Effects of specific disease mutations in non-muscle myosin 2A on its structure and function.
Effects of specific disease mutations in non-muscle myosin 2A on its structure and function.
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DOI：
10.1016/j.jbc.2023.105514
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发表时间：
2024-01
影响因子：
4.8
通讯作者：
Peckham, Michelle
中科院分区：
文献类型：
--
作者：
Casas-Mao, David;Carrington, Glenn;Pujol, Marta Giralt;Peckham, Michelle
非肌凝蛋白2A （NM2A）是一种广泛表达的2类肌凝蛋白，对细胞中肌动蛋白丝的组织起重要作用。它在紧凑的无活性10S状态和丝状状态之间循环，在这种状态下，它的调节轻链（RLC）被去磷酸化，而在丝状状态下，肌凝蛋白头部与肌动蛋白相互作用，RLC被磷酸化。在编码NM2A重链的基因MYH9中，已有超过170个错义突变被描述。这些导致MYH9疾病，一种常染色体显性疾病，可导致出血性疾病、肾病、白内障和耳聋。大约三分之二的突变发生在盘绕的尾巴上。这些突变可能破坏10S状态和/或破坏丝的形成，或两者兼而有之。为了验证这一点，我们使用多种方法确定了六种特定突变的影响，包括圆二色性检测二级结构的变化，阴性染色电镜分析体外10S和丝的形成，以及GFP-NM2A在固定细胞和活细胞中的成像以确定丝的组装和动力学。D1424的两个突变（D1424G和D1424N）和V1516M强烈降低了10S稳定性，对体外成丝的影响有限。相比之下，D1447和E1841K突变对10S稳定性的影响较小，但在体外增加了丝长。所有突变体在细胞中的动态行为都发生了改变。因此，突变残基的位置及其在灯丝形成和10S稳定中的作用是了解它们在疾病中对NM2A的贡献的关键。
Non-muscle myosin 2A (NM2A), a widely expressed class 2 myosin, is important for organizing actin filaments in cells. It cycles between a compact inactive 10S state in which its regulatory light chain (RLC) is dephosphorylated and a filamentous state in which the myosin heads interact with actin, and the RLC is phosphorylated. Over 170 missense mutations in MYH9, the gene that encodes the NM2A heavy chain, have been described. These cause MYH9 disease, an autosomal-dominant disorder that leads to bleeding disorders, kidney disease, cataracts, and deafness. Approximately two-thirds of these mutations occur in the coiled-coil tail. These mutations could destabilize the 10S state and/or disrupt filament formation or both. To test this, we determined the effects of six specific mutations using multiple approaches, including circular dichroism to detect changes in secondary structure, negative stain electron microscopy to analyze 10S and filament formation in vitro, and imaging of GFP-NM2A in fixed and live cells to determine filament assembly and dynamics. Two mutations in D1424 (D1424G and D1424N) and V1516M strongly decrease 10S stability and have limited effects on filament formation in vitro. In contrast, mutations in D1447 and E1841K, decrease 10S stability less strongly but increase filament lengths in vitro. The dynamic behavior of all mutants was altered in cells. Thus, the positions of mutated residues and their roles in filament formation and 10S stabilization are key to understanding their contributions to NM2A in disease.