Institute of Neuropathology, University Hospital Erlangen, Erlangen, Germany.
Neuromuscular Research Department, Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria.
Neuropathol Appl Neurobiol. 2019 Aug;45(5):476-494. doi: 10.1111/nan.12516. Epub 2018 Sep 26.
We investigated newly generated immortalized heterozygous and homozygous R349P desmin knock-in myoblasts in conjunction with the corresponding desminopathy mice as models for desminopathies to analyse major protein quality control processes in response to the presence of R349P mutant desmin.
We used hetero- and homozygous R349P desmin knock-in mice for analyses and for crossbreeding with p53 knock-out mice to generate immortalized R349P desmin knock-in skeletal muscle myoblasts and myotubes. Skeletal muscle sections and cultured muscle cells were investigated by indirect immunofluorescence microscopy, proteasomal activity measurements and immunoblotting addressing autophagy rate, chaperone-assisted selective autophagy and heat shock protein levels. Muscle sections were further analysed by transmission and immunogold electron microscopy.
We demonstrate that mutant desmin (i) increases proteasomal activity, (ii) stimulates macroautophagy, (iii) dysregulates the chaperone assisted selective autophagy and (iv) elevates the protein levels of αB-crystallin and Hsp27. Both αB-crystallin and Hsp27 as well as Hsp90 displayed translocation patterns from Z-discs as well as Z-I junctions, respectively, to the level of sarcomeric I-bands in dominant and recessive desminopathies.
Our findings demonstrate that the presence of R349P mutant desmin causes a general imbalance in skeletal muscle protein homeostasis via aberrant activity of all major protein quality control systems. The augmented activity of these systems and the subcellular shift of essential heat shock proteins may deleteriously contribute to the previously observed increased turnover of desmin itself and desmin-binding partners, which triggers progressive dysfunction of the extrasarcomeric cytoskeleton and the myofibrillar apparatus in the course of the development of desminopathies.
我们研究了新生成的杂合和纯合 R349P 中间丝蛋白肌营养不良 knock-in 成肌细胞以及相应的中间丝蛋白病小鼠模型，以分析主要的蛋白质质量控制过程，以应对 R349P 突变中间丝蛋白的存在。
我们使用杂合和纯合 R349P 中间丝蛋白 knock-in 小鼠进行分析，并与 p53 knock-out 小鼠杂交，以生成永生化 R349P 中间丝蛋白 knock-in 骨骼肌成肌细胞和肌管。通过间接免疫荧光显微镜、蛋白酶体活性测量和免疫印迹分析自噬率、伴侣辅助选择性自噬和热休克蛋白水平，研究骨骼肌切片和培养的肌肉细胞。肌肉切片进一步通过透射和免疫金电子显微镜分析。
我们证明突变中间丝蛋白（i）增加蛋白酶体活性，（ii）刺激巨自噬，（iii）失调伴侣辅助选择性自噬，（iv）增加 αB-晶体蛋白和 Hsp27 的蛋白水平。αB-晶体蛋白和 Hsp27 以及 Hsp90 均显示出从 Z 盘以及 Z-I 连接部向肌节 I 带水平的转位模式，分别在显性和隐性中间丝蛋白病中。
我们的研究结果表明，R349P 突变中间丝蛋白的存在导致骨骼肌蛋白质动态平衡的普遍失衡，通过所有主要蛋白质质量控制系统的异常活性。这些系统的增强活性和必需热休克蛋白的亚细胞移位可能对先前观察到的中间丝蛋白本身和中间丝蛋白结合伴侣的周转率增加产生有害影响，这触发了中间丝蛋白病发展过程中细胞外肌细胞骨架和肌原纤维装置的进行性功能障碍。
