Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.
Cardiovasc Res. 2011 Jun 1;90(3):513-20. doi: 10.1093/cvr/cvr026. Epub 2011 Jan 24.
In this paper, we tested the hypothesis that different binding affinities of the C-terminus of human cardiac alkali (essential) myosin light chain (A1) isoforms to the IQ1 motif of the myosin lever arm provide a molecular basis for distinct sarcomeric sorting and inotropic activity.
We employed circular dichroism and surface plasmon resonance spectroscopy to investigate structural properties, secondary structures, and protein-protein interactions of a recombinant head-rod fragments of rat cardiac β-myosin heavy chain aa664-915 with alanine-mutated IQ2 domain (rβ-MYH(664-915)IQ(ala4)) and A1 isoforms [human atrial (hALC1) and human ventricular (hVLC-1) light chains]. Double epitope-tagging competition was used to monitor the intracellular localization of exogenously introduced hALC-1 and hVLC-1 constructs in neonatal rat cardiomyocytes. Contractile functions of A1 isoforms were investigated by monitoring shortening and intracellular-free Ca(2+) (Fura-2) of adult rat cardiomyocytes infected with adenoviral (Ad) vectors using hALC-1 or β-galactosidase as expression cassettes. hALC-1 bound more strongly (greater than three-fold lower K(D)) to rβ-MYH(664-915) than did hVLC-1. Sorting specificity of A1 isoforms to sarcomeres of cardiomyocytes rose in the order hVLC-1 to hALC-1. Replacement of endogenous VLC-1 by hALC-1 in adult rat cardiomyocytes increased contractility while the systolic Ca(2+) signal remained unchanged.
Intense myosin binding of hALC-1 provides a mechanism for preferential sarcomeric sorting and Ca(2+)-independent positive inotropic activity.
在本文中，我们检验了这样一个假设，即人源心脏碱（必需）肌球蛋白轻链（A1）同工型 C 端与肌球蛋白杆臂的 IQ1 基序不同的结合亲和力为人源心脏碱（必需）肌球蛋白轻链（A1）同工型提供了一种分子基础，以实现独特的肌节分拣和变力活性。
我们采用圆二色性和表面等离子体共振光谱法研究了重组大鼠心脏β-肌球蛋白重链 aa664-915 的头-杆片段与丙氨酸突变的 IQ2 结构域（rβ-MYH（664-915）IQ（ala4））和 A1 同工型[人心房（hALC1）和人心室（hVLC-1）轻链]的结构特性、二级结构和蛋白质-蛋白质相互作用。采用双表位标记竞争法监测外源性引入的 hALC-1 和 hVLC-1 构建体在新生大鼠心肌细胞中的细胞内定位。通过监测感染腺病毒（Ad）载体的成年大鼠心肌细胞的缩短和细胞内游离 Ca（2+）（Fura-2），研究 A1 同工型的收缩功能，并用 hALC-1 或β-半乳糖苷酶作为表达盒。hALC-1 与 rβ-MYH（664-915）的结合力更强（K（D）低 3 倍以上），而 hVLC-1 的结合力则较弱。A1 同工型对心肌细胞肌节的分拣特异性按 hVLC-1 至 hALC-1 的顺序递增。成年大鼠心肌细胞中内源性 VLC-1 被 hALC-1 取代后，心肌收缩力增加，而收缩期 Ca（2+）信号不变。
hALC-1 与肌球蛋白的强烈结合提供了一种优先肌节分拣和 Ca（2+）独立正变力活性的机制。
