关键词： IQGAP1   glioblastoma multiforme (GBM)   scaffold protein   podosome/invadosome  

摘要： Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumor. GBM is formed by a very heterogeneous astrocyte population, neurons, neovascularization and infiltrating myeloid cells (microglia and monocyte derived macrophages). The IQGAP1 scaffold protein interacts with components of the cytoskeleton, cell adhesion molecules, and several signaling molecules to regulate cell morphology and motility, cell cycle and other cellular functions. IQGAP1 overexpression and delocalization has been observed in several tumors, suggesting a role for this protein in cell proliferation, transformation and invasion. IQGAP1 has been identified as a marker of amplifying cancer cells in GBMs. To determine the involvement of IQGAP1 in the onco-biology of GBM, we performed immunohistochemical confocal microscopic analysis of the IQGAP1 protein in human GBM tissue samples using cell type-specific markers. IQGAP1 immunostaining and subcellular localization was heterogeneous;the protein was located in the plasma membrane and, at variable levels, in nucleus and/or cytosol. Moreover, IQGAP1 positive staining was found in podosome/invadopodia-like structures. IQGAP1(+) staining was observed in neurons (Map2(+) cells), in cancer stem cells (CSC;nestin(+)) and in several macrophages (CD31(+) or Iba1(+)). Our results indicate that the IQGAP1 protein is involved in normal cell physiology as well as oncologic processes.

关键词： IQGAP1   足细胞   细胞骨架重组   嘌呤霉素  

摘要： 目的:通过体外培养小鼠足细胞进行实验研究,采用分子生物学研究方法探讨IQGAP1对足细胞细胞骨架重组的调节作用。方法:嘌呤霉素(PAN)诱导的微小病变性肾病动物模型在临床表现和病理改变上与人类相比具有一定的相似性,而微小病变性肾病最具特征性的病理改变就是足突广泛融合,足细胞细胞骨架蛋白的重组在足突融合中起到关键作用。使用PAN50mg刺激小鼠足细胞后,运用实时定量real-timePCR和WesternBlot印迹法分别检测细胞内IQGAP1 mRNA和蛋白表达水平,激光共聚焦显微镜观察足细胞骨架F-actin的分布,F-actin肌动蛋白评分系统(CFS)对骨架重组进行定量分析,并进一步利用质粒转染技术和基因沉默技术分别观察上调和下调足细胞IQGAPI表达后对PAN诱导的足细胞骨架重组的影响。运用SPSS22.5统计软件处理和分析数据,计量资料采用均数土标准差(x±s)表示,参数资料采用单因素方差分析,LSD法进行组间比较,偏相关分析表示两变量之间的相关性,P<0.05表示差异有统计学意义。结果:重复三次试验,均顺利进行。(1)与对照组相比,PAN刺激组足细胞内IQGAP1 mRNA和蛋白表达明显下降(P<0.05),足细胞F-actin排列紊乱,CFS评分明显升高;(2)IQGAP1 siRNA进一步抑制IQGAP1表达后,PAN引起的上述足细胞F-actin重组更加明显(P<0.05);CFS评分更加升高(3)转染pCantag-myc-IQGAP1质粒促进IQGAP1表达后,PAN诱导的足细胞F-actin排列紊乱显著减轻,CFS评分明显下降(P<0.05)。结论:IQGAP1能抑制嘌呤霉素诱导的足细胞骨架重组,对维持足细胞正常骨架结构起着重要作用。

关键词： IQGAP1   thyroid cancer   proliferation   epithelial-mesenchymal transition   Wnt/beta-catenin  

摘要： Background: Thyroid cancer is the most common endocrine malignant disease with a high incidence rate. The expression of IQGAP1 is upregulated in various cancers, including thyroid cancer. However, the role and underlying mechanism of IQGAP1 in thyroid cancer are still not clear. Materials and methods: The expression of IQGAP1 in thyroid cancer tissues and cells was determined by reverse transcription polymerase chain reaction and Western blot analysis. Cells were transfected with different siRNAs using Lipofectamine 2000 or were treated with various concentrations of XAV939. The effects of IQGAP1 knockdown on proliferation and epithelial-mesenchymal transition (EMT) of thyroid cancer cells were determined by MTT assay and Western blot analysis. Animal experiments were performed to investigate the effects of IQGAP1 knockdown on the growth of tumors in vivo. Results: High IQGAP1 expression is found in thyroid cancer tissues and cells. Knockdown of IQGAP1 had inhibitory effects on cell proliferation and EMT, as well as on the Wnt/beta-catenin pathway. Additionally, inactivation of the Wnt/beta-catenin pathway by XAV939 or si-beta-catenin suppressed cell proliferation and EMT. Furthermore, suppression of the Wnt/beta-catenin pathway reversed the positive effects of pcDNA-IQGAP1 on cell proliferation and EMT in vitro. Moreover, downregulation of IQGAP1 suppressed tumor growth and EMT in SW579 tumor xenografts through the Wnt/a-catenin pathway in vivo. Conclusion: Our study demonstrated that knockdown of IQGAP1 inhibited cell proliferation and EMT through blocking the Wnt/a-catenin pathway in thyroid cancer.

关键词： 结直肠癌   miR-124   IQ结构域GTP酶激活蛋白1   细胞增殖   肿瘤转移  

摘要： 目的:结直肠癌是由正常细胞突变导致其增殖失控后肿瘤细胞扩散引起的疾病,其形成是一个复杂的多因素共同作用的结果,涉及多种基因、多种途径、多个步骤。据相关研究报道,结直肠癌的发病率及死亡率呈逐年升高趋势。microRNA是一类细胞内源性的非编码小RNA,在人体组织中广泛表达,参与对增殖、分化、死亡等基本细胞过程的调控。在肿瘤发展过程中,异常表达的miRNAs导致功能基因的表达失调,是引起肿瘤恶性生物学行为的重要原因。miR-124被发现在多种肿瘤中表达降低或缺失。体内外研究发现,恢复miR-124的表达能够抑制肿瘤细胞的生长、侵袭、EMT等恶性行为,提示其作为抑癌性miRNA的功能。因此,鉴定miR-124的靶基因是揭示以miR-124为中心的肿瘤调控网络的必要工作。IQGAP1(IQ Motif Containing GTPase Activating Protein 1)是 IQGAP 支架蛋白家族的一员,广泛表达于人体组织器官中。IQGAP1能与actin、E-cadherin等细胞骨架蛋白相互结合,在调节细胞粘附、细胞相互作用、细胞迁移等生物学行为中起重要作用。此外,IQGAP1还在MAPK、Wnt/β-catenin等信号途径的级联传导中起促进作用,影响细胞的增殖和分化。目前,在包括结直肠癌在内的多种肿瘤中均发现IQGAP1的异常高表达,且其表达水平与肿瘤的转移程度呈正相关,提示其在肿瘤进展中的重要作用。因此,我们拟探讨miR-124是否通过靶向调控IQGAP1而影响结直肠癌细胞的增殖和侵袭转移。方法:Real-time PCR检测结直肠癌组织中miR-124以及IQGAP1的表达水平,Western blot检测IQGAP1及其下游相关基因的蛋白表达水平,慢病毒感染方式建立稳定敲低IQGAP1的结直肠癌细胞株,CCK-8实验评价细胞的增殖能力,细胞克隆形成试验评价细胞生长能力,划痕实验评价细胞的迁移能力,Transwell侵袭实验评价细胞侵袭能力。生物信息学分析预测miR-124下游调控的靶基因,双荧光素酶报告试验进一步验证潜在靶向关系。结果:miR-124在结直肠癌中表达异常降低,而IQGAP1表达异常升高。恢复miR-124的表达能够显著降低ERK1/2的磷酸化水平并抑制β-catenin和IQGAP1的表达,体外功能试验证实恢复miR-124的表达均能够显著抑制结直肠癌细胞的增殖、克隆形成、侵袭和转移。双荧光素酶报告系统证实miR-124可以靶向结合IQGAP1。稳定敲低IQGAP1表达后证实敲低IQGAP1同样能够显著降低ERK1/2的磷酸化水平和抑制β-catenin的表达,体外功能试验证实敲低IQGAP1也能够显著抑制结直肠癌细胞的增殖、克隆形成、侵袭和转移。结论:在结直肠癌中miR-124通过影响MAPK以及Wnt/β-catenin信号的活性发挥抑癌作用;敲低IQGAP1同样能够通过影响MAPK以及Wnt/β-catenin信号的活性发挥抑癌作用;结合miR-124靶向结合IQGAP1的双荧光素酶报告结果,我们明确miR-124通过靶向调控IQGAP1抑制结直肠癌细胞的增殖和侵袭转移。

关键词： IQGAP1   食管癌   血管生成   机制   p-VEGFR2  

摘要： 目的:1.构建IQGAP1基因过表达和基因干扰的稳转细胞系及其对照细胞系。2.通过体内外实验分别检测IQGAP1基因对血管生成的影响。3.研究IQGAP1对血管生成影响的分子机制。方法:1.利用Lipofectamine 2000将绿色荧光蛋白(green fluorescent protein,GFP)标记的IQGAP1过表达质粒和它所对应的对照质粒分别转染到人食管癌细胞EC9706,用G418耐药单克隆细胞的筛选方法,等到稳定生长后,荧光显微镜下观察其细胞定位,并通过Western blot检测GFP-IQGAP1融合蛋白的表达,确定IQGAP1过表达的稳转细胞系是否构建成功。2.利用Lipofectamine 2000将IQGAP1干扰质粒及其对照质粒转染进人食管癌细胞KYSE150内,采用G418筛选耐药克隆,待其稳定生长后,荧光显微镜下观察KYSE150细胞GFP的表达,并通过Western blot检测对照组与干扰组IQGAP1蛋白的表达情况,确定IQGAP1干扰的稳转细胞系是否构建成功。3.分别通过体外HUVECs小管形成实验和体内的鸡胚尿囊膜实验来检测IQGAP1基因表达对肿瘤血管生成的影响。4.采用Western blot技术研究IQGAP1对食管癌细胞中VEGFA与其受体VEGFR2、p-VEGFR2血管生成相关因子表达的影响。5.通过Western blot技术研究IQGAP1对Akt和ERK活化的影响。6.在IQGAP1过表达组细胞中加入Akt和ERK抑制剂进行干预处理后,观察抑制剂对VEGFA和p-VEGFR2蛋白表达的影响及对血管生成的影响。结果:1.荧光显微镜下观察发现,在转染含GFP-IQGAP1重组质粒的EC9706细胞中观察到其为胞质定位;Western blot结果显示,在IQGAP1过表达细胞中有GFP-IQGAP1融合蛋白的表达,而在其对照组内则没有,说明IQGAP1过表达的稳转细胞系已构建成功。2.在荧光显微镜下可观察到,GFP在转染IQGAP1干扰质粒及对照质粒的KYSE150细胞中为全细胞定位;对支架蛋白IQGAP1在干扰组细胞内的表达量与其在对照组细胞内的表达量通过Western blot进行比较,发现其表达量显著降低,有统计学意义(P<0.0001)。3.体外HUVECs小管形成的实验结果显示,与对照组相比较,IQGAP1过表达组小管生成数更多,而且所生成小管的管腔结构更加完整,两者差异有统计学意义(P<0.0001),表明IQGAP1过表达在体外可以促进血管的生成;体内鸡胚尿囊膜的实验结果也显示,IQGAP1过表达组生成的血管数量较对照组多,并且所生成血管的形态舒展,颜色较为鲜红,经统计学分析,差异有统计学意义(P<0.001),说明IQGAP1过表达可以促进体内血管的生成;体内外实验结果一致,表明IQGAP1过表达后会促进肿瘤血管的生成。***1干扰组采用体外HUVECs小管生成实验,结果显示,与对照组相比较,发现IQGAP1基因干扰后小管生成数较少,生成小管的管腔结构也不完整,差异有统计学意义(P<0.0001),表明IQGAP1干扰后在体外可以抑制血管的生成;体内鸡胚尿囊膜实验结果显示,IQGAP1基因干扰后所生成的血管数量较对照组少,生成的血管色泽灰暗并且主血管较少,经数据统计得,两者之间的差异有统计学意义(P<0.001);上述体内外实验结果一致,表明干扰IQGAP1基因表达后能够明显抑制肿瘤的血管生成。5.在IQGAP1过表达的细胞中,经Western blot检测发现,VEGFA与p-VEGFR2蛋白表达量与其在对照细胞内的表达量相比都升高,差异有统计学意义(P<0.0001),而VEGFR2蛋白的表达量则在IQGAP1过表达组与其对照组细胞中无明显差异,表明IQGAP1可能通过VEGFA与VEGFR2相互作用后促进VEGFR2发生磷酸化,进而促进血管的生成。6.在IQGAP1干扰组细胞中,经Western blot检测发现,VEGFA与p-VEGFR2蛋白的表达量与其在对照细胞内相比,表达量都降低,差异有统计学方面的意义(P<0.0001),而VEGFR2的表达量则在IQGAP1干扰组与其对照组中无明显差异,表明IQGAP1可能通过阻断了VEGFA与其受体VEGFR2的相互作用进而抑制血管生成的过程。7.经Western blot检测发现,IQGAP1过表达组细胞中p-Akt、p-ERK蛋白的表达量与其在对照组的结果比较,发现两者的表达量都升高,差异有统计学的意义(P<0.0001);而IQGAP1干扰组细胞p-Akt、p-ERK的表达量均低于其在对照组内的表达量,差异有统计学意义(P<0.0001);说明IQGAP1对肿瘤细胞血管生成的发生机制的调

关键词： Asparaginyl endopeptidase   MAP kinase signaling pathway   drug resistance   stomach cancer  

摘要： Purpose: In recent years, understanding of the role of asparaginyl endopeptidase (AEP) in tumorigenesis has steadily increased. In this study, we investigated whether AEP expression correlates with sensitivity to chemotherapeutic drugs in gastric cancer and explored the mechanism. Patients and methods: AEP expression in the serum of patients' peripheral blood was measured by enzyme-linked immunosorbent assay. Patient survival time was evaluated using univariate and multivariate analyses. Mass spectrometry and co-immunoprecipitation assays were utilized to discover proteins that interact with AEP. Gastric cancer cell lines were established, in which AEP was overexpressed or knocked out using lentiviral CRISPR. The proliferative abilities of these cell lines in response to chemotherapy agents were evaluated using the Cell Counting Kit-8 method. Gene expression changes in these lines were assessed by real-time polymerase chain reaction and Western blot. Results: Patients with low expression of AEP were significantly more likely to have a good prognosis and experience complete response or partial response after treatment with docetaxel/ S-1 regimen. Mass spectrum analysis showed that several proteins in the focal adhesion and mitogen-activated protein kinase signaling pathways interacted with AEP. IQGAP1 was confirmed to be one of the proteins interacting with AEP, and its protein level increased when AEP was knocked out. AEP knockout decreased resistance to microtubule inhibitors, including paclitaxel, docetaxel, and T-DM1. The expression levels of MDR1, p-EGFR, p-JNK, p-ERK, and p-Rac1/cdc42 were decreased in AEP knockout gastric cancer cell lines, and inhibitors of both JNK and ERK could block AEP-induced expression of MDR1. Conclusion: AEP was not only a prognostic factor but also a predictive marker. AEP knockout could inhibit the activity of the EGFR/JNK/ERK signaling pathway and improve sensitivity to microtubule inhibitors through interacting with IQGAP1.

关键词： CDC42   fluorescence anisotropy   GTPase activating protein (GAP)   protein-protein interaction   Ras homolog gene family   member A (RHOA)   Ras-related C3 botulinum toxin substrate 1 (Rac1)   Rho (Rho GTPase)   signal transduction   IQGAP1   binding affinity  

摘要： IQ motif-containing GTPase activating protein 1 (IQGAP1) plays a central role in the physical assembly of relevant signaling networks that are responsible for various cellular processes, including cell adhesion, polarity, and transmigration. The RHO family proteins CDC42 and RAC1 have been shown to mainly interact with the GAP-related domain (GRD) of IQGAP1. However, the role of its RASGAPC-terminal (RGCT) and C-terminal domains in the interactions with RHO proteins has remained obscure. Here, we demonstrate that IQGAP1 interactions with RHO proteins underlie a multiple-step binding mechanism: (i) a high affinity, GTP-dependent binding of RGCT to the switch regions of CDC42 or RAC1 and (ii) a very low affinity binding of GRD and a C terminus adjacent to the switch regions. These data were confirmed by phosphomimetic mutation of serine 1443 to glutamate within RGCT, which led to a significant reduction of IQGAP1 affinity for CDC42 and RAC1, clearly disclosing the critical role of RGCT for these interactions. Unlike CDC42, an extremely low affinity was determined for the RAC1-GRD interaction, suggesting that the molecular nature of IQGAP1 interaction with CDC42 partially differs from that of RAC1. Our study provides new insights into the interaction characteristics of IQGAP1 with RHO family proteins and highlights the complementary importance of kinetic and equilibrium analyses. We propose that the ability of IQGAP1 to interact with RHO proteins is based on a multiple-step binding process, which is a prerequisite for the dynamic functions of IQGAP1 as a scaffolding protein and a critical mechanism in temporal regulation and integration of IQGAP1-mediated cellular responses.

关键词： Cell signalling   Phosphoinositol signalling   Phosphorylation  

摘要： Despite being one of the most studied signalling pathways, precisely how phospholipid synthesis is regulated in the phosphoinositide signalling cascade remains unclear. The scaffold protein IQGAP1 is now shown to orchestrate the assembly of a multi-enzyme complex that streamlines PtdIns(3,4,5)P-3 synthesis to facilitate Akt activation in response to extracellular stimuli.

关键词： Burkholderia pseudomallei   melioidosis   intracellular bacterial pathogen   actin-based motility   BimA   IQGAP1  

摘要： Intracellular actin-based motility of the melioidosis pathogen Burkholderia pseudomallei requires the bacterial factor BimA. Located at one pole of the bacterium, BimA recruits and polymerizes cellular actin to promote bacterial motility within and between cells. Here, we describe an affinity approach coupled with mass spectrometry to identify cellular proteins recruited to BimA-expressing bacteria under conditions that promote actin polymerization. We identified a group of cellular proteins that are recruited to the B. pseudomallei surface in a BimA-dependent manner, a subset of which were independently validated with specific antisera including the ubiquitous scaffold protein Ras GTPase-activating-like protein (IQGAP1). IQGAP1 integrates several key cellular signaling pathways including those involved in actin dynamics and has been shown to be involved in the adhesion of attaching and effacing Escherichia coli to infected cells and invasion of host cells by Salmonella enterica serovar Typhimurium. Although a direct interaction between BimA and IQGAP1 could not be detected using either conventional pulldown or yeast two hybrid techniques, confocal microscopy revealed that IQGAP1 is recruited to B. pseudomallei actin tails in infected cells, and siRNA-mediated knockdown highlighted a role for this protein in controlling the length and actin density of B. pseudomallei actin tails.

关键词： Interfaces   Peptides and proteins   Monomers   Crystal structure   Oligomers  

摘要： IQGAP1 is a multidomain scaffold protein involved in many cellular processes. We have determined the crystal structure of an N-terminal fragment spanning residues 1-191 (CHDF hereafter) that contains the entire calponin homology,domain. The structure of the CHDF is very similar to those of other type 3 calponin homology domains like those from calponin, Vav, and the yeast IQGAP1 ortholog Rng2. However, in the crystal, two CHDF molecules form a "head-to-head" or parallel dimer through mostly hydrophobic interactions. Binding experiments indicate that the CHDF binds to both F-actin and Ca2+/calmodulin, but binding is mutually exclusive. On the basis of the structure, two dimer interface substitutions were introduced. While CHDFL157D disrupts the dimer in gel filtration experiments, oxidized CHDFK161C stabilizes the dimer. These results imply that the CHDF forms the same dimer in solution that is seen in the crystal structure. The disulfide-stabilized dimer displays a reduced level of F-actin binding in sedimentation assays and shows no binding to Ca2+/calmodulin in isothermal titration calorimetry (ITC) experiments, indicating that interface residues are utilized for both binding events. The Calmodulin Target Database predicts that residues (KK94)-K-93 are important for CaM binding, and indeed, the (EE94)-E-93 double mutation displays a reduced level of binding to Ca2+/calmodulin in ITC experiments. Our results indicate that the CHDF dimer interface is used for both F-actin and Ca2+/calmodulin binding, and the (KK94)-K-93 pair, near the interface, is also used for Ca2+/calmodulin binding. These results are also consistent with full-length IQGAP1 forming a parallel homodimer.