关键词： RhoC   IQGAP1   肝细胞性肝癌   免疫组化  

摘要： 背景与目的 肝细胞性肝癌(hepatocellular carcinoma, HCC)是我国常见的恶性肿瘤之一,因其早期诊断困难、易发生转移,并缺乏有效的治疗方法,预后极差,死亡率高。故探讨肝细胞性肝癌的发生及转移机制,寻找有效预防及治疗的手段,是目前迫切需要解决的问题。HCC的发生、发展是一个多阶段、多基因参与的连续性过程,机制十分复杂。Ras同源基因(Rho)家族蛋白是一类与ras同源的小GTP结合蛋白,包括Rho (RhoA, RhoB, RhoC),Rac,Cdc42等家族成员,它循环于活性GTP结合型与失活性GDP结合型之间,作为分子开关控制细胞信号转导途径,产生多种生物学效应。RhoC (Ras homologous C)属于小分子G蛋白超家族中的Rho亚家族,亦是Rho信号转导通路的重要分子。文献报道,RhoC在肝癌、胃癌、结肠癌等多种恶性肿瘤中呈高表达,并与肿瘤的浸润转移高度相关。IQGAP1(IQ motif containing GTPase activating protein1)是一种Ras鸟苷三磷酸酶活化蛋白,因其含有类似于Ras GAPs催化域的广泛序列和位于N端的4个可与钙调蛋白相互作用的IQ模体而被命名。研究表明,IQGAP1通过多种转导通路在肝癌、胃癌、结肠癌等多种肿瘤的发生及转移中发挥了显著的促进作用,被认为是一种致癌基因。目前已有在胃癌中的研究发现,RhoC蛋白和IQGAP1蛋白均呈高表达,且二者呈正相关关系。但有关二者在肝癌中的表达情况尚无人报道。为了解二者在HCC发生、发展中的作用及各种可能的机制,本文通过观察HCC组织、癌旁组织和正常肝组织中RhoC和IQGAP1的表达情况以及二者与HCC临床病理特征的关系,初步探讨RhoC和IQGAP1在HCC组织中的相关性。 方法 随机取郑州大学第一附属医院2010年3月-2011年3月手术切除的56例肝癌标本。肝癌患者中男48例,女8例,年龄34-65岁。术前均未接受化疗放疗及免疫治疗,病理学均确诊为原发性肝细胞癌,其中高分化癌14例,中分化癌22例,低分化癌20例；有门脉浸润14例,无浸润42例；TNM分期参照AJCC肝癌肿瘤分期标准,Ⅰ～Ⅱ期17例,Ⅲ～Ⅳ期39例；肿瘤大于或等于5cm者22例,小于5cm者34例。随机选25例性别与年龄构成与上述无显著差异并经病理学证实为肝硬化的癌旁组织做对照,另选15例同期肝血管瘤旁病理证实为正常肝组织的组织为正常对照。以上所有标本组织均经过甲醛溶液固定,石蜡包埋,4μm厚连续切片。载玻片经泡酸、高温灭菌及多聚赖氨酸涂片处理。采用免疫组化PV-9000二步法,具体步骤参照试剂盒说明。用PBS代替一抗做阴性对照。以已知的RhoC和IQGAP1阳性切片作为阳性对照。结果采用SPSS17.0软件分析数据,对HCC组织、癌旁组织及正常组织中RhoC和IQGAP1的差异性采用χ2检验和Fisher确切概率法,对HCC组织中二者的相关性行spearman相关分析,以α=0.05作为检验水准。 结果 1、RhoC表达与HCC临床病理因素的关系 RhoC阳性染色主要定位于细胞浆,呈弥漫性或灶性分布,以弥漫性为主。RhoC在HCC组织、癌旁组织及正常肝组织中的阳性表达率分别为66.1%、40.0%、20.0%,RhoC在HCC中的阳性表达高于癌旁组织(χ2=4.824,P=0.028)及正常肝组织(χ2=10.209,P=0.001),且三者比较具有显著性差异(χ2=12.040,P=0.002)。HCC组织中RhoC在高分化组、中分化组和低分化组中的阳性表达率分别为28.6%、63.6%和95.0%。RhoC在低分化组织中的阳性表达高于中分化组织(Fisher确切概率法,P=0.022)及高分化组(χ2=13.708,P=0.000),且三者比较具有显著性差异(χ2=16.307,P<0.001)。HCC组织中RhoC在临床分期Ⅰ～Ⅱ组与Ⅲ-Ⅳ组的阳性率分别为35.3%与79.5%,两者的差异性比较具有统计学意义(χ2=10.315,P=0.001)。HCC组织中有门脉浸润组RhoC的表达显著高于无门脉浸润组(92.9%vs57.1%),两组的差异具有统计学意义(χ2=4.487,P=0.034)。HCC组织中RhoC的表达与性别、年龄、肿瘤大小无关(均P>0.05) 2、IQGAP1表达与HCC临床病理因素的关系 IQGAP1的阳性染色主要定位于细胞浆或细胞膜,呈弥漫分布。IQGAP1在HCC组织、癌旁组织及正常肝组织中的阳性表达率分别为75.0%、44.0%、13.3%；IQGAP1在HCC组织中的阳性表达显著高于癌旁组织(χ2=7.343,P=0.007),而在癌旁组

摘要： Alterations in the strength of glutamatergic synaptic activity shape the synaptic localization of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors (AMPARs), and this process is thought to underlie higher brain functions such as learning and memory. During excitatory synaptic transmission, the presynaptic neuron releases glutamate which activates AMPARs that, in turn, conduct postsynaptic synaptic transmission. Concomitantly, excitatory amino acid transporters (EAATs) re-uptake released glutamate, thereby curtailing the strength of AMPAR synaptic activation. Although the coordination of glutamate receptors and transporters likely occurs, whether and how transporter activity affects AMPAR synaptic expression remains less clear. Using cultured hippocampal neurons, I show that inhibition of glutamate transporters leads to a rapid redistribution and dramatic reduction in AMPAR synaptic accumulation. I also find that EAAT inactivity causes AMPAR internalization and reduction of surface AMPAR expression. AMPAR reduction can be blocked by suppression of proteasome activity and results in the induction of receptor ubiquitination, strongly indicating the involvement of proteasome-mediated receptor degradation. In searching for the molecular components involved in activity-induced receptor endocytosis, I identified the molecular scaffold, IQGAP1. IQGAP1 enhances AMPAR turnover through binding to ubiquitinated AMPARs and promoting receptor internalization. In addition, I also found a decrease in AMPAR abundance following enhanced expression of IQGAP1. Furthermore, activation of parasynaptically localized NR2B-containing NMDA receptors (NMDARs) is required for AMPAR degradation, suggesting glutamate spillover from the synaptic cleft occurs during EAAT inhibition. Interestingly, I found that the neuronal, but not the glial glutamate transporters are responsible for the observed AMPAR regulation. Thus, my results strongly indicate a role for ne

关键词： Aurora-A激酶   骨架蛋白   蛋白稳定性   肿瘤细胞  

摘要： IQGAP1是一种进化保守的多结构域蛋白，它是到目前为止IQGAP(IQ-domainGTPase-activatingprotein)蛋白家族三个成员IQGAP1、IQGAP2和IQGAP3中被研究的最多也是最深入的一员，IQGAP1于1994年首次被克隆出。其独特的分子结构赋予了IQGAP1多种功能，从而使其成为细胞黏附、细胞迁移、细胞外信号转导及细胞分裂等多种功能活动的重要调控分子。研究发现，许多人类组织中都能检测到骨架蛋白IQGAP1的表达。在细胞中，IQGAP1能通过与多种细胞信号通路中重要蛋白分子相互作用，这些相互作用使得IQGAP1能够参与调控许多细胞内及细胞外事件。目前越来越多的研究发现，IQGAP1在肿瘤的发生发展中扮演着越来越重要的角色。因此，IQGAP1对肿瘤的研究具有十分重要的意义。　　 本研究中，我们在肿瘤细胞内发现了一个新的可以与IQGAP1相结合的蛋白分子-Aurora-A。Aurora-A定位于人类染色体20q13，属于丝氨酸/苏氨酸蛋白激酶家族-Aurora家族的成员之一，到目前为止该蛋白激酶家族共分为三类：Aurora-A、Aurora-B和***丝氨酸/苏氨酸蛋白激酶家族在中心体成熟、分离，双极纺锤体组装，染色体分离和胞质分裂过程中都发挥重要作用。Aurora-A作为一个重要的癌基因，其编码的蛋白在多种人类肿瘤中都存在过表达的现象，如结肠癌、卵巢癌、胃癌、乳腺癌以及食管癌等。我们验通过体外实验研究发现，GST-Aurora-A融合蛋白可以结合细胞内源性IQGAP1蛋白。同时免疫共沉淀实验也发现在HeLa细胞中，内源性Aurora-A与IQGAP1蛋白也可以相互作用。在乳腺癌MCF-7细胞中瞬时过表达IQGAP1可以引起Aurora-A蛋白水平的增高和激酶活性的增加。进一步的研究还发现，在IQGAP1过表达的细胞中，Aurora-A蛋白的半衰期延长、稳定性增加、其与后期促进复合体(theanaphasepromotingcomplex/cyclosome)中的各个组成蛋白(APC2、CDC27和CDH1)之间的相互作用也明显减弱。与此同时瞬时特异性敲降MCF-7细胞内源性IQGAP1的表达可以有效的降低细胞内Aurora-A的蛋白水平。但是通过逆转录PCR以及实时荧光定量PCR实验研究发现，特异性敲降细胞内IQGAP1并不能引起Aurora-AmRNA水平的变化。这一结果提示，IQGAP1对Aurora-A的调控主要体现在转录后水平。由于过表达IQGAP1可以增强肿瘤细胞的增殖能力，因此本实验利用Aurora-AsiRNA特异性敲降IQGAP1过表达MCF-7细胞中内源性Aurora-A发现，Aurora-AsiRNA可以有效的抑制由IQGAP1过表达引起的细胞增殖。此结果提示我们，IQGAP1有可能通过影响细胞内Aurora-A的水平和活性，参与调控肿瘤细胞的增殖。　　 综上所述，IQGAP1可以通过调节细胞内Aurora-A的蛋白稳定性及激酶活性，参与细胞增殖，从而影响肿瘤的发生发展。　　 真核细胞中细胞分裂周期是一个严格调控、高度有序的过程，细胞的有丝分裂对肿瘤细胞的扩增起着关键的调节作用，有丝分裂的偏差会导致基因组不稳定，从而引发肿瘤。Aurora激酶家族又被称作极光激酶家族，是一组调控细胞有丝分裂的丝/苏氨酸蛋白激酶，包括三个成员：AuroraA、B、C。在低等生物（酵母）在进化早期就独立出来，因此它们仅有一个Aurora基因。较高等的动物细胞由于基因复制的结果，它们有Aurora-A和Aurora-B两个同源基因。脊椎动物的Aurora-A和Aurora-B可能来自于无脊椎动物的Aurora-A复制后的独立进化。哺乳动物Aurora-B在进化中可能发生了重组或复制，衍生出Aurora-C。Aurora激酶三亚群在结构上具有高度保守性，其蛋白质的一级结构含有两个结构域，C末端结构即催化区和N末端即可变区，其中人类Aurora-A和Aurora-B催化区的氨基酸序列保守性达到71％，然而它们三者的N末端即可变区却不相同。这种催化区结构的保守性表明Aurora激酶的底物在结构上具有特异性，而且催化区的晶体结构也已完全揭示，为研究其特异性底物和调控物提供了条件。Aurora-A蛋白激酶在中心体复制、两极纺锤体的形成、染色体的重排和染色体检查点的监测等重要的有丝分裂事件中发挥着至关重要的作用。本文就近年来有关Aurora-A蛋白激酶的研究进展进行综述。

关键词： IQ motif-containing GTPase-activating protein 1 (IQGAP1)   microbial pathogenesis   protein scaffold   Salmonella  

摘要： Salmonella enterica serotype Typhimurium invades eukaryotic cells by re-arranging the host-cell cytoskeleton. However, the precise mechanisms by which Salmonella induces cytoskeletal changes remain undefined. IQGAP1 (IQ motif-containing GTPase-activating protein 1) is a scaffold protein that binds multiple proteins including actin, the Rho GTPases Rac1 and Cdc42 (cell division cycle 42), and components of the MAPK (mitogen-activated protein kinase) pathway. We have shown previously that optimal invasion of Salmonella into HeLa cells requires IQGAP1. In the present paper, we use IQGAP1-null MEFs (mouse embryonic fibroblasts) and selected well-characterized IQGAP1 mutant constructs to dissect the molecular determinants of Salmonella invasion. Knockout of IQGAP1 expression reduced Salmonella invasion into MEFs by 75%. Reconstituting IQGAP1-null MEFs with wild-type IQGAP1 completely rescued invasion. By contrast, reconstituting IQGAP1-null cells with mutant

关键词： 肝细胞癌   RhoC   IQGAP1   免疫组织化学  

摘要： 目的:探讨RhoC和IQGAP1蛋白在原发性肝细胞癌(hepatocellular carcinoma,HCC)组织中的表达及其临床病理意义.方法:采用免疫组织化学法分别检测56例原发性肝细胞癌和15例正常肝组织中RhoC及IQGAP1蛋白的表达,并分析两者的相关性及与临床病理因素的关系.结果:RhoC和IQGAP1在原发性肝细胞癌组织中的表达明显高于正常组织(66%vs20%;75%vs13%,均P<0.05),且两者的表达呈正相关(r=0.631,P<0.05),并与肿瘤组织的分化程度及门脉浸润等均密切相关(均P<0.05).结论:RhoC及IQGAP1在原发性肝细胞癌的发生、发展中发挥重要的促进作用,可考虑作为HCC临床评价肿瘤生物学行为及评估预后的指标.

关键词： calcium   calmodulin   in vitro   IQ motif   IQGAP1  

摘要： IQ motif-containing GTPase-activating protein 1 (IQGAP1), which is a well-known calmodulin (CaM) binding protein, is involved in a wide range of cellular processes including cell proliferation, tumorigenesis, adhesion, and migration. Interaction of IQGAP1 with CaM is important for its cellular functions. Although each IQ domain of IQGAP1 for CaM binding has been characterized in a Ca(2+)-dependent or -independent manner, it was not clear which IQ motifs are physiologically relevant for CaM binding in the cells. In this study, we performed immunoprecipitation using 3xFLAGhCaM in mammalian cell lines to characterize the domains of IQGAP1 that are key for CaM binding under physiological conditions. Interestingly, using this method, we identified two novel domains, IQ(2.7-3) and IQ(3.5-4.4), within IQGAP1 that were involved in Ca(2+)-independent or -dependent CaM binding, respectively. Mutant analysis clearly showed that the hydrophobic regions within IQ(2.7-3) were mainly involved in apoCaM binding, while the basic amino acids and hydrophobic region of IQ(3.5-4.4) were required for Ca(2+)/CaM binding. Finally, we showed that IQ(2.7-3) was the main apoCaM binding domain and both IQ(2.7-3) and IQ(3.5-4.4) were required for Ca(2+)/CaM binding within IQ(1-2-3-4). Thus, we identified and characterized novel direct CaM binding motifs essential for IQGAP1. This finding indicates that IQGAP1 plays a dynamic role via direct interactions with CaM in a Ca(2+)-dependent or -independent manner.

关键词： calcium   calmodulin   in vitro   IQ motif   IQGAP1  

摘要： IQ motif-containing GTPase-activating protein 1 (IQGAP1), which is a well-known calmodulin (CaM) binding protein, is involved in a wide range of cellular processes including cell proliferation, tumorigenesis, adhesion, and migration. Interaction of IQGAP1 with CaM is important for its cellular functions. Although each IQ domain of IQGAP1 for CaM binding has been characterized in a Ca-dependent or -independent manner, it was not clear which IQ motifs are physiologically relevant for CaM binding in the cells. In this study, we performed immunoprecipitation using 3xFLAGhCaM in mammalian cell lines to characterize the domains of IQGAP1 that are key for CaM binding under physiological conditions. Interestingly, using this method, we identified two novel domains, IQ(2.7-3) and IQ(3.5-4.4), within IQGAP1 that were involved in Ca-independent or -dependent CaM binding, respectively. Mutant analysis clearly showed that the hydrophobic regions within IQ(2.7-3) were mainly involved in apoCaM binding, while the basic amino acids and hydrophobic region of IQ(3.5-4.4) were required for Ca/CaM binding. Finally, we showed that IQ(2.7-3) was the main apoCaM binding domain and both IQ(2.7-3) and IQ(3.5-4.4) were required for Ca/CaM binding within IQ(1-2-3-4). Thus, we identified and characterized novel direct CaM binding motifs essential for IQGAP1. This finding indicates that IQGAP1 plays a dynamic role via direct interactions with CaM in a Ca-dependent or -independent manner. [ABSTRACT FROM AUTHOR]

关键词： ERK1/2   FAK   IQGAP1   Melusin   Heart hypertrophy  

摘要： Extracellular signal-regulated kinase 1/2 (ERK1/2) signalling is a key pathway in cardiomyocyte hypertrophy and survival in response to many different stress stimuli. We have previously characterized melusin as a muscle-specific chaperone protein capable of ERK1/2 signalling activation in the heart. Here, we show that in the heart, melusin forms a supramolecular complex with the proto-oncogene c-Raf, MEK1/2 (also known as MAPKK1/2) and ERK1/2 and that melusin-bound mitogen-activated protein kinases (MAPKs) are activated by pressure overload. Moreover, we demonstrate that both focal adhesion kinase (FAK) and IQ motif-containing GTPase activating protein 1 (IQGAP1), a scaffold protein for the ERK1/2 signalling cascade, are part of the melusin complex and are required for ERK1/2 activation in response to pressure overload. Finally, analysis of isolated neonatal cardiomyocytes indicates that both FAK and IQGAP1 regulate melusin-dependent cardiomyocyte hypertrophy and survival through ERK1/2 activation.

关键词： ACTIN   CELL migration   CYTOKINESIS   CYTOSKELETON   CALMODULIN  

摘要： IQGAP1 is a large modular protein that displays multiple partnership and is thought to act as a scaffold in coupling cell signaling to the actin and microtubule cytoskeletons in cell migration, adhesion, and cytokinesis. However the molecular mechanisms underlying the activities of IQGAP1 are poorly understood in part because of its large size, poor solubility and lack of functional assays to challenge biochemical properties in various contexts. We have purified bacterially expressed recombinant human IQGAP1. The protein binds Cdc42, Rac1, and the CRIB domain of N-WASP in a calmodulin-sensitive fashion. We further show that in addition to bundling of filaments via a single N-terminal calponin-homology domain, IQGAP1 actually regulates actin assembly. It caps barbed ends, with a higher affinity for ADP-bound terminal subunits (K-B = 4 nM). The barbed end capping activity is inhibited by calmodulin, consistent with calmodulin binding to IQGAP1 with a K-C of 40 nM, both in the absence and presence of Ca2+ ions. The barbed end capping activity resides in the C-terminal half of IQGAP1. It is possible that the capping activity of IQGAP1 accounts for its stimulation of cell migration. We further find that bacterially expressed recombinant IQGAP1 fragments easily co-purify with nucleic acids that turn out to activate N-WASP protein to branch filaments with Arp2/3 complex. The present results open perspectives for tackling the function of IQGAP1 in more complex reconstituted systems.

关键词： IQGAP1   肝癌细胞   mTOR  

摘要： 目的:研究IQGAP1(IQ domain GTPase-activating proteins,IQGAP1)对肝癌细胞株增殖及mTOR信号转导通路的影响,阐述其诱导细胞增殖的机制。方法:Western blot检测IQGAP1在肝癌细胞株HepG2、Huh-7中的表达。选择高表达IQGAP1的细胞株,针对IQGAP1基因的mRNA序列合成siRNA,采用脂质体转染法将其转入HepG2细胞;采用MTT法分析细胞的增殖;流式细胞仪检测细胞周期变化;免疫印迹法观测IQGAP1、mTOR和p-mTOR蛋白表达的变化。结果:IQGAP1在HepG2和Huh-7细胞中均表达,且在HepG2细胞中表达高于Huh-7细胞。IQGAP1 siRNA转染HepG2细胞后,抑制细胞的增殖;DNA合成前期(G_0/G_1期)细胞比例上升,合成期(S期)细胞比例下降;mTOR表达变化不明显,而p-mTOR的表达下降。结论:IQGAP1促进肝癌细胞的增殖,其机制可能是通过mTOR相关信号通路实现的。