关键词： Actins   Amorphous solids   Biochemistry   Biological cultures   Biology and life sciences   Cancers and neoplasms   Cell biology   Cell cultures   Cellular structures and organelles   Contractile proteins   Cultured tumor cells   Cytoskeletal proteins   Cytoskeleton   Deletion mutagenesis   Engineering and technology   Equipment   Gels   Laboratory equipment   Laboratory glassware   Materials by structure   Materials science   Medicine and health sciences   Melanoma cells   Melanomas   Microtubules   Mixtures   Molecular biology   Molecular biology techniques   Mutagenesis and gene deletion techniques   Oncology   Physical sciences   Proteins   Research and analysis methods   Research Article  

摘要： IQGAP1 interacts with a number of binding partners through a calponin homology domain (CHD), aWWmotif, IQ repeats, a Ras GAP-related domain (GRD), and a conserved C-terminal (CT) domain. Among various biological and cellular functions, IQGAP1 is known to play a role in actin cytoskeleton dynamics during membrane ruffling and lamellipodium protrusion. In addition, phosphorylation near the CT domain is thought to control IQGAP1 activity through regulation of intramolecular interaction. In a previous study, we discovered that IQGAP1 preferentially localizes to retracting areas in B16F10 mouse melanoma cells, not areas of membrane ruffling and lamellipodium protrusion. Nothing is known of the domains needed for retraction localization and very little is known of IQGAP1 function in the actin cytoskeleton of melanoma cells. Thus, we examined localization of IQGAP1 mutants to retracting areas, and characterized knock down phenotypes on tissue culture plastic and physiologic-stiffness hydrogels. Localization of IQGAP1 mutants (S1441E/S1443D, S1441A/S1443A, Delta CHD, Delta GRD or Delta CT) to retracting and protruding cell edges were measured. In retracting areas there was a decrease in S1441A/S1443A, Delta GRD and Delta CT localization, a minor decrease in Delta CHD localization, and normal localization of the S1441E/S1443D mutant. In areas of cell protrusion just behind the lamellipodium leading edge, we surprisingly observed both Delta GRD and Delta CT localization, and increased number of microtubules. IQGAP1 knock down caused loss of cell polarity on laminin-coated glass, decreased proliferation on tissue culture polystyrene, and abnormal spheroid growth on laminin-coated hydrogels. We propose that the GRD and CT domains regulate IQGAP1 localization to retracting actin networks to promote a tumorigenic role in melanoma cells.

摘要： Introduction: While AML is phenotypically diverse, genome-wide sequencing studies indicate that the median number of non-synonymous mutations is eight (Science 339,1546, 2013) and that the same pathways are affected in tumors with distinct genetic alterations (Nature 453,1112-1116, 2008). These insights provided the impetus to immunoaffinity purify shared antigenic moieties in human AML and examine their prevalence as well as function. Methods: We employed a previously described method wherein immunization of rabbits with normal human peripheral blood cells whose surface charge had been modified by incubation with fluorodinitrobenzene (FDNB) elicited an antibody response that cross reacted against a broad range of human leukemias (Nature 232,197, 1971). Replicating the described methods WBCs from healthy volunteer donors were suspended at 1x107 cells per ml of PBS and mixed with equal volume of 0.86 mM FDNB for 12 to 15 minutes at room temperature. Three rabbits were immunized with FDNB-treated cells (experimental rabbits). A control rabbit was immunized with sham-treated cells (control rabbit). After complement inactivation, immune sera were absorbed against WBCs from healthy donors. Absorbed immune sera were tested in clinical AML samples by Western blotting. Immunoprecipitation of antigens from whole cell lysates of clinical AML samples was performed using IgG adsorbed on protein A/G Agarose beads. Liquid chromatography and mass spectrometry of the immunoprecipitated material was performed. Fold change in IQGAP1 expression in normal vs AML bone marrow was determined by qRT-PCR and from raw data from Gene Expression Omnibus at the NCBI. IQGAP1 expression was evaluated by immunohistochemistry in cell lines, bone marrow biopsies from AML patients and normal healthy volunteers. IQGAP1 expression was modulated by RNAi and the effect on proliferation and colony formation was measured in cell lines. Results: Western blotting of whole cell lysates of clinical AML samples

关键词： IQGAP1   VCP   Proteomics  

摘要： Scaffold proteins play a pivotal role in making protein complexes, and organize binding partners into a functional unit to enhance specific signaling pathways. IQ motif-containing GTPase activating protein 1 (IQGAP1) is an essential protein for spine formation due to its role in scaffolding multiple signal complexes. However, it remains unclear how IQGAP1 interacts within the brain. In the present study, we screened novel IQGAP1-interacting proteins by a proteomic approach. As a novel IQGAP1-interacting protein, we identified valosin-containing protein (VCP) which is a causative gene in patients with inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD). The physiological interaction of IQGAP1 with VCP was confirmed by an immunoprecipitation assay. Both the N-terminal (N-half) and C-terminal (C-half) fragments of IQGAP1 interacted with the N-terminal region of VCP. Co-localization of IQGAP1 and VCP was observed in the growth corn, axonal shaft, cell body, and dendrites in cultured hippocampal neurons at 4 days in vitro (DIV4). In cultured neurons at DIV14, IQGAP1 co-localized with VCP in dendrites. When HEK293T cells were co-transfected with IQGAP1 and VCP, an immunoprecipitation assay revealed that binding of IQGAP1 with disease-related mutant (R155H or A232E) VCP was markedly reduced compared to wild-type (WT) VCR These results suggest that reduction of IQGAP1 and VCP interaction may be associated with the pathophysiology of IBMPFD. (C) 2017 Elsevier Inc. All rights reserved.

关键词： IQ结构域GTP酶激活蛋白1   胰腺癌   免疫组织化学   生存分析  

摘要： 目的检测胰腺癌组织中IQ结构域GTP酶激活蛋白1(IQGAP1)的表达与临床病理特征及与预后的关系。方法采用免疫组织化学法检测66例胰腺癌组织和49例癌旁组织中IQGAP1的表达情况,分析IQGAP1与胰腺癌临床病理特征及其与预后的相关性。结果胰腺癌组织和癌旁组织中IQGAP1的阳性表达率分别为63.636%和12.245%,胰腺癌组织高于癌旁组织(P<0.05);IQGAP1的表达与肿瘤分化程度、淋巴结转移有关(P<0.05),与患者年龄、性别、肿瘤部位、大小、神经浸润及临床分期差异无统计学意义(P>0.05)。IQGAP1阳性患者的总生存期短于IQGAP1阴性患者(P=0.002),Cox多因素分析显示IQGAP1阳性表达是独立预后危险因素(^HR=2.128,95%CI=1.127,4.019,P=0.020)。结论胰腺癌组织中高表达的IQGAP1可能参与胰腺癌的发生、发展及转移过程,并可作为判断胰腺癌预后的重要指标。

关键词： radial glia   outer radial glia   basal radial glia   human neocortex   gyrification   neurogenesis   neural progenitor   intermediate progenitor   TMEM14B   IQGAP1  

摘要： Human brain evolution is associated with expansion and folding of the neocortex. Increased diversity in neural progenitor (NP) populations (such as basally located radial glia [RG], which reside in an enlarged outer subventricular zone [OSVZ]) likely contributes to this evolutionary expansion, although their characteristics and relative contributions are only partially understood. Through single-cell transcriptional profiling of sorted human NP subpopulations, we identified the primate-specific TMEM14B gene as a marker of basal RG. Expression of TMEM14B in embryonic NPs induces cortical thickening and gyrification in postnatal mice. This is accompanied by SVZ expansion, the appearance of outer RG-like cells, and the proliferation of multiple NP subsets, with proportional increases in all cortical layers and normal lamination. TMEM14B drives NP proliferation by increasing the phosphorylation and nuclear translocation of IQGAP1, which in turn promotes G1/S cell cycle transitions. These data show that a single primate-specific gene can drive neurodevelopmental changes that contribute to brain evolution.

关键词： TISSUE scaffolds   NUCLEOTIDES   LEUCINE   CYTOSOL   FIBROBLASTS  

摘要： Sensing of cytosolic nucleotides is a critical initial step in the elaboration of type I IFN. One of several upstream receptors, cyclic GMP-AMP synthase, binds to cytosolic DNA and generates dicyclic nucleotides that act as secondary messengers. These secondary messengers bind directly to stimulator of IFN genes (STING). STING recruits TNFR-associated NF-kB kinase-binding kinase 1 which acts as a critical node that allows for efficient activation of IFN regulatory factors to drive the antiviral transcriptome. NLRC3 is a recently characterized nucleotide-binding domain, leucine-rich repeat containing protein (NLR) that negatively regulates the type I IFN pathway by inhibiting subcellular redistribution and effective signaling of STING, thus blunting the transcription of type I IFNs. NLRC3 is predominantly expressed in lymphoid and myeloid cells. IQGAP1 was identified as a putative interacting partner of NLRC3 through yeast two-hybrid screening. In this article, we show that IQGAP1 associates with NLRC3 and can disrupt the NLRC3-STING interaction in the cytosol of human epithelial cells. Furthermore, knockdown of IQGAP1 in THP1 and HeLa cells causes significantly more IFN-beta production in response to cytosolic nucleic acids. This result phenocopies NLRC3-deficient macrophages and fibroblasts and short hairpin RNA knockdown of NLRC3 in THP1 cells. Our findings suggest that IQGAP1 is a novel regulator of type I IFN production, possibly via interacting with NLRC3 in human monocytic and epithelial cells.

关键词： 7-helix receptor   adhesion   cancer stem cells   G protein-coupled receptor (GPCR)   intestine  

摘要： Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a bona fide marker of adult stem cells in several epithelial tissues, most notably in the intestinal crypts, and is highly up-regulated in many colorectal, hepatocellular, and ovarian cancers. LGR5 activation by R-spondin (RSPO) ligands potentiates Wnt/beta-catenin signaling in vitro;however, deletion of LGR5 in stem cells has little or no effect on Wnt/beta-catenin signaling or cell proliferation in vivo. Remarkably, modulation of LGR5 expression has a major impact on the actin cytoskeletal structure and cell adhesion in the absence of RSPO stimulation, but the molecular mechanism is unclear. Here, we show that LGR5 interacts with IQ motif-containing GTPase-activating protein 1 (IQGAP1), an effector of Rac1/CDC42 GTPases, in the regulation of actin cytoskeleton dynamics and cell-cell adhesion. Specifically, LGR5 decreased levels of IQGAP1 phosphorylation at Ser-1441/1443, leading to increased binding of Rac1 to IQGAP1 and thus higher levels of cortical F-actin and enhanced cell-cell adhesion. LGR5 ablation in colon cancer cells and crypt stem cells resulted in loss of cortical F-actin, reduced cell-cell adhesion, and disrupted localization of adhesion-associated proteins. No evidence of LGR5 coupling to any of the four major subtypes of heterotrimeric G proteins was found. These findings suggest that LGR5 primarily functions via the IQGAP1-Rac1 pathway to strengthen cell-cell adhesion in normal adult crypt stem cells and colon cancer cells.

关键词： colorectal cancer   oxaliplatin   IQGAP1   scaffold protein  

摘要： IQGAP1 is a scaffolding protein that serves a key role in cell dynamics by integrating internal and external stimuli to distinct signal outputs. Previous studies have identified several genes that are significantly up-or downregulated in the peripheral white cells (PWCs) of patients with colorectal adenocarcinoma (CRC), who underwent oxaliplatin-based chemotherapy (CT). In addition, screening studies have reported that IQ-motif containing GTPase activating protein 1 (IQGAP1) transcriptional expression levels varied from 'off' to 'on' following oxaliplatin CT. In order to determine if variations previously described in PWCs are able to be observed at the protein level in tumors and in metastases following CT, the present study performed an immunohistochemical analysis of IQGAP1 in CRC and primary metastases. IQGAP1 expression was observed in the nuclear envelope and in lateral cell membranes and cytoplasm in normal colon tissue. However, in tumor tissue, cells exhibited a diffuse pattern, with variable expression levels of staining in the nuclear membrane and cytoplasm, with the highest expression intensity observed at the invasive front. In healthy and metastasized liver tissue and in the metastases themselves, expression levels varied from cell to cell from no expression to a high level. In the majority of cells, IQGAP1 co-localized with microtubules at the cytoplasmic face of the nuclear envelope. Strong positive expression was observed in areas of the lesion where cells were detaching from the lesion into the lumen. Despite the homogeneous IQGAP1 staining pattern observed in healthy colon tissue sections, CRC demonstrated heterogeneity in staining, which was more marked in metastasized liver tissue resected following CT. However, the most notable findings were the observed effects on the cellular and subcellular distribution and its implications for cancer biology. These results suggest that IQGAP1 may be a putative biomarker, a candidate for clinical diagnostics

摘要： Ceramide, a bioactive lipid and signaling molecule associated with cardiovascular disease, is known to activate extracellular signal regulated kinases 1 and 2 (ERK1/2). Here, we determined that the effect of ceramide on ERK1/2 is mediated by ceramide signaling on an ERK scaffold protein, IQ motif containing GTPase activating protein 1 (IQGAP1). Experiments were performed with aortic smooth muscle cells using inhibitor screening, small interfering RNA (siRNA), immunoprecipitation (IP), immunoblots and bioinformatics. We report here that C6 ceramide increases serum-stimulated ERK1/2 activation in a manner dependent on the ERK1/2 scaffold IQGAP1. C6 ceramide increases IQGAP1 protein levels by preventing its cleavage. Bioinformatic analysis of the IQGAP1 amino acid sequence revealed potential cleavage sites for proteases of the proprotein convertase family that match the cleavage products. These potential cleavage sites overlap with known motifs for lysine acetylation. Deacetylase inhibitor treatment increased IQGAP1 acetylation and reduced IQGAP1 cleavage. These data are consistent with a model in which IQGAP1 cleavage is regulated by acetylation of the cleavage sites. Activation of ERK1/2 by ceramide, known to increase lysine acetylation, appears to be mediated by acetylationdependent stabilization of IQGAP1. This novel mechanism could open new possibilities for therapeutic intervention in cardiovascular diseases.

关键词： IQGAP1   足细胞   细胞骨架重组   嘌呤霉素  

摘要： 目的 :探讨IQ结构域GTP酶活化蛋白1(IQ domain GTPase-activating protein 1,IQGAP1)对足细胞细胞骨架重组的调节作用。方法:嘌呤霉素(PAN)刺激小鼠足细胞后,运用实时定量PCR和Western blot印迹法分别检测细胞内IQGAP1m RNA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)对骨架重组进行分析,并进一步观察上调和下调足细胞IQGAP1表达对PAN诱导的足细胞骨架重组的影响。结果:(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明显升高;(2)IQGAP1 si RNA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);(3)转染p Cantag-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。