Biochemical and Biological Properties of Actins and Myosins

今年的研究重点是：（1）阿米巴肌球蛋白IC（AMIC）定位于特定膜的分子基础，（2）S-腺苷高半胱氨酸水解酶（SAHH）在趋化性中的作用，以及（3）Tyr53-磷酸化肌动蛋白（pY53-actin）的性质。 (1)AMIC的466个残基的尾部具有N-末端碱性区，随后是Gly/Pro/Ala富集（GPA）区、SH3结构域和C-末端GPA区。我们之前已经证明（Hwang et al.，2007），碱性区含有plekstrin同源（PH）结构域，并推测PH结构域可能参与AMIC与膜和酸性磷脂囊泡的高亲和力结合，已知这是通过碱性区介导的（Doberstein & Pollard，1992）。 我们发现AMIC与含有磷酸肌醇二磷酸（PIP2）的单层磷脂酰胆碱囊泡的亲和力高于与含有磷脂酰丝氨酸（PS）的囊泡的亲和力。更紧密的结合完全归因于PIP2的5倍更高的负电荷，例如，AMIC以相等的亲和力与含有5%PIP2或25%PS的囊泡结合。正如预期的那样，尾部的碱性区域对于结合是必要的，也是足够的。 正在进行的工作与适当的缺失突变体将确定是否PH结构域在碱性区域所需的高亲和力结合，或如果结合是由于简单的基本电荷。 我们还发现，AMIC与含有5%PIP2 + 25%PS的囊泡的亲和力比仅含有25%PS的囊泡高10倍，这表明AMIC可能靶向阿米巴中膜的PIP2富集区域。与此假设一致，我们发现AMIC和PIP2共定位在内吞杯，伪足，膜皱褶和运动细胞的前缘。 (2)去年，我们报道了S-腺苷高半胱氨酸水解酶（SAHH），所有真核细胞中转甲基化途径的重要组分，在静息细胞的细胞质中扩散，但在化学趋化性网柄阿米巴和人嗜中性粒细胞的前部与聚合肌动蛋白一起定位（Shu et al.，2006年）。这些结果将SAHH添加到重新定位到趋化细胞的前部或后部的蛋白质列表中，并表明甲基化在趋化性中的作用，这进一步得到了杀结核菌素（抑制SAHH的腺苷类似物）抑制趋化性的观察结果的支持。 今年更广泛的研究表明，杀结核菌素对cAMP诱导的个体Dictyosteomyomeoeclavia的趋化性几乎没有影响，但延迟了细胞流的形成，这是Dictyosteomyoeclavia发育周期中的一个重要步骤。分离SAHH敲除细胞的尝试是不成功的，但是创建了仅表达正常量的SAHH的25%的网骨藻细胞系。SAHH敲低细胞的表型不同于暴露于杀结核菌素的野生型细胞的表型，表明杀结核菌素对趋化性的抑制可能不是由于SAHH的抑制。这并不奇怪，因为腺苷类似物如杀结核菌素可能抑制许多生化途径。 (3)酪氨酸53-磷酸化肌动蛋白（pY53-actin）在网骨藻发育周期的后期形成，并且也在变形阶段的细胞受到应激时形成。去年，我们纯化了pY53-肌动蛋白，并表明Tyr53-磷酸化增加了肌动蛋白的临界浓度，降低了聚合速率，基本上抑制了肌动蛋白丝尖端的成核和伸长，并适度降低了倒刺末端的伸长速率（Liu et al.，2006年）。在聚合条件下，pY53-肌动蛋白形成小的寡聚体，在加入肌球蛋白亚片段1后，这些寡聚体转化为典型的长丝，肌球蛋白亚片段1通常被丝状pY53-肌动蛋白激活。 今年的努力集中在为我们在宾夕法尼亚大学的合作者Roberto Dominguez和Kyuwon Baek博士获得足够的pY53-actin，以通过X射线晶体学确定其原子结构。分析了未磷酸化肌动蛋白与profilin I和VASP肽的三元复合物的晶体结构。 已经获得了pY53-肌动蛋白的小晶体，但尚未获得适于衍射研究的pY53-肌动蛋白晶体。

Research this year was focused on: (1) the molecular basis of the localization of Acanthamoeba myosin IC (AMIC) to specific membranes, (2) the role of S-adenosylhomocysteine hydrolase (SAHH) in chemotaxis, and (3) the properties of Tyr53-phosphorylated actin (pY53-actin). (1) The 466-residue tail of AMIC has an N-terminal basic region followed by a Gly/Pro/Ala-rich (GPA) region, an SH3 domain, and a C-terminal GPA region. We had shown previously (Hwang et al., 2007) that the basic region contains a plekstrin homology (PH) domain, and speculated that the PH domain might be involved in the high-affinity binding of AMIC to membranes and acidic phospholipid vesicles, which is known to be mediated through the basic region (Doberstein & Pollard, 1992). We found that AMIC binds with higher affinity to unilamellar phosphatidylcholine vesicles containing phosphoinositol bisphosphate (PIP2)than to vesicles containing phosphatidylserine (PS). The tighter binding is fully attributable to the 5-fold higher negative charge of PIP2, for example, AMIC binds with equal affinity to vesicles containing 5% PIP2 or 25% PS. As expected, the basic region in the tail is both necessary and sufficient for binding. Ongoing work with appropriate deletion mutants will determine if the PH domain in the basic region is required for high affinity binding, or if binding is due simply to the basic charge. We found also that AMIC binds with 10-fold higher affinity to vesicles containing 5% PIP2+25% PS than to vesicles containing only 25% PS, which suggests that AMIC might be targeted to PIP2-enriched regions of membranes in the amoebae. Consistent with this hypothesis, we find that AMIC and PIP2 co-localize at endocytic cups, pseudopods, membrane ruffles and the leading edge of motile cells. (2) Last year we reported that S-adenosylhomocysteine hydrolase (SAHH), an essential component of the trans-methylation pathway in all eukaryotic cells, is diffuse in the cytoplasm of resting cells but localizes with polymerized actin at the front of both chemotaxing Dictyostelium amoebae and human neutrophils (Shu et al., 2006). These results add SAHH to the list of proteins that relocate to either the front or rear of a chemotaxing cell, and suggest a role for methylation in chemotaxis, which was further supported by the observation that tubercidin (an adenosine analogue that inhibits SAHH) inhibits chemotaxis. More extensive work this year showed that tubercidin has little, if any, effect on cAMP-induced chemotaxis of individual Dictyostelium amoebae, but delays the formation of cell streams, an essential step in the developmental cycle of Dictyostelium. Attempts to isolate SAHH-knockout cells were unsuccessful, but Dictyostelium cell lines were created that express only 25% of the normal amount of SAHH. The phenotype of the SAHH-knockdown cells is different than the phenotype of wild-type cells exposed to tubercidin, suggesting that tubercidin inhibition of chemotaxis may not be due to inhibition of SAHH. This is not surprising given the many biochemical pathways that an adenosine analogue like tubercidin might inhibit. (3) Tyrosine53-phosphorylated actin (pY53-actin) forms late in the Dictyostelium developmental cycle, and also when cells in the amoeboid stage are subjected to stress. Last year, we purified pY53-actin and showed that Tyr53-phosphorylation increases actins critical concentration, reduces the rate of polymerization, substantially inhibits nucleation and elongation from the pointed-end of actin filaments, and moderately reduces the rate of elongation from the barbed-end (Liu et al., 2006). Under polymerization conditions, pY53-actin forms small oligomers that are converted to typical long filaments upon addition of myosin subfragment 1, which is activated normally by filamentous pY53-actin. Efforts this year were concentrated on obtaining sufficient pY53-actin for our collaborators at the University of Pennsylvania, Drs. Roberto Dominguez and Kyuwon Baek, to determine its atomic structure by X-ray crystallography. The crystal structure of unphosphorylated actin in a ternary complex with profilin I and a VASP peptide has been analyzed. Small crystals of pY53-actin have been obtained, but crystals of pY53-actin suitable for diffraction studies have not yet been obtained.