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.

关键词： 子宫颈肿瘤   IQGAP1   免疫组织化学  

摘要： 目的 探讨子宫颈癌组织和癌旁组织中IQ结构域GTP酶激活蛋白1（IQ-domain GTPase-activating protein1,IQGAP1）的表达及其意义。方法 采用免疫组化SP法检测48例子宫颈癌组织和23例癌旁组织中IQGAP1的表达,并分析IQGAP1表达与子宫颈癌临床病理特征的关系。结果 IQGAP1在子宫颈癌中的阳性率为77.1%（37/48）,明显高于癌旁组织（30.4%,7/23）,两组相比差异有统计学意义（χ2=14.285,P〈0.01）。IQGAP1表达与子宫颈癌组织分化程度（χ2=3.102,P〈0.05）、临床分期（χ2=7.111,P〈0.05）和淋巴结转移（χ2=6.553,P〈0.05）相关;与患者年龄（χ2=0.112,P〉0.05）无关。结论 子宫颈癌组织中IQGAP1蛋白阳性率明显高于癌旁组织,且与肿瘤分化程度、临床分期及有无淋巴结转移密切相关,有望成为子宫颈癌生物学行为的有效指标之一。

关键词： Myeloproliferative neoplasms   Erythrocyte proteome   IQGAP1   Rho GTPases  

摘要： Besides genetic abnormalities in MPN patients, several studies have reported alterations in protein expression that could contribute towards the clinical phenotype. However, little is known about protein modifications in Ph- MPN erythrocytes. In this context, we used a quantitative mass spectrometry proteomics approach to study the MPN erythrocyte proteome. LC-MS/MS (LTQ Orbitrap) analysis led to the identification of 51 and 86 overexpressed proteins in Polycythemia Vera and Essential Thrombocythemia respectively, compared with controls. Functional comparison using pathway analysis software showed that the Rho GTPase family signaling pathways were deregulated in MPN patients. In particular, IQGAP1 was significantly overexpressed in MPNs compared with controls. Additionally, Western-blot analysis not only confirmed IQGAP1 overexpression, but also showed that IQGAP1 levels depended on the patient's genotype. Moreover, we found that in JAK2V617F patients IQGAP1 could bind RhoA, Rac1 and Cdc42 and consequently recruit activated GTP-Rac1 and the cytoskeleton motility protein PAK1. In CALR( +) patients, IQGAP1 was not overexpressed but immunoprecipitated with RhoGDI. In JAK2V617F transduced Ba/F3 cells we confirmed JAK2 inhibitor-sensitive overexpression of IQGAP1/PAK1. Altogether, our data demonstrated alterations of IQGAP1/Rho GTPase signaling in MPN erythrocytes dependent on JAIC2/CALR status, reinforcing the hypothesis that modifications in erythrocyte signaling pathways participate in Ph- MPN pathogenesis. (C) 2016 Elsevier B.V. All rights reserved.

关键词： Erythrocyte proteome   IQGAP1   Myeloproliferative neoplasms   Rho GTPases  

摘要： Besides genetic abnormalities in MPN patients, several studies have reported alterations in protein expression that could contribute towards the clinical phenotype. However, little is known about protein modifications in Ph − MPN erythrocytes. In this context, we used a quantitative mass spectrometry proteomics approach to study the MPN erythrocyte proteome. LC-MS/MS (LTQ Orbitrap) analysis led to the identification of 51 and 86 overexpressed proteins in Polycythemia Vera and Essential Thrombocythemia respectively, compared with controls. Functional comparison using pathway analysis software showed that the Rho GTPase family signaling pathways were deregulated in MPN patients. In particular, IQGAP1 was significantly overexpressed in MPNs compared with controls. Additionally, Western-blot analysis not only confirmed IQGAP1 overexpression, but also showed that IQGAP1 levels depended on the patient's genotype. Moreover, we found that in JAK2V617F patients IQGAP1 could bind RhoA, Rac1 and Cdc42 and consequently recruit activated GTP-Rac1 and the cytoskeleton motility protein PAK1. In CALR(+) patients, IQGAP1 was not overexpressed but immunoprecipitated with RhoGDI. In JAK2V617F transduced Ba/F3 cells we confirmed JAK2 inhibitor-sensitive overexpression of IQGAP1/PAK1. Altogether, our data demonstrated alterations of IQGAP1/Rho GTPase signaling in MPN erythrocytes dependent on JAK2/CALR status, reinforcing the hypothesis that modifications in erythrocyte signaling pathways participate in Ph − MPN pathogenesis. [ABSTRACT FROM AUTHOR]

关键词： IQGAP1   Dvl   免疫组化   人非小细胞肺癌  

摘要： 目的探讨非小细胞肺癌细胞中IQ-domain GTPase-activatingprotein 1(IQGAP1)与Dishevelled(Dvl)的相关性。方法应用免疫组化法检测109例非小细胞肺癌组织中IQGAP1和Dvl的表达,分析与临床病理因素的关联。结果 IQGAP1在肺癌组织中的表达水平显著高于癌旁正常组织。IQGAP1与Dvl在肺癌细胞浆及核的共定位表达显著正相关,而核共定位组的c-myc和cyclin D1表达率明显高于浆共定位组(P<0.05)。淋巴结转移组中二者浆/核共定位表达率明显高于未转移组(P<0.05),且与生存时间呈正比。结论 IQGAP1可能在胞浆促进Dvl入核从而激活经典Wnt通路,促进市癌的恶性表型。

关键词： adipocyte   caveolae   caveolin-1   cytoskeletal proteins   IQGAP1  

摘要： The ubiquitously expressed IQ motif-containing GTPase activating protein-1 (IQGAP1) is a scaffolding protein implicated in an array of cellular functions, in particular by binding to cytoskeletal elements and signaling proteins. A role of IQGAP1 in adipocytes has not been reported. We therefore investigated the cellular IQGAP1 interactome in primary human adipocytes. Immunoprecipitation and quantitative mass spectrometry identified caveolae and caveolae-associated proteins as the major IQGAP1 interactors alongside cytoskeletal proteins. We confirmed co-localization of IQGAP1 with the defining caveolar marker protein caveolin-1 by confocal microscopy and proximity ligation assay. Most interestingly, insulin enhanced the number of IQGAP1 interactions with caveolin-1 by fivefold. Moreover, we found a significantly reduced abundance of IQGAP1 in adipocytes from patients with type 2 diabetes compared with cells from nondiabetic control subjects. Both the abundance of IQGAP1 protein and mRNA were reduced, indicating a transcriptional defect in diabetes. Our findings suggest a novel role of IQGAP1 in insulin-regulated interaction between caveolae and cytoskeletal elements of the adipocyte, and that this is quelled in the diabetic state.