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Expression Studies of Other Unconventional Myosins

其他非常规肌球蛋白的表达研究

基本信息

批准号：
7734993
负责人：
JAMES R. SELLERS
金额：
$ 56.33万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7734993
关键词：
ATP phosphohydrolase Actins Affinity Amino Acids Amoeba genus Behavior Binding Biochemical Biological Assay Bite C-terminal Cell Adhesion Cells Charge Class Cyclic AMP-Dependent Protein Kinases Drosophila genus Electron Microscope Electrons Excision Exhibits Filament Goals Human Image Image Analysis Kinetics Length Limulus MYO7A gene Mammals Mechanics Mediating Microscopic Modification Molecular Molecular Conformation Motor Mus Mutation Myosin ATPase Myosin III Myosin Type II N-terminal Numbers Organism Phagocytosis Phosphoproteins Phosphorylation Phosphorylation Site Phosphotransferases Photoreceptors Play Point Mutation Property Proteins Range Regulation Role Second Messenger Systems Signal Transduction Signaling Protein Site Structure System Tail Techniques Translations actin kinase base circadian pacemaker deafness human RHOK protein interest member mutant null mutation optical traps particle protein structure response rhodopsin kinase second messenger single molecule

项目摘要

Myosin VIIa is an unconventional myosin widely expressed in organisms ranging from amoebae to mammals that has been shown to play vital roles in cell adhesion and phagocytosis. We have studied Drosophila myosin VIIa that was expressed in Sf9 cells. We have shown that this myosin has high duty ratio kinetics similar to that of processive motors, but we also showed that it did not easily dimerize even if the full lenght molecule was expressed in Sf9 cells. We have examined the regulation of enzymatic activity of full length myosin VIIa and various C-terminally truncated fragments. Full length myosin VIIa (FLM7a) has a Vmax of about 1 per sec, but has a low apparent affinity for actin, requiring 30-50 uM acin for half-maximal activation. Various C-terminally truncated fragments have a similar Vmax, but much less actin is required for half maximal activation (0.5-1 uM). This means that at 5 uM actin the activity of the fragments is near maximal whereas that of the FLM7a is still barely activated. Removal of even the last 99 amino acids is sufficient to cause this remarkable change in activity. We explored the structural basis for this regulation by using single particle analysis in the electron microscope. We find that in the presence of ATP FLM7a is tightly folded into a compact structure such that the myosin motor domain cannot be discerned whereas in the absence of ATP the molecule is more extended and shows a clearly distinguishable motor domain. Removable of small bits of the tail also is accompanied by the extended conformation leading us to suggest that the compact structure represents an inhibited state of the molecule. Point mutations of a pair of conserved charged groups in the C-terminal tail also result in loss of regulation and unfolding of the molecule. We have expressed the second FERM domain of myosin VIIa and find that it can bind to actin in an ATP dependent manner. We are now searching for binding partners or post-translation modifications that will convert the foled, inactive FLM7a into an extend, active form. We are currently examining the mechanical ability of myosin VIIa using optical trapping nanometry. Preliminary results reveal that this myosin has a long attachment lifetime. We are also studying the localization of GFP-tagged myosin VII and truncation mutants in S2 cells. We are also attempting to make a null mutation in Drosophila myosin VIIb. We have examined the effect of some deafness-associated mutations in the motor domain of human myosin VIIa on the actin activated MgATPase activity and are attempting to express a full length construct of this myosin in the Sf9 system. Myosin XVIIIa has a large predicted coiled-coil forming sequence, but the structure of the myosin has not been examined on a single molecule level. We expressed full length myosin XVIIIa from mouse and showed that it has a long tail similar to that of myosin II. We are examining whether this tail can form filaments. Little is known about the functions of class III unconventional myosins although, with an N-terminal kinase domain, they are potentially both signaling and motor proteins. Limulus myosin III is particularly interesting because it is a phosphoprotein abundant in photoreceptors that becomes more heavily phosphorylated at night by protein kinase A. This enhanced nighttime phosphorylation occurs in response to signals from an endogenous circadian clock and correlates with dramatic changes in photoreceptor structure and function. We seek to understand the role of Limulus myosin III and its phosphorylation in photoreceptors. Here we determined the sites that become phosphorylated in Limulus myosin III and investigated its kinase, actin binding, and myosin ATPase activities. We show that Limulus myosin III exhibits kinase activity and that a major site for both protein kinase A and autophosphorylation is located within loop 2 of the myosin domain, an important actin binding region. We also identify the phosphorylation of an additional protein kinase A and autophosphorylation site near loop 2, and a predicted phosphorylation site within loop 2. We show that the kinase domain of Limulus myosin III shares some pharmacological properties with protein kinase A, and that it is a potential opsin kinase. Finally, we demonstrate that Limulus myosin III binds actin but lacks ATPase activity. We conclude that Limulus myosin III is an actin-binding and signaling protein and speculate that interactions between actin and Limulus myosin III are regulated by both second messenger mediated phosphorylation and autophosphorylation of its myosin domain within and near loop 2.

肌球蛋白VIIa是一种非传统的肌球蛋白，在从阿米巴到哺乳动物的各种生物中广泛表达，已被证明在细胞黏附和吞噬过程中发挥重要作用。我们研究了在Sf9细胞中表达的果蝇肌球蛋白VIIa。我们已经证明了这种肌球蛋白具有类似于前进马达的高占空比动力学，但我们也发现即使全长分子在Sf9细胞中表达，它也不容易二聚化。我们研究了全长肌球蛋白VIIa和各种C末端截短片段的酶活性调节。全长肌球蛋白VIIa(FLM7a)的Vmax约为每秒1个，但对肌动蛋白的表观亲和力较低，需要30-50微米Ac才能达到一半最大激活。各种C末端截短的片段具有相似的Vmax，但需要的肌动蛋白要少得多，才能达到一半的最大激活(0.5-1um)。这意味着在5微米肌动蛋白时，片段的活性接近最大，而FLM7a的活性仍然几乎不被激活。即使去除最后99个氨基酸也足以导致这种显著的活性变化。我们用电子显微镜中的单颗粒分析方法探索了这一规律的结构基础。我们发现，在有ATP存在的情况下，FLM7a被紧密折叠成一个紧凑的结构，使得肌球蛋白的运动域不能被识别，而在没有ATP的情况下，分子更加伸展，显示出一个明显可区分的运动域。尾部小片段的可移除也伴随着延伸的构象，这使得我们认为紧凑的结构代表了分子的抑制状态。C末端一对保守的带电基团的点突变也会导致分子调控的丧失和分子的展开。我们已经表达了肌球蛋白VIIa的第二个FERM结构域，并发现它可以以一种依赖于ATP的方式与肌动蛋白结合。我们现在正在寻找结合伙伴或翻译后修改，以将折叠的、非活性的FLM7a转换为扩展的、活性的形式。我们目前正在使用光学捕获纳米计量学来检测肌球蛋白VIIa的机械能力。初步结果表明，该肌球蛋白具有较长的附着寿命。我们还在研究GFP标记的肌球蛋白VII和截断突变体在S2细胞中的定位。我们还试图在果蝇肌球蛋白VIIB中进行零突变。我们已经检测了人肌球蛋白VIIa运动域中一些与耳聋相关的突变对肌动蛋白激活的镁ATPase活性的影响，并试图在Sf9系统中表达这种肌球蛋白的全长结构。肌球蛋白XVIIIa有一个预测的大卷曲形成序列，但肌球蛋白的结构还没有在单分子水平上进行研究。我们从小鼠中表达了全长的肌球蛋白XVIIIa，并发现它有一条与肌球蛋白II类似的长尾。我们正在研究这条尾巴是否能形成细丝。人们对III类非常规肌球蛋白的功能知之甚少，尽管它们具有N-末端的激酶域，可能既是信号蛋白又是马达蛋白。肌球蛋白III特别有趣，因为它是一种在光感受器中丰富的磷蛋白，在夜间被蛋白激酶A更严重地磷酸化。这种增强的夜间磷酸化发生在响应来自内源生物钟的信号，并与光感受器结构和功能的戏剧性变化相关。我们试图了解蛙肌球蛋白III的作用及其在光感受器中的磷酸化。在这里，我们确定了肌球蛋白III中的磷酸化位点，并研究了它的激酶、肌动蛋白结合和肌球蛋白ATPase活性。我们发现，肌球蛋白III具有激酶活性，蛋白激酶A和自磷酸化的一个主要部位位于肌球蛋白结构域的第二环内，这是一个重要的肌动蛋白结合区。我们还鉴定了一个额外的蛋白激酶A的磷酸化和环2附近的自动磷酸化位点，以及在环2内的一个预测的磷酸化位点。我们表明，肌球蛋白III的激动域与蛋白激酶A具有一些药理特性，是一种潜在的视蛋白激酶。最后，我们证明了肌球蛋白III能结合肌动蛋白，但缺乏ATPase活性。我们推测肌球蛋白III是一种肌动蛋白结合和信号传递蛋白，推测肌动蛋白与肌球蛋白III之间的相互作用受第二信使介导的肌球蛋白结构域在环2及其附近的磷酸化和自磷酸化的调节。