关键词： Biochemistry   Cell biology  

摘要： The small GTPase Cdc42 is an integral component of the cytoskeleton, and its dysregulation leads to pathophysiological conditions, such as cancer. Binding of Cdc42 to the scaffold protein IQGAP1 stabilizes Cdc42 in its active form. The interaction between Cdc42 and IQGAP1 enhances migration and invasion of cancer cells. Disrupting this association could impair neoplastic progression and metastasis;however, no effective means to achieve this has been described. Here, we screened 78,500 compounds using a homogeneous time resolved fluorescence-based assay to identify small molecules that disrupt the binding of Cdc42 to IQGAP1. From the combined results of the validation assay and counter-screens, we selected 44 potent compounds for cell-based experiments. Immunoprecipitation and cell viability analysis rendered four lead compounds, namely NCGC00131308, NCGC00098561, MLS000332963 and NCGC00138812, three of which inhibited proliferation and migration of breast carcinoma cells. Microscale thermophoresis revealed that two compounds bind directly to Cdc42. One compound reduced the amount of active Cdc42 in cells and effectively impaired filopodia formation. Docking analysis provided plausible models of the compounds binding to the hydrophobic pocket adjacent to the GTP binding site of Cdc42. In conclusion, we identified small molecules that inhibit binding between Cdc42 and IQGAP1, which could potentially yield chemotherapeutic agents.

关键词： 肌萎缩侧索硬化症   脊髓   RAC1   RAC3   IQGAP1   SOD1^(G93A)突变小鼠  

摘要： 目的:检测Ras相关C3肉毒菌毒物底物1(RAC1)、Ras相关C3肉毒菌毒物底物3(RAC3)和IQ结构域GTP酶活化蛋白1(IQGAP1)在SOD1^(G93A)突变小鼠脊髓中的表达变化。方法:选取SOD1^(G93A)突变小鼠与野生型(WT)小鼠作为动物模型,将其分为症状前期(出生70 d)、症状早期(出生95 d)、症状中期(出生108 d)和症状晚期(出生122 d)4个组别,利用RT-PCR检测小鼠脊髓中RAC1、RAC3和IQGAP1 mRNA表达,利用Western Blot和免疫荧光染色检测RAC1、RAC3和IQGAP1蛋白表达与定位。结果:与同窝WT小鼠相比,RAC1 mRNA表达水平在出生不同时间点SOD1^(G93A)突变小鼠脊髓中无明显变化;在出生95、108和122 d RAC3 mRNA均明显降低,IQGAP1 mRNA均明显升高;RAC1、RAC3和IQGAP1蛋白在出生95、108和122 d表达均明显降低;RAC1、RAC3、IQGAP1免疫阳性细胞主要分布在脊髓前角,即运动神经元所在的部位,RAC1、RAC3和IQGAP1均与神经元特异性核抗原(NeuN)标记的神经元共表达。结论:SOD1^(G93A)突变小鼠脊髓中RAC1、RAC3和IQGAP1的表达异常与肌萎缩侧索硬化症(ALS)的发病密切相关。

关键词： IQGAP1   phosphoproteomics   HNSCC   PI3K pathway   alternative splicing  

摘要： that are implicated in carcinogenesis, including the RAS and PI3K pathways that involve multiple protein kinases. IQGAP1 has been shown to promote head and neck squamous cell carcinoma (HNSCC);however, the underlying mechanism(s) remains unclear. Here, we report a mass spectrometry-based analysis identifying differences in phosphorylation of cellular proteins in vivo and in vitro in the presence or absence of IQGAP1. By comparing the esophageal phosphoproteome profiles between Iqgap1+/+ and Iqgap1-/- mice, we identified RNA splicing as one of the most altered cellular processes. Serine/arginine-rich splicing factor 6 (SRSF6) was the protein with the most downregulated levels of phosphorylation in Iqgap1-/- tissue. We confirmed that the absence of IQGAP1 reduced SRSF6 phosphorylation both in vivo and in vitro. We then expanded our analysis to human normal oral keratinocytes. Again, we found factors involved in RNA splicing to be highly altered in the phosphoproteome profile upon genetic disruption of IQGAP1. Both the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and the Cancer Genome Atlas (TCGA) data sets indicate that phosphorylation of splicing-related proteins is important in HNSCC prognosis. The Biological General Repository for Interaction Datasets (BioGRID) repository also suggested multiple interactions between IQGAP1 and splicingrelated proteins. Based on these collective observations, we propose that IQGAP1 regulates the phosphorylation of splicing proteins, which potentially affects their splicing activities and, therefore, contributes to HNSCC. Raw data are available from the MassIVE database with identifier MSV000087770.

摘要： Introduction Il existe une atteinte des podocytes dans le syndrome néphrotique résistant aux corticostéroïdes. L’objectif de ce travail est la détermination de la mutation rare de la protéine IQGAP1, qui est une protéine impliquée dans le remodelage du cytosquelette d’actine du podocyte. Description L’étude de la mutation de la proteine IQGAP1, qui engendrera sur le plan clinique un syndrome néphrotique cortico-résistant qui se transmet d’une façon récessive. Méthodes Nous avons identifié 13 patients dans 3 familles atteintes d’un syndrome néphrotique cortico-résistant, dont 2 décès dans les deux premières familles alors que la 3 e famille : pas de notion de décès. Les adultes étaient aussi atteints que les enfants. Les trois familles étaient issues d’un mariage avec consanguin à transmission autosomique récessive. Résultats Les patients avaient un syndrome néphrotique résistant aux corticostéroïdes. Le syndrome néphrotique est découvert à l’âge de 3 ans en moyenne pour les enfants et 25 ans pour les adultes. La biopsie rénale a montré des lésions glomérulaires minimes à 16 %, une prolifération mésangiale à 2 % et une hyalinose segmentaire et focale à 72 %. Les manifestations extrarénales observées étaient liées à des complications de l’insuffisance rénale chronique. L’altération de l’expression d’IQGAP1 ou de sa localisation cellulaire, ainsi que des altérations structurelles ou fonctionnelles de la protéine pourraient conduire à des interactions anormales entre les différents complexes du podocyte et du cytosquelette ; la spécificité de l’étude est la découverte d’un SNCRF chez des patients présentant une seule mutation du gène IQGAP1  sans mutation des autres IQGAP (2 et 3) ( Figure 1 ). Conclusion La mutation de la protéine IQGAP1 est héritée de façon autosomique récessive. Une enquête génétique est indispensable. IQGAP1 peut donc être considéré comme un facteur influençant la progression des glomérulopathies.

关键词： 宫颈癌组织   激活的白激酶C受体1   IQ序列三磷酸鸟苷酶激活蛋白   病理特征   β-连环蛋白   相关性  

摘要： 目的:分析宫颈癌组织中激活的白激酶C受体1(RACK1)、IQ序列三磷酸鸟苷酶激活蛋白(IQGAPl)表达及与临床病理特征和β-连环蛋白(β-catenin)的相关性。方法:收集2018年1月-2020年12月本院行宫颈癌手术治疗的患者106例临床资料,采用实时荧光定量PCR(RT-PCR)和免疫组化技术检测RACK1、IQGAP1、β-catenin mRNA和蛋白表达,分析宫颈癌组织中RACK1、IQGAP1表达及与临床病理特征和β-catenin的相关性。结果:宫颈癌组织中RACK1 mRNA的相对表达量(0.27±0.11)低于癌旁组织(0.75±0.19),IQGAP1 mRNA(0.65±0.18)、β-catenin mRNA(0.61±0.21)高于癌旁组织(0.22±0.13、0.32±0.17),RACK1蛋白阳性率(18.9%)低于癌旁组织(63.2%),IQGAP1(76.4%)、β-catenin(68.9%)蛋白阳性率高于癌旁组织(28.3%、31.1%)(均P<0.05)。RACK1在高分化、TNM分期为I期、无淋巴结转移的宫颈癌组织中阳性率高于中低分化、TNM分期为II-III期、有淋巴结转移患者的组织,IQGAP1在中低分化、TNM分期为II-III期的宫颈癌组织中阳性率高于高分化、TNM分期为I期组织(均P<0.05)。宫颈癌中RACK1表达与β-catenin呈负相关(r=-0.405,P<0.05),IQGAP1表达与β-catenin呈显著正相关(r=0.346,P<0.05)。结论:宫颈癌组织RACK1低表达,IQGAP1高表达;RACK1蛋白表达异常可能与患者宫颈癌分化程度、TNM分期、淋巴结转移及β-catenin表达有关,IQGAP1蛋白表达异常可能与分化程度、TNM分期及β-catenin表达有关。

关键词： IQGAP1   EGF   EGFR   PD-L1   Cancer   IQGAP1   EGF   EGFR   PD-L1   Cancer  

摘要： IQ motif containing GTPase activating protein 1 (IQGAP1) is a scaffold protein to transduce multiple signals from the extracellular microenvironment. However, whether IQGAP1 mediates EGF-induced cancer progression has been not observed. In this research, we explored the immunological correlations of IQGAP1. IQGAP1 was upregulated in most cancer and related to poor prognosis in ovarian cancer. In addition, IQGAP1 was correlated with immune-related genes and tumor-infiltrating lymphocytes. IQGAP1 was a novel EGF-response gene, which was up-regulated by EGF. As the downstream of EGFR, IQGAP1 mediated EGF-induced proliferation, migration and invasion of tumor cells and was essential for EGF-induced PD-L1 expression as well. Mechanically, IQGAP1 interacted with STATs and mediated their phosphorylation. Furthermore, Curcumin inhibited EGFR/IQGAP1 axis to down-regulate PD-L1 and suppress cancer progression. To sum up, IQGAP1 responds to EGF stimulation and mediates EGF-induced phosphorylation of STATs, which is a novel promising target for blocking the EGFinduced cancer progression.

关键词： human Fas-associated factor 1(FAF1)   heat shock protein 70(Hsp70)   adherens junction   RhoA activation   IQGAP1   X-ray crystallography   FAF1–Hsp70 complex  

摘要： Fas-associated factor 1 (FAF1) is a scaffolding protein that plays multiple functions, and dysregulation of FAF1 is associated withmany types of diseases such as cancers. FAF1 contains multiple ubiquitin-related domains (UBA, UBL1, UBL2, UAS, and UBX), eachdomain interacting with a specific partner. In particular, the interaction of UBL1 with heat shock protein 70 (Hsp70) is associatedwith tumor formation, although the molecular understanding remains unknown. In this study, the structural analysis revealed thatHis160 of FAF1 is important for its interaction with Hsp70. The association of Hsp70 with FAF1 is required for the interaction withIQGAP1. FAF1 negatively regulates RhoA activation by FAF1–Hsp70 complex formation, which then interacts with IQGAP1. Thesesteps play a key role in maintaining the stability of cell-to-cell junction. We conclude that FAF1 plays a critical role in the structureand function of adherens junction during tissue homeostasis and morphogenesis by suppressing RhoA activation, which induces theactivation of Rho-associated protein kinase, phosphorylation of myosin light chain, formation of actin stress fiber, and disruptionof adherens junction. In addition, depletion of FAF1 increased collective invasion in a 3D spheroid cell culture. These results provideinsightinto how the FAF1–Hsp70 complex acts as a novelregulator ofthe adherens junction integrity. The complex can be a potentialtherapeutic target to inhibit tumorigenesis and metastasis.

关键词： IQGAP1   MMP2   Invasion   NF-kappa B  

摘要： Esophageal squamous cell carcinoma (ESCC) is one type of the most common malignancies, yet the overall survival rate is still not ideal. IQ motif containing GTPase activating protein 1 (IQGAP1) participates in cell biological functions of various tumors as an oncogene. However, the mechanisms of IQGAP1 affecting malignant development of ESCC are still unclear. In this study, the expression and correlation of IQGAP1 and MMP2 in esophageal cancer tissues were evaluated by online databases and immunohistochemistry. Stably transfected cell lines with IQGAP1 overexpression and knockdown were constructed. Cell growth, migration and invasion ability, the expression of MMP2 and NF-kappa B expression were examined in ESCC cells. Furthermore, the cellular malignant phenotypes of ESCC and MMP2 expression in IQGAP1 overexpressing cells after treatment with the NF-kappa B inhibitor pyrrolidinecarbodithioic acid (PDTC) or JSH-23 were detected. We found that the expression of IQGAP1 and MMP2 were up-regulated and positively correlated in ESCC tissues. IQGAP1 overexpression promoted the growth, migration and invasion of ESCC cells, and up-regulated the expression of MMP2, and increased the expression and the nuclear localization level of NF-kappa B. Treating with PDTC or JSH-23 reversed IQGAP1-mediated cell migration and invasion ability, as well as the expression of MMP2. In summary, IQGAP1 plays a tumor promotion role to regulate the migration and invasion of ESCC cells and the expression of MMP2 through upregulating NF-kappa B activity, supporting a promising therapeutic target against ESCC.

关键词： IQGAP1   sphingosine 1-phosphate receptor 3   bone marrow mesenchymal stromal cells   migration   liver fibrosis   Cdc42   Rac1   F-actin  

摘要： IQ motif-containing guanosine triphosphatase (GTPase)-activating protein 1 (IQGAP1) is a cytosolic scaffolding protein involved in cell migration. Our previous studies suggest sphingosine 1-phosphate (S1P) triggers bone marrow (BM) mesenchymal stromal cells (BMSCs) to damaged liver, thereby promoting liver fibrosis. However, the role of IQGAP1 in S1P-induced BMSC migration and liver fibrogenesis remains unclear. Chimeric mice of BM cell labeled by EGFP were used to build methionine-choline-deficient and high-fat (MCDHF)-diet-induced mouse liver fibrosis. IQGAP1 small interfering RNA (siRNA) was utilized to silence IQGAP1 in vivo. IQGAP1 expression is significantly elevated in MCDHF-diet-induced mouse fibrotic livers. Positive correlations are presented between IQGAP1 and fibrosis hallmarks expressions in human and mouse fibrotic livers. In vitro, depressing IQGAP1 expression blocks S1P-induced motility and cytoskeleton remodeling of BMSCs. S1P facilitates IQGAP1 aggregating to plasma membrane via S1P receptor 3 (S1PR(3)) and Cdc42/Rac1. In addition, IQGAP1 binds to Cdc42/Rac1, regulating S1P-induced activation of Cdc42/Rac1 and mediating BMSC migration in concert. In vivo, silencing IQGAP1 reduces the recruitment of BMSCs to impaired liver and effectively alleviates liver fibrosis induced by MCDHF diet. Together, silencing IQGAP1 relieves liver fibrosis by blocking BMSC migration, providing an effective therapeutic strategy for liver fibrosis.

摘要： IQGAP1 is a multidomain scaffold protein that coordinates the direction and impact of multiple signaling pathways by scaffolding its various binding partners. However, the spatial and temporal resolution of IQGAP1 scaffolding remains unclear. Here, we use fluorescence imaging and correlation methods that allow for real-time live-cell changes in IQGAP1 localization and complex formation during signaling. We find that IQGAP1 and PIPKI gamma interact on both the plasma membrane and in cytosol. Epidermal growth factor (EGF) stimulation, which can initiate cytoskeletal changes, drives the movement of the cytosolic pool toward the plasma membrane to promote cytoskeletal changes. We also observe that a significant population of cytosolic IQGAP1-PIPKI gamma complexes localize to early endosomes, and in some instances form aggregated clusters which become highly mobile upon EGF stimulation. Our imaging studies show that PIPKI gamma and PI3K bind simultaneously to IQGAP1, which may accelerate conversion of PI4P to PI(3,4,5)P-3 that is required for cytoskeletal changes. Additionally, we find that IQGAP1 is responsible for PIPKI gamma association with two proteins associated with cytoskeletal changes, talin and Cdc42, during EGF stimulation. These results directly show that IQGAP1 provides a physical link between phosphoinositides (through PIPKI gamma), focal adhesion formation (through talin), and cytoskeletal reorganization (through Cdc42) upon EGF stimulation. Taken together, our results support the importance of IQGAP1 in regulating cell migration by linking phosphoinositide lipid signaling with cytoskeletal reorganization.