Niba Emma Tabe Eko, Nishio Hisahide, Wijaya Yogik Onky Silvana, Ar Rochmah Mawaddah, Takarada Toru, Takeuchi Atsuko, Kimizu Tomokazu, Okamoto Kentaro, Saito Toshio, Awano Hiroyuki, Takeshima Yasuhiro, Shinohara Masakazu
Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Hyogo, Japan.
Department of Occupational Therapy, Faculty of Rehabilitation, Kobe Gakuin University, 518 Arise, Ikawadani-cho, Nishi-ku, Kobe 651-2180, Hyogo, Japan.
Genes (Basel). 2022 Jan 24;13(2):205. doi: 10.3390/genes13020205.
Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disease characterized by defects of lower motor neurons. Approximately 95% of SMA patients are homozygous for () gene deletion, while ~5% carry an intragenic mutation. Here, we investigated the stability and oligomerization ability of mutated SMN1 proteins. Plasmids containing wild- and mutant-type cDNA were constructed and transfected into HeLa cells. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated similar abundances of transcripts from the plasmids containing cDNA, but Western blotting showed different expression levels of mutated SMN1 proteins, reflecting the degree of their instability. A mutated SMN1 protein with T274YfsX32 exhibited a much lower expression level than other mutated SMN1 proteins with E134K, Y276H, or Y277C. In immunoprecipitation analysis, the mutated SMN1 protein with T274YfsX32 did not bind to endogenous SMN1 protein in HeLa cells, suggesting that this mutation completely blocks the oligomerization with full-length SMN2 protein in the patient. The patient with T274YfsX32 showed a much more severe phenotype than the other patients with different mutations. In conclusion, the stability and oligomerization ability of mutated SMN1 protein may determine the protein stability and may be associated with the clinical severity of SMA caused by intragenic mutation.
脊髓性肌萎缩症（SMA）是一种常见的常染色体隐性神经肌肉疾病，其特征为下运动神经元缺陷。约95%的SMA患者为（）基因缺失的纯合子，而约5%携带基因内突变。在此，我们研究了突变型SMN1蛋白的稳定性和寡聚化能力。构建了包含野生型和突变型cDNA的质粒，并将其转染至HeLa细胞中。逆转录-聚合酶链反应（RT-PCR）显示，含有cDNA的质粒转录本丰度相似，但蛋白质印迹法显示突变型SMN1蛋白的表达水平不同，反映了它们的不稳定程度。具有T274YfsX32突变的SMN1蛋白的表达水平远低于具有E134K、Y276H或Y277C突变的其他突变型SMN1蛋白。在免疫沉淀分析中，具有T274YfsX32突变的SMN1蛋白在HeLa细胞中不与内源性SMN1蛋白结合，这表明该突变完全阻断了患者体内全长SMN2蛋白的寡聚化。具有T274YfsX32突变的患者表现出比具有不同突变的其他患者严重得多的表型。总之，突变型SMN1蛋白的稳定性和寡聚化能力可能决定蛋白质稳定性，并可能与基因内突变导致的SMA临床严重程度相关。
