胰岛素受体的配体结合结构域的克隆与表达

用ＰＣＲ方法从人胎盘ｃＤＮＡ 中获得编码胰岛素受体α亚基中结合胰岛素的相对独立的结构域Ｌ１、Ｌ２以及人工设计的Ｌ１－（Ａｌａ）１０－Ｌ２的基因,克隆入含Ｔ７噬菌体ＲＮＡ聚合酶启动子的表达质粒ｐＥＴ－３ａ中,转化大肠杆菌ＢＬ２１（ＤＥ３）,用ＩＰＴＧ诱导表达成功。ＤＮＡ测序、氨基酸组成分析以及蛋白质Ｎ端测序证明所表达的蛋白质正确。经过包涵体的分离、洗涤、溶解和纯化,得到了纯的变性状态受体的胰岛素高亲     

胰岛素受体底物PH结构域相互作用蛋白结构和功能研究进展

PHIP是一种与胰腺β细胞中胰岛素受体底物（IRS）的PH结构域相互作用的蛋白。根据小鼠PHIP（mPHIP）mRNA翻译的不同起始位点,除全长的PHIP1外,mPHIP基因还编码其他3种不同变异体。在胰岛素诱导的信号途径中,主要分布于细胞核的PHIP1和IRS-1的PH结构域相互作用,介导IRS蛋白酪氨酸的磷酸化。IRS-2和PHIP1的共表达能诱导IRS在细胞膜上的定位,促进葡萄糖转运蛋白4（GLUT4）向细胞质膜的转移。PHIP1的表达能提高β-细胞内细胞周期蛋白D2的表达,促进β细胞的生长。PHIP1的表达活化蛋白激酶B（PKB）,活化的PKB能明显抑制β细胞的凋亡。PHIP与胰岛素信号传导途径中其他信号分子的相互作用机制尚不明确。     

胰岛素受体底物家族中的两个新成员

胰岛素受体底物蛋白参与多种激素,细胞因子的信号转导,可联系下游含ＳＨ２结构域的蛋白,它是一种极为重要的信号传递中介分子,胰岛素受体底物家族有４个成员。本文从基因结构,蛋白质结构介绍新发现的两个成员（ＩＲＳ－３,ＩＲＳ－４）的一些基本特征。     

胰岛素受体底物活性调控的分子机制

胰岛素受体底物(insulin receptor substrate,IRS)是胰岛素信号转导通路中一个极其重要的信号分子,对胰岛素信号级联效应具有至关重要的作用。目前有关胰岛素受体底物活性调节的研究主要集中在两个方面,一方面是磷酸化水平的调节机制,另一方面是细胞因子信号阻抑剂(suppressor of cytokine signaling,COCS)所介导的直接和间接调控。了解胰岛素受体底物活性调节机制将有助于进一步探索胰岛素抵抗和Ⅱ型糖尿病的发病机制。     

水杨酸钠对胰岛素抵抗大鼠胰岛素受体底物-1的影响

目的探讨抗炎药水杨酸钠对胰岛素抵抗大鼠胰岛素敏感性的影响及其作用机制。方法分别给大鼠静脉输注脂肪乳+肝素,脂肪乳+肝素+水杨酸钠和生理盐水7 h,并在输注的最后2 h,行清醒状态高胰岛素-正血糖钳夹试验,测定血浆葡萄糖、游离脂肪酸(FFA)、胰岛素和C-肽水平,检测肝脏、肌肉中胰岛素受体底物-1(IRS-1)及307位丝氨酸磷酸化的IRS-1表达。结果输注脂肪乳大鼠葡萄糖输注率(GIR)是输注生理盐水大鼠的45%,水杨酸钠可使GIR提高1.3倍(P0.01)。脂肪乳输注组大鼠肝脏及肌肉中307位丝氨酸磷酸化的IRS-1分别为生理盐水输注组大鼠的3倍和3.8倍(P0.001),输注水杨酸钠,肝脏、肌肉307位丝氨酸磷酸化的IRS-1下降45%、20%(P0.05)。结论 FFA增高引起肝脏及肌肉中307位丝氨酸磷酸化的IRS-1水平增高,可能是导致胰岛素抵抗发生的机制之一,应用水杨酸钠,大鼠肝脏及肌肉组织中IRS-1丝氨酸磷酸化水平下降,胰岛素抵抗改善。抗炎药物水杨酸钠可能通过抑制FFA引起的IRS-1丝氨酸磷酸化,而发挥改善胰岛素抵抗的作用。     

胰岛素受体底物家族与Ⅱ型糖尿病

胰岛素受体底物(insulin receptor substrate,IRS)家族是胰岛素／类胰岛素生长因子信号系统上游通路的关键介导者,在维持细胞生长、分裂和代谢中起着重要作用。已有四个成员被鉴定出：IRS-1、IRS-2、IRS-3和IRS-4,其中IRS-1和IRS-2在许多不同的组织细胞中起着特异性作用。IRS介导的胰岛素信号通路与很多其他信号通路存在交叉,它们能干扰胰岛素发挥效应,导致胰岛素抵抗,从而引发糖尿病。     

胰岛素受体及其底物与血管内皮生长因子

VEGF参与肿瘤发生与发展、缺血性血管病变、糖尿病小血管异常增生等多个病理生理过程,胰岛素受体及其底物作为VEGF的调节因素之一可通过多种途径影响VEGF的表达。文章综述了在糖尿病视网膜病变、肿瘤、缺血性血管病等不同病理生理条件下胰岛素受体及其底物对VEGF表达的影响,推测两者之间可能存在的调节机制。     

Toll样受体与核苷酸结合寡聚化结构域蛋白在防御反应中的相互作用

哺乳动物具有两个微生物识别系统,一个系统是由Toll样受体（TLRs）的膜结合受体家族组成;另一个系统是由位于胞浆的核苷酸结合寡聚化结构域（NOD）蛋白家族组成。入侵机体的微生物可能通过不同抗原激活多个模式识别受体,导致免疫反应的产生。本文将对TLRs及NODs信号通路在防御反应中的相互作用进行综述。     

引发细胞凋亡受体的死亡结构域

死亡结构域是存在于肿瘤于死因因子受体Ⅰ型和Ｆａｓ等能引起细胞凋亡的细胞膜表面受包浆区的一段氨基酸序列,它通过聚合针这些膜表面受体与胞浆信号蛋白联系起来,成为引起细胞凋亡或活化的信号转导通路中重要的一个环节。本文综述了死亡结构域及其在细胞信号转导过程中所起作用的最新进展。     

胰岛素信号转导障碍与胰岛素抵抗的形成

胰岛素生理作用的发挥,起始于胰岛素与其受体的结合,并由此引起细胞内一系列信号转导,最终到达各效应器产生各种生理效应。胰岛素信号转导在胰岛素生理作用的发挥中起着至关重要的作用。胰岛素信号转导减弱或受阻,使得胰岛素生理作用减弱,导致胰岛素抵抗形成。本文综述了胰岛素信号转导失调在胰岛素抵抗形成中的作用。     

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     

Insulin and Metabolic Stress Stimulate Multisite Serine/Threonine Phosphorylation of Insulin Receptor Substrate 1 and Inhibit Tyrosine Phosphorylation

IRS1 and IRS2 are key substrates of the insulin receptor tyrosine kinase. Mass spectrometry reveals more than 50 phosphorylated IRS1 serine and threonine residues (Ser(P)/Thr(P) residues) in IRS1 from insulin-stimulated cells or human tissues. We investigated a subset of IRS1 Ser(P)/Thr(P) residues using a newly developed panel of 25 phospho-specific monoclonal antibodies (αpS/TmAb^Irs1). CHO cells overexpressing the human insulin receptor and rat IRS1 were stimulated with insulin in the absence or presence of inhibitors of the PI3K → Akt → mechanistic target of rapamycin (mTOR) → S6 kinase or MEK pathways. Nearly all IRS1 Ser(P)/Thr(P) residues were stimulated by insulin and significantly suppressed by PI3K inhibition; fewer were suppressed by Akt or mTOR inhibition, and none were suppressed by MEK inhibition. Insulin-stimulated Irs1 tyrosine phosphorylation (Tyr(P)^Irs1) was enhanced by inhibition of the PI3K → Akt → mTOR pathway and correlated with decreased Ser(P)-302^Irs1, Ser(P)-307^Irs1, Ser(P)-318^Irs1, Ser(P)-325^Irs1, and Ser(P)-346^Irs1. Metabolic stress modeled by anisomycin, thapsigargin, or tunicamycin increased many of the same Ser(P)/Thr(P) residues as insulin, some of which (Ser(P)-302^Irs1, Ser(P)-307^Irs1, and four others) correlated significantly with impaired insulin-stimulated Tyr(P)^Irs1. Thus, IRS1 Ser(P)/Thr(P) is an integrated response to insulin stimulation and metabolic stress, which associates with reduced Tyr(P)^Irs1 in CHO^IR/IRS1 cells.     

非典型性肺炎病毒S蛋白受体结合域的可溶性表达

试验目的是获得S蛋白受体结合域基因,并获得其高效表达,为SARS病毒受体的进一步研究奠定一定的基础。首先通过P（取方法获得了S蛋白起始密码和SalⅠ限制性内切酶之间包含受体结合区的片段,然后将该基因定向克隆到pET-22b原核表达载体,构建了per—22b—S1重组质粒,转化大肠杆菌并诱导目的蛋白的表达,经SDS—PAGE没有发现明显的目的蛋白带。利用载体和S1基因上的NeoⅠ酶切位点,将S1基因的信号肽序列和部分疏水序列切掉后,构建pET—22b—SNS重组质粒。pET—22b—SNS重组质粒仍然包含受体结合域序列,并且阅读框没有改变。将pET—22b—SNS转化大肠杆菌,发现明显的目的蛋白带。Westem blot结果表明表达蛋白为SARS病毒S1蛋白的一部分。     

Sequence-specific 1H, 13C, and 15N resonance assignments of GRP1 PH domain

The carboxy-terminal pleckstrin homology (PH) domain recruits GRP1 to the plasma membrane through the specific binding to phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P₃]. Here, we describe backbone and side chain assignments of the GRP1 PH domain determined by triple resonance experiments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tyr728 in the Kinase Domain of the Murine Kinase Suppressor of RAS 1 Regulates Binding and Activation of the Mitogen-activated Protein Kinase Kinase

In metazoans, the highly conserved MAPK signaling pathway regulates cell fate decision. Aberrant activation of this pathway has been implicated in multiple human cancers and some developmental disorders. KSR1 functions as an essential scaffold that binds the individual components of the cascade and coordinates their assembly into multiprotein signaling platforms. The mechanism of KSR1 regulation is highly complex and not completely understood. In this study, we identified Tyr⁷²⁸ as a novel regulatory phosphorylation site in KSR1. We show that Tyr⁷²⁸ is phosphorylated by LCK, uncovering an additional and unexpected link between Src kinases and MAPK signaling. To understand how phosphorylation of Tyr⁷²⁸ may regulate the role of KSR1 in signal transduction, we integrated structural modeling and biochemical studies. We demonstrate that Tyr⁷²⁸ is involved in maintaining the conformation of the KSR1 kinase domain required for binding to MEK. It also affects phosphorylation and activation of MEK by RAF kinases and consequently influences cell proliferation. Moreover, our studies suggest that phosphorylation of Tyr⁷²⁸ may affect the intrinsic kinase activity of KSR1. Together, we propose that phosphorylation of Tyr⁷²⁸ may regulate the transition between the scaffolding and the catalytic function of KSR1 serving as a control point used to fine-tune cellular responses.     

Down-regulation of S1P1 Receptor Surface Expression by Protein Kinase C Inhibition

The sphingosine 1-phosphate receptor type 1 (S1P₁) is important for the maintenance of lymphocyte circulation. S1P₁ receptor surface expression on lymphocytes is critical for their egress from thymus and lymph nodes. Premature activation-induced internalization of the S1P₁ receptor in lymphoid organs, mediated either by pharmacological agonists or by inhibition of the S1P degrading enzyme S1P-lyase, blocks lymphocyte egress and induces lymphopenia in blood and lymph. Regulation of S1P₁ receptor surface expression is therefore a promising way to control adaptive immunity. Hence, we analyzed potential cellular targets for their ability to alter S1P₁ receptor surface expression without stimulation. The initial observation that preincubation of mouse splenocytes with its natural analog sphingosine was sufficient to block Transwell^TM chemotaxis to S1P directed subsequent investigations to the underlying mechanism. Sphingosine is known to inhibit protein kinase C (PKC), and PKC inhibition with nanomolar concentrations of staurosporine, calphostin C, and GF109203X down-regulated surface expression of S1P₁ but not S1P₄ in transfected rat hepatoma HTC₄ cells. The PKC activator phorbol 12-myristate 13-acetate partially rescued FTY720-induced down-regulation of the S1P₁ receptor, linking PKC activation with S1P₁ receptor surface expression. FTY720, but not FTY720 phosphate, efficiently inhibited PKC. Cell-based efficacy was obvious with 10 nm FTY720, and in vivo treatment of mice with 0.3–3 mg/kg/day FTY720 showed increasing concentration-dependent effectiveness. PKC inhibition therefore may contribute to lymphopenia by down-regulating S1P₁ receptor cell surface expression independently from its activation.     

Par14 Protein Associates with Insulin Receptor Substrate 1 (IRS-1), Thereby Enhancing Insulin-induced IRS-1 Phosphorylation and Metabolic Actions

Pin1 and Par14 are parvulin-type peptidyl-prolyl cis/trans isomerases. Although numerous proteins have been identified as Pin1 substrates, the target proteins of Par14 remain largely unknown. Par14 expression levels are increased in the livers and embryonic fibroblasts of Pin1 KO mice, suggesting a compensatory relationship between the functions of Pin1 and Par14. In this study, the association of Par14 with insulin receptor substrate 1 (IRS-1) was demonstrated in HepG2 cells overexpressing both as well as endogenously in the mouse liver. The analysis using deletion-mutated Par14 and IRS-1 constructs revealed the N-terminal portion containing the basic domain of Par14 and the two relatively C-terminal portions of IRS-1 to be involved in these associations, in contrast to the WW domain of Pin1 and the SAIN domain of IRS-1. Par14 overexpression in HepG2 markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events, PI3K binding with IRS-1 and Akt phosphorylation. In contrast, treating HepG2 cells with Par14 siRNA suppressed these events. In addition, overexpression of Par14 in the insulin-resistant ob/ob mouse liver by adenoviral transfer significantly improved hyperglycemia with normalization of hepatic PEPCK and G6Pase mRNA levels, and gene suppression of Par14 using shRNA adenovirus significantly exacerbated the glucose intolerance in Pin1 KO mice. Therefore, although Pin1 and Par14 associate with different portions of IRS-1, the prolyl cis/trans isomerization in multiple sites of IRS-1 by these isomerases appears to be critical for efficient insulin receptor-induced IRS-1 phosphorylation. This process is likely to be one of the major mechanisms regulating insulin sensitivity and also constitutes a potential therapeutic target for novel insulin-sensitizing agents.     

PH结构域与细胞方向感觉

PH（pleckstrin homology）结构域与细胞方向感觉关系密切,目前已发现,PH结构域存在于60多种蛋白质中,这些蛋白质能与趋化细胞胞膜表面的相关结合位点结合,进而激发信号转导的下游事件.这种结合有以下特点：a.迅速而短暂;b.只与外界环境中两点间浓度梯度差相关,据此提出了“空间模式”;c.改变趋化剂的位置时,结合位点在胞膜上的分布也随之改变,由此提出了“时间模式”.深入而全面地探讨各种PH结构域及其结合位点在细胞方向感觉中的作用,对于细胞方向感觉的研究具有巨大的推动作用和深远的理论意义.