泛素E3连接酶RBCK1基因突变导致葡聚糖体累积症的分子机制研究

Polyglucosan body diseases are characterized by the pathological accumulation of polyglucosan bodies, which consist of unspecific polysaccharides as a consequence of a defective glycogen metabolism. A subset of patients with glycogen storage diseases was found to have underlying mutations in the RBCK1 gene. Patients with biallelic loss-of-function mutations resulting in RBCK1 deficiency usually suffer from progressive muscular weakness and dilated cardiomyopathy. However, the molecular mechanism of RBCK1 mutation-caused glycogen storage diseases is unknown. Alpha amylase is an oligosaccharide endoglycosidase, an enzyme that catalyzes the hydrolysis of starch into sugars. In our previous work, we identified the α-amylase as an interaction protein of RBCK1 by BioID methods. RBCK1 promotes the non-degradable ubiquitination modification of α-amylase and enhances the activity of α-amylase to degrade intracellular glycogen. The glycogen storage diseases-derived RBCK1 mutants lost the capacity to promote α-amylase ubiquitination. We also found that α-amylase is expressed in muscle tissues. Taken together, these results suggest that alpha-amylase may have a new role in promoting glycogenolysis in muscle cells, in addition to digesting starch in secreted form. This study intends to reveal the key role of the "RBCK1-α-amylase" regulatory axis in muscle tissue glycogen metabolism at the biochemical, cellular and animal levels, and provide a theoretical basis for targeted therapy in patients of glycogen storage diseases with RBCK1 mutations.

葡聚糖体累积症是一类糖原代谢异常的遗传性疾病。泛素E3连接酶RBCK1突变可导致糖原累积症，病人肌肉组织中糖原异常累积，导致肌无力和心肌病。但RBCK1突变致病的分子机理不明。α-淀粉酶（α-amylase）是一类水解淀粉的内切糖苷酶。前期工作中，我们通过BioID技术鉴定到α-amylase是RBCK1的互作蛋白，RBCK1介导了α-amylase的非降解型泛素化修饰，增强了α-amylase对胞内糖原的分解作用，糖原累积症来源的RBCK1突变体则丧失了对α-amylase的泛素化修饰。同时我们发现α-amylase在肌肉组织中表达。这些结果提示α-amylase除了以分泌形式消化淀粉外，还可能具有促进肌肉细胞内糖原分解的新作用。本项研究拟从生化、细胞和动物水平揭示“RBCK1-α-amylase”调节轴在肌肉组织糖原代谢中的关键作用，为后续RBCK1突变患者的靶向治疗提供理论依据。

葡聚糖体累积症是一类糖原代谢异常的遗传性疾病。泛素E3连接酶RBCK1突变可导致免疫系统缺陷，这和其作为LUBAC复合体成分之一调控NF-κB信号通路有关。另外RBCK1突变可以导致糖原累积症，病人肌肉组织中糖原异常累积，导致肌无力和心肌病。但RBCK1突变代谢疾病的分子机理不明。α-淀粉酶（α-amylase）是一类水解淀粉的内切糖苷酶。通常认为分泌到消化道起到分解进食的淀粉的作用。在本项目中，我们通过BioID 技术鉴定RBCK1的互作蛋白来解析其参与的生物学过程。我们发现了和糖原代谢相关的蛋白α-amylase是RBCK1的互作蛋白，后续实验证实RBCK1介导了α-amylase的非降解型泛素化修饰，增强了α-amylase对胞内糖原的分解作用，糖原累积症来源的RBCK1突变体则丧失了对α-amylase的泛素化修饰。同时我们发现α-amylase在肌肉组织中表达。这些结果提示α-amylase除了以分泌形式消化淀粉外，还具有促进肌肉细胞内糖原分解的新作用。我们构建了RBCK的基因敲除小鼠，发现小鼠的肌肉组织糖原积累。本项研究从生化、细胞和动物水平揭示了“RBCK1-α-amylase”调节轴在肌肉组织糖原代谢中的关键作用，为后续RBCK1突变患者的靶向治疗提供理论依据。

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