Amarasinghe Chinthaka, Hossain M Moazzem, Jin J-P
Department of Physiology, Wayne State University School of Medicine , Detroit, Michigan 48201, United States.
Biochemistry. 2016 Aug 16;55(32):4560-7. doi: 10.1021/acs.biochem.6b00577. Epub 2016 Aug 2.
Troponin T (TnT) is the tropomyosin (Tm)-binding and thin filament-anchoring subunit of troponin and plays a central role in striated muscle contraction. A nonsense mutation in exon 11 of the TNNT1 gene encoding slow skeletal muscle troponin T (ssTnT) truncating the polypeptide chain at Glu(180) causes a lethal recessive nemaline myopathy (NM) in the Amish (ANM). More TNNT1 NM mutations have been reported recently with similar recessive phenotypes. A nonsense mutation in exon 9 causes truncation at Ser(108), and a splicing site mutation causes truncation at Leu(203). Another splicing site mutation causes an internal deletion of the 39 exon 8-encoded amino acids. We engineered and characterized these ssTnT mutants to demonstrate that the Ser(108) truncation exhibits a Tm binding affinity lower than that of the ANM Glu(180) truncation, indicating a partial loss of Tm-binding site 1. Despite the presence of Tm-binding sites 1 and 2, ssTnT truncated at Leu(203) binds Tm with decreased affinity, consistent with its recessive NM phenotype and the requirement of troponin complex formation for high-affinity binding of TnT to Tm. The exon 8-deleted ssTnT has a partial loss of Tm-binding site 1 but retains high-affinity Tm-binding site 2. However, exon 8-deleted ssTnT exhibits a dramatically diminished Tm binding affinity, indicating a long-range conformational effect of this middle region deletion. Predicted from the TnT structure-function relationship, removal of the N-terminal variable region partially rescued this negative impact. These novel findings lay a foundation for understanding the pathogenesis of TNNT1 myopathies and provide insights into the development of targeted treatment.
肌钙蛋白T（TnT）是肌钙蛋白中与原肌球蛋白（Tm）结合并锚定细肌丝的亚基，在横纹肌收缩中起核心作用。编码慢肌骨骼肌肌钙蛋白T（ssTnT）的TNNT1基因第11外显子中的一个无义突变，导致多肽链在Glu（180）处截断，在阿米什人（ANM）中引起致死性隐性杆状体肌病（NM）。最近报道了更多具有相似隐性表型的TNNT1 NM突变。第9外显子中的一个无义突变导致在Ser（108）处截断，一个剪接位点突变导致在Leu（203）处截断。另一个剪接位点突变导致第8外显子编码的39个氨基酸内部缺失。我们构建并表征了这些ssTnT突变体，以证明Ser（108）截断表现出比ANM Glu（180）截断更低的Tm结合亲和力，表明Tm结合位点1部分丧失。尽管存在Tm结合位点1和2，但在Leu（203）处截断的ssTnT与Tm的结合亲和力降低，这与其隐性NM表型以及TnT与Tm高亲和力结合所需的肌钙蛋白复合物形成一致。缺失第8外显子的ssTnT具有部分丧失的Tm结合位点1，但保留了高亲和力的Tm结合位点2。然而，缺失第8外显子的ssTnT表现出显著降低的Tm结合亲和力，表明该中间区域缺失具有远距离构象效应。根据TnT结构-功能关系预测，去除N端可变区部分挽救了这种负面影响。这些新发现为理解TNNT1肌病的发病机制奠定了基础，并为靶向治疗的开发提供了见解。
