Biochemical and Biological Properties of Actins and Myos

肌动蛋白和肌动蛋白的生化和生物学特性

• 批准号: 7321511
• 负责人: EDWARD D KORN
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 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/7321511
• 关键词: Biochemical; Biological; Properties; Actins; Myos

Chemotaxis of bacteria requires regulated methylation of chemoreceptors. However, despite considerable effort in the 1980s, transmethylation has never been established as a component of eukaryotic cell chemotaxis. S-adenosylhomocysteine (SAH), the product formed when the methyl group of the universal donor S-adenosylmethionine (SAM) is transferred to an acceptor molecule, is a potent inhibitor of all transmethylation reactions. In eukaryotic cells, this inhibition is relieved by hydrolysis of SAH to adenosine and homocysteine catalyzed by S-adenosylhomocysteine hydrolase (SAHH), which is an important component of the methylation machinery. We now report that SAHH, which is diffuse in the cytoplasm of non-motile Dictyostelium amoebae and human neutrophils, concentrates with F-actin in pseudopods at the front of motile, chemotaxing cells, but is not present in filopodia or at the very leading edge. Tubercidin, an inhibitor of SAHH, substantially inhibits chemotaxis in both Dictyostelium and neutrophils at concentrations that have little effect on cell viability. Tubercidin does not inhibit starvation-induced expression of the cAMP receptor, cAR1, nor does it inhibit the G protein-mediated stimulation of adenylyl cyclase activity and actin polymerization in Dictyostelium. Tubercidin has no effect on either capping of concanavalin A receptors or phagocytosis in Dictyostelium. These results add SAHH to the list of proteins that redistribute in response to chemotactic signals in Dictyostelium and neutrophils, and strongly suggest a role for transmethylation in chemotaxis of eukaryotic cells. Dictyostelium actin was previously shown to become phosphorylated on Tyr53 late in the developmental cycle and when cells in the amoeboid stage are subjected to stress, but the phosphorylated actin had not been purified and characterized. We have separated phosphorylated and unphosphorylated actin and shown that Tyr53-phosphorylation substantially reduces actin's ability to inactivate DNase I, increases actin's critical concentration, and greatly reduces its rate of polymerization. Tyr53-phosphorylation substantially, if not completely, inhibits nucleation and elongation from the pointed-end of actin filaments, and reduces the rate of elongation from the barbed-end. Negatively stained electron microscopic images of polymerized Tyr53-phosphorylated actin show a variable mixture of small oligomers and filaments, which are converted to more typical, long filaments upon addition of myosin subfragment 1. Tyr53-phosphorylated and unphosphorylated actin co-polymerize in vitro, and phosphorylated and unphosphorylated actin co-localize in amoebae. Tyr53-phosphorylation does not affect the ability of filamentous actin to activate myosin ATPase. The structure of the tail of the heavy chain of Acanthamoeba myosin IC (AMIC), a 466 amino acid residue protein, was investigated to determine if there is any interaction between its N-terminal and C-terminal regions of the protein, as was suggested by cryo-electron microscopy (Ishikawa, T. et al., 2004). The tail comprises an N-terminal 220-residue basic region (BR) followed by a 56-residue Gly/Pro/Ala-rich region (GPA1), a 55-residue SH3 domain and a C-terminal 135-residue region (GPA2). Comparison of NMR spectra of the full-length tail and the individually expressed BR and GPA1-SH3-GPA2 regions showed some specific differences, implying an interaction between the N-terminal and C-terminal regions of the full-length tail. From the results of the NMR experiments, we assigned many of the BR and GPA1-SH3-GPA2 amino acid residues that are critical for the interaction. By combining homology modeling with the NMR data, we suggest how the two regions might interact in the full length AMIC tail. Further, we suggest that the N-terminal basic region contains a PH (pleckstrin homology) domain, consistent with the predominant association of AMIC with plasma membranes and large vacuole and contractile vacuole membranes.

细菌的趋化性需要化学感受器的甲基化。然而，尽管在20世纪80年代做出了相当大的努力，反甲基化从未被确立为真核细胞趋化性的一个组成部分。S-腺苷同型半胱氨酸是万能供体S-腺苷甲硫氨酸的甲基转移到受体分子上形成的产物，是所有转甲基化反应的有效抑制物。在真核细胞中，这种抑制是通过S同型半胱氨酸腺苷水解酶催化的腺苷和同型半胱氨酸的水解酶来解除的，腺苷同型半胱氨酸水解酶是甲基化机制的重要组成部分。我们现在报告SAHH，它弥漫在非运动性的Dictyostelialamombae和人中性粒细胞的细胞质中，与F-肌动蛋白一起集中在运动的趋化细胞的前部的伪足中，但不存在于丝状伪足或最前缘。结节杀菌素是一种SAHH的抑制剂，在对细胞活力几乎没有影响的浓度下，它实质上抑制了网囊和中性粒细胞的趋化作用。结节菌素不抑制饥饿诱导的cAMP受体cAR1的表达，也不抑制G蛋白介导的腺酰环化酶活性的刺激和网柄苔藓中肌动蛋白的聚合。结节菌素对刀豆球蛋白A受体的封闭性或对网柄苔藓的吞噬作用均无影响。这些结果将SAHH添加到Dictyostelials和中性粒细胞中响应趋化信号而重新分布的蛋白质列表中，并强烈表明反甲基化在真核细胞趋化中的作用。 以往的研究表明，在发育周期的晚期和阿米巴阶段的细胞中，Dictyostelialactin在Tyr53上发生了磷酸化，但这种磷酸化的肌动蛋白尚未得到纯化和鉴定。我们已经分离了磷酸化和非磷酸化的肌动蛋白，并表明Tyr53-磷酸化大大降低了肌动蛋白灭活DNase I的能力，增加了肌动蛋白的临界浓度，并大大降低了其聚合速度。Tyr53的磷酸化即使不能完全抑制肌动蛋白细丝尖端的成核和伸长，也会降低从带刺末端的伸长率。聚合的Tyr53-磷酸化肌动蛋白的负染电子显微镜图像显示了一种不同的小寡聚体和细丝的混合物，当加入肌球蛋白亚片段1后，它们被转化为更典型的长丝。Tyr53-磷酸化和非磷酸化的肌动蛋白在体外共聚合，磷酸化和非磷酸化的肌动蛋白在阿米巴中共定位。Tyr53-磷酸化不影响丝状肌动蛋白激活肌球蛋白ATPase的能力。 研究了棘阿米巴肌球蛋白IC(AMIC)重链尾部的结构，以确定其N端和C端之间是否存在冷冻电子显微镜所提示的相互作用(Ishikawa，T.等，2004)。该尾部包括N-末端220-残基碱性区域(BR)，随后是56-残基Gly/Pro/Ala富集区(GPA1)、55-残基SH3结构域和C-末端135-残基区域(GPA2)。将全长尾部与单独表达的BR和GPA1-SH3-GPA2区域的核磁共振谱进行了比较，发现了一些特定的差异，表明全长尾部的N-末端和C-末端之间存在相互作用。根据核磁共振实验的结果，我们指定了许多对相互作用至关重要的BR和GPA1-SH3-GPA2氨基酸残基。通过将同源建模与核磁共振数据相结合，我们建议了这两个区域如何在全长AMIC尾部相互作用。此外，我们认为N-末端碱性区域包含一个PH(Pleckstrin Homology)结构域，这与AMIC与质膜以及大液泡和收缩液泡膜的主要结合一致。