Biochemical and Biological Properties of Myosins

我们感兴趣的是肌球蛋白I类和II类肌球蛋白肌动蛋白激活的ATPase活性调节的分子基础，这些肌球蛋白的生物学作用及其生物活性的调节。一项研究探讨了心肌病(CM)环对网柄菌肌球蛋白II的生化和生物学特性的影响。所有II类肌球蛋白重链的CM环始于Arg残基(其在人类心肌肌球蛋白II中的突变导致家族性肥厚性心肌病。Dictyostelialmyosin II(Arg397-Gln407)的CM环是其生物学功能和生化活动所必需的，即CM环缺失突变体具有生化和生物活性。我们发现，肌球蛋白II的CM环部分替代了肌球蛋白II的CM环，而心肌肌球蛋白II的CM环在支持悬浮培养中的生长、刀豆蛋白A表面受体的封端和体内子实体的丘状发育以及肌动蛋白激活的镁ATPase和体外运动活性方面表现不佳。平滑肌和心肌嵌合体和19个CM-环点突变体支持生长和发育的能力彼此之间有很好的相关性，但与支持刀豆蛋白A受体封顶的能力无关。肌球蛋白的生化和生物活性之间几乎没有总体相关性，但只有支持基本完整生物功能的五个突变结构(测试的21个结构)具有kcat/Kactin值，这是一种催化效率的衡量标准，相当于野生型肌球蛋白。生物和生化功能尤其依赖于第400位的ALA，在那里几乎所有其他II类肌球蛋白都有Val。Arg397的三个点突变相当于那些导致人类肥厚性心肌病的突变，具有最小的生物学影响和不同的生化影响。我们现在正在研究S1结构，以确定肌动球蛋白ATPase循环中的哪个步骤(S)受到ALA/VAL突变的影响。 棘阿米巴肌球蛋白IC的尾部结构域由一个碱性区域(BR)和两个富含Gly/Pro/Ala的区域(GPA1和GPA2)组成，中间隔着一个Src同源区域(SH3)。用WT和截尾突变体修饰的重建冷冻电子显微镜图像显示，BR形成一个约40埃长的椭圆形，从头部区域倾斜发散，GPA1和GPA2区域折叠回到BR区域的肌动蛋白近端。这些研究表明尾部的N端和C端之间存在相互作用，后续正在进行N15和C13标记的尾部结构的核磁共振，以确定这些相互作用的性质。 目前正在进行的有关blbbistatin对网柄苔藓生物学影响的研究与其他学者发表的关于它是肌球蛋白II的特异性抑制剂的研究是一致的，但也表明酶失活的肌球蛋白II可以影响不直接涉及肌球蛋白II的细胞过程。这些结果对于解释blblebistatin对细胞的影响具有重要意义，因为在这些细胞中，肌球蛋白II的生物学作用并不像网柄苔藓中的那样明确。 作为对棘阿米巴PAK(肌球蛋白I重链激酶)研究的延伸，我们发现在HeLa细胞中表达具有结构性活性的RAC显著地增加了肌球蛋白II调节轻链的磷酸化水平，但并不完全是通过激活PAK来实现的，这一点通过使用激酶特异性抑制剂来证明。这是RAC激活肌球蛋白II可能是RAC影响细胞骨架的方式之一的第一个证据。

We are interested in the molecular bases of the regulation of the actin-activated ATPase activities of class-I and class-II myosins, the biological roles of these myosins and the regulation of their biological activities. One study investigated the role of the cardiomyopathy (CM)-loop on the biochemical and biological properties of Dictyostelium myosin II. The CM-loop of the heavy chain of all class-II myosins begins with an Arg residue (whose mutation in human ?-cardiac myosin II results in familial hypertrophic cardiomyopathy. The CM-loop of Dictyostelium myosin II (Arg397-Gln407) is essential for its biological functions and biochemical activities, i.e. the CM-loop deletion mutant is biochemically and biologically inactive. We found that the CM-loop of smooth muscle myosin II substitutes partially and the CM-loop of ?-cardiac myosin II much less well in supporting growth in suspension culture, capping of concanavalin A surface receptors and development of mounds to fruiting bodies in vivo and the actin-activated MgATPase and in vitro motility activities of purified myosins. The abilities of the smooth and cardiac muscle chimeras and 19 CM-loop point mutants to support growth and development correlate well with each other but not with the ability to support capping of concanavalin A receptors. There is little overall correlation between the biochemical and biological activities of the myosins but only the five mutant constructs (of 21 constructs tested) that support essentially full biological function have kcat/Kactin values, a measure of catalytic efficiency, equivalent to wild-type myosin. Biological and biochemical functions are particularly dependent on Ala at position 400, where almost all other class-II myosins have Val. The three point mutations of Arg397 equivalent to those that result in hypertrophic cardiomyopathy in humans had minimal biological effects and different biochemical effects. We are now studying S1 constructs to determine which step(s) in the actomyosin ATPase cycle is affected by the Ala/Val mutation. The tail domain of Acanthamoeba myosin IC consists of a basic region (BR) and two Gly/Pro/Ala-rich regions (GPA1 and GPA2) separated by a Src homology (SH3) region. Reconstructed cryo-electron microscopic images F-actin decorated with WT and truncated tail mutants show that the BR forms a ~40 angstrom-long oval diverging obliquely from the head domain and that the GPA1 and GPA2 regions are folded back on the actin-proximal side of the BR region. These studies, which imply interactions between the N-terminal and C-terminal halves of the tail, are being followed up by NMR of N15 and C13 labeled tail constructs to determine the nature of these interactions. Ongoing studies on the affect of blebbistatin on the biology of Dictyostelium are consistent with it being a specific inhibitor of myosin II, as published by others, but also show that the enzymatically inactivated myosin II can affect cellular processes that do not directly involve myosin II. These results have significant implications for interpreting the effects of blebbistatin on cells in which the biological roles of myosin II are not as clearly defined as they are in Dictyostelium. As an extension of studies of Acanthamoeba PAK (myosin I heavy chain kinase), we found that expression of constitutively active Rac in HeLa cells increases the level of phosphorylation of the regulatory light chain of myosin II substantially, but not exclusively, by activation of PAK, as shown by the use of kinase-specific inhibitors. This is the first evidence that Rac-activation of myosin II may be one of the ways that Rac affects the cytoskeleton,