5DFRXXL region of long myosin light chain kinase causes F-actin bundle formation

Long myosin light chain kinase （L-MLCK） contains five DFRXXL motifs with ability to bind F-actin. Binding stoichiometry data indicated that each DFRXXL motif might bind each G-actin, but its biological significance remained unknown. We hypothesized that L-MLCK might act as an F-actin bundle peptides by its multiple binding sites of 5DFRXXL motifs to actin. In order to characterize F-actin-bundle formation properties of 5DFRXXL region of long myosin light chain kinase, we expressed and purified 5DFRXXL peptides tagged with HA in vitro. The properties of 5DFRXXL peptides binding to myofilaments or F-actin were analyzed by binding stoichiometries assays. The results indicated that 5DFRXXL peptides bound to myofilaments or F-actin with high affinity. KD values of 5DFRXXL binding to myofilaments and F-actin were 0.45 and 0.41 μmol/L, respectively. Cross-linking assay demonstrated that 5DFRXXL peptides could bundle F-actin efficiently. Typical F-actin bundles were observed morphologically through determination of confocal and electron microscopy after adding 5DFRXXL peptides. After transfection of pEGFP-5DFRXXL plasmid into eukaryocyte, spike structure was observed around cell membrane edge. We guess that such structure formation may be attributable to F-actin over-bundle formation caused by 5DFRXXL peptides. Therefore, we suppose that L-MLCK may be a new bundling protein and somehow play a certain role in organization of cell skeleton besides mediating cell contraction by it kinase activity.     

拟穴青蟹细丝蛋白C的生物信息学分析及重组表达

为了探讨抗原表位与细丝蛋白C（filamin C,FLN c）结构之间的构效关系，对FLN c进行生物信息学分析及分段重组表达。生物信息学分析结果表明，FLN c二级结构以β折叠及无规则卷曲为主，确定其具有9个结构域，三级结构呈免疫球蛋白样折叠，具有典型的细丝蛋白家族特征。基于生物信息学分析，将拟穴青蟹（Scylla paramamosain）FLN c通过分段原核表达，纯化出带有麦芽糖结合蛋白（maltose binding protein，MBP）标签的重组表达蛋白rFLN c1（AA:1-335）、rFLN c2（AA:336-531）、rFLN c3（AA:532-847）。血清学分析结果显示，rFLN c2与蟹类过敏患者血清IgE结合能力最强，推测此区域含有大部分抗原表位。因此，定位拟穴青蟹FLN c氨基酸336-531结构域为抗原表位优势区，rFLN c2可用于FLN c的晶体学研究。     

Mechanical strain in actin networks regulates FilGAP and integrin binding to filamin A

Mechanical stresses elicit cellular reactions mediated by chemical signals. Defective responses to forces underlie human medical disorders such as cardiac failure and pulmonary injury. The actin cytoskeleton's connectivity enables it to transmit forces rapidly over large distances, implicating it in these physiological and pathological responses. Despite detailed knowledge of the cytoskeletal structure, the specific molecular switches that convert mechanical stimuli into chemical signals have remained elusive. Here we identify the actin-binding protein filamin A (FLNA) as a central mechanotransduction element of the cytoskeleton. We reconstituted a minimal system consisting of actin filaments, FLNA and two FLNA-binding partners: the cytoplasmic tail of β-integrin, and FilGAP. Integrins form an essential mechanical linkage between extracellular and intracellular environments, with β-integrin tails connecting to the actin cytoskeleton by binding directly to filamin. FilGAP is an FLNA-binding GTPase-activating protein specific for RAC, which in vivo regulates cell spreading and bleb formation. Using fluorescence loss after photoconversion, a novel, high-speed alternative to fluorescence recovery after photobleaching, we demonstrate that both externally imposed bulk shear and myosin-II-driven forces differentially regulate the binding of these partners to FLNA. Consistent with structural predictions, strain increases β-integrin binding to FLNA, whereas it causes FilGAP to dissociate from FLNA, providing a direct and specific molecular basis for cellular mechanotransduction. These results identify a molecular mechanotransduction element within the actin cytoskeleton, revealing that mechanical strain of key proteins regulates the binding of signalling molecules.     

超薄层等电聚焦电泳在人群运铁蛋白遗传多态性研究中的应用

本文应用改良超薄层等电聚焦技术对海南岛543个无关中国人个体(汉族)的血清运铁蛋白的多态性进行分析。研究发现海南岛汉族人群 Tf 亚型的基因型频率为:TfCl=0.710,TfC2=0.027,TfC3=0.258。在该人群中还发现三种罕见基因型,其基因型频率为0.005。该群体表型的分布符合 Hardy-Weinberg 平衡定律。本研究证明采用改良超簿层等电聚焦技术于人运铁蛋白多态性研究,能有效地分离各种运铁蛋白亚型,各亚型的基因频率分配在不同人种中有显著性差异。本文还就超薄层等电聚焦区带的分布及鉴别提出新的见解。     

hASB-8相互作用蛋白的初步研究

hASB-8基因是对肿瘤细胞生长具有明显抑制作用的人类新基因．其编码蛋白属于人ASB蛋白家族中的一个成员,与小鼠中的ASB-8蛋白同源性达96％．保守结构域分析显示hASB-8在N端包含4个Ankyrin repeats,在C端包含了一个SOCS box．利用酵母双杂交技术,筛选了人的胎盘(Placenta)cDNA文库,获得了与KASB-8相互作用的2个蛋白,Elongin C和CDK4 binding protein;并在二倍体酵母体内进行了验证．这些试验提示hASB-8蛋白可能介导肿瘤细胞中靶蛋白和泛素复合体之间的相互作用,并与肿瘤细胞靶蛋白转录调节有关．     

环境污染物PFOS可致人脑微血管内皮细胞骨架蛋白改变

目的:探讨全氟辛烷磺酰基化合物(PFOS)引起的人脑微血管内皮细胞(HBMEC)骨架蛋白(F-actin)排列变化.方法:培养正常的人脑毛细血管内皮细胞,不同浓度PFOS作用30分钟,利用免疫荧光染色的方法检测细胞骨架蛋白F-actin的变化.结果:PFOS可使细胞骨架蛋白F-actin发生重排,这与PFOS的浓度有关.     

抗人骨桥蛋白单克隆抗体制备及其特异性研究

利用RT-PCR技术克隆人骨桥蛋白(hOPN)基因,构建OPN原核表达质粒pET-32a(+)-hOPN,转化BL21菌株,经IPTG诱导表达重组人骨桥蛋白(rhOPN).以纯化的rhOPN为免疫原,免疫BABL/c小鼠,取其脾细胞与小鼠骨髓瘤细胞NS1融合.通过有限稀释法进行克隆和间接ELISA筛选,获得抗人OPN蛋白单克隆抗体杂交瘤细胞株,以ELISA、Western blot对抗体特异性进行鉴定.通过竞争抑制试验对单克隆抗体识别抗原位点进行分析.结果共获得2株抗人OPN单克隆抗体,分别命名为8F1和2B5,亚型测定皆为IgG1.通过细胞侵袭抑制试验检测,2株抗人OPN mAb皆能很好地抑制细胞迁移.本研究成功获得了抗人骨桥蛋白的特异性单克隆抗体,为进一步研究OPN蛋白在自身免疫病和肿瘤中的功能提供了重要的工具.     

苯环喹溴铵血浆蛋白结合率测定

为了测定苯环喹溴铵大鼠和人的血浆蛋白结合率,将大鼠和人的血浆样品加入内标后用甲醇直接沉淀法进行LC/ESI/MS分析,然后采用平衡透析法测定大鼠和人血浆中苯环喹溴铵的血浆蛋白结合率。结果表明,苯环喹溴铵大鼠血浆中药物浓度为105.2~1842ng/ml,血浆蛋白结合是线性的,苯环喹溴铵与大鼠血浆蛋白结合率约为42.0%;苯环喹溴铵人血浆中药物浓度为305.3~3813ng/ml,血浆蛋白结合是线性的,与人血浆蛋白结合率约为71.4%。苯环喹溴铵在不同种属中的血浆蛋白结合率是不同的,与人的血浆蛋白结合率较大鼠的血浆蛋白结合率高,且与人的血浆蛋白具有中等强度的结合。     

分泌癌胚抗原肿瘤靶向性Cu,Zn-SOD的基因克隆及表达

将Cu,Zn-SOD与抗癌胚抗原（CEA）单链抗体基因（ScFvgene)融合,重组到含T7启动子的表达载体p ET-22b(＋）中,构建表达质粒pETSOD－ScFv,并转化大肠杆菌BL21（ED3）,进行了高效表达,表达物占菌体可溶性总蛋白的18％。SDS－PAGE和蛋白质印迹图谱显示表达物相对分子质量为41kD,与融合基因编码蛋白质的理论值相符。该蛋白质在磊肠杆菌中为分泌型表达,有利于纯化。RIA测定表明产物能特异性地与抗原CEA结合,邻苯三酚法测定也表明产物具有SOD酶的活性,该融合蛋白为分泌CEA肿瘤的靶向性治疗及放疗、化疗后的恢复提供新的途径。     

人Rab蛋白cDNA的克隆和表达

从人胎脑cDNA文库中克隆到一种新的Rab cDNA,全长920bp,以编码213个氨基酸残基,该蛋白预测的分子质量为24567u,等电点7．34,经同源比较,该cDNA与GenBank数据库中登录号为X14964的Rab蛋白有83％的相似性和76％的相同性,将该cDNA克隆到经改造的PBV220表达质粒,转化DH5a菌株诱导表达出该蛋白,取24种不同组织的总cDNA各100ng,用该基因序列设计引物作PCR,结果在胎肝组织中检测到有明显条带,表明该Rab基因相对在胎肝有高表达。     

Structural basis for vinculin activation at sites of cell adhesion

Vinculin is a highly conserved intracellular protein with a crucial role in the maintenance and regulation of cell adhesion and migration. In the cytosol, vinculin adopts a default autoinhibited conformation. On recruitment to cell-cell and cell-matrix adherens-type junctions, vinculin becomes activated and mediates various protein-protein interactions that regulate the links between F-actin and the cadherin and integrin families of cell-adhesion molecules. Here we describe the crystal structure of the full-length vinculin molecule (1,066 amino acids), which shows a five-domain autoinhibited conformation in which the carboxy-terminal tail domain is held pincer-like by the vinculin head, and ligand binding is regulated both sterically and allosterically. We show that conformational changes in the head, tail and proline-rich domains are linked structurally and thermodynamically, and propose a combinatorial pathway to activation that ensures that vinculin is activated only at sites of cell adhesion when two or more of its binding partners are brought into apposition.     

A single amino-acid difference confers major pharmacological variation between human and rodent 5-HT1B receptors.

Neuropsychiatric disorders such as anxiety, depression, migraine, vasospasm and epilepsy may involve different subtypes of the 5-hydroxytryptamine (5-HT) receptor. The 1B subtype, which has a unique pharmacology, was first identified in rodent brain. But a similar receptor could not be detected in human brain, suggesting the absence in man of a receptor with equivalent function. Recently a human receptor gene was isolated (designated 5-HT1B receptor, 5-HT1D beta receptor, or S12 receptor) which shares 93% identity of the deduced protein sequence with rodent 5-HT1B receptors. Although this receptor is identical to rodent 5-HT1B receptors in binding to 5-HT, it differs profoundly in binding to many drugs. Here we show that replacement of a single amino acid in the human receptor (threonine at residue 355) with a corresponding asparagine found in rodent 5-HT1B receptors renders the pharmacology of the receptors essentially identical. This demonstrates that the human gene does indeed encode a 1B receptor, which is likely to have the same biological functions as the rodent 5-HT1B receptor. In addition, these findings show that minute sequence differences between homologues of the same receptor from different species can cause large pharmacological variation. Thus, drug-receptor interactions should not be extrapolated from animal to human species without verification.     

Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product

The proteins encoded by the myc gene family are involved in the control of cell proliferation and differentiation, and aberrant expression of myc proteins has been implicated in the genesis of a variety of neoplasms. In the carboxyl terminus, myc proteins have two domains that encode a basic domain/helix-loop-helix and a leucine zipper motif, respectively. These motifs are involved both in DNA binding and in protein dimerization. In addition, myc protein family members share several regions of highly conserved amino acids in their amino termini that are essential for transformation. We report here that an N-terminal domain present in both the c-myc and N-myc proteins mediates binding to the retinoblastoma gene product, pRb. We show that the human papilloma virus E7 protein competes with c-myc for binding to pRb, indicating that these proteins share overlapping binding sites on pRb. Furthermore, a mutant Rb protein from a human tumour cell line that carried a 35-amino-acid deletion in its C terminus failed to bind to c-myc. Our results suggest that c-myc and pRb cooperate through direct binding to control cell proliferation.     

Requirement of yeast fimbrin for actin organization and morphogenesis in vivo

The SAC6 gene was found by suppression of a yeast actin mutation. Its protein product, Sac6p (previously referred to as ABP67), was independently isolated by actin-filament affinity chromatography and colocalizes with actin in vivo. Thus Sac6p binds to actin in vitro, and functionally associates with actin structures involved in the development and maintenance of cell polarity in vivo. We report here that Sac6p is an actin-filament bundling protein 43% identical in amino-acid sequence to the vertebrate bundling protein fimbrin. This yeast fimbrin homologue contains two putative actin-binding regions homologous to domains of dystrophin, beta-spectrin, filamin, actin-gelation protein and alpha-actinin. Mutants lacking Sac6p do not form normal actin structures and are defective in morphogenesis. These findings demonstrate an in vivo role for the well-documented biochemical interaction between fimbrin and actin.     

一个新C2H2型锌指蛋白的功能的初步研究

通过筛选人18周胎脑cDNA文库,得到一条编码332AA的全长新基因,生物信息学研究表明,该蛋白质序列有2个C2H2型锌指结构,其中1个锌指结构有RNA－binding蛋白特异锌指的特征,虽然同源比较发现与多种蛋白质精氨酸N端转甲基酶（protein arginine N-methyltransferase) 有一定的同源性,但新锌指蛋白不含转甲基酶的活性功能区域,属功能未知的基因,利用芯片研究功能未知基因的表达是一种较好的手段,通过代谢增强剂PMA（phorbol myristae acetate)刺激培养的血管内皮细胞,观察细胞受激活后新锌指蛋白基因的表达变化,结果表明新基因表达量提高了11倍以上,证实新基因属内皮细胞的极早期应答基因（Immediate early response gene,ERG)。     

Correlation between platelet gelsolin levels and different types of coronary heart disease

Gelsolin is an important cytoskeletal protein of platelets and studies have shown a close relationship between gelsolin and cardiovascular disease.However,the role of gelsolin in the development of coronary heart disease(CHD) is unclear.In this study,we record the distribution of gelsolin in human platelets and plasma and its association with different types of CHD.This study included 114 cases,with 33 stable angina pectoris(SAP) cases,81 acute coronary syndrome(ACS) cases—composed of 39 unstable angina pectoris(UAP) and 42 acute myocardial infarction(AMI) cases,and 31 healthy control participants.Gelsolin concentration in platelet rich plasma(PRP) and platelet poor plasma(PPP),actin filament(F-actin) and Gc-globulin of PPP were determined by enzyme-linked immunoadsorbent assay(ELISA).The fluorescence intensity of CD62p and cytoplasmic calcium([Ca2+] i) in human platelets measured by flow cytometry.We also used turbidimetry to detect the platelet aggregation rate(PAR).We analyzed the correlation between platelet gelsolin concentration and CD62p or plasma F-actin levels among each different patient group.Compared with the control group,the gelsolin level in PRP of UAP and AMI groups increased significantly(P<0.01),while the gelsolin level in PPP of all the three patient groups decreased markedly(P<0.01),and the CD62p,PAR,[Ca2+] i of platelets,F-actin and Gc-globulin of the UAP and AMI groups increased significantly(P<0.01).Compared with the SAP group,the gelsolin level in PRP,the PAR,[Ca2+] i of platelets and CD62p of other two groups increased significantly(P<0.01),F-actin of the AMI group increased markedly(P<0.01).Platelet cytoskeleton protein dynamics vary among the different types of CHD.Platelet gelsolin levels are markedly increased and accompanied by increased platelet activity,F-actin and [Ca2+] i of ACS patients,while gelsolin levels in PPP are markedly lower.Abnormally increased platelet gelsolin levels show high positive correlation with the level of platelet activity.Therefore,platelet gelsolin might be a novel molecular marker and/or a new potential therapeutic target of anti-platelet therapy of ACS.     

Binding of disease-associated prion protein to plasminogen

Transmissible spongiform encephalopathies are associated with accumulation of PrP(Sc), a conformer of a cellular protein called PrP(C). PrP(Sc) is thought to replicate by imparting its conformation onto PrP(C) (ref. 1), yet conformational discrimination between PrP(C) and PrP(Sc) has remained elusive. Because deposition of PrP(Sc) alone is not enough to cause neuropathology, PrP(Sc) probably damages the brain by interacting with other cellular constituents. Here we find activities in human and mouse blood which bind PrP(Sc) and prion infectivity, but not PrP(C). We identify plasminogen, a pro-protease implicated in neuronal excitotoxicity, as a PrP(Sc)-binding protein. Binding is abolished if the conformation of PrP(Sc) is disrupted by 6M urea or guanidine. The isolated lysine binding site 1 of plasminogen (kringles I-III) retains this binding activity, and binding can be competed for with lysine. Therefore, plasminogen represents the first endogenous factor discriminating between normal and pathological prion protein. This unexpected property may be exploited for diagnostic purposes.     

Molecular structure of the chick cerebellar kainate-binding subunit of a putative glutamate receptor

Kainate receptors mediate some of the excitatory transactions carried out in the central nervous system by the neurotransmitter glutamate. They are involved in neurotoxicity, possibly in neurodegenerative disorders and it has been suggested that they have a role in long-term potentiation. Kainate receptors are present both on neuronal and glial cell membranes where they regulate the gating of a voltage-independent ion channel. Nothing is known about their molecular structure. Taking advantage of the unusually high abundance of 3H-kainate binding sites in the chick cerebellum, we have isolated an oligomeric protein that displays a pharmacological profile similar to that of a kainate receptor, and have demonstrated, using the monoclonal antibody IX-50, that this protein is composed of a single polypeptide of Mr 49,000 which harbours the specific kainate recognition site. The structure of this kainate binding protein (KBP) is also of interest because of its exclusive cerebellar localization on Bergmann glial membrane in close proximity to established glutamatergic synapses. We now report the isolation of the complementary DNA containing the complete coding region of the kainate binding protein. The predicted structure of the mature protein has four putative transmembrane domains with a topology analogous to that found in the superfamily of ligand-gated ion channels. This raises the possibility, that kainate binding protein may form part of an ion channel and may be a subunit of a kainate subtype of glutamate receptor.