肌切蛋白的结构与功能多样性

肌切蛋白（scinderin）是一种重要的肌动蛋白结合蛋白,在哺乳动物和脊椎动物中广泛表达.肌切蛋白作为凝溶胶蛋白超家族的成员之一,通过肌动蛋白丝切割、肌动蛋白聚集等方式来控制肌动蛋白的结构.肌切蛋白生物活性具有多样性,除影响肌动蛋白丝重组外,肌切蛋白还参与细胞胞吐作用、调节细胞运动、细胞分化等细胞活动.此外,肌切蛋白在慢性炎症、凝血过程、免疫性疾病和肿瘤发生发展中也发挥了重要作用.本文对肌切蛋白的结构特点、参与调节细胞的功能和机制进行概述.     

凝溶胶蛋白基因的生物学功能及其应用研究进展

凝溶胶蛋白(gelsolin,GSN)是Gelsolin/Villin超家族的核心成员,是一种多功能的钙依赖性肌动蛋白结合蛋白,在细胞中Ca^2+和PIP2等多因素的调控下,对细胞凋亡、吞噬功能、肌动蛋白微丝切割、细胞信号转导等方面起着重要的作用。近年来,凝溶胶蛋白还被频繁用于相关疾病的预防、诊断与治疗,但其在调控细胞凋亡、炎症等病理生理中的作用机制还存在些许争议。本研究综述了凝溶胶蛋白的结构特点、生物学功能以及对疾病的诊断和治疗,旨在了解凝溶胶蛋白在生物医学及动物科学等领域的应用以及未来凝溶胶蛋白的发展前景。     

凝溶胶蛋白对植物微丝的调节作用及在花粉中的定位

以植物花粉为实验材料, 研究了来源于动物的凝溶胶蛋白对植物肌动蛋白丝的作用及凝溶胶蛋白在植物细胞中的定位. 利用离心沉淀丝状肌动蛋白及电子显微镜负染的实验结果显示, 花粉纯化肌动蛋白丝及粗提液中肌动蛋白丝可被凝溶胶蛋白剪切, 而且这种剪切作用依赖于Ca²⁺. 另外, 利用免疫沉淀测定凝溶胶蛋白与花粉肌动蛋白单体结合的实验表明, 当溶液中有Ca²⁺. 存在时, 花粉肌动蛋白与凝溶胶蛋白的摩尔比值约为2.0 ± 0.21; 用EGTA去除Ca²⁺. 后, 该比值有所降低, 减至1.2 ± 0.23; 而加入PIP2后, 这种结合比值又降至0.8 ± 0.10, 表明植物肌动蛋白与凝溶胶蛋白的结合有类似于动物肌动蛋白的特性, 受到Ca²⁺. 和PIP2的调节. 花粉中凝溶胶蛋白的免疫鉴定及免疫荧光定位实验结果表明, 花粉中存在凝溶胶蛋白, 在未萌发的花粉粒中凝溶胶蛋白主要集中在萌发沟处, 而在萌发2 h的花粉管中, 花粉管胞质内均可看到荧光分布, 但花粉管顶端荧光较强, 推测凝溶胶蛋白可能参与花粉的萌发和花粉管的生长.     

凝溶胶蛋白与相关疾病的关系

凝溶胶蛋白（gelsolin,GSN）是一种在机体内普遍存在的,对细胞结构和代谢功能具有多种调节作用的蛋白。GSN作为凝溶胶蛋白超家族的成员之一,是一种重要的肌动蛋白（actin）结合蛋白,可通过切断、封闭肌动蛋白丝,或使actin聚集成核等方式来调控actin的结构与代谢功能.GSN不仅能在重组的肌动蛋白细丝（F-actin）中发挥作用,而且在细胞运动、细胞凋亡等细胞活动中也发挥着重要的作用。GSN有血浆型（plasma gelsolin,pGSN）和细胞质型（cytoplasmic gelsolin,cGSN）两种亚型,它们在淀粉样变性、炎症、癌症、心血管疾病、阿茨海默病（AD）及肾脏疾病中都起着重要的作用,GSN可能成为多种疾病的一个新的生物标记物或者治疗靶点。本文将就GSN与相关疾病的关系的研究进展做一综述。     

凝溶胶蛋白治疗阿尔茨海默症的研究进展

阿尔茨海默症(Alzheimer’s disease,AD)的病理学特征之一是患者脑内存在以β-淀粉样肽(Aβ)为主要成分的老年斑。大量的实验证据表明,以Aβ为靶目标,清除老年斑有助于提高患者的认知能力,是防治AD的一个重要研究方向。凝溶胶蛋白在细胞骨架结构重排和细胞运动等过程中都发挥重要作用。目前多个小组的研究成果显示,凝溶胶蛋白与AD的发生、发展密切相关。凝溶胶蛋白能够抑制Aβ积聚形成纤维,也能够引发已形成的Aβ纤维发生解聚。更重要的是,凝溶胶蛋白能够清除转基因AD模型小鼠脑内的老年斑和降低Aβ的水平。未来凝溶胶蛋白有可能被应用于AD的预防和治疗。     

植物微丝骨架动态变化的调节

由球形肌动蛋白聚合而成的微丝骨架,又称肌动蛋白纤维,它在细胞运动、细胞形态建成以及物质运输等诸多生命活动中发挥重要作用。细胞内微丝的解聚和聚合动态特性是微丝骨架行使功能的重要基础,并受到如微丝结合蛋白、金属离子、小G蛋白等各种因素的严格控制。植物细胞微丝骨架的研究虽然晚于动物细胞,但也取得了飞速发展。本文对植物细胞内微丝骨架动态变化的作用机制及一些主要调节因子的最新研究进展做一介绍。     

肌动蛋白相关蛋白2/3复合体的结构、功能与调节

微丝参与了细胞形态维持及细胞运动等多种重要的细胞过程。微丝由肌动蛋白单体组装而成 ,肌动蛋白相关蛋白 2 / 3(Arp2 /Arp3,Arp2 / 3)复合体在微丝形成过程中起重要作用。Arp2 / 3复合体由 7个亚单位组成 ,在细胞内受到多种核化促进因子的调节 ,并与这些因子协同作用来调节肌动蛋白的核化。Arp2 / 3复合体结构、功能及调节的研究对于阐明微丝形成机制及细胞骨架与某些信号分子的关系有重要意义。     

α辅肌动蛋白的结构和功能

α辅肌动蛋白是近年来在细胞骨架与细胞运动研究中的热点蛋白 .目前发现有α辅肌动蛋白 1、2、3和 4四种类型 ,呈细胞或组织特异性分布 .这四种蛋白的共同结构特征是在细胞内均为反向平行的二聚体 ,并具有N末端肌动蛋白结合结构域 (ABD)、血影蛋白样中央重复结构域和C末端“EF手”结构域 .作为细胞骨架中一种重要的肌动蛋白交联蛋白 ,α辅肌动蛋白通过与其相关蛋白包括整合素 (integrins)、钙粘素 (cadherin)以及细胞信号传导通路中的信号分子等的协同作用 ,在稳定细胞粘附、调节细胞形状及细胞运动中发挥着重要作用 .因此 ,肿瘤的发生、发展和恶化与α辅肌动蛋白的结构、功能密切相关 .本文结合本实验室的研究工作 ,综述了α辅肌动蛋白家族成员的结构、功能及其与肿瘤发生的相关性 .     

植物微丝骨架的研究进展

微丝骨架是细胞骨架的重要组成部分,在各种细胞活动中都发挥着重要作用。微丝骨架的主要组成部分是肌动蛋白和肌动蛋白结合蛋白,参与细胞形态建成、物质运输和信号转导等生命活动。通过鬼笔环肽标记或表达荧光融合蛋白等方法,国内外许多学者对植物微丝骨架的组成、功能等进行了大量的研究,并取得了一些成果。基于前人的研究,本研究从组成、功能及研究方法三个方面对植物微丝骨架的进行概述。     

植物细胞中的前纤维蛋白

肌动蛋白组成的微丝骨架是真核细胞中的重要结构,在体内处于高度动态变化之中,受多种肌动蛋白结合蛋白（actin-binding proteins）的调节。前纤维蛋白（profilin）是一种单体肌动蛋白结合蛋白,存在于所有的真核细胞中,在植物细胞中也得到较多的研究。前纤维蛋白除可以结合单体肌动蛋白之外,还可以与磷脂酰肌醇及富含多聚脯氨酸的蛋白质等多种分子结合,在细胞信号转导中行使着重要的功能。本文结合本实验室的研究结果,概述了前纤维蛋白的最新研究进展。     

Identification of a polyphosphoinositide-binding sequence in an actin monomer-binding domain of gelsolin.

Gelsolin is an actin filament-severing and -capping protein that has profound effects on actin filament organization and assembly. It is activated by Ca2+ and inhibited by polyphosphoinositides (PPI). We have previously shown that PPI inhibit actin filament severing by the amino-terminal half of gelsolin and hypothesized that this is mediated through inhibition of actin filament side binding (by domains II-III of gelsolin), a requisite first step in severing. In this paper, we report that the subsequent step in severing, which is mediated by an actin monomer binding site located in domain I of gelsolin, is also regulated by PPI. We used deletional mutagenesis and a synthetic peptide to locate the sequence required for high affinity PPI binding in domain I. Our results show that the PPI-binding sequence has a basic charge distribution that is also present in the PPI-regulated actin filament side binding domain, and the two gelsolin PPI-binding sites have similar PPI-binding affinities. In addition, a similar motif is present in several other PPI-binding proteins, including a highly conserved region in the phospholipase C family. We propose that the sequences identified in gelsolin may represent a consensus for PPI binding in a variety of proteins.     

Identification of critical functional and regulatory domains in gelsolin

Gelsolin can sever actin filaments, nucleate actin filament assembly, and cap the fast-growing end of actin filaments. These functions are activated by Ca2+ and inhibited by polyphosphoinositides (PPI). We report here studies designed to delineate critical domains within gelsolin by deletional mutagenesis, using COS cells to secrete truncated plasma gelsolin after DNA transfection. Deletion of 11% of gelsolin from the COOH terminus resulted in a major loss of its ability to promote the nucleation step in actin filament assembly, suggesting that a COOH-terminal domain is important in this function. In contrast, derivatives with deletion of 79% of the gelsolin sequence exhibited normal PPI-regulated actin filament-severing activity. Combined with previous results using proteolytic fragments, we deduce that an 11-amino acid sequence in the COOH terminus of the smallest severing gelsolin derivative identified here mediates PPI-regulated binding of gelsolin to the sides of actin filaments before severing. Deletion of only 3% of gelsolin at the COOH terminus, including a dicarboxylic acid sequence similar to that found on the NH2 terminus of actin, resulted in a loss of Ca2+-requirement for filament severing and monomer binding. Since these residues in actin have been implicated as potential binding sites for gelsolin, our results raise the possibility that the analogous sequence at the COOH terminus of gelsolin may act as a Ca2+-regulated pseudosubstrate. However, derivatives with deletion of 69-79% of the COOH-terminal residues of gelsolin exhibited normal Ca2+ regulation of severing activity, establishing the intrinsic Ca2+ regulation of the NH2-terminal region. One or both mechanisms of Ca2+ regulation may occur in members of the gelsolin family of actin-severing proteins.     

Gelsolin Induces Colorectal Tumor Cell Invasion via Modulation of the Urokinase-Type Plasminogen Activator Cascade

Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin's influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix-degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites.     

Gelsolin: calcium- and polyphosphoinositide-regulated actin-modulating protein

Receptor-mediated stimulation induces massive actin polymerization and cyto-skeletal reorganization. The activity of a potent actin-modulating protein, gelsolin, is regulated both by Ca²⁺ and polyphos-phoinositides, and it may have a pivotal role in restructuring the actin cytoskeleton in response to agonist stimulation. Structure-function analysis of gelsolin has (1) indicated that its NH₂-terminal half is primarily responsible for modulating actin filament length and polymerization; and (2) elucidated mechanisms by which Ca²⁺ and phospholipids may regulate such functions. Gelsolin is functionally and structurally similar to villin, another Ca²⁺-activated actin-severing protein found in microvilli, suggesting that gelsolin may be a prototype of this family of actin-modulating proteins. A molecular variant of gelsolin is secreted and may be involved in the clearance of actin filaments released during tissue damage. The two forms of gelsolin are encoded by a single gene, and distinct messages are derived by alternative message splicing.     

Interactions between the actin filament capping and severing protein gelsolin and the molecular chaperone CCT: evidence for nonclassical substrate interactions

CCT is a member of the chaperonin family of molecular chaperones and consists of eight distinct subunit species which occupy fixed positions within the chaperonin rings. The activity of CCT is closely linked to the integrity of the cytoskeleton as newly synthesized actin and tubulin monomers are dependent upon CCT to reach their native conformations. Furthermore, an additional role for CCT involving interactions with assembling/assembled microfilaments and microtubules is emerging. CCT is also known to interact with other proteins, only some of which will be genuine folding substrates. Here, we identify the actin filament remodeling protein gelsolin as a CCT-binding partner, and although it does not behave as a classical folding substrate, gelsolin binds to CCT with a degree of specificity. In cultured cells, the levels of CCT monomers affect levels of gelsolin, suggesting an additional link between CCT and the actin cytoskeleton that is mediated via the actin filament severing and capping protein gelsolin.     

Functional comparison of villin and gelsolin. Effects of Ca2+, KCl, and polyphosphoinositides

A family of homologous actin-binding proteins sever and cap actin filaments and accelerate actin filament assembly. The functions of two of these proteins, villin and gelsolin, and of their proteolytically derived actin binding domains were compared directly by measuring their effects, under various ionic conditions, on the rates and extents of polymerization of pyrene-labeled actin. In 1 mM Ca2+ and 150 mM KCl, villin and gelsolin have similar severing and polymerization-accelerating properties. Decreasing [Ca2+] to 25 microM greatly reduces severing by villin but not gelsolin. Decreasing [KCl] from 150 to 10 mM at 25 microM Ca2+ increases severing by villin, but not gelsolin, over 10-fold. The C-terminal half domains of both proteins have Ca2+-sensitive actin monomer-binding properties, but neither severs filaments nor accelerates polymerization. The N-terminal halves of villin and gelsolin contain all the filament-severing activity of the intact proteins. Severing by gelsolin's N-terminal half is Ca2+-independent, but that of villin has the same Ca2+ requirement as intact villin. The difference in Ca2+ sensitivity extends to 14-kDa N-terminal fragments which bind actin monomers and filament ends, requiring Ca2+ in the case of villin but not gelsolin. Severing of filaments by villin and its N-terminal half is shown to be inhibited by phosphatidylinositol 4,5-bisphosphate, as shown previously for gelsolin (Janmey, P.A., and Stossel, T.P. (1987) Nature 325, 362-364). The functional similarities of villin and gelsolin correlate with known structural features, and the greater functional dependence of villin on Ca2+ compared to gelsolin is traced to differences in their N-terminal domains.     

Tropomyosin and gelsolin cooperate in controlling the microfilament system

Tropomyosin has been shown to cause annealing of gelsolin-capped actin filaments. Here we show that tropomyosin is highly efficient in transforming even the smallest gelsolin-actin complexes into long actin filaments. At low concentrations of tropomyosin, the effect of tropomyosin depends on the length of the actin oligomer, and the cooperative nature of the process is a direct indication that tropomyosin induces a conformational change in the gelsolin-actin complexes, altering the structure at the actin (+) end such that capping by gelsolin is abolished. At increased concentrations of tropomyosin, heterodimers, trimers, and tetramers are converted to actin filaments. In addition, evidence is presented demonstrating that gelsolin, once removed from the (+) end of the actin, can reassociate with the newly formed tropomyosin-decorated actin filaments. Interestingly, the binding of gelsolin to the tropomyosin-actin filament complexes saturates at 2 gelsolin molecules per 14 actin and 2 tropomyosins, i.e. two gelsolins per tropomyosin-regulatory unit along the filament. These observations support the view that both tropomyosin and gelsolin are likely to have important functions in addition to those proposed earlier.     

Chimeric and truncated gCap39 elucidate the requirements for actin filament severing and end capping by the gelsolin family of proteins.

gCap39 is an actin filament end-capping protein which has a threefold repeated domain structure similar to the N-terminal half of gelsolin. However, unlike gelsolin, gCap39 does not sever actin filaments and dissociates completely from filament ends after calcium removal. We have capitalized on these differences to explore the structural basis for actin filament capping, severing, and their regulation. Using truncated gCap39, generated by limited proteolysis or deletion mutagenesis, we found that actin filament capping requires multiple gCap domains, and almost the entire molecule is necessary for optimal activity. gCap39 domain I, like the equivalent domain in gelsolin, contains an actin monomer binding site. gCap39 domains II-III are, however, different from gelsolin in that they do not bind to the side of actin filaments. Since filament side binding is hypothesized to be the first step in severing, lack of side binding may explain why gCap39 does not sever. This is confirmed directly by swapping gCap39 domains II-III for the side-binding gelsolin domains to generate a chimera which severs actin filaments. The chimera is Ca2+ independent in actin filament severing and capping, although gCap39 domain I itself is regulated by Ca2+.