Proteasomal Inhibition Restores Biological Function of Mis-sense Mutated Dysferlin in Patient-derived Muscle Cells (Retracted article. See vol. 292, pg. 12542, 2017)
Proteasomal Inhibition Restores Biological Function of Mis-sense Mutated Dysferlin in Patient-derived Muscle Cells (Retracted article. See vol. 292, pg. 12542, 2017)
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DOI：
10.1074/jbc.m111.329078
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发表时间：
2012-03-23
影响因子：
4.8
通讯作者：
Sinnreich, Michael
中科院分区：
文献类型：
--
作者：
Azakir, Bilal A.;Di Fulvio, Sabrina;Sinnreich, Michael
Dysferlin是一种跨膜蛋白，参与肌肉细胞的表面膜修复。dysferlin的突变导致进行性肌营养不良Miyoshi肌病、肢带型肌营养不良2B和远端前室肌病。Dysferlinopathies以常染色体隐性方式遗传，并且许多患有这种疾病的患者在其两个致病性DYSF等位基因中的至少一个中具有错义突变。这些患者骨骼肌中dysferlin水平显著降低或缺失，表明由错义等位基因编码的dysferlin被细胞质量控制系统快速降解。我们推断，如果从降解中拯救出来，错义突变的dysferlin可能具有生物学功能。我们使用了dysferlin缺陷的人成肌细胞培养物，其含有常见的R555W错义等位基因和DYSF无效等位基因，以及对照人成肌细胞培养物，其含有两个野生型或两个无效等位基因。我们测量了dysferlin蛋白和mRNA水平，激光诱导的质膜伤口的再密封动力学，肌管形成，以及用蛋白酶体抑制剂lactacystin或bortezelatin（万珂）处理人成肌细胞培养物后的细胞活力。我们发现内源性R555W错义突变dysferlin被蛋白酶体系统降解。lactacystin或万珂对蛋白酶体的抑制增加了R555W错义突变dysferlin的水平。这种挽救的蛋白质是功能性的，因为它恢复患者来源的成肌细胞中的质膜重新密封，并逆转它们在肌管形成中的缺陷。硼替佐米和lactacystin在使用的方案中没有引起细胞毒性。我们的研究结果提高了抑制错义突变dysferlin的降解途径的可能性，可用作对具有某些dysferlin错义突变的患者的治疗策略。
Dysferlin is a transmembrane protein implicated in surface membrane repair of muscle cells. Mutations in dysferlin cause the progressive muscular dystrophies Miyoshi myopathy, limb girdle muscular dystrophy 2B, and distal anterior compartment myopathy. Dysferlinopathies are inherited in an autosomal recessive manner, and many patients with this disease harbor mis-sense mutations in at least one of their two pathogenic DYSF alleles. These patients have significantly reduced or absent dysferlin levels in skeletal muscle, suggesting that dysferlin encoded by mis-sense alleles is rapidly degraded by the cellular quality control system. We reasoned that mis-sense mutated dysferlin, if salvaged from degradation, might be biologically functional. We used a dysferlin-deficient human myoblast culture harboring the common R555W mis-sense allele and a DYSF-null allele, as well as control human myoblast cultures harboring either two wild-type or two null alleles. We measured dysferlin protein and mRNA levels, resealing kinetics of laser-induced plasmalemmal wounds, myotube formation, and cellular viability after treatment of the human myoblast cultures with the proteasome inhibitors lactacystin or bortezomib (Velcade). We show that endogenous R555W mis-sense mutated dysferlin is degraded by the proteasomal system. Inhibition of the proteasome by lactacystin or Velcade increases the levels of R555W mis-sense mutated dysferlin. This salvaged protein is functional as it restores plasma membrane resealing in patient-derived myoblasts and reverses their deficit in myotube formation. Bortezomib and lactacystin did not cause cellular toxicity at the regimen used. Our results raise the possibility that inhibition of the degradation pathway of mis-sense mutated dysferlin could be used as a therapeutic strategy for patients harboring certain dysferlin mis-sense mutations.