关键词： 气道上皮损伤   修复机制   细胞质连接蛋白   GTP酶激活蛋白1   恒定链短肽   气态甲醛  

摘要： 室温下甲醛是一种刺激性气体,可造成气道(气管和支气管)上皮损伤。在修复过程中,损伤周围的上皮细胞(airway epithelial cell,AEC)分裂增殖、分化和迁移,重新覆盖受损裸露区域,这一系列细胞活动过程依赖于微丝和微管的协调运动。包含IQ序列的GTP酶激活蛋白1(IQ motif containing GTPase activating protein1,IQGAP1)及其众多靶蛋白可以调节微管和微丝的活动,在其中起了关键的作用。IQGAP1是Rho家族GTPases成员Cdc42的一个重要的效应因子,属于细胞骨架蛋白,可以直接与肌动蛋白结合交联微丝,也能通过细胞质连接蛋白-170(cytoplasmic linker protein-170,CLIP-170)募集微管,从而参与细胞损伤修复、组织发展等多种活动。CLIP-170属于微管正极示踪蛋白(microtubule plus-end-tracking proteins,+TIPs),参与微管组装过程,并能稳定微管结构,促进微管延长。基于以上资料,推测IQGAP1和CLIP-170可能参与修复过程中的细胞极化和定向迁移等活动,从而在气道上皮损伤修复过程中发挥重要作用。　　目的:本研究通过建立小鼠甲醛吸入动物模型,检测IQGAP1和CLIP-170在模型小鼠气道上皮损伤修复不同阶段的动态变化,初步探讨二者在甲醛致气道上皮损伤修复过程中的作用及机制,继而为进一步深入探讨气道上皮损伤后的修复机制提供理论和实验依据。　　方法:选用健康昆明种小鼠24只,随机分成4组(甲醛吸入3、7、60天组和对照组),用5mg/m3气态甲醛分别使小鼠吸入3天、7天和60天,每天吸入4小时,建立甲醛致气道上皮损伤修复的动物模型,以未吸甲醛小鼠为对照。采用HE染色观察损伤修复过程中气道上皮的形态学变化,免疫组织化学染色检测IQGAP1和CLIP-170在气道上皮内表达的动态变化,Western印迹技术检测肺组织中IQGAP1和CLIP-170的总表达量,免疫荧光共聚焦成像观察IQGAP1和CLIP-170在气道上皮细胞内的分布变化。　　结果:①HE染色结果显示:对照组小鼠气道粘膜上皮结构完整,纤毛排列整齐,细胞间连接紧密;甲醛吸入3天组小鼠气道上皮细胞的纤毛粘连,倒伏,上皮细胞部分坏死脱落并有炎症细胞浸润;甲醛吸入7天组小鼠气道上皮出现修复现象,细胞-细胞间黏附变得松散,大部分气道被上皮细胞覆盖并在细支气管周围有大量的炎症细胞浸润;甲醛吸入60天组小鼠气道上皮细胞间粘附紧密,并可见部分气道上皮基底层细胞增生,细胞层次增多,管壁慢性炎症细胞浸润。②免疫组化染色结果:甲醛吸入3天、7天时IQGAP1表达均较对照组显著增强(P<0.01),60天时IQGAP1表达较7天组减弱(P<0.05),但与对照组相比无显著差异;CLIP-170在甲醛吸入3、7天时表达高于对照组(分别为P<0.01,P<0.05),60天时CLIP-170表达水平与对照组比较无显著性差异。③ Western印迹显示:IQGAP1和 CLIP-170蛋白含量在各实验组肺组织的变化与免疫组化表达变化一致。④荧光共聚焦成像显示:IQGAP1红色荧光颗粒在对照组气道上皮细胞胞质内呈均匀弥散分布,甲醛吸入3、7天时红色荧光颗粒明显增强,主要聚集在气道上皮细胞腔面和侧面质膜下的胞质中,60天时荧光分布与对照组相似;CLIP-170红色荧光颗粒在对照组气道上皮细胞胞质内呈均匀弥散分布,甲醛吸入3天和7天时红色荧光颗粒在气道上皮细胞腔面和侧面质膜下聚集成簇状,60天时荧光分布与对照组相似。　　结论:①气态甲醛对气道上皮存在持续性的损害作用,早期以气道上皮损伤性变化和炎症反应为主,长期吸入可引起气道慢性炎症及黏膜上皮细胞增生。②IQGAP1和CLIP-170可能通过表达量及分布变化参与甲醛引起的小鼠气道上皮损伤后修复过程。　　由上述结果推测:IQGAP1和CLIP-170可能通过对微管及微丝活动的调节,影响修复过程中细胞极化和定向迁移等生物学活动,从而在气道上皮损伤修复过程中发挥着重要的作用。

关键词： IQGAP1   磷酸化   气道上皮细胞   损伤修复   Rho GTPases   IQGAP1   气道上皮细胞   损伤修复  

摘要： 第一部分气道上皮细胞中机械划伤致IQGAP1磷酸化对损伤修复的影响 实验背景: 由于气道(气管和支气管)粘膜暴露于外界环境,易受到损伤。气道上皮受损后将立即启动修复过程,从而保持正常的肺功能。这个损伤修复过程需要气道上皮细胞伸展,迁移和增生最终覆盖受损区域。气道受损是气道疾病例如慢性支气管炎和COPD的重要的发病过程。因此气道上皮自身的修复能力是解决这些气道疾病的关键步骤。然而,参与气道上皮损伤修复的分子机制尚未完全明了。 IQGAP1(IQ domain GTPase-activating protein)在许多细胞生命活动中是一个非常有意义的蛋白,但是尚未有人阐述其在气道上皮损伤修复中的作用。磷酸化作为翻译后的主要手段,参与调节蛋白质及细胞骨架的功能。有报导显示,IQGAP1丝氨酸残基能被高磷酸化,结果能促进神经元轴突外生性生长。PKC的一个新的亚型PKCε可能有助于IQGAP1的磷酸化。我们推测,在气道上皮损伤修复过程中IQGAP1磷酸化可能发挥重要作用。 目的: 使用目前广泛应用的刮伤气道上皮细胞的体外模型,初步探讨机械划伤后IQGAP1的磷酸化水平以及对损伤修复的影响。 方法: 建立机械划痕引起体外气道上皮损伤模型,采用损伤分析实验、免疫组化、western blot、免疫荧光共聚焦成像、免疫沉淀、瞬时转染等方法观测IQGAP1磷酸化、IQGAP1与PKCε在损伤前后的变化,并通过激活和抑制PKCε活性,观察对损伤愈合的影响。 结果: 形态学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。在机械性损伤后,气道上皮细胞可成片状的单向迁移,且其迁移方向垂直于划痕方向。损伤6 h细胞形态即发生显著改变,形成具有特征性的极化形态学改变,损伤12 h更为显著。损伤24 h后迁移的气道上皮细胞能掩盖划线部分。 本实验发现,IQGAP1在多种动物气道上皮细胞中表达丰富。它与微管骨架共定位,但能被微管解聚剂nocodazole破坏。机械划伤后,IQGAP1磷酸化水平迅速增加,在损伤后1小时达到峰值,持续9小时。过表达PKCε或添加PKC活化剂PMA,能促进IQGAP1磷酸化。相反,PKC抑制剂GF109203X能消除划伤引起的IQGAP1磷酸化。通过免疫荧光共聚焦和免疫共沉淀方法,我们发现机械划伤能加强IQGAP1与PKCε结合。损伤分析实验结果显示,刮擦24h后,正常对照组细胞划痕已经闭合,而过表达PKCε或PMA处理组细胞划痕提前愈合。相反,GF109203X处理组不仅没有闭合,而且其划痕宽度仍占原始划痕的32%,提示IQGAP1磷酸化介导了气道上皮细胞的划痕愈合。 结论 本实验表明,IQGAP1在气道上皮细胞中有丰富的表达,且PKCε介导的IQGAP1磷酸化参与了机械划伤引起的气道上皮细胞损伤愈合过程。 第二部分IQGAP1以不依赖于Rac1和Cdc42的方式参与气道上皮损伤修复的增殖过程 实验背景: 香烟烟雾、大气污染、感染等多种致病因素在机体抵抗力降低时,都能引起气道上皮损伤,导致肺疾病的发生。气道损伤后立即启动修复过程,这是一个复杂的多步骤过程,包括细胞延伸、迁移和增殖。Rho GTPases是小G蛋白超家族的主要成员,其中RhoA,Rac1和Cdc42研究最为广泛。Rho GTPases在GTP结合的活化状态和GDP结合的失活状态间转换。通过与许多效应子的作用,如IQGAP1、PAK(p21 activated kinase)和WASP(Wiskott-Aldrich syndrome protein),Rho GTPases在组织形成、细胞极化和迁移过程中发挥重要作用。作为Rac1和Cdc42的效应子,IQGAP1能与细胞骨架成分、细胞黏附复合体及微管相关蛋白结合,参与了细胞黏附、极化和定向迁移过程。目前尚未有人阐述Rho GTPases及IQGAP1在肺上皮细胞中的作用。本研究推断上述信号可能在气道上皮细胞损伤修复中发挥作用,并探讨其可能的机制。 目的: 使用了体外损伤模型来研究Rho GTPases及IQGAP1的功能。检测上述因子在气道上皮不同损伤修复时期的动态表达变化,初步研究其在气道上皮损伤修复过程中的作用,继而为深入探讨损伤修复机制提供理论和实验依据。 方法: 在机械划伤单层细胞建立的体外损伤模型中,通过pull-down方法检测Rac1及Cdc42的活性。采用免疫细胞荧光、RT-PCR、荧光定量PCR、western blot、分离核浆蛋白、质粒瞬时转染和RNA干

关键词： IQGAP1   肝癌   胰腺癌   表达   免疫组织化学  

摘要： 目的：探讨IQGAP1蛋白在肝癌、胰腺癌中的表达,及与临床、病理的关系。 方法：采用免疫组织化学方法,观察IQGAP1蛋白在肝癌、胰腺癌中的表达,及与临床病理的联系。 结果：肝癌中IQGAP1蛋白阳性染色率52.7％(19/36),胞膜型30.6％(11/36)及细胞浆型16.6％(6/36),胞核型5.5％(2/36);胰腺癌组织中IQGAP1蛋白阳性表达率21.9％(7/32),胞膜表达型12.5％(4/32),胞浆表达型6.3％(2/32),胞核表达型3.1％(1/32)。IQGAP1蛋白在肝癌、胰腺癌临床分期、血管浸润及淋巴结转移方面有统计学意义。 结论:IQGAP1蛋白在肝癌、胰腺癌中表达部位有差别,与临床分期、血管浸润及淋巴结转移具有相关性。

关键词： PROTEINS   SALMONELLA   ACTIN   CYTOSKELETAL proteins   RHO GTPases   ENTEROBACTERIACEAE  

摘要： To infect host cells, Salmonella utilizes an intricate system to manipulate the actin cytoskeleton and promote bacterial uptake. Proteins injected into the host cell by Salmonella activate the Rho GTPases, Rac1 and Cdc42, to induce actin polymerization. Following uptake, a different set of proteins inactivates Rac1 and Cdc42, returning the cytoskeleton to normal. Although the signaling pathways allowing Salmonella to invade host cells are beginning to be understood, many of the contributing factors remain to be elucidated. IQGAP1 is a multidomain protein that influences numerous cellular functions, including modulation of Rac1/Cdc42 signaling and actin polymerization. Here, we report that IQGAP1 regulates Salmonella invasion. Through its interaction with actin, IQGAP1 co-localizes with Rac1, Cdc42, and actin at sites of bacterial uptake, whereas infection promotes the interaction of IQGAP1 with both Rac1 and Cdc42. Knockdown of IQGAP1 significantly reduces Salmonella invasion and abrogates activation of Cdc42 and Rac1 by Salmonella. Overexpression of IQGAP1 significantly increases the ability of Salmonella to enter host cells and required interaction with both actin and Cdc42/Rac1. Together, these data identify IQGAP1 as a novel regulator of Salmonella invasion.

关键词： APC   Wnt signalling pathway   colon cancer   chromosome instability   cell migration   cell adhesion   beta-catenin   Asef   IQGAP1   actin   EB1   microtubules  

摘要： The adenomatous polyposis coli ( APC) gene is a key tumor suppressor gene. Mutations in the gene have been found not only in most colon cancers but also in some other cancers, such as those of the liver. The APC gene product is a 312 kDa protein that has multiple domains, through which it binds to various proteins, including beta-catenin, axin, CtBP, Asefs, IQGAP1, EB1 and microtubules. Studies using mutant mice and cultured cells have demonstrated that APC suppresses canonical Wnt signalling, which is essential for tumorigenesis, development and homeostasis of a variety of cell types, such as epithelial and lymphoid cells. Further studies have suggested that APC plays roles in several other fundamental cellular processes. These include cell adhesion and migration, organization of the actin and microtubule networks, spindle formation and chromosome segregation. Deregulation of these processes caused by mutations in APC is implicated in the initiation and expansion of colon cancer.

关键词： MOLNEURO   SYSNEURO   SIGNALING  

摘要： Cadherin-mediated interactions are integral to synapse formation and potentiation. Here we show that N-cadherin is required for memory formation and regulation of a subset of underlying biochemical processes. N-cadherin antagonistic peptide containing the His-Ala-Val motif (HAV-N) transiently disrupted hippocampal N-cadherin dimerization and impaired the formation of long-term contextual fear memory while sparing short-term memory, retrieval, and extinction. HAV-N impaired the learning-induced phosphorylation of a distinctive, cytoskeletally associated fraction of hippocampal Erk-1/2 and altered the distribution of IQGAP1, a scaffold protein linking cadherin-mediated cell adhesion to the cytoskeleton. This effect was accompanied by reduction of N-cadherin/ IQGAP1/Erk-2 interactions. Similarly, in primary neuronal cultures, HAV-N prevented NMDA-induced dendritic Erk-1/2 phosphorylation and caused relocation of IQGAP1 from dendritic spines into the shafts. The data suggest that the newly identified role of hippocampal N-cadherin in memory consolidation may be mediated, at least in part, by cytoskeletal IQGAP1/Erk signaling.

关键词： IQGAP1   calmodulin   scaffold   cell migration   actin  

摘要： Cell migration, a highly complex physiological phenomenon that requires the co-ordinated and tightly regulated function of several proteins, is mediated by a number of signalling pathways. Elucidation of the molecular mechanisms of cell migration impacts our comprehension of numerous cell functions, ranging from development and immune surveillance to angiogenesis and metastasis. The scaffold protein IQGAP1, which binds multiple proteins and regulates their functions, promotes cell motility. Many of the IQGAP1 binding proteins have been implicated in cell migration. In this study, we employed a multifaceted strategy to identify proteins that contribute to IQGAP1-stimulated cell migration. Using specific IQGAP1 point mutant constructs, an interaction with actin was shown to be essential for IQGAP1 to increase cell migration. In contrast, eliminating the binding of Ca2+/calmodulin, but not Ca2+-free calmodulin, augmented the ability of IQGAP1 to stimulate cell migration. Consistent with these findings, selective inhibition of calmodulin function at the plasma membrane with a specific peptide inhibitor enhanced cell migration mediated by IQGAP1. Interestingly, immunofluorescence staining and confocal microscopy suggest that localization of Cdc42 at the leading edge is not necessary for maximal migration of epithelial cells. Coupled with the observations that Cdc42 and Rac1 contribute to IQGAP1-stimulated cell migration, these data suggest that IQGAP1 serves as a junction to integrate multiple signalling molecules to facilitate cell migration. (c) 2007 Elsevier Inc. All rights reserved.

关键词： DIAPHANOUS-RELATED FORMINS   ACTIN-FILAMENTS   HOMOLOGY-2 DOMAIN   NUCLEATION   MDIA1   AUTOINHIBITION   POLARIZATION   DYNAMICS   GTPASES   ALPHA  

摘要： The Diaphanous-related formin Dial nucleates actin polymerization, thereby regulating cell shape and motility. Mechanisms that control the cellular location of Dial to spatially define actin polymerization are largely unknown. In this study, we identify the cytoskeletal scaffold protein IQGAP1 as a Dial-binding protein that is necessary for its subcellular location. IQGAP1 interacts with Dial through a region within the Diaphanous inhibitory domain after the RhoA-mediated release of Dial autoinhibition. Both proteins colocalize at the front of migrating cells but also at the actin-rich phagocytic cup in macrophages. We show that IQGAP1 interaction with Dial is required for phagocytosis and phagocytic cup formation. Thus, we identify IQGAP1 as a novel component involved in the regulation of phagocytosis by mediating the localization of the actin filament nucleator Dial.

关键词： Calmodulin   findings   Proteins   GTP Phosphohydrolases  

摘要： IQGAP1 is a scaffolding protein involved in multiple fundamental cellular activities, including transcription, cell-cell attachment, and regulation of the cytoskeleton. To function in these pathways, IQGAP1 associates with numerous proteins such as actin, calmodulin, E-cadherin, beta-catenin, CLIP-170, and components of the mitogen-activated protein kinase pathway. Moreover, IQGAP1 binds to active Cdc42 and Rac1 but not RhoA or Ras. Here we show that IQGAP1 also binds to the small GTPase Rap1. In vitro analysis demonstrates a direct interaction between Rap1 and IQGAP1, which is augmented by activation (GTP loading) of Rap1. Cdc42 does not modulate the interaction between Rap1 and IQGAP1. In contrast, the association is eliminated by calmodulin both in the absence and presence of Ca2+. The binding of Rap1 to a point mutant IQGAP1 construct that is unable to interact with calmodulin is 2.5-fold more than to wild type IQGAP1. Consistent with these findings, Rap1 binds to the IQ region of IQGAP1. Confocal microscopy demonstrates that Rap1 and IQGAP1 co-localize at the periphery of human epithelial cells but not in the cytoplasm. The interaction has functional sequelae. Overexpression of IQGAP1 substantially reduces adhesion-mediated activation of Rap1. In addition, Rap1 activation by cAMP is attenuated in cells that overexpress IQGAP1 and enhanced in cells lacking IQGAP1. These findings reveal that the interaction of IQGAP1 with Rap1 differs in several respects from its interaction with other small GTPases. Furthermore, our data suggest that IQGAP1 may link the calmodulin and Rap1 signaling pathways.

关键词： EGF   MAP kinase   signalling  

摘要： IQGAP1 modulates several cellular functions, including cell-cell adhesion, transcription, cytoskeletal architecture, and selected signaling pathways. We previously documented that IQGAP1 binds ERK and MAPK kinase (MEK) and regulates EGF-stimulated MEK and ERK activity. Here we characterize the interaction between IQGAP1 and B-Raf, the molecule immediately upstream of MEK in the Ras/MAPK signaling cascade. B-Raf binds directly to IQGAP1 in vitro and coimmunoprecipitates with IQGAP1 from cell lysates. Importantly, IQGAP1 modulates B-Raf function. EGF is unable to stimulate B-Raf activity in IQGAP1-null cells and in cells transfected with an IQGAP1 mutant construct that is unable to bind B-Raf. Interestingly, binding to IQGAP1 significantly enhances B-Raf activity in vitro. Our data identify a previously unrecognized interaction between IQGAP1 and B-Raf and suggest that IQGAP1 is a scaffold necessary for activation of B-Raf by EGF.