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1.
Cell Prolif ; 53(1): e12630, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31713929

RESUMO

OBJECTIVES: miR-92b has been reported to play critical roles in several carcinomas; however, our understanding of the mechanisms by which miR-92b stimulates gastric cancer (GC) is incomplete. The aim of this study was to investigate the clinical significance and functional relevance of miR-92b in GC. MATERIALS AND METHODS: Expression of miR-92b in GC and peritumoural tissues was determined using qRT-PCR, in situ hybridization and bioinformatics. CCK-8, colony formation and fluorescence-activated cell sorting assays were utilized to explore the effect of miR-92b on GC cells. A luciferase reporter assay and Western blotting were employed to verify miR-92b targeting of DAB2IP. Furthermore, Western blotting was used to evaluate the levels of DAB2IP and PI3K/Akt signalling pathway-related proteins. RESULTS: In this study, we found that miR-92b was upregulated in GC tissues compared with peritumoural tissues. Overexpression of miR-92b promoted cell proliferation, colony formation, and G0 /G1 transition and decreased apoptosis. Our results indicated that miR-92b repressed the expression of DAB2IP and that loss of DAB2IP activated the PI3K/AKT signalling pathway. Overexpression of DAB2IP rescued the effects of miR-92b in GC cells. Finally, our results demonstrated a significant correlation between miR-92b expression and DAB2IP expression in GC tissues. CONCLUSIONS: Our results suggest that miR-92b promotes GC cell proliferation by activating the DAB2IP-mediated PI3K/AKT signalling pathway. The miR-92b/DAB2IP/PI3K/AKT signalling axis may be a potential therapeutic target to prevent GC progression.


Assuntos
MicroRNAs/metabolismo , RNA Neoplásico/metabolismo , Transdução de Sinais , Neoplasias Gástricas/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , MicroRNAs/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Neoplásico/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Proteínas Ativadoras de ras GTPase/genética
2.
Biol Pharm Bull ; 42(9): 1532-1537, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31474712

RESUMO

Glucose-stimulated insulin secretion is controlled by both exocytosis and endocytosis in pancreatic ß-cells. Although endocytosis is a fundamental step to maintain cellular responses to the secretagogue, the molecular mechanism of endocytosis remains poorly defined. We have previously shown that in response to high concentrations of glucose, guanosine 5'-diphosphate (GDP)-bound Rab27a is recruited to the plasma membrane where IQ motif-containing guanosine 5'-triphosphatase (GTPase)-activating protein 1 (IQGAP1) is expressed, and that complex formation promotes endocytosis of secretory membranes after insulin secretion. In the present study, the regulatory mechanisms of dissociation of the complex were investigated. Phosphorylation of IQGAP1 on serine (Ser)-1443, a site recognized by protein kinase Cε (PKCε), inhibited the interaction of GDP-bound Rab27a with IQGAP1 in a Cdc42-independent manner. Glucose stimulation caused a translocation of PKCε from the cytosol to the plasma membrane. In addition, glucose-induced endocytosis was inhibited by the knockdown of IQGAP1 with small interfering RNA (siRNA). However, the expression of the non-phosphorylatable or phosphomimetic form of IQGAP1 could not rescue the inhibition, suggesting that a phosphorylation-dephosphorylation cycle of IQGAP1 is required for endocytosis. These results suggest that IQGAP1 phosphorylated by PKCε promotes the dissociation of the IQGAP1-GDP-bound Rab27a complex in pancreatic ß-cells, thereby regulating endocytosis of secretory membranes following insulin secretion.


Assuntos
Endocitose , Guanosina Difosfato/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Proteínas rab27 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Sítios de Ligação , Células COS , Membrana Celular/metabolismo , Citosol/metabolismo , Glucose/farmacologia , Proteínas de Fluorescência Verde/genética , Guanosina Difosfato/genética , Imunoprecipitação , Células Secretoras de Insulina/efeitos dos fármacos , Fosforilação , Ligação Proteica , Proteínas rab27 de Ligação ao GTP/genética , Proteínas Ativadoras de ras GTPase/genética
3.
Nat Commun ; 10(1): 3975, 2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31484924

RESUMO

Rho family proteins are prenylated by geranylgeranyltransferase type I (GGTase-I), which normally target proteins to membranes for GTP-loading. However, conditional deletion of GGTase-I in mouse macrophages increases GTP-loading of Rho proteins, leading to enhanced inflammatory responses and severe rheumatoid arthritis. Here we show that heterozygous deletion of the Rho family gene Rac1, but not Rhoa and Cdc42, reverses inflammation and arthritis in GGTase-I-deficient mice. Non-prenylated Rac1 has a high affinity for the adaptor protein Ras GTPase-activating-like protein 1 (Iqgap1), which facilitates both GTP exchange and ubiquitination-mediated degradation of Rac1. Consistently, inactivating Iqgap1 normalizes Rac1 GTP-loading, and reduces inflammation and arthritis in GGTase-I-deficient mice, as well as prevents statins from increasing Rac1 GTP-loading and cytokine production in macrophages. We conclude that blocking prenylation stimulates Rac1 effector interactions and unleashes proinflammatory signaling. Our results thus suggest that prenylation normally restrains innate immune responses by preventing Rac1 effector interactions.


Assuntos
Imunidade Inata/genética , Prenilação de Proteína , Transdução de Sinais/genética , Proteínas rac1 de Ligação ao GTP/genética , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Animais , Citocinas/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ligação Proteica , Células RAW 264.7 , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo
4.
Biomed Pharmacother ; 119: 109434, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31536933

RESUMO

Our previous research had firstly shown that MM cells overexpressed IQGAP1 gene and activated Ras/Raf/MEK/ERK pathway. But the mechanism of IQGAP1 overexpression and IQGAP1 gene transcription regulation remains uncertain. The mechanism of IQGAP1 overexpression and transcriptional regulation of IQGAP1 gene in myeloma cells was explored in the study. Through bioinformatics analysis and prediction we predicted and screened transcription factor Sp1 as a possible upstream regulator of IQGAP1.The proliferation, cell cycle and downstream ERK1/2 and p-ERK1/2 proteins were detected after siRNA-IQGAP1 was transfected to myeloma cells. The expression of Sp1, p300, IQGAP1, p-ERK1/2 and ERK1/2 were detected after Sp1 and p300 were inhibited or overexpressed respectively. The dual-luciferase reporter system was used to detect the activity of IQGAP1 gene promoter. CHIP was used to detect the binding of the Sp1 and IQGAP1 promoter regions.CO-IP was used to explore the interaction between Sp1 and p300.The mRNA expression levels of Sp1,p300 and IQGAP1 of the myeloma patients were detected, and the correlation analysis of their mRNA expression levels were carried out. The results showed IQGAP1-siRNA inhibits cell proliferation, cell cycle, IQGAP1 expression and phosphorylation of ERK1/2 protein. Inhibition of Sp1 or p300 down-regulated ERK1/2 and IQGAP1 expression; overexpression of Sp1 or p300 up-regulated ERK1/2 and IQGAP1 expression; Sp1 and p300 had a positive regulation effect on IQGAP1.Over expression of Sp1 or p300 significantly increased activity of IQGAP1 gene promoter. The transcription factor Sp1 plays a regulatory role in the IQGAP1 promoter region. There is an interaction between Sp1 and p300 in myeloma cells. The mRNA expression levels of Sp1, IQGAP1 and p300 in MM samples showed a positive correlation. In summary IQGAP1 is required for cell proliferation in MM cells, and the transcription of Sp1/p300 complex regulates expression of IQGAP1 gene.


Assuntos
Proteína p300 Associada a E1A/metabolismo , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Fator de Transcrição Sp1/metabolismo , Transcrição Genética , Proteínas Ativadoras de ras GTPase/genética , Medula Óssea/metabolismo , Medula Óssea/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo/genética , Proteína p300 Associada a E1A/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Fator de Transcrição Sp1/genética , Proteínas Ativadoras de ras GTPase/metabolismo
5.
Int J Med Sci ; 16(8): 1149-1156, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31523178

RESUMO

Background Sepsis, a leading cause of death in intensive care units, is generally associated with vascular dysfunction. However, its pathophysiological process has not been fully clarified, lacking in-depth knowledge of its pathophysiological process may hinder the improvement of diagnosis and therapy for sepsis. Hence, as the key parts of the vascular wall, the interaction between endothelial cells (ECs) and smooth muscle cells (SMCs) under septic situation need to be further studied. Methods ECs and SMCs were co-cultured using Transwell plates. Lipopolysaccharide (LPS) was used to induce sepsis. A scratch-wound assay was used to assess cell migration, and western blotting was used to assess the level of redifferentiation of SMCs as well as the expression of PDGFR-ß and IQGAP1. Results Co-culture with ECs reduced the redifferentiation of SMCs induced by LPS (10 µg/ml), which was characterized by increased migration ability and decreased expression of contractile proteins (e.g., SM22 and α-SMA). The production of TNF-α could decrease the level of PDGFR-ß in SMCs. Treatment of SMCs with the PDGFR-ß inhibitor imatinib (5 µM) was able to counteract LPS-induced SMC redifferentiation and reduce IQGAP1 protein expression, especially when SMCs were co-cultured with ECs. Conclusion The phenotype of vascular SMCs co-cultured with ECs was modulated by IQGAP1 through the PDGFR-ß pathway, which may lead to vascular remodeling and homeostasis in LPS-induced intravascular injury. This pathway could be a novel target for the treatment of vascular damage.


Assuntos
Músculo Liso Vascular/citologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Técnicas de Cocultura , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Humanos , Lipopolissacarídeos/toxicidade , Músculo Liso Vascular/efeitos dos fármacos , Fenótipo , Sepse/metabolismo , Sepse/patologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
6.
Expert Opin Pharmacother ; 20(17): 2115-2120, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31446808

RESUMO

Introduction: Atypical absences are generalized epileptic seizures typically affecting children with severe epilepsies and learning difficulties along with other seizure types. Video-EEG is essential for their diagnosis. Recently, atypical absence seizures have been reported as a hallmark of some developmental and epileptic encephalopathies.Areas covered: This is a narrative review of the literature which describes the electroclinical features of atypical seizures, the characteristics of developmental epileptic encephalopathies in which this seizure type can occur, and the evidence supporting the use of individual antiseizure drugs for the treatment of atypical absences.Expert opinion: Treatment of absence seizures typically relies on ethosuximide (ineffective against tonic-clonic seizures), valproate (associated with larger proportion of adverse events), or lamotrigine (less effective than the other two). However, unlike typical absences, atypical absences are usually intractable, persist lifetime, and their prognosis depends on the underlying etiology or associated epilepsy syndrome. Besides efficacy, other relevant factors, such as drug formulation, ease of titration and dosing, and drug interactions, should be considered. Drugs that may worsen epilepsy, cognition and behavior should be avoided. In the vast majority of patients, a polytherapy is required, although usually with limited efficacy. Finally, epilepsy syndromes featuring atypical absences require a multidisciplinary approach.


Assuntos
Anticonvulsivantes/uso terapêutico , Epilepsia Generalizada/tratamento farmacológico , Etossuximida/uso terapêutico , Humanos , Lamotrigina/uso terapêutico , Síndrome de Lennox Gastaut/tratamento farmacológico , Síndrome de Lennox Gastaut/patologia , Ácido Valproico/uso terapêutico , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo
7.
Med Sci Monit ; 25: 5170-5180, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31298226

RESUMO

BACKGROUND Medullary thyroid carcinoma (MTC), a rare type of thyroid cancer, is a big challenge in clinical treatment. However, the pathogenesis of MTC remains poorly understand. MicroRNAs (miRNAs) were previously demonstrated to be involved in the pathogenesis of MTC, however, the roles of majority of miRNAs in MTC are still undetermined. MATERIAL AND METHODS Two GEO miRNA expression profiles (GSE40807, GSE97070) were downloaded, and the differentially expressed miRNAs (DEmiRNAs) of GSE40807 and GSE97070 were analyzed by bioinformatics methods. Expressions of miRNAs were detected by quantitative real-time polymerase chain reaction; cell proliferation was examined through Cell Counting Kit-8, colony formation and in vivo tumor growth assays; the interaction between miRNA and mRNA was verified by dual-luciferase reporter assay; functional analysis of target genes was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID, www.david.ncifcrf.gov) software. RESULTS Ten miRNAs were identified to be dysregulated in both GSE40807 and GSE97070 datasets, and miR-31-3p showed the highest change fold (Log fold change=-3.460625 in GSE40807 and Log fold change=-0.07084374 in GSE97070). MiR-31-3p expression was significantly downregulated in MTC, and low miR-31-3p expression showed a poor prognosis relative to high miR-31-3p expression (P<0.05). Functionally, miR-31-3p inhibited MTC cell proliferation in vitro and in vivo. Functional analysis also showed that the target genes of miR-31-3p were involved in numerous of biochemical processes and pathways, of which Ras signaling pathway was selected for further study. RASA2, overexpressed in MTC, were negatively regulated by miR-31-3p. In addition, we found that knockdown of RASA2 inhibited MTC cell proliferation. CONCLUSIONS Reduced expression level of miR-31-3p might play a key role in the tumorigenesis of MTC by targeting critical pathways, especially Ras signaling pathway.


Assuntos
Carcinoma Neuroendócrino/genética , MicroRNAs/genética , Neoplasias da Glândula Tireoide/genética , Proteínas Ativadoras de ras GTPase/genética , Carcinoma Neuroendócrino/metabolismo , Carcinoma Neuroendócrino/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , MicroRNAs/metabolismo , RNA Mensageiro/genética , Transdução de Sinais , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Transcriptoma , Proteínas Ativadoras de ras GTPase/metabolismo
8.
BMC Dev Biol ; 19(1): 15, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31277570

RESUMO

BACKGROUND: Prostate androgen-regulated mucin-like protein 1 (PARM1) is a pro-proliferative and anti-apoptotic glycoprotein involved in the endoplasmic reticulum (ER) stress response. A single nucleotide polymorphism in the coding region of PARM1 has been associated with competence of bovine embryos to develop to the blastocyst stage. Here we tested the importance of PARM1 for development by evaluating consequences of reducing PARM1 mRNA abundance on embryonic development and differentiation, gene expression and resistance to ER stress. RESULTS: Knockdown of PARM1 using an anti-PARM1 GapmeR did not affect competence of embryos to develop into blastocysts but decreased the number of trophectoderm (TE) cells in the blastocyst and tended to increase the number of cells in the blastocyst inner cell mass (ICM). Treatment of embryos with anti-PARM1 GapmeR affected expression of 4 and 3 of 90 genes evaluated at the compact-morula and blastocyst stage of development at days 5.5 and 7.5 after fertilization, respectively. In morulae, treatment increased expression of DAB2, INADL, and STAT3 and decreased expression of CCR2. At the blastocyst stage, knockdown of PARM1 increased expression of PECAM and TEAD4 and decreased expression of CCR7. The potential role of PARM1 in ER stress response was determined by evaluating effects of knockdown of PARM1 on development of embryos after exposure to heat shock or tunicamycin and on expression of ATF6, DDIT3 and EIF2AK3 at the compact morula and blastocyst stages. Both heat shock and tunicamycin reduced the percent of embryos becoming a blastocyst but response was unaffected by PARM1 knockdown. Similarly, there was no effect of knockdown on steady-state amounts of ATF6, DDIT3 or EIF2AK3. CONCLUSION: PARM1 participates in formation of TE and ICM cells in early embryonic development but there is no evidence for the role of PARM1 in the ER stress response.


Assuntos
Proteína de Ligação a Androgênios/genética , Blastocisto/citologia , Desenvolvimento Embrionário/genética , Estresse do Retículo Endoplasmático/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Animais , Bovinos , Diferenciação Celular/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/genética , Receptores CCR2/metabolismo , Receptores CCR7/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteínas de Junções Íntimas/metabolismo , Tunicamicina/farmacologia , Proteínas Ativadoras de ras GTPase/metabolismo
9.
Oncogene ; 38(36): 6370-6381, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31312020

RESUMO

Inactivation of the tumor suppressor NF2/merlin underlies neurofibromatosis type 2 (NF2) and some sporadic tumors. Previous studies have established that merlin mediates contact inhibition of proliferation; however, the exact mechanisms remain obscure and multiple pathways have been implicated. We have previously reported that merlin inhibits Ras and Rac activity during contact inhibition, but how merlin regulates Ras activity has remained elusive. Here we demonstrate that merlin can directly interact with both Ras and p120RasGAP (also named RasGAP). While merlin does not increase the catalytic activity of RasGAP, the interactions with Ras and RasGAP may fine-tune Ras signaling. In vivo, loss of RasGAP in Schwann cells, unlike the loss of merlin, failed to promote tumorigenic growth in an orthotopic model. Therefore, modulation of Ras signaling through RasGAP likely contributes to, but is not sufficient to account for, merlin's tumor suppressor activity. Our study provides new insight into the mechanisms of merlin-dependent Ras regulation and may have additional implications for merlin-dependent regulation of other small GTPases.


Assuntos
Neurofibromina 2/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Células Cultivadas , Proteínas Ativadoras de GTPase/metabolismo , Genes Supressores de Tumor , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neurofibromatose 2/genética , Neurofibromatose 2/metabolismo , Neurofibromina 2/metabolismo , Ligação Proteica , Transdução de Sinais/genética
10.
Biomed Pharmacother ; 117: 109055, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31176165

RESUMO

Increasing studies provide evidence to support that microRNAs (miRNAs) play important roles in regulating hepatocellular carcinoma (HCC) initiation and progression. However, whether miR-1307-3p is aberrantly expressed in HCC and affects malignant behaviors of cancer cells remain unknown. In this study, we found that miR-1307-3p expression was obviously up-regulated in HCC compared to adjacent nontumor tissues. Moreover, miR-1307-3p expression was prominently higher in HCC cells compared with the normal hepatic cell line LO2. Patients with venous infiltration, tumor size ≥5 cm and advanced tumor stages (III + IV) had significant higher levels of miR-1307-3p in HCC tissues. Notably, the high level of miR-1307-3p predicted poor clinical outcomes of HCC patients. Functionally, miR-1307-3p knockdown inhibited the proliferation, migration and invasion of MHCC97H and HCCLM3 cells, and suppressed the in vivo growth and metastasis of HCCLM3 cells. Conversely, overexpression of miR-1307-3p facilitated Hep3B cell proliferation, migration and invasion. Mechanistically, DAB2 interacting protein (DAB2IP) was screened as a direct target of miR-1307-3p. The expression of DAB2IP mRNA was down-regulated and inversely correlated with miR-1307-3p level in HCC tissues. miR-1307-3p knockdown increased the level of DAB2IP in HCC cells. Luciferase reporter assay confirmed the direct interaction between miR-1307-3p and 3'UTR of DAB2IP. Importantly, DAB2IP overexpression significantly suppressed the proliferation, migration and invasion of HCCLM3 cells. DAB2IP knockdown rescued miR-1307-3p silencing-attenuated HCC cell proliferation, migration and invasion. Taken together, our findings suggest that miR-1307-3p plays a driving role in HCC progression by targeting DAB2IP. Our study may provide new therapeutic targets for HCC treatment.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , MicroRNAs/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Sequência de Bases , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Invasividade Neoplásica , Metástase Neoplásica , Prognóstico
11.
Org Biomol Chem ; 17(19): 4879-4891, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31041977

RESUMO

The mechanism of the deceptively simple reaction of guanosine triphosphate (GTP) hydrolysis catalyzed by the cellular protein Ras in complex with the activating protein GAP is an important issue because of the significance of this reaction in cancer research. We show that molecular modeling of GTP hydrolysis in the Ras-GAP active site reveals a diversity of mechanisms of the intrinsic chemical reaction depending on molecular groups at position 61 in Ras occupied by glutamine in the wild-type enzyme. First, a comparison of reaction energy profiles computed at the quantum mechanics/molecular mechanics (QM/MM) level shows that an assignment of the Gln61 side chain in the wild-type Ras either to QM or to MM parts leads to different scenarios corresponding to the glutamine-assisted or the substrate-assisted mechanisms. Second, replacement of Gln61 by the nitro-analog of glutamine (NGln) or by Glu, applied in experimental studies, results in two more scenarios featuring the so-called two-water and the concerted-type mechanisms. The glutamine-assisted mechanism in the wild-type Ras-GAP, in which the conserved Gln61 plays a decisive role, switching between the amide and imide tautomer forms, is consistent with the known experimental results of structural, kinetic and spectroscopy studies. The results emphasize the role of the Ras residue Gln61 in Ras-GAP catalysis and explain the retained catalytic activity of the Ras-GAP complex towards GTP hydrolysis in the Gln61NGln and Gln61Glu mutants of Ras.


Assuntos
Biocatálise , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Proteínas Ativadoras de ras GTPase/metabolismo , Sítios de Ligação , Guanosina Trifosfato/química , Hidrólise , Conformação Molecular , Mutação , Teoria Quântica , Proteínas Ativadoras de ras GTPase/química , Proteínas Ativadoras de ras GTPase/genética
12.
Elife ; 82019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31025938

RESUMO

It remains unclear to what extent neurodevelopmental disorder (NDD) risk genes retain functions into adulthood and how they may influence disease phenotypes. SYNGAP1 haploinsufficiency causes a severe NDD defined by autistic traits, cognitive impairment, and epilepsy. To determine if this gene retains therapeutically-relevant biological functions into adulthood, we performed a gene restoration technique in a mouse model for SYNGAP1 haploinsufficiency. Adult restoration of SynGAP protein improved behavioral and electrophysiological measures of memory and seizure. This included the elimination of interictal events that worsened during sleep. These events may be a biomarker for generalized cortical dysfunction in SYNGAP1 disorders because they also worsened during sleep in the human patient population. We conclude that SynGAP protein retains biological functions throughout adulthood and that non-developmental functions may contribute to disease phenotypes. Thus, treatments that target debilitating aspects of severe NDDs, such as medically-refractory seizures and cognitive impairment, may be effective in adult patients.


Assuntos
Envelhecimento/metabolismo , Comportamento , Encéfalo/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Potenciais de Ação , Animais , Comportamento Animal , Eletroencefalografia , Feminino , Humanos , Masculino , Memória , Camundongos , Camundongos Mutantes , Convulsões/metabolismo , Convulsões/fisiopatologia , Sono , Vigília
13.
In Vivo ; 33(3): 737-742, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028191

RESUMO

BACKGROUND/AIM: Nine genetic loci have been associated with abdominal aortic aneurysm (AAA) susceptibility, including DAB2IP. This gene is playing a role in apoptosis, cell proliferation and epithelial-to-mesenchymal transition in cancers. This study aimed to elucidate the differential expression levels of DAB2IP in AAA tissues and investigate whether mir-363-3p and EZH2 can be considered as potential mediators of its expression. MATERIALS AND METHODS: 18 AAA samples and 15 non-aneurysmatic controls were collected. Relative mRNA expression levels of DAB2IP, EZH2 and mir-363-3p were measured using qPCR. RESULTS: DAB2IP was significant up-regulated (~2.29 fold) in AAA tissues, while EZH2 and mir-363-3p were down-regulated (3.28 and 3.62-fold, respectively). A limited negative correlation was found between the DAB2IP and EZH2 expression and between DAB2IP and the mir-363-3p. CONCLUSION: An increased expression of DAB2IP in AAA tissues was shown. We suggest 2 potential mediators of DAB2IP expression in abdominal aortic aneurysm, EZH2 and mir-363-3p.


Assuntos
Aneurisma da Aorta Abdominal/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Expressão Gênica , MicroRNAs/genética , Proteínas Ativadoras de ras GTPase/genética , Idoso , Idoso de 80 Anos ou mais , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Biomarcadores , Comorbidade , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Interferência de RNA , Proteínas Ativadoras de ras GTPase/metabolismo
15.
PLoS One ; 14(1): e0209179, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30640896

RESUMO

Certain organs are capable of containing the replication of various types of viruses. In the liver, infection of Hepatitis B virus (HBV), the etiological factor of Hepatitis B and hepatocellular carcinoma (HCC), often remains asymptomatic and leads to a chronic carrier state. Here we investigated how hepatocytes contain HBV replication and promote their own survival by orchestrating a translational defense mechanism via the stress-sensitive SUMO-2/3-specific peptidase SENP3. We found that SENP3 expression level decreased in HBV-infected hepatocytes in various models including HepG2-NTCP cell lines and a humanized mouse model. Downregulation of SENP3 reduced HBV replication and boosted host protein translation. We also discovered that IQGAP2, a Ras GTPase-activating-like protein, is a key substrate for SENP3-mediated de-SUMOylation. Downregulation of SENP3 in HBV infected cells facilitated IQGAP2 SUMOylation and degradation, which leads to suppression of HBV gene expression and restoration of global translation of host genes via modulation of AKT phosphorylation. Thus, The SENP3-IQGAP2 de-SUMOylation axis is a host defense mechanism of hepatocytes that restores host protein translation and suppresses HBV gene expression.


Assuntos
Cisteína Endopeptidases/metabolismo , Vírus da Hepatite B/fisiologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Animais , Cisteína Endopeptidases/genética , Regulação para Baixo , Regulação Viral da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Hep G2 , Hepatite B/metabolismo , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Especificidade por Substrato , Sumoilação , Replicação Viral/fisiologia , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo
16.
J Mol Histol ; 50(2): 119-127, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30659402

RESUMO

IQGAP1 is a multifunctional, 190-kDa scaffolding protein that plays an important role in the regulation of cell adhesion, migration, proliferation, differentiation, polarization and cytoskeletal remodeling. IQGAP1 is ubiquitously expressed in human organs and is highly expressed in the kidney. Currently, the site-specific expression of IQGAP1 in the human nephrons is unclear. We performed Western blotting analysis, immunohistochemistry and double-immunolabeling confocal microscopic analysis of IQGAP1 with specific biomarkers of each nephron segment to study the expression and distribution of IQGAP1 in human nephrons. We found that IQGAP1 was strongly expressed in human podocytes and glomerular endothelial cells, but weakly expressed in glomerular mesangial cells. In human renal tubules, IQGAP1 was strongly expressed in the collecting duct, moderately expressed in the proximal tubule, medullary loop, distal convoluted tubule and connecting tubule. IQGAP1 staining was much stronger in the apical membrane in the proximal tubule, thick descending limb and thick ascending limb of medullary loop and collecting duct. However, the expression of IQGAP1 was mainly in the basolateral membrane of the connecting tubule, and diffusely in the thin limb of medullary loop and distal convoluted tubule. The interaction between IQGAP1 and F-actin suggested that cytoskeleton regulation may be the underlying mechanism mediating the effect of IQGAP1 in human nephrons. To the best of our knowledge, this is the first report of specific expression and differential subcellular location of IQGAP1 in human nephrons. The site-specific expression pattern of IQGAP1 suggests that IQGAP1 may play diverse roles in various human nephron segments.


Assuntos
Néfrons/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Humanos , Medula Renal/química , Túbulos Renais Coletores/química , Túbulos Renais Proximais/química , Distribuição Tecidual
17.
Proc Natl Acad Sci U S A ; 116(4): 1289-1298, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30622175

RESUMO

Macropinocytosis and phagocytosis are evolutionarily conserved forms of bulk endocytosis used by cells to ingest large volumes of fluid and solid particles, respectively. Both processes are regulated by Ras signaling, which is precisely controlled by mechanisms involving Ras GTPase activating proteins (RasGAPs) responsible for terminating Ras activity on early endosomes. While regulation of Ras signaling during large-scale endocytosis in WT Dictyostelium has been, for the most part, attributed to the Dictyostelium ortholog of human RasGAP NF1, in commonly used axenic laboratory strains, this gene is mutated and inactive. Moreover, none of the RasGAPs characterized so far have been implicated in the regulation of Ras signaling in large-scale endocytosis in axenic strains. In this study, we establish, using biochemical approaches and complementation assays in live cells, that Dictyostelium IQGAP-related protein IqgC interacts with active RasG and exhibits RasGAP activity toward this GTPase. Analyses of iqgC - and IqgC-overexpressing cells further revealed participation of this GAP in the regulation of both types of large-scale endocytosis and in cytokinesis. Moreover, given the localization of IqgC to phagosomes and, most prominently, to macropinosomes, we propose IqgC acting as a RasG-specific GAP in large-scale endocytosis. The data presented here functionally distinguish IqgC from other members of the Dictyostelium IQGAP family and call for repositioning of this genuine RasGAP outside of the IQGAP group.


Assuntos
Dictyostelium/metabolismo , Endocitose/fisiologia , Proteínas de Protozoários/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Sequência de Aminoácidos , Citocinese/fisiologia , Humanos , Fagocitose/fisiologia , Fagossomos/metabolismo , Pinocitose/fisiologia , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Proteínas ras/metabolismo
19.
Cell Cycle ; 18(2): 204-225, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30560710

RESUMO

Numerous studies exploring oncogenic Ras or manipulating physiological Ras signalling have established an irrefutable role for Ras as driver of cell cycle progression. Despite this wealth of information the precise signalling timeline and effectors engaged by Ras, particularly during G1, remain obscure as approaches for Ras inhibition are slow-acting and ill-suited for charting discrete Ras signalling episodes along the cell cycle. We have developed an approach based on the inducible recruitment of a Ras-GAP that enforces endogenous Ras inhibition within minutes. Applying this strategy to inhibit Ras stepwise in synchronous cell populations revealed that Ras signaling was required well into G1 for Cyclin D induction, pocket protein phosphorylation and S-phase entry, irrespective of whether cells emerged from quiescence or G2/M. Unexpectedly, Erk, and not PI3K/Akt or Ral was activated by Ras at mid-G1, albeit PI3K/Akt signalling was a necessary companion of Ras/Erk for sustaining cyclin-D levels and G1/S transition. Our findings chart mitogenic signaling by endogenous Ras during G1 and identify limited effector engagement restricted to Raf/MEK/Erk as a cogent distinction from oncogenic Ras signalling.


Assuntos
Ciclina D/metabolismo , Fase G1/fisiologia , Sistema de Sinalização das MAP Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fase S/fisiologia , Proteínas ras/metabolismo , Proliferação de Células/fisiologia , Células HEK293 , Células HeLa , Humanos , Multimerização Proteica , Transdução de Sinais/fisiologia , Transdução Genética , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Ativadoras de ras GTPase/farmacologia , Proteínas ras/antagonistas & inibidores
20.
Pancreas ; 48(1): 94-98, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30540680

RESUMO

OBJECTIVES: IQ motif containing GTPase-activating protein 1 (IQGAP1) acts as a scaffold for aberrant mitogen-activated protein kinase (MAPK) signaling driven by KRAS mutations in pancreatic ductal adenocarcinoma (PDAC). We determined the role of IQGAP1 in clonogenic growth and metastasis in PDAC. METHODS: We inhibited IQGAP1 expression using shRNA and assessed clonogenic growth, cell migration, and MAPK signaling in vitro and tumor initiation and metastasis in vivo. The efficacy of a peptide mimicking the IQGAP1 WW domain that binds and inhibits ERK1/2 was determined in vitro and in vivo. RESULTS: IQGAP1 loss inhibited clonogenic growth and migration of KRAS-dependent PDAC cells by disrupting MAPK signaling. In mice, IQGAP1 knockdown decreased tumor-initiating cell frequency and metastasis. WW peptide treatment inhibited clonogenic growth and in vivo tumor growth. CONCLUSIONS: Pancreatic ductal adenocarcinoma clonogenic growth, metastasis, and tumor initiation are dependent on MAPK signaling via IQGAP1. Treatment with a WW peptide disrupts IQGAP1 function and represents a novel targeting strategy for PDAC.


Assuntos
Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Proteínas Ativadoras de ras GTPase/genética , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/terapia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Metástase Neoplásica , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapia , Interferência de RNA , Terapêutica com RNAi/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Proteínas Ativadoras de ras GTPase/metabolismo
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