Tec家族蛋白酪氨酸激酶Itk PH结构域与OS-9蛋白的相互作用

用酵母双杂交技术筛选与ItkPH结构域相互作用的蛋白分子 ,以了解Itk的功能及其在T细胞信号转导中的位置与作用 .Itk的PH结构域扩增后克隆入酵母双杂交系统的pLexA载体 ,转化酵母细胞EGY4 8(p8op lacZ) ,经检测PH结构域无自激活作用 ,且对酵母细胞无毒性作用 .用PH结构域作为“钓饵”蛋白 ,在酵母双杂交系统中筛选构建于AD载体的T细胞cDNA文库 .将PH结构域及筛库所得基因片段分别进行融合表达 ,用于体外结合实验 ,进一步证实二者的相互作用 .经营养缺陷选择、诱导筛选和鉴定确证 ,筛库所得的插段约 15 0 0bp的文库质粒为一真阳性克隆 .经blast比较分析为骨肉瘤、横纹肌肉瘤等肿瘤组织中高表达的os 9基因 .体外结合实验也表明 ,ItkPH结构域可与该基因表达产物结合 .Itk的PH结构域可与OS 9蛋白相互作用 .二者结合的意义有待进一步研究     

蛋白激酶B的PH结构域可溶性表达与纯化及其二级结构分析

蛋白激酶B(亦称为Akt)是一种蛋白质丝氨酸 苏氨酸激酶 ,因其与蛋白激酶A(PKA)和蛋白激酶C(PKC)具有相对高的同源性 ,而被命名为PKB .作为磷脂酰肌醇 3激酶 (PI3 kinase)的下游分子 ,它广泛参与细胞各种功能的调节 .PKB对代谢的影响主要表现为促进蛋白质的合成 ,促进糖原的转运[1] .同时 ,PKB还在细胞增殖与调控中发挥重要作用[2 ] .PH结构域 (pleckstrinhomologydomain)是一种存在于多种信号蛋白质和细胞骨架相关蛋白质中的功能性区域 .它通常由 7个反向平行的 β片层和C末端的一个α螺旋构成 .PH结构域的配体具有一定的多样性…     

拟南芥中PAP特异磷酸酶(AtAHL)的折叠识别与结构预测

通过现有的序列同源性比较、二级结构预测、三维结构预测和模拟方法,得到了拟南芥中PAP特异磷酸酶的三维结构.这是一种与酵母中的Hal2p蛋白质类似,并且N端为α＋β,C端为α/β结构域的多结构域蛋白质.分析预测所得结构,发现了Mg²⁺等金属离子的结合位点,推测了对Na⁺敏感的结构基础.这些结合位点与其生化功能相关.而且,通过结构与功能分析,讨论了蛋白质数据库(PDB)中同一个酶已有理论结构的不合理性.     

RNA和蛋白质的相互作用

RNA与蛋白质的相互作用是许多基本的细胞生理过程得以实现的决定性因素.近年来,随着技术的改进和新方法的建立,RNA和蛋白质的相互作用研究取得了长足进步.目前科研人员已经鉴定了许多RNA上的蛋白质结合位点,也发现了许多蛋白质中的RNA结合结构域,并对它们的结构特征进行了比较详细的研究.这些都为最终探明RNA和蛋白质相互作用的分子机制,从而从本质上认识相关的细胞生理过程打下了坚实的基础.     

LNX1基因的研究

LNX1基因编码的蛋白质含有2个异形体(isoform),分别称为LNX1-p70和LNX1-p80,其中LNX1-p80具有E3泛素连接酶活性。LNX1蛋白异形体都含有一个NAPY结构域和4个PDZ结构域,其中PDZ结构域是多种蛋白质中具有的结构,主要介导蛋白质相互作用。LNX1可以和细胞中多种蛋白质相互作用,改变被结合蛋白质在细胞中的数量与位置,参与调节生物体胚胎发育,在细胞紧密接界的重构中具有重要作用,可能对肿瘤的形成具有阻抑作用。     

SH2结构域:一个潜在的药靶

众多包含SH2结构域(src homology 2 domain)的蛋白质在细胞内信号传导过程中起到重要作用,它们通过SH2结构域特异性结合包含磷酸化酪氨酸的蛋白质.sH2结构域保守的结构特征和重要的功能使它成为一种潜在的药物作用靶标.基于其结合靶蛋白序列设计的sH2结构域的抑制剂为治疗胞内信号通路异常引起的疾病提供了很好的方向.本文简述了sH2结构域的结构特征,结合特异性和抑制剂设计的进展.     

hhLIM与actin相互作用依赖其C端LIM结构域

hhLIM是LIM蛋白家族成员之一,该蛋白质含有两个LIM结构域,在基因表达调节、细胞骨架组构及细胞肥大过程中发挥重要作用.构建hhLIM不同LIM结构域的突变体,探讨其两个LIM结构域在与actin相互结合中的作用及其可能机制.GST-pull down和hhLIM及其突变体与actin细胞定位关系的免疫荧光分析结果表明,C端的LIM结构域2是hhLIM与actin结合所必需的,该结构域中的两个Cys置换为Ser后可使hhLIM结合actin的功能完全丧失,N端的LIM结构域1突变使hhLIM结合actin的能力下降.F-actin交联实验结果显示,hhLIM通过LIM结构域2与actin直接结合并起到交联F-actin的作用.结果表明,LIM结构域2在hhLIM与actin相互作用及调节actin细胞骨架组构中起决定性作用.     

DDX21 RNA解旋酶不同结构域的功能特点解析

DEAD-box家族是在生物体内普遍存在的一类高度保守的RNA解旋酶,在RNA的合成和加工、细胞发育和细胞代谢等过程中都发挥着重要作用。DDX21 RNA解旋酶是DEAD-box家族成员之一,而目前为止DDX21的酶学功能及结构特征尚未被完全了解。本研究运用生物化学与生物物理学前沿技术,系统地研究了DDX21各结构域在不同功能中发挥的作用。首先重组构建并纯化了人的DDX21 RNA解旋酶及不同的截短蛋白质,利用动态激光散射和凝胶层析技术分析各蛋白质的寡聚形态,发现N-端的非功能区（N-端181aa）与C-端的4个FRGQR重复结构域对其结构有较大的影响;利用荧光偏振技术比较分析了各蛋白质与单链RNA的结合反应,结果显示,仅保留DEADc和HELICc结构域的截短蛋白质与单链RNA完全没有亲和性,缺失N-端181aa的截短蛋白质对ssRNA的结合能力与全长蛋白质基本一致,而仅缺失C-端的4个重复FRGQR结构域的截短蛋白质与单链RNA的亲和能力将显著下降;利用快速停流检测技术分析各截短蛋白质的解旋及退火活性,发现DEADc、HELICc及GUCT_RHII三个结构域共同参与DDX21的解旋功能,另一方面,缺失C-端4个FRGQR重复结构域的截短蛋白质导致退火能力的丧失。本研究揭示了DDX21的GUCT_RHII结构域及C-端4个FRGQR重复结构域在其结构及功能中发挥的重要作用,为今后研究DDX21的结构及其细胞功能提供了重要的理论依据。     

基因表达调控中的核因子作用

利用病毒和动物系统对基因表达调控进行了广泛和深入的研究,发现了顺式作用调节序列,鉴定了序列专一的DNA结合蛋白,DNA与蛋白质相互识别、结合及蛋白质与蛋白质相互作用中起作用的蛋白质结构域,并且对调节蛋白基因的克隆和序列进行了分析．基因表达调控领域又由于植物基因调控机制取得的发展而得到了补充,文章着重介绍植物基因中的DNA与蛋白质间的作用;植物调节蛋白基因的分离;这一领域的今后研究方向及展望．     

东方鲎Factor C中的结构域在结合脂多糖中的作用

Factor C是鲎血细胞中的一种丝氨酸蛋白酶原,因其能以高亲和力结合脂多糖(LPS),在医药产品的内毒素检测中起着重要的作用.以往研究表明处于N端的3个Sushi结构域对Factor C的LPS结合活性起着关键作用, 而Factor C N端的其他3个结构域,包括Cys-rich结构域、EGF-like结构域和Lectin-like结构域对LPS结合活性的影响尚不清楚.利用Bac-to-Bac昆虫细胞表达系统可使rFactor C及其4个截短的片段rCES123L,rCES123,rS123L 和rS123获得表达,并且可采用亲和层析柱将这5个重组表达产物纯化.通过测定5个重组表达产物的LPS结合活性及抑菌活性,可以确定Factor C的LPS结合位点位于S123区域.尽管Cys-rich结构域、EGF-like结构域和Lectin-like结构域中不存在LPS结合位点,但当这3个结构域同时存在时,可提高Factor C或CES123L的LPS结合能力,因此rCES123L具有与rFactor C非常相近的LPS结合能力.实验结果表明,rCES123L在昆虫细胞中的表达量比rFactor C高出4倍,预示出rCES123L在医药领域的应用前景.     

Phosphoinositide-dependent activation of the ADP-ribosylation factor GTPase-activating protein ASAP1. Evidence for the pleckstrin homology domain functioning as an allosteric site

The ADP-ribosylation factor (Arf) family of GTP-binding proteins are regulators of membrane traffic and the actin cytoskeleton. Both negative and positive regulators of Arf, the centaurin beta family of Arf GTPase-activating proteins (GAPs) and Arf guanine nucleotide exchange factors, contain pleckstrin homology (PH) domains and are activated by phosphoinositides. To understand how the activities are coordinated, we have examined the role of phosphoinositide binding for Arf GAP function using ASAP1/centaurin beta4 as a model. In contrast to Arf exchange factors, phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) specifically activated Arf GAP. D3 phosphorylated phosphoinositides were less effective. Activation involved PtdIns-4,5-P(2) binding to the PH domain; however, in contrast to the Arf exchange factors and contrary to predictions based on the current paradigm for PH domains as independently functioning recruitment signals, we found the following: (i) the PH domain was dispensable for targeting to PDGF-induced ruffles; (ii) activation and recruitment could be uncoupled; (iii) the PH domain was necessary for activity even in the absence of phospholipids; and (iv) the Arf GAP domain influenced localization and lipid binding of the PH domain. Furthermore, PtdIns-4,5-P(2) binding to the PH domain caused a conformational change in the Arf GAP domain detected by limited proteolysis. Thus, these data demonstrate that PH domains can function as allosteric sites. In addition, differences from the published properties of the Arf exchange factors suggest a model in which feedforward and feedback loops involving lipid metabolites coordinate GTP binding and hydrolysis by Arf.     

A phosphoinositide-binding sequence is shared by PH domain target molecules—a model for the binding of PH domains to proteins

Pleckstrin homology (PH) domains have been proven to bind phosphoinositides (PI) and inositolphosphates (IP). On the other hand, a binding of PH domains to proteins is still a matter of debate. The goal of this work was to identify potential PH domain protein target sites and to build a model for PH domain–protein binding. A candidate sequence, called HIKE, was identified by sequence homology analysis of the proteins that are considered the strongest PH binding candidates, i.e., G_β, PKC, and Akt. HIKE contains a PI binding sequence and fulfills several criteria for a potential PH-binding site, i.e., it is present in other PH-binding candidates, lies in regulatory regions independently predicted to bind PH domains, and is conserved in 3-D structure among different molecules. These findings and the similarities with the mode of binding of PTB and PDZ domains suggest a β strand–β strand coordination model for PH–protein binding. The HIKE model predicts that membrane anchoring of PH domains and their targets could be a critical step in their interaction, which would consistently explain why PH–protein binding has only been detected in the presence of PI. Proteins 31:1–9, 1998. © 1998 Wiley-Liss, Inc.     

Distinct polyphosphoinositide binding selectivities for pleckstrin homology domains of GRP1-like proteins based on diglycine versus triglycine motifs

GRP1 and the related proteins ARNO and cytohesin-1 are ARF exchange factors that contain a pleckstrin homology (PH) domain thought to target these proteins to cell membranes through binding polyphosphoinositides. Here we show the PH domains of all three proteins exhibit relatively high affinity for dioctanoyl phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P(3)), with K(D) values of 0.05, 1.6 and 1.0 micrometer for GRP1, ARNO, and cytohesin-1, respectively. However, the GRP1 PH domain was unique among these proteins in its striking selectivity for PtdIns(3,4, 5)P(3) versus phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P(2)), for which it exhibits about 650-fold lower apparent affinity. Addition of a glycine to the Gly(274)-Gly(275) motif in GRP1 greatly increased its binding affinity for PtdIns(4,5)P(2) with little effect on its binding to PtdIns(3,4,5)P(3), while deletion of a single glycine in the corresponding triglycine motif of the ARNO PH domain markedly reduced its binding affinity for PtdIns(4,5)P(2) but not for PtdIns(3,4,5)P(3). In intact cells, the hemagglutinin epitope-tagged PH domain of GRP1 was recruited to ruffles in the cell surface in response to insulin, as were full-length GRP1 and cytohesin-1, but the PH domain of cytohesin-1 was not. These data indicate that the unique diglycine motif in the GRP1 PH domain, as opposed to the triglycine in ARNO and cytohesin-1, directs its remarkable PtdIns(3,4,5)P(3) binding selectivity.     

The Kindlin 3 Pleckstrin Homology Domain Has an Essential Role in Lymphocyte Function-associated Antigen 1 (LFA-1) Integrin-mediated B Cell Adhesion and Migration

The protein kindlin 3 is mutated in the leukocyte adhesion deficiency III (LAD-III) disorder, leading to widespread infection due to the failure of leukocytes to migrate into infected tissue sites. To gain understanding of how kindlin 3 controls leukocyte function, we have focused on its pleckstrin homology (PH) domain and find that deletion of this domain eliminates the ability of kindlin 3 to participate in adhesion and migration of B cells mediated by the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1). PH domains are often involved in membrane localization of proteins through binding to phosphoinositides. We show that the kindlin 3 PH domain has binding affinity for phosphoinositide PI(3,4,5)P₃ over PI(4,5)P₂. It has a major role in membrane association of kindlin 3 that is enhanced by the binding of LFA-1 to intercellular adhesion molecule 1 (ICAM-1). A splice variant, kindlin 3-IPRR, has a four-residue insert in the PH domain at a critical site that influences phosphoinositide binding by enhancing binding to PI(4,5)P₂ as well as by binding to PI(3,4,5)P₃. However kindlin 3-IPRR is unable to restore the ability of LAD-III B cells to adhere to and migrate on LFA-1 ligand ICAM-1, potentially by altering the dynamics or PI specificity of binding to the membrane. Thus, the correct functioning of the kindlin 3 PH domain is central to the role that kindlin 3 performs in guiding lymphocyte adhesion and motility behavior, which in turn is required for a successful immune response.     

Probing the importance and potential roles of the binding of the PH-domain protein Boi1 to acidic phospholipids

Background  

The related proteins Boi1 and Boi2, which appear to promote polarized growth in S. cerevisiae, both contain a PH (pleckstrin homology) and an SH3 (src homology 3) domain. Previously, we gained evidence that a PH domain-bearing segment of Boi1, which we call Boi1-PH, is sufficient and necessary for function. In the current study, we investigate the binding of Boi1's PH domain to the acidic phospholipids PIP₂ (phosphatidylinositol-4,5-bisphosphate) and PS (phosphatidylserine).     

Enigmatic relationship of Pleckstrin homology domain with phospholipid breakdown mediated signal transduction.

Research into phospholipid signaling continues to flourish, as more and more bioactive lipids and proteins are being identified and their actions characterised. The Pleckstrin homology (PH) domain is one such newly recognized protein module thought to play an important role in intracellular signal transduction. The tertiary structures of several PH domains have been determined, some of them complexed with ligands and on the basis of structural similarities between PH domains and lipid binding proteins it has been suggested that PH domains may be binding to lipophilic molecules. In fact many of the proteins that contain this domain can interfere with the membrane association. This review examines the specificity of this binding and illustrates the importance of charge-charge interactions in PIP2-PH domain complex formation. The precise physiological functions of PH domain in vivo remains to be explored therefore this review examines the biochemical aspects of the interaction of PH domains with phospholipid breakdown mediated products and proto-oncogenic serine-threonine kinase (Akt), protein tyrosine kinases, which have been found to be a target of phospholipid second messengers. Thus, number of cellular processes mediated by this way, ranging from insulin signaling and protein synthesis to differentiation and cell survival are regulated by this intracellular signaling protein module.     

In vivo functional analysis of the daughter of sevenless protein in receptor tyrosine kinase signaling

One mechanism used by receptor tyrosine kinases to relay a signal to different downstream effector molecules is to use adaptor proteins that provide docking sites for a variety of proteins. The daughter of sevenless (dos) gene was isolated in a genetic screen for components acting downstream of the Sevenless (Sev) receptor tyrosine kinase. Dos contains a N-terminally located PH domain and several tyrosine residues within consensus binding sites for a number of SH2 domain containing proteins. The structural features of Dos and experiments demonstrating tyrosine phosphorylation of Dos upon Sev activation suggested that Dos belongs to the family of multisite adaptor proteins that include the Insulin Receptor Substrate (IRS) proteins, Gab1, and Gab2. Here, we studied the structural requirements for Dos function in receptor tyrosine kinase mediated signaling processes by expressing mutated dos transgenes in the fly. We show that mutant Dos proteins lacking the putative binding sites for the SH2 domains of Shc, PhospholipaseC-γ (PLC-γ) and the regulatory subunit of Phosphoinositide 3-kinase (PI3-K) can substitute the loss of endogenous Dos function during development. In contrast, tyrosine 801, corresponding to a predicted Corkscrew (Csw) tyrosine phosphatase SH2 domain binding site, is essential for Dos function. Furthermore, we assayed whether the Pleckstrin homology (PH) domain is required for Dos function and localization. Evidence is provided that deletion or mutation of the PH domain interferes with the function but not with localization of the Dos protein. The Dos PH domain can be replaced by the Gab1 PH domain but not by a heterologous membrane anchor, suggesting a specific function of the PH domain in regulating signal transduction.     

The intermolecular interaction between the PH domain and the C-terminal domain of Arabidopsis dynamin-like 6 determines lipid binding specificity

Dynamin and its related proteins are a group of mechanochemical proteins involved in the modulation of lipid membranes in various biological processes. Here we investigate the nature of membrane binding of the Arabidopsis dynamin-like 6 (ADL6) involved in vesicle trafficking from the trans-Golgi network to the central vacuole. Fractionation experiments by continuous sucrose gradients and gel filtration revealed that the majority of ADL6 is associated with membranes in vivo. Amino acid sequence analysis revealed that ADL6 has a putative pleckstrin homology (PH) domain. In vitro lipid binding assays demonstrated that ADL6 showed high affinity binding to phosphatidylinositol 3-phosphate (PtdIns-3-P) and that the PH domain was responsible for this interaction. However, the PH domain alone binds equally well to both PtdIns-3-P and phosphatidylinositol 4-phosphate (PtdIns-4-P). Interestingly, the high affinity binding of the PH domain to PtdIns-3-P was restored by a protein-protein interaction between the PH domain and the C-terminal region. In addition, deletion of the inserted regions within the PH domain results in high affinity binding of the PH domain to PtdIns-3-P. These results suggest that ADL6 binds specifically to PtdIns-3-P and that the lipid binding specificity is determined by the interaction between the PH domain and the C-terminal domain of ADL6.