关键词： DP1   GPCR   IQGAP1   Interactome   Signaling   Trafficking  

摘要： Background: Mechanisms governing localization, trafficking and signaling of G protein-coupled receptors (GPCRs) are critical in cell function. Protein-protein interactions are determinant in these processes. However, there are very little interacting proteins known to date for the DP1 receptor for prostaglandin D-2. Methods: We performed LC-MS/MS analyses of the DP1 receptor interactome in HEK293 cells. To functionally validate our LC-MS/MS data, we studied the implications of the interaction with the IQGAP1 scaffold protein in the trafficking and signaling of DP1. Results: In addition to expected interacting proteins such as heterotrimeric G protein subunits, we identified proteins involved in signaling, trafficking, and folding localized in various cell compartments. Endogenous DP1-IQGAP1 co-immunoprecipitation was observed in colon cancer HT-29 cells. The interaction was augmented by DP1 agonist activation in HEK293 cells and GST-pulldown assays showed that IQGAP1 binds to intracellular loops 2 and 3 of DP1. Co-localization of the two proteins was observed by confocal microscopy at the cell periphery and in intracellular vesicles in the basal state. PGD2 treatment resulted in the redistribution of the DP1-IQGAP1 co-localization in the perinuclear vicinity. DP1 receptor internalization was promoted by overexpression of IQGAP1, while it was diminished by IQGAP1 knockdown with DsiRNAs. DP1-mediated ERK1/2 activation was augmented and sustained overtime by overexpression of IQGAP1 when compared to DP1 expressed alone. IQGAP1 knockdown decreased ERK1/2 activation by DP1 stimulation. Interestingly, ERK1/2 signaling by DP1 was increased when IQGAP2 was silenced, while it was impaired by IQGAP3 knockdown. Conclusions: Our findings define the putative DP1 interactome, a patho-physiologically important receptor, and validated the interaction with IQGAP1 in DP1 function. Our data also reveal that IQGAP proteins may differentially regulate GPCR signaling. General significanc

关键词： Gastric cancer   RNA splicing   Stress signalling  

摘要： In response to oncogenic signals, Alternative Splicing (AS) regulators such as SR and hnRNP proteins show altered expression levels, subnuclear distribution and/or post-translational modification status, but the link between signals and these changes remains unknown. Here, we report that a cytosolic scaffold protein, IQGAP1, performs this task in response to heat-induced signals. We show that in gastric cancer cells, a nuclear pool of IQGAP1 acts as a tethering module for a group of spliceosome components, including hnRNPM, a splicing factor critical for the response of the spliceosome to heat-shock. IQGAP1 controls hnRNPM's sumoylation, subnuclear localisation and the relevant response of the AS machinery to heat-induced stress. Genome-wide analyses reveal that IQGAP1 and hnRNPM co-regulate the AS of a cell cycle-related RNA regulon in gastric cancer cells, thus favouring the accelerated proliferation phenotype of gastric cancer cells. Overall, we reveal a missing link between stress signals and AS regulation.

关键词： 甲状腺癌   miR-506-3p   IQ结构域GTP酶激活蛋白(IQGAP)1   细胞增殖   迁移   侵袭  

摘要： 目的探讨miR-506-3p靶向IQ结构域GTP酶激活蛋白(IQGAP)1调控甲状腺癌细胞增殖、迁移和侵袭的分子机制。方法采用qRT-PCR和Western印迹检测甲状腺癌细胞SW579、FTC-133、KAT-18和正常甲状腺上皮细胞TEC中miR-506-3p、IQGAP1 mRNA和IQGAP1蛋白的表达;建立miR-506-3p过表达或IQGAP1抑制表达的SW579细胞株。采用噻唑蓝(MTT)法检测细胞增殖活力,Transwell法检测细胞迁移和侵袭能力,Western印迹检测细胞周期蛋白(Cyclin)D1、P21、P27、基质金属蛋白酶(MMP)-2、MMP-9和MMP-14蛋白表达水平。采用双荧光素酶报告基因实验验证miR-506-3p对IQGAP1的靶向作用。结果与正常甲状腺上皮细胞TEC相比,甲状腺癌细胞SW579、FTC-133和KAT-18中miR-506-3p表达显著降低,而IQGAP1表达显著升高。miR-506-3p过表达或IQGAP1抑制表达均可抑制SW579细胞的增殖、迁移和侵袭。IQGAP1是miR-506-3p的靶基因,miR-506-3p可负向调控IQGAP1表达。IQGAP1过表达可显著逆转miR-506-3p对SW579细胞的增殖、迁移和侵袭的抑制作用。结论miR-506-3p可通过下调IQGAP1表达抑制甲状腺癌细胞的增殖、迁移和侵袭。

关键词： PUMA蛋白   IQGAP1蛋白   胰腺癌   临床病理特征  

摘要： 目的探讨PUMA、IQGAP1蛋白在胰腺癌中的表达及与临床病理、预后生存的相关性。方法选取2016年10月至2018年10月本院收治的胰腺癌患者胰腺癌组织标本、远离胰腺肿瘤3 cm的癌旁组织标本各74份,比较两组织中PUMA、IQGAP1蛋白的表达情况,并探讨其与病理特征的关系。随访2年,了解74例患者生存率,采用ROC曲线分析不同PUMA、IQGAP1表达的胰腺癌患者预后2年生存情况,采用Logistic回归模型分析影响胰腺癌患者2年预后的独立危险因素。结果胰腺癌组织中的PUMA阳性率显著低于癌旁组织,IQGAPl阳性率显著高于癌旁组织,差异均有统计学意义(P<0.05)。TNM分期为Ⅲ~Ⅵ期、有淋巴结转移、分化程度为中-低分化的胰腺癌患者PUMA阴性率、IQGAPl阳性率更高(P<0.05)。74例胰腺癌患者2年生存率为16.22%。PUMA阴性表达、IQGAPl阳性表达的胰腺癌患者死亡率高于PUMA阳性表达、IQGAPl阴性表达的患者,差异有统计学意义(P<0.05)。生存分析显示PUMA(表达阳性)、IQGAPl(表达阴性)者生存期较长,Keplan-meier生存曲线明显不同(P<0.05)。TNM(Ⅲ~Ⅵ期)、淋巴结转移(有)、分化程度(中-低分化)、PUMA(表达阴性)、IQGAPl(表达阳性)为影响胰腺癌患者预后生存的独立危险因素(P<0.05)。结论 PUMA阴性、IQGAPl阳性为影响胰腺癌患者预后生存的危险因素,临床可通过加强监测二者表达情况,以评估胰腺癌患者病情进展情况及预后。

关键词： IQGAP1   Microvascular endothelium   Cytoskeleton   Acute lung injury   Rap1   ICAM-1  

摘要： Pulmonary microvascular barrier dysfunction is a hallmark feature of acute lung injury (ALI). IQGAP1 is a ubiquitously expressed scaffolding protein known to regulate cancer metastasis, angiogenesis, and barrier stability. However, the function of IQGAP1 in lipopolysaccharide (LPS)-induced microvascular endothelial hyperpermeability remains poorly understood. In the present study, we demonstrated that IQGAP1 was markedly upregulated in LPS-induced ALI models and rat pulmonary microvascular endothelial cells (RPMVECs). Lentivirus-mediated knockdown of IQGAP1 significantly attenuated the formation of actin stress fibers, phosphorylation of myosin light chain (MLC), and disruption of VE-cadherin, thereby protecting the RPMVECs barrier failure from LPS damage. In addition, IQGAP1 depletion reduced the reactive oxygen species (ROS)-mediated increase in intracellular adhesion molecule-1 (ICAM-1) in RPMVECs stimulated with LPS. Mechanistically, we found that the upregulation of IQGAP1 affected the activity of Rap1 and the downstream phosphorylation of Src. In conclusion, these findings reveal an essential mechanism by which increased IQGAP1 in LPS-treated RPMVECs promotes barrier dysfunction and ICAM-1 upregulation, at least in part by regulating Rap1/Src signalling, indicating that IQGAP1 may be a potential therapeutic target to prevent endothelial hyperpermeability and inflammation in ALI.

关键词： choroidal neovascularization   IQGAP1   Rac1GTP   Rap1GTP   vascular endothelial growth factor  

摘要： Neovascular age-related macular degeneration (nAMD) is a leading cause of blindness. The pathophysiology involves activation of choroidal endothelial cells (CECs) to transmigrate the retinal pigment epithelial (RPE) monolayer and form choroidal neovascularization (CNV) in the neural retina. The multidomain GTPase binding protein, IQGAP1, binds active Rac1 and sustains activation of CECs, thereby enabling migration associated with vision-threatening CNV. IQGAP1 also binds the GTPase, Rap1, which when activated reduces Rac1 activation in CECs and CNV. In this study, we tested the hypothesis that active Rap1 binding to IQGAP1 is necessary and sufficient to reduce Rac1 activation in CECs, and CNV. We found that pharmacologic activation of Rap1 or adenoviral transduction of constitutively active Rap1a reduced VEGF-mediated Rac1 activation, migration, and tube formation in CECs. Following pharmacologic activation of Rap1, VEGF-mediated Rac1 activation was reduced in CECs transfected with an IQGAP1 construct that increased active Rap1-IQGAP1 binding but not in CECs transfected with an IQGAP1 construct lacking the Rap1 binding domain. Specific knockout of IQGAP1 in endothelial cells reduced laser-induced CNV and Rac1 activation in CNV lesions, but pharmacologic activation of Rap1 did not further reduce CNV compared to littermate controls. Taken together, our findings provide evidence that active Rap1 binding to the IQ domain of IQGAP1 is sufficient to interfere with active Rac1-mediated CEC activation and CNV formation.

关键词： Liver fibrosis   miR-124   IQGAP1   Inflammation   NF-kappa B  

摘要： Liver fibrosis is a health concern that leads to organ failure mediated via production of inflammatory cytokines and fibrotic biomarkers. To date, there was no direct approved antifibrotic therapy, and current treatment was mainly the removal of the causative factor. Recent studies demonstrated that aberrant expression of miR-124 was involved in the progression of various liver diseases including hepatocellular carcinoma (HCC). However, whether miR-124 could function as a transcriptional regulator in the inflammatory cytokines secretion of liver fibrosis remains unclear. In this study, we demonstrated that the expression of miR-124 was downregulated in liver fibrosis tissues and TNF-alpha-induced LX-2 cells, concomitant with the upregulated expression of IQGAP1, suggesting that miR-124 and IQGAP1 might be associated with the development of inflammation in liver fibrosis. Therefore, we demonstrated that the overexpression of miR-124 and knockdown of IQGAP1 could lead to the downregulation of TNF-alpha, IL-1 beta and IL-6. While knockdown of miR-124 or overexpression of IQGAP1 showed reversed results. Moreover, dual luciferase reporter assays demonstrated that miR-124 specifically targeted the 3 '-UTR of IQGAP1, and thus inhibited the expression of IQGAP1. Mechanistically, we found that the expression changes of miR-124 and IQGAP1 could be involved in inhibition or activation of NF-kappa B signaling pathway in response to TNF-alpha. In conclusion, these results indicated that miR-124 plays a crucial role in TNF-alpha-induced LX-2 cells via regulating NF-kappa B signaling pathway.

关键词： MAL2   IQGAP1   ERK   Pancreatic cancer  

摘要： Pancreatic cancer is a digestive tract malignancy characterized by an occult onset and rapid progression. The genetic heterogeneity of pancreatic cancer is closely related to its highly malignant biological behavior. The myelin and lymphocyte protein 2 (MAL2) is upregulated in multiple cancers at the transcriptional level. However, the exact role of MAL2 in pancreatic cancer remains elusive. In this study, we demonstrated that MAL2 protein and mRNA levels were upregulated in pancreatic cancer. MAL2 over expression was significantly associated with poor prognosis in patients with pancreatic cancer. We further showed that MAL2 interacted with IQGAP1 to increase ERK1/2 phosphorylation levels, which promoted pancreatic cancer progression. Therefore, these results suggest that MAL2 could be a novel therapeutic target for pancreatic cancer. ? 2021 Elsevier Inc. All rights reserved.