摘要： Enteropathogenic Escherichia coli (EPEC), atypical enteropathogenic Escherichia coli (ATEC) and enterohemorrhagic Escherichia coli (EHEC) belong to the family of attaching and effacing (A/E) pathogens. Pathogenicity is mediated by subversion of host cell functions involving type III secretion system (TTSS)-dependent effector proteins. In this study, we have identified and characterized a novel TTSS-dependent effector protein encoded at the 5'-end of the locus of enterocyte effacement (LEE) pathogenicity island (PAI) of ATEC strain 3431-4/86 (O8:H-). Using affinity purification we identified IQGAP1, a scaffolding protein involved in the regulation of the actin cytoskeleton, as a putative host cell target. Accordingly, we termed the novel effector protein 'Ibe' for IQGAP1-binding effector. The interaction of Ibe and IQGAP1 was confirmed by co-immunoprecipitation from ATEC-infected cells and immunofluorescence analysis, which revealed colocalization of Ibe and IQGAP1 in ATEC-induced pedestals and actin-rich membrane ruffles. This suggests that the putative effector function of Ibe is mediated via IQGAP1. The Ibe-independent recruitment of IQGAP1 to ATEC-induced pedestals implies a general role for IQGAP1 in the subversion of host cell functions during infection. Homologues of the novel effector Ibe are widely distributed among EPEC, ATEC and EHEC strains but are not necessarily genetically linked to the LEE as they have occasionally also been found to be encoded within lambdoid prophages.

关键词： Proteomics   Quercetin  

摘要： Quercetin, a wild distributed bioflavonoid, exhibits antitumor effects on murine models by inducing apoptosis and inhibiting growth of many cancer cell lines, while proteins involved in antitumor effects at proteomic level are still unclear. In our study, we used a quantitative proteomic strategy termed stable isotope labeling by amino acids in cell culture (SILAC)-mass spectrometry (MS) to study the differential proteomic profiling of HepG2 cells treated by quercetin. In all, there were 70 changed proteins among those quantified proteins in HepG2 cells treated by 50 mu M quercetin for 48 h, and 14 proteins showed significant upregulation, whereas 56 proteins were downregulated. The functional classification of changed proteins includes signaling protein, protein synthesis, cytoskeleton, metabolism, etc. Of these, Ras GTPase-activating-like protein (IQGAP1) and beta-tubulin were found to be reduced at a large degree. The migration inhibition of HepG2 cells can be induced by quercetin, and the RNA and protein expression level of IQGAP1 and beta-tubulin were respectively decreased obviously in HepG2 cells exposed to quercetin for 48 h in the scratch migration assay. The downregulated expression of IQGAP1 and beta-tubulin by quercetin treatment correlated with cell migration ability, and quercetin probably inhibits HepG2 proliferation and migration through IQGAP1 and beta-tubulin expression changes and their interactions with other proteins.

关键词： menin   IQGAP1   intercellular adhesion   migration  

摘要： Multiple endocrine neoplasia type 1 (MEN1) is a dominantly inherited tumor syndrome that results from the mutation of the MEN1 gene that encodes protein menin. Stable overexpression of MEN1 has been shown to partially suppress the Ras-mediated morphological changes of fibroblast cells. Little is known about the molecular mechanisms by which menin decreases the oncogenic effects on cell morphology and other phenotypes. Here we showed that ectopic expression of menin in pretumor beta-cells increases islet cell adhesion and reduces cell migration. Our further studies revealed that menin interacts with the scaffold protein, IQ motif containing GTPase activating protein 1 (IQGAP1), reduces GTP-Rac1 interaction with IQGAP1 but increases epithelial cadherin (E-cadherin)/beta-catenin interaction with IQGAP1. Consistent with an essential role for menin in regulating beta-cell adhesion in vivo, accumulations of beta-catenin and E-cadherin are reduced at cell junctions in the islets from Men1-excised mice. Together, these results de. ne a novel menin-IQGAP1 pathway that controls cell migration and cell-cell adhesion in endocrine cells.

关键词： beta-catenin   Membrane ruffle   FRAP   IQGAP1  

摘要： beta-catenin is a key mediator of the Wnt signaling process and accumulates in the nucleus and at the membrane in response to Wnt-mediated inhibition of GSK-3 beta. In this study we used live cell photobleaching experiments to determine the dynamics and rate of recruitment of beta-catenin at membrane adherens junctions (cell adhesion) and membrane ruffles (cell migration). First, we confirmed the nuclear-cytoplasmic shuttling of GFP-tagged beta-catenin, and found that a small mobile pool of beta-catenin can move from the nucleus to membrane ruffles in NIH 3T3 fibroblasts with a t(0.5) of similar to 30 s. Thus, beta-catenin can shuttle between the nucleus and plasma membrane. The localized recruitment of beta-catenin-GFP to membrane ruffles was more rapid, and the strong recovery observed after bleaching (mobile fraction 53%, t(0.5) similar to 5 s) is indicative of high turnover and transient association. In contrast, beta-catenin-GFP displayed poor recovery at adherens junctions in MDCK epithelial cells (mobile fraction 10%, t(0.5) similar to 8 s), indicating stable retention at these membrane structures. We previously identified IQGAP1 as an upstream regulator of beta-catenin at the membrane, and this is supported by photobleaching assays which now reveal IQGAP1 to be more stably anchored at membrane ruffles than beta-catenin. Further analysis showed that LiCl-mediated inactivation of the kinase GSK-3 beta increased beta-catenin membrane ruffle staining;this correlated with a faster rate of recruitment and not increased membrane retention of beta-catenin. In summary, beta-catenin displays a high turnover rate at membrane ruffles consistent with its dynamic internalization and recycling at these sites by macropinocytosis. (c) 2008 Elsevier Inc. All rights reserved.

关键词： 博莱霉素   Akt   GSK3β   NF-κB   气道上皮细胞   甲醛   IQGAP1   β-连环素/Tcf信号   APC   气道上皮细胞  

摘要： 第一部分 博莱霉素致气道上皮细胞毒性损伤中GSK3β的活性变化对NF-κB信号通路的影响 实验背景 博莱霉素是一种有效的化疗药物，但所引起的急性肺损伤与肺纤维化的副作用却不容忽视。气道上皮是呼吸系统对抗毒性刺激的第一道防线，因此气道上皮细胞通常被认为是博莱霉素肺毒性的主要靶点。暴露于博莱霉素的细胞可发生各种细胞反应，包括细胞凋亡、细胞坏死及前炎症因子分泌等，这些细胞反应共同促进了博莱霉素导致的肺损伤与肺纤维化的发展过程。大量研究显示，核因子-κB(nuclear factor-κB，NF-κB)信号参与调节细胞存活、细胞分化、增殖、凋亡及炎症过程的多种基因的转录，与博莱霉素引起的肺毒性密切相关。 正常情况下，NF-κB与抑制蛋白IκB相结合并隐匿于静息细胞的胞浆，保持无活性的状态。外界刺激可引起IκB进入泛素化降解途径从而使NF-κB被释放并转位入核，激活下游靶基因的转录。糖原合成酶激酶3(glycogen synthase kinase 3，GSK3)是一种多功能的丝／苏氨酸蛋白激酶，与NF-κB不同，GSK3β在静息细胞内呈组成性激活的状态，其N末端丝氨酸残基可发生PI3K(phosphatidylinositol3-kinase)／Akt(also called protein kinase B，PKB)依赖性的磷酸化并导致其活性抑制。近年来的研究显示，GSK3β可能参与调节与NF-κB信号相关的多种疾病的病理生理过程，但具体机制并不明确。因此，本研究针对GSK3β在博莱霉素肺毒性中的活性变化及其对NF-κB信号通路的影响进行了初步探讨。 实验目的 本实验使用博莱霉素刺激建立体外肺毒性损伤模型，初步探讨在博莱霉素作用下气道上皮细胞中GSK3β的活性变化对NF-κB信号通路的影响，从而进一步分析肺纤维化发生发展的机制。 实验方法 使用博莱霉素刺激建立体外肺毒性损伤模型，首先采用相差显微镜及细胞荧光染色观察气道上皮细胞在博莱霉素刺激下的形态变化，并进一步使用MTT方法检测不同浓度下细胞活力的变化。然后使用Western blot、免疫荧光共聚焦成像、核浆分离、免疫共沉淀、瞬时转染及报告基因分析等方法观测博莱霉素处理后NF-κB信号的活化状态、Akt／GSK3β通路的活性变化及其对NF-κB信号的影响。 实验结果 相差显微镜下观察显示培养的气道上皮细胞呈现上皮细胞典型的铺路石状形态，其特点是立体的，稍微凸起的，细胞间紧密连接。经博莱霉素毒性刺激后，细胞间隙增宽，伸展扁平，立体感消失，死细胞增多，在荧光显微镜下可见大量细胞核呈现皱缩变圆、亮蓝色的凋亡形态。MTT结果显示博莱霉素可引起浓度依赖性的细胞活力降低。 然后，我们使用瞬时转染及报告基因分析方法发现博莱霉素可引起气道上皮细胞中NF-κB信号的活化，而且在博莱霉素刺激2 h时，NF-κB的抑制蛋白IκBα迅速发生降解并几近消失，同时核浆分离实验及免疫荧光共聚焦结果均显示NF-κB亚单位p65发生了向核内的转位，与文献所报道的体内实验的结果相符。接着我们发现，博莱霉素刺激以后，Akt与GSK3β发生了明显的磷酸化，而使用抑制剂LY294002抑制二者的磷酸化以后，NF-κB信号的活性也随之降低。因此我们推测，博莱霉素所引起的NF-κB信号的活化必与GSK3β的活化状态有着密切的关系。于是我们进一步使用持续激活型GSK3βS9A质粒转染入细胞后再使用博莱霉素刺激，结果发现GSK3β的持续活化抑制了博莱霉素诱导的NF-κB信号活化，并保护内源性IκBα不受降解，继而阻止了NF-κB亚单位p65转位入核。为了更深入的探究这种现象的可能原因所在，我们使用免疫共沉淀的方法将GSK3β与IκBα互相沉淀，发现二者可以共同沉淀下来，并在博莱霉素刺激后共同沉淀的蛋白减少，而在沉淀的IκBα蛋白中并未检测到磷酸化的GSK3β，提示GSK3β与IκBα存在于同一个复合物中，并且很有可能对IκBα有着某种机制的保护作用，进一步说明GSK3β可能通过与IκBα的这种相互作用从而影响了博莱霉素诱导的NF-κB信号活化。 实验结论 本实验表明：(1)博莱霉素可导致气道上皮细胞发生形态学改变及细胞活力降低。(2)博莱霉素可诱导气道上皮细胞NF-κB信号的活化。(3)博莱霉素诱导的气道上皮细胞中Akt及GSK3β的磷酸化可能参与了NF-κB信号的活化。(4)GSK3β的活性改变可能通过与NF-κB抑制蛋白IκBα的相互作用从而负向调节博莱霉素诱导的气道上皮细胞中NF-κB信号的活化过程。 第二部分 甲醛暴露致气道上皮毒性损伤中IQGAP1的表达变化对APC蛋白及

关键词： Cell culture  

摘要： During infection, enteropathogenic Escherichia coli (EPEC) injects effector proteins into the host cell to manipulate the actin cytoskeleton and promote formation of actin pedestals. IQGAP1 is a multidomain protein that participates in numerous cellular functions, including Rac1/Cdc42 and Ca2+/calmodulin signaling and actin polymerization. Here we report that IQGAP1, Ca2+, and calmodulin modulate actin pedestal formation by EPEC. Infection with EPEC promotes both the interaction of IQGAP1 with calmodulin and the localization of IQGAP1 and calmodulin to actin pedestals while reducing the interaction of IQGAP1 with Rac1 and Cdc42. IQGAP1-null fibroblasts display a reduced polymerization of actin in response to EPEC. In addition, antagonism of calmodulin or chelation of intracellular Ca2+ reduces EPEC-dependent actin polymerization. Furthermore, IQGAP1 specifically interacts with Tir in vitro and in cells. Together these data identify IQGAP1, Ca2+, and calmodulin as a novel signaling complex regulating actin pedestal formation by EPEC.

关键词： Phosphorylation   Endothelial cells   Immunoprecipitation   Small interfering RNA   Angiogenesis   Tyrosine   VEGF signaling   Protein kinases  

摘要： Background: Vascular endothelial growth factor receptor-2 (VEGFR-2) signaling is an obligate requirement for normal development and pathological angiogenesis such as cancer and age-related macular degeneration. Although autophosphorylation of tyrosine 1173 (Y1173) of VEGFR-2 is considered a focal point for its angiogenic signal relay, however, the mechanism of phosphorylation of Y1173, signaling proteins that are recruited to this residue and their role in angiogenesis is not fully understood. Methodology/Principal Findings: In this study we demonstrate that c-Src kinase directly through its Src homology 2 (SH2) domain and indirectly via c-Cbl binds to phospho-Y1057 of VEGFR-2. Activation of c-Src kinase by a positive feedback mechanism phosphorylates VEGFR-2 at multi-docking site, Y1173. c-Src also catalyzes tyrosine phosphorylation of IQGAP1 and acts as an adaptor to bridge IQGAP1 to VEGFR-2. In turn, IQGAP1 activates b-Raf and mediates proliferation of endothelial cells. Silencing expression of IQGAP1 and b-Raf revealed that their activity is essential for VEGF to stimulate angiogenesis in an in vivo angiogenesis model of chicken chorioallantoic membrane (CAM). Conclusions/Significance: Angiogenesis contributes to the pathology of numerous human diseases ranging from cancer to age-related macular degeneration. Determining molecular mechanism of tyrosine phosphorylation of VEGFR-2 and identification of molecules that are relaying its angiogenic signaling may identify novel targets for therapeutic intervention against angiogenesis-associated diseases. Our study shows that recruitment and activation of c-Src by VEGFR-2 plays a pivotal role in relaying angiogenic signaling of VEGFR-2;it phosphorylates VEGFR-2 at Y1173, facilitates association and activation of IQGAP1 and other signaling proteins to VEGFR-2. IQGAP1-dependent signaling, in part, is critically required for endothelial cell proliferation, a key step in angiogenesis. Thus, Y1057 of VEGFR-2 serves to regul

关键词： Ovarian Cancer   Ovarian Carcinoma   Ovarian Cancer Cell   Ovarian Cancer Cell Line   Cell Migration Assay  

摘要： Background: IQGAP1 is a scaffolding protein and overexpressed in many human tumors, including ovarian cancer. However, the contribution of IQGAP1 to invasive properties of ovarian cancer cells remains unknown. Here, we investigated the effect of IQGAP1-specific short hairpin RNA (shRNA) expressing plasmids on metastatic potential of ovarian cancer HO-8910PM cells. Methods: We used RT-PCR and Western blot analysis to characterize expression of IQGAP1 in three human ovarian cancer-derived cell lines SK-OV-3, HO-8910 and HO-8910PM. We then determined whether expression of endogenous IQGAP1 correlated with invasive and migratory ability by using an in vitro Matrigel assay and cell migration assay. We further knocked down IQGAP1 using shRNA expressing plasmids controlled by U1 promoter in HO-8910PM cells and examined the proliferation activity, invasive and migration potential of IQGAP1 shRNA transfectants using MTT assay, in vitro Matrigel-coated invasion assay and migration assay. Results: IQGAP1 expression level seemed to be closely associated with the enhanced invasion and migration in ovarian cancer cell lines. Levels of both IQGAP1 mRNA and protein were significantly reduced in HO-8910PM cells transfected with plasmid-based IQGAP1-specific shRNAs. RNAimediated knockdown of IQGAP1 expression in HO-8910PM cells resulted in a significant decrease in cell invasion and migration. Conclusion: Our findings support the hypothesis that IQGAP1 promotes tumor progression and identify IQGAP1 as a potential therapeutic strategy for ovarian cancer and some other tumors with over-expression of the IQGAP1 gene.

关键词： IQ序列三磷酸鸟苷酶激活蛋白   细胞分裂周期42蛋白   气态甲醛   气道上皮   损伤修复  

摘要： 目的探讨包含IQ序列的三磷酸鸟苷酶(GTP)激活蛋白1(IQGAP1)和细胞分裂周期42蛋白(Cdc42)在气道上皮细胞损伤修复不同阶段的作用。方法选用健康昆明种小鼠24只,5 mg/m3气态甲醛,染毒4 h/d,分别染毒3,7和60 d后处死,采用免疫组织化学染色、蛋白印迹(Western blot)检测和免疫荧光共聚焦成像方法,观察IQ-GAP1和Cdc42蛋白在染毒小鼠气道上皮细胞损伤修复过程中的表达和定位变化。结果IQGAP1在染毒3和7 d时表达明显增强(P<0.01),60 d时恢复至对照组水平;Cdc42蛋白在染毒3 d时表达明显减弱(P<0.01),7 d时表达有所恢复。对照组中IQGAP1主要定位于气道上皮细胞的胞膜处,而Cdc42蛋白弥散分布于气道上皮细胞(AECs)胞质中;3 d和7 d时共同定位于细胞极化前缘;60 d时其定位与对照组相似。结论Cdc42蛋白和IQGAP1在气道上皮细胞损伤修复过程中的表达及定位呈动态变化,提示其在这一过程中发挥着重要作用。