糖基转移酶的变体也会引起肌营养不良。此外，影响内质网（ER）到高尔基体膜运输机制的组分的变体，如TRAPPC 11，GOSR 2和BET 1，最近已被添加到遗传性CMD的列表中。GOSR 2和BET 1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，其对于ER、ER-高尔基体中间室（ERGIC）和高尔基体之间的囊泡介导的膜运输的对接和融合是必需的。我们和其他人已经表明，BET 1和GOSR 2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。 在这个建议中的实验将集中在BET 1和GOSR 2在肌管和体内DG复合物的膜运输中的作用，以提供对肌肉疾病的发病机制的分子见解。特别是，我们将在分子水平上分析在患有先天性小头畸形、癫痫和伴有肌酸激酶和乳酸水平升高的肌病的患者中在GOSR 2中鉴定的意义未知的新型双等位基因变体。我们将对GOSR 2和BET 1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GOSR 2缺陷小鼠的表型的表征将使我们能够分析ER-高尔基体SNARE在体内的作用。我们希望这项提议的结果将确定由ER-高尔基体陷阱BET 1和GOSR 2引起的CMD是否属于肌营养不良聚糖病或另一种亚型。

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.