RESUMO
Disruption of SYNGAP1 directly causes a genetically identifiable neurodevelopmental disorder (NDD) called SYNGAP1-related intellectual disability (SRID). Without functional SynGAP1 protein, individuals are developmentally delayed and have prominent features of intellectual disability (ID), motor impairments, and epilepsy. Over the past two decades, there have been numerous discoveries indicating the critical role of Syngap1. Several rodent models with a loss of Syngap1 have been engineered, identifying precise roles in neuronal structure and function, as well as key biochemical pathways key for synapse integrity. Homozygous loss of SYNGAP1/Syngap1 is lethal. Heterozygous mutations of Syngap1 result in a broad range of behavioral phenotypes. Our in vivo functional data, using the original mouse model from the Huganir laboratory, corroborated behaviors including robust hyperactivity and deficits in learning and memory in young adults. Furthermore, we described impairments in the domain of sleep, characterized using neurophysiological data that was collected with wireless, telemetric electroencephalography (EEG). Syngap1+/- mice exhibited elevated spiking events and spike trains, in addition to elevated power, most notably in the delta power frequency. For the first time, we illustrated that primary neurons from Syngap1+/- mice displayed: 1) increased network firing activity, 2) greater bursts, 3) and shorter inter-burst intervals between peaks, by utilizing high density microelectrode arrays (HD-MEA). Our work bridges in vitro electrophysiological neuronal activity and function with in vivo neurophysiological brain activity and function. These data elucidate quantitative, translational biomarkers in vivo and in vitro that can be utilized for the development and efficacy assessment of targeted treatments for SRID.
Assuntos
Modelos Animais de Doenças , Deficiência Intelectual , Fenótipo , Proteínas Ativadoras de ras GTPase , Animais , Deficiência Intelectual/genética , Deficiência Intelectual/fisiopatologia , Camundongos , Proteínas Ativadoras de ras GTPase/genética , Eletroencefalografia , Neurônios/fisiologia , Masculino , Comportamento Animal , FemininoRESUMO
De novo mutations in the synaptic GTPase activating protein (SynGAP) are associated with neurological disorders like intellectual disability, epilepsy, and autism. SynGAP is also implicated in Alzheimer's disease and cancer. Although pathogenic variants are highly penetrant in neurodevelopmental conditions, a substantial number of them are caused by missense mutations that are difficult to diagnose. Hence, in silico mutagenesis was performed for probing the missense effects within the N-terminal region of SynGAP structure. Through extensive molecular dynamics simulations, encompassing three 150-ns replicates for 211 variants, the impact of missense mutations on the protein fold was assessed. The effect of the mutations on the folding stability was also quantitatively assessed using free energy calculations. The mutations were categorized as potentially pathogenic or benign based on their structural impacts. Finally, the study introduces wild-type-SynGAP in complex with RasGTPase at the inner membrane, while considering the potential effects of mutations on these key interactions. This study provides structural perspective to the clinical assessment of SynGAP missense variants and lays the foundation for future structure-based drug discovery.
Assuntos
Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Proteínas Ativadoras de ras GTPase , Humanos , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/química , Proteínas Ativadoras de ras GTPase/metabolismo , Dobramento de Proteína , Relação Estrutura-AtividadeRESUMO
Humans with monogenic inborn errors responsible for extreme disease phenotypes can reveal essential physiological pathways. We investigated germline mutations in GNAI2, which encodes Gαi2, a key component in heterotrimeric G protein signal transduction usually thought to regulate adenylyl cyclase-mediated cyclic adenosine monophosphate (cAMP) production. Patients with activating Gαi2 mutations had clinical presentations that included impaired immunity. Mutant Gαi2 impaired cell migration and augmented responses to T cell receptor (TCR) stimulation. We found that mutant Gαi2 influenced TCR signaling by sequestering the guanosine triphosphatase (GTPase)-activating protein RASA2, thereby promoting RAS activation and increasing downstream extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)-AKT S6 signaling to drive cellular growth and proliferation.
Assuntos
Subunidade alfa Gi2 de Proteína de Ligação ao GTP , Mutação em Linhagem Germinativa , Receptores de Antígenos de Linfócitos T , Linfócitos T , Proteínas Ativadoras de ras GTPase , Humanos , Movimento Celular/genética , Proliferação de Células , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/genética , Imunidade/genética , Sistema de Sinalização das MAP Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Proteínas ras/metabolismo , Proteínas ras/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , LinhagemRESUMO
Mutations in SYNGAP1 are a common genetic cause of intellectual disability (ID) and a risk factor for autism. SYNGAP1 encodes a synaptic GTPase-activating protein (GAP) that has both signaling and scaffolding roles. Most pathogenic variants of SYNGAP1 are predicted to result in haploinsufficiency. However, some affected individuals carry missense mutations in its calcium/lipid binding (C2) and GAP domains, suggesting that many clinical features result from loss of functions carried out by these domains. To test this hypothesis, we targeted the exons encoding the C2 and GAP domains of SYNGAP. Rats heterozygous for this deletion exhibit reduced exploration and fear extinction, altered social investigation, and spontaneous seizures-key phenotypes shared with Syngap heterozygous null rats. Together, these findings indicate that the reduction of SYNGAP C2/GAP domain function is a main feature of SYNGAP haploinsufficiency. This rat model provides an important system for the study of ID, autism, and epilepsy.
Assuntos
Proteínas Ativadoras de ras GTPase , Animais , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Ratos , Domínios Proteicos , Haploinsuficiência , Masculino , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Humanos , Convulsões/metabolismo , Convulsões/genética , Heterozigoto , Medo/fisiologia , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Modelos Animais de DoençasRESUMO
Human brain ontogeny is characterized by a considerably prolonged neotenic development of cortical neurons and circuits. Neoteny is thought to be essential for the acquisition of advanced cognitive functions, which are typically altered in intellectual disability (ID) and autism spectrum disorders (ASDs). Human neuronal neoteny could be disrupted in some forms of ID and/or ASDs, but this has never been tested. Here, we use xenotransplantation of human cortical neurons into the mouse brain to model SYNGAP1 haploinsufficiency, one of the most prevalent genetic causes of ID/ASDs. We find that SYNGAP1-deficient human neurons display strong acceleration of morphological and functional synaptic formation and maturation alongside disrupted synaptic plasticity. At the circuit level, SYNGAP1-haploinsufficient neurons display precocious acquisition of responsiveness to visual stimulation months ahead of time. Our findings indicate that SYNGAP1 is required cell autonomously for human neuronal neoteny, providing novel links between human-specific developmental mechanisms and ID/ASDs.
Assuntos
Córtex Cerebral , Neurônios , Proteínas Ativadoras de ras GTPase , Animais , Humanos , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/deficiência , Proteínas Ativadoras de ras GTPase/metabolismo , Neurônios/metabolismo , Camundongos , Córtex Cerebral/metabolismo , Córtex Cerebral/citologia , Haploinsuficiência , Plasticidade Neuronal/fisiologia , Sinapses/metabolismo , Sinapses/fisiologia , Deficiência Intelectual/genética , Masculino , FemininoRESUMO
BACKGROUND: SYNGAP1 variants are associated with varying degrees of intellectual disability (ID), developmental delay (DD), epilepsy, autism, and behavioural difficulties. These features may also be observed in other monogenic conditions. There is a need to systematically compare the characteristics of SYNGAP1 with other monogenic causes of ID and DD to identify features unique to the SYNAGP1 phenotype. We aimed to contrast the neurodevelopmental and behavioural phenotype of children with SYNGAP1-related ID (SYNGAP1-ID) to children with other monogenic conditions and a matched degree of ID. METHODS: Participants were identified from the IMAGINE-ID study, a UK-based, national cohort study of neuropsychiatric risk in children with ID of known genetic origin. Thirteen children with SYNGAP1 variants (age 4-16 years; 85% female) were matched (2:1) with 26 controls with other monogenic causes of ID for chronological and mental age, sex, socio-economic deprivation, adaptive behaviour, and physical health difficulties. Caregivers completed the Development and Wellbeing Assessment (DAWBA) and physical health questionnaires. RESULTS: Our results demonstrate that seizures affected children with SYNGAP1-ID (84.6%) more frequently than the ID-comparison group (7.6%; p = < 0.001). Fine-motor development was disproportionally impaired in SYNGAP1-ID, with 92.3% of children experiencing difficulties compared to 50% of ID-comparisons(p = 0.03). Gross motor and social development did not differ between the two groups. Children with SYNGAP1-ID were more likely to be non-verbal (61.5%) than ID-comparisons (23.1%; p = 0.01). Those children able to speak, spoke their first words at the same age as the ID-comparison group (mean = 3.25 years), yet achieved lower language competency (p = 0.04). Children with SYNGAP1-ID compared to the ID-comparison group were not more likely to meet criteria for autism (SYNGAP1-ID = 46.2%; ID-comparison = 30.7%; p = .35), attention-deficit hyperactivity disorder (15.4%;15.4%; p = 1), generalised anxiety (7.7%;15.4%; p = .49) or oppositional defiant disorder (7.7%;0%; p = .15). CONCLUSION: For the first time, we demonstrate that SYNGAP1-ID is associated with fine motor and language difficulties beyond those experienced by children with other genetic causes of DD and ID. Targeted occupational and speech and language therapies should be incorporated early into SYNGAP1-ID management.
Assuntos
Deficiência Intelectual , Proteínas Ativadoras de ras GTPase , Humanos , Feminino , Proteínas Ativadoras de ras GTPase/genética , Masculino , Criança , Adolescente , Pré-Escolar , Deficiência Intelectual/genética , Deficiência Intelectual/etiologia , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/etiologia , Reino Unido , Transtornos do Neurodesenvolvimento/genética , Estudos de Coortes , Fenótipo , Epilepsia/genética , Convulsões/genéticaRESUMO
Ras has been extensively studied as a promoter of cell proliferation, whereas few studies have explored its role in migration. To investigate the direct and immediate effects of Ras activity on cell motility or polarity, we focused on RasGAPs, C2GAPB in Dictyostelium amoebae and RASAL3 in HL-60 neutrophils and macrophages. In both cellular systems, optically recruiting the respective RasGAP to the cell front extinguished pre-existing protrusions and changed migration direction. However, when these respective RasGAPs were recruited uniformly to the membrane, cells polarized and moved more rapidly, whereas targeting to the back exaggerated these effects. These unexpected outcomes of attenuating Ras activity naturally had strong, context-dependent consequences for chemotaxis. The RasGAP-mediated polarization depended critically on myosin II activity and commenced with contraction at the cell rear, followed by sustained mTORC2-dependent actin polymerization at the front. These experimental results were captured by computational simulations in which Ras levels control front- and back-promoting feedback loops. The discovery that inhibiting Ras activity can produce counterintuitive effects on cell migration has important implications for future drug-design strategies targeting oncogenic Ras.
Assuntos
Actomiosina , Movimento Celular , Polaridade Celular , Dictyostelium , Proteínas ras , Dictyostelium/metabolismo , Dictyostelium/genética , Células HL-60 , Actomiosina/metabolismo , Humanos , Proteínas ras/metabolismo , Proteínas ras/genética , Macrófagos/metabolismo , Miosina Tipo II/metabolismo , Miosina Tipo II/genética , Neutrófilos/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Animais , Quimiotaxia , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Actinas/metabolismo , Simulação por Computador , Camundongos , Transdução de SinaisRESUMO
Human papillomavirus (HPV)-related cervical and nasopharyngeal cancers differ in molecular mechanisms underlying the oncogenic processes. The disparity may be attributed to differential expression of oncoproteins. The current study investigated the host oncogenes expression pattern in HPV-associated cervical and nasopharyngeal cancer. Formalin-fixed paraffin-embedded tissues originating from the nasopharyngeal and cervical regions were screened using Hematoxylin and Eosin staining. Genomic DNA and total RNA were extracted from confirmed cancer biopsies and non-cancer tissues (NC). HPV was detected by PCR using MY09/GP5+/6+ primers. Protein expression levels of AKT, IQGAP1, and MMP16 in HPV-infected cancers and controls were determined by immunohistochemistry. RT-qPCR was used to profile mRNAs of the oncogenes. AKT and IQGAP1 proteins were highly expressed in the epithelial cancers compared with the non-cancer tissues (p < 0.05). IQGAP1 and MMP16 mRNAs level was significantly higher in the cancers than in the NC (p < 0.05), but not AKT mRNA levels. MMP16 protein was ubiquitously expressed in all tissues. AKT mRNA level was significantly elevated in CC compared with NPC (p < 0.001). However, the difference in AKT, IQGAP1 and MMP16 proteins level between CC and NPC was not significant (p > 0.05). The oncoproteins expression level between the HPV-positive and HPV-negative cancer biopsies showed no significant difference (p < 0.05). Current study reports AKT but not IQGAP1 and MMP16 mRNAs differentially expression in cervical and nasopharyngeal cancers, independent of HPV infection status.
Assuntos
Neoplasias Nasofaríngeas , Neoplasias do Colo do Útero , Humanos , Feminino , Neoplasias do Colo do Útero/virologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Neoplasias Nasofaríngeas/virologia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Oncogenes , Regulação Neoplásica da Expressão Gênica , Papillomaviridae/genética , Papillomaviridae/patogenicidade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adulto , Masculino , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo , Idoso , Papillomavirus HumanoRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is a global life-threatening problem and therapeutic interventions are still encountered. IQGAP genes are involved in HCC oncogenesis. The modulatory effect of statins on the expression of IQGAP genes is still unclear. This study aims to study the effect of free SV and chitosan (CS) decorated simvastatin (SV) loaded solid lipid nanoparticles (C-SV-SLNs) on HCC mortality. METHODS AND RESULTS: Plain, SV-SLN, and C-SV- SLN were prepared and characterized in terms of particle size (PS), zeta potential (ZP), and polydispersity index (PDI). The biosafety of different SLN was investigated using fresh erythrocytes, moreover, cytotoxicity was investigated using HepG2 cell lines. The effect of SLNs on IQGAPs gene expression as well as JNK, HDAC6, and HDAC8 activity was investigated using PCR and MOE-docking. The current results displayed that SV-SLNs have nanosized, negative ZP and are homogenous, CS decoration shifts the ZP of SLN into cationic ZP. Furthermore, all SLNs exhibited desirable biosafety in terms of no deleterious effect on erythrocyte integrity. SV solution and SV-SLN significantly increase the mortality of HepG2 compared to undertreated cells, however, the effect of SV-SLN is more pronounced compared to free SV. Remarkably, C-SV-SLN elicits high HepG2 cell mortality compared to free SV and SV-SLN. The treatment of HepG2 cells with SV solution, SV-SLN, or C-SV-SLN significantly upregulates the IQGAP2 gene with repression of IQGAP1 and IQGAP3 genes. MOE-docking studies revealed both SV and tenivastatin exhibit interactions with the active sites of JNK, HDAC6, and HDAC8. Moreover, tenivastatin exhibited greater interactions with magnesium and zinc compared to SV. CONCLUSIONS: This research provides novel insights into the therapeutic potential of SV, SV-SLN and C-SV-SLNs in HCC treatment, modulating critical signaling cascades involving IQGAPs, JNK, and HDAC. The development of C-SV-SLNs presents a promising strategy for effective HCC therapy.
Assuntos
Carcinoma Hepatocelular , Quitosana , Histona Desacetilases , Neoplasias Hepáticas , Nanopartículas , Proteínas Ativadoras de ras GTPase , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Células Hep G2 , Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Quitosana/farmacologia , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo , Nanopartículas/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Tamanho da Partícula , Lipossomos , Proteínas RepressorasRESUMO
BACKGROUND: Synaptic Ras GTPase activating protein 1 (SYNGAP1)-related non-specific intellectual disability is a neurodevelopmental disorder caused by an insufficient level of SynGAP1 resulting in a dysfunction of neuronal synapses and presenting with a wide array of clinical phenotypes. Hematopoietic stem cell gene therapy has the potential to deliver therapeutic levels of functional SynGAP1 to affected neurons upon transduction of hematopoietic stem and progenitor cells with a lentiviral vector. METHODS: As a novel approach toward the treatment of SYNGAP1, we have generated a lentiviral vector expressing a modified form of SynGAP1 for transduction of human CD34+ hematopoietic stem and progenitor cells. The gene-modified cells were then transplanted into adult immunodeficient SYNGAP1+/- heterozygous mice and evaluated for improvement of SYNGAP1-related clinical phenotypes. Expression of SynGAP1 was also evaluated in the brain tissue of transplanted mice. RESULTS: In our proof-of-concept study, we have demonstrated significant improvement of SYNGAP1-related phenotypes including an improvement in motor abilities observed in mice transplanted with the vector transduced cells because they displayed decreased hyperactivity in an open field assay and an increased latency to fall in a rotarod assay. An increased level of SynGAP1 was also detected in the brains of these mice. CONCLUSIONS: These early-stage results highlight the potential of this stem cell gene therapy approach as a treatment strategy for SYNGAP1.
Assuntos
Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Deficiência Intelectual , Animais , Humanos , Camundongos , Encéfalo/metabolismo , Modelos Animais de Doenças , Terapia Genética/métodos , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Deficiência Intelectual/terapia , Deficiência Intelectual/genética , Lentivirus/genética , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo , Transdução GenéticaRESUMO
Chemoresistance is one of the major hindrances to many cancer therapies, including esophageal squamous cell carcinoma (ESCC). Ferroptosis, a new programmed cell death, plays an essential role in chemoresistance. IQ-domain GTPase activating protein 1 (IQGAP1) is a scaffold protein and functions as an oncogene in various human malignancies. However, the underlying effect and molecular mechanisms of IQGAP1 on paclitaxel (PTX) resistance and ferroptosis in ESCC remain to be elucidated. In this study, we found that IQGAP1 was highly expressed in ESCC tissues and could as a potential biomarker for diagnosis and predicting the prognosis of ESCC. Functional studies revealed that IQGAP1 overexpression reduced the sensitivity of ESCC cells to PTX by enhancing ESCC cell viability and proliferation and inhibiting cell death, and protected ESCC cells from ferroptosis, whereas IQGAP1 knockdown exhibited contrary effects. Importantly, reductions of chemosensitivity and ferroptosis caused by IQGAP1 overexpression were reversed with ferroptosis inducer RSL3, while the increases of chemosensitivity and ferroptosis caused by IQGAP1 knockdown were reversed with ferroptosis inhibitor ferrostatin-1 (Fer-1) in ESCC cells, indicating that IQGAP1 played a key role in resistance to PTX through regulating ferroptosis. Mechanistically, we demonstrated that IQGAP1 overexpression upregulated the expression of Yes-associated protein (YAP), the central mediator of the Hippo pathway. YAP inhibitor Verteporfin (VP) could reverse the effects of IQGAP1 overexpression on ESCC chemoresistance and ferroptosis. Taken together, our findings suggest that IQGAP1 promotes chemoresistance by blocking ferroptosis through targeting YAP. IQGAP1 may be a novel therapeutic target for overcoming chemoresistance in ESCC.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Ferroptose , Paclitaxel , Proteínas Ativadoras de ras GTPase , Humanos , Ferroptose/efeitos dos fármacos , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Paclitaxel/farmacologia , Proteínas de Sinalização YAP/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacosRESUMO
Acute myeloid leukemia (AML) is fatal in the majority of adults. Identification of new therapeutic targets and their pharmacologic modulators are needed to improve outcomes. Previous studies had shown that immunization of rabbits with normal peripheral WBCs that had been incubated with fluorodinitrobenzene elicited high titer antibodies that bound to a spectrum of human leukemias. We report that proteomic analyses of immunoaffinity-purified lysates of primary AML cells showed enrichment of scaffolding protein IQGAP1. Immunohistochemistry and gene-expression analyses confirmed IQGAP1 mRNA overexpression in various cytogenetic subtypes of primary human AML compared to normal hematopoietic cells. shRNA knockdown of IQGAP1 blocked proliferation and clonogenicity of human leukemia cell-lines. To develop small molecules targeting IQGAP1 we performed in-silico screening of 212,966 compounds, selected 4 hits targeting the IQGAP1-GRD domain, and conducted SAR of the 'fittest hit' to identify UR778Br, a prototypical agent targeting IQGAP1. UR778Br inhibited proliferation, induced apoptosis, resulted in G2/M arrest, and inhibited colony formation by leukemia cell-lines and primary-AML while sparing normal marrow cells. UR778Br exhibited favorable ADME/T profiles and drug-likeness to treat AML. In summary, AML shows response to IQGAP1 inhibition, and UR778Br, identified through in-silico studies, selectively targeted AML cells while sparing normal marrow.
Assuntos
Proliferação de Células , Leucemia Mieloide Aguda , Proteínas Ativadoras de ras GTPase , Humanos , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/genética , Proliferação de Células/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Simulação por Computador , Antineoplásicos/farmacologia , Domínios Proteicos , Animais , Proteômica/métodosRESUMO
Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), still ranks among the leading causes of annual human death by infectious disease. Mtb has developed several strategies to survive for years at a time within the host despite the presence of a robust immune response, including manipulating the progression of the inflammatory response and forming granulomatous lesions. Here we demonstrate that IQGAP1, a highly conserved scaffolding protein, compartmentalizes and coordinates multiple signaling pathways in macrophages infected with Mycobacterium marinum (Mm or M.marinum), the closest relative of Mtb. Upregulated IQGAP1 ultimately suppresses TNF-α production by repressing the MKK3 signal and reducing NF-κBp65 translocation, deactivating the p38MAPK pathway. Accordingly, IQGAP1 silencing and overexpression significantly alter p38MAPK activity by modulating the production of phosphorylated MKK3 during mycobacterial infection. Pharmacological inhibition of IQGAP1-associated microtubule assembly not only alleviates tissue damage caused by M.marinum infection but also significantly decreases the production of VEGF-a critical player for granuloma-associated angiogenesis during pathogenic mycobacterial infection. Similarly, IQGAP1 silencing in Mm-infected macrophages diminishes VEGF production, while IQGAP1 overexpression upregulates VEGF. Our data indicate that mycobacteria induce IQGAP1 to hijack NF-κBp65 activation, preventing the expression of proinflammatory cytokines as well as promoting VEGF production during infection and granuloma formation. Thus, therapies targeting host IQGAP1 may be a promising strategy for treating tuberculosis, particularly in drug-resistant diseases.
Assuntos
Macrófagos , NF-kappa B , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular , Proteínas Ativadoras de ras GTPase , Animais , Camundongos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , MAP Quinase Quinase 3/metabolismo , MAP Quinase Quinase 3/genética , Camundongos Endogâmicos C57BL , Infecções por Mycobacterium não Tuberculosas/imunologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Infecções por Mycobacterium não Tuberculosas/metabolismo , NF-kappa B/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Células RAW 264.7 , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVE: This study aims to elucidate the functional role of IQGAP1 phosphorylation modification mediated by the SOX4/MAPK1 regulatory axis in developing pancreatic cancer through phosphoproteomics analysis. METHODS: Proteomics and phosphoproteomics data of pancreatic cancer were obtained from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database. Differential analysis, kinase-substrate enrichment analysis (KSEA), and independent prognosis analysis were performed on these datasets. Subtype analysis of pancreatic cancer patients was conducted based on the expression of prognostic-related proteins, and the prognosis of different subtypes was evaluated through prognosis analysis. Differential analysis of proteins in different subtypes was performed to identify differential proteins in the high-risk subtype. Clinical correlation analysis was conducted based on the expression of prognostic-related proteins, pancreatic cancer typing results, and clinical characteristics in the pancreatic cancer proteomics dataset. Functional pathway enrichment analysis was performed using GSEA/GO/KEGG, and most module proteins correlated with pancreatic cancer were selected using WGCNA analysis. In cell experiments, pancreatic cancer cells were grouped, and the expression levels of SOX4, MAPK1, and the phosphorylation level of IQGAP1 were detected by RT-qPCR and Western blot experiments. The effect of SOX4 on MAPK1 promoter transcriptional activity was assessed using a dual-luciferase assay, and the enrichment of SOX4 on the MAPK1 promoter was examined using a ChIP assay. The proliferation, migration, and invasion functions of grouped pancreatic cancer cells were assessed using CCK-8, colony formation, and Transwell assays. In animal experiments, the impact of SOX4 on tumor growth and metastasis through the regulation of MAPK1-IQGAP1 phosphorylation modification was studied by constructing subcutaneous and orthotopic pancreatic cancer xenograft models, as well as a liver metastasis model in nude mice. RESULTS: Phosphoproteomics and proteomics data analysis revealed that the kinase MAPK1 may play an important role in pancreatic cancer progression by promoting IQGAP1 phosphorylation modification. Proteomics analysis classified pancreatic cancer patients into two subtypes, C1 and C2, where the high-risk C2 subtype was associated with poor prognosis, malignant tumor typing, and enriched tumor-related pathways. SOX4 may promote the occurrence of the high-risk C2 subtype of pancreatic cancer by regulating MAPK1-IQGAP1 phosphorylation modification. In vitro cell experiments confirmed that SOX4 promoted IQGAP1 phosphorylation modification by activating MAPK1 transcription while silencing SOX4 inhibited the proliferation, migration, and invasion of pancreatic cancer cells by reducing the phosphorylation level of MAPK1-IQGAP1. In vivo, animal experiments further confirmed that silencing SOX4 suppressed the growth and metastasis of pancreatic cancer by reducing the phosphorylation level of MAPK1-IQGAP1. CONCLUSION: The findings of this study suggest that SOX4 promotes the phosphorylation modification of IQGAP1 by activating MAPK1 transcription, thereby facilitating the growth and metastasis of pancreatic cancer.
Assuntos
Progressão da Doença , Neoplasias Pancreáticas , Proteômica , Fatores de Transcrição SOXC , Proteínas Ativadoras de ras GTPase , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Camundongos Nus , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Fosfoproteínas/metabolismo , Fosforilação , Prognóstico , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Transdução de Sinais , Fatores de Transcrição SOXC/metabolismo , Fatores de Transcrição SOXC/genéticaRESUMO
The dynamic crosstalk between tumor and stromal cells is a major determinant of cancer aggressiveness. The tumor-suppressor DAB2IP (Disabled homolog 2 interacting protein) plays an important role in this context, since it modulates cell responses to multiple extracellular inputs, including inflammatory cytokines and growth factors. DAB2IP is a RasGAP and negatively controls Ras-dependent mitogenic signals. In addition, it modulates other major oncogenic pathways, including TNFα/NF-κB, WNT/ß-catenin, PI3K/AKT, and androgen receptor signaling. In line with its tumor-suppressive role, DAB2IP is frequently inactivated in cancer by transcriptional and post-transcriptional mechanisms, including promoter methylation, microRNA-mediated downregulation, and protein-protein interactions. Intriguingly, some observations suggest that downregulation of DAB2IP in cells of the tumor stroma could foster establishment of a pro-metastatic microenvironment. This review summarizes recent insights into the tumor-suppressive functions of DAB2IP and the consequences of its inactivation in cancer. In particular, we explore potential approaches aimed at reactivating DAB2IP, or augmenting its expression levels, as a novel strategy in cancer treatment. We suggest that reactivation or upregulation of DAB2IP would concurrently attenuate multiple oncogenic pathways in both cancer cells and the tumor microenvironment, with implications for improved treatment of a broad spectrum of tumors.
Assuntos
Neoplasias , Proteínas Ativadoras de ras GTPase , Humanos , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Animais , Transdução de SinaisRESUMO
We previously found IQ motif containing GTPase activating protein (IQGAP1) to be consistently elevated in lung fibroblasts (LF) isolated from patients with scleroderma (systemic sclerosis, SSc)-associated interstitial lung disease (ILD) and reported that IQGAP1 contributed to SSc by regulating expression and organization of α-smooth muscle actin (SMA) in LF. The aim of this study was to compare the development of ILD in the presence and absence of IQGAP1. Pulmonary fibrosis was induced in IQGAP1 knockout (KO) and wild-type (WT) mice by a single-intratracheal instillation of bleomycin. Two and three weeks later, mice were euthanized and investigated. We observed that the IQGAP1 KO mouse was characterized by a reduced rate of actin polymerization with reduced accumulation of actin in the lung compared to the WT mouse. After exposure to bleomycin, the IQGAP1 KO mouse demonstrated decreased contractile activity of LF, reduced expression of SMA, TGFß, and collagen, and lowered overall fibrosis scores compared to the WT mouse. The numbers of inflammatory cells and expression of pro-inflammatory cytokines in lung tissue were not significantly different between IQGAP1 KO and WT mice. We conclude that IQGAP1 plays an important role in the development of lung fibrosis induced by bleomycin, and the absence of IQGAP1 reduces the contractile activity of lung fibroblast and bleomycin-induced pulmonary fibrosis. Thus, IQGAP1 may be a potential target for novel anti-fibrotic therapies for lung fibrosis.
Assuntos
Actinas , Bleomicina , Fibroblastos , Fibrose Pulmonar , Proteínas Ativadoras de ras GTPase , Animais , Camundongos , Actinas/metabolismo , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Pulmão/patologia , Pulmão/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polimerização , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
The activation of the angiopoietin (Angpt)-Tie system is linked to endothelial dysfunction during sepsis. Bacterial quorum-sensing molecules function as pathogen-associated molecular patterns. However, their impact on the endothelium and the Angpt-Tie system remains unclear. Therefore, this study investigated whether treatment with N-3-oxododecanoyl homoserine lactone (3OC12-HSL), a quorum-sensing molecule derived from Pseudomonas aeruginosa, impaired endothelial function in human umbilical vein endothelial cells. 3OC12-HSL treatment impaired tube formation even at sublethal concentrations, and immunocytochemistry analysis revealed that it seemed to reduce vascular endothelial-cadherin expression at the cell-cell interface. Upon assessing the mRNA expression patterns of genes associated with the Angpt-Tie axis, the expressions of Angpt2, Forkhead box protein O1, Tie1, and vascular endothelial growth factor 2 were found to be upregulated in the 3OC12-HSL-treated cells. Moreover, western blot analysis revealed that 3OC12-HSL treatment increased Angpt2 expression. A co-immunoprecipitation assay was conducted to assess the effect of 3OC12-HSL on the IQ motif containing GTPase activating protein 1 (IQGAP1) and Rac1 complex and the interaction between these proteins was consistently maintained regardless of 3OC12-HSL treatment. Next, recombinant human (rh)-Angpt1 was added to assess whether it modulated the effects of 3OC12-HSL treatment. rh-Angpt1 addition increased cellular viability, improved endothelial function, and reversed the overall patterns of mRNA and protein expression in endothelial cells treated with 3OC12-HSL. Additionally, it was related to the increased expression of phospho-Akt and the IQGAP1 and Rac1 complex. Collectively, our findings indicated that 3OC12-HSL from Pseudomonas aeruginosa can impair endothelial integrity via the activation of the Angpt-Tie axis, which appeared to be reversed by rh-Angpt1 treatment.
Assuntos
4-Butirolactona , Angiopoietina-2 , Homosserina , Células Endoteliais da Veia Umbilical Humana , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Angiopoietina-2/metabolismo , Angiopoietina-2/genética , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacologia , Homosserina/análogos & derivados , Homosserina/farmacologia , Homosserina/metabolismo , Caderinas/metabolismo , Caderinas/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Transdução de Sinais/efeitos dos fármacos , Antígenos CD/metabolismo , Antígenos CD/genética , Angiopoietina-1/metabolismo , Angiopoietina-1/genéticaRESUMO
SYNGAP1-ID is a neurodevelopmental disorder caused by a mutation of the SYNGAP1 gene. Characterized by moderate to severe developmental delay, it is associated with several physical and behavioral issues as well as additional diagnoses, including autism. However, it is not known whether social cognitive differences seen in SYNGAP1-ID are similar to those previously identified in idiopathic or other forms of autism. This study therefore investigated visual social attention in SYNGAP1-ID. Eye movements were recorded across three passive viewing tasks (face scanning, pop-out, and social preference) of differing social complexity in 24 individuals with SYNGAP1-ID and 12 typically developing controls. We found that SYNGAP1-ID participants looked at faces less than the controls, and when they did look at faces, they had less time looking at and fewer fixations to the eyes. For the pop-out task, where social and nonsocial objects (Phone, car, face, bird, and face-noise) were presented in an array, those with SYNGAP1-ID spent significantly less time looking at the phone stimulus as well as fewer fixations to the face compared with the typically developing controls. When looking at two naturalistic scenes side by side, one social in nature (e.g., with children present) and the other not, there were no differences between the SYNGAP1-ID group and typically developing controls on any of the examined eye tracking measures. This study provides novel findings on the social attention of those with SYNGAP1-ID and helps to provide further evidence for using eye tracking as an objective measure of the social phenotype in this population in future clinical trials.
Assuntos
Atenção , Deficiência Intelectual , Proteínas Ativadoras de ras GTPase , Humanos , Masculino , Feminino , Proteínas Ativadoras de ras GTPase/genética , Atenção/fisiologia , Deficiência Intelectual/genética , Deficiência Intelectual/fisiopatologia , Criança , Adolescente , Adulto , Adulto Jovem , Movimentos Oculares/fisiologia , Percepção Visual/fisiologia , Comportamento SocialRESUMO
AIDA-1, encoded by ANKS1B, is an abundant postsynaptic scaffold protein essential for brain development. Mutations of ANKS1B are closely associated with various psychiatric disorders. However, very little is known regarding the molecular mechanisms underlying AIDA-1's involvements under physiological and pathophysiological conditions. Here, we discovered an interaction between AIDA-1 and the SynGAP family Ras-GTPase activating protein (GAP) via affinity purification using AIDA-1d as the bait. Biochemical studies showed that the PTB domain of AIDA-1 binds to an extended NPx[F/Y]-motif of the SynGAP family proteins with high affinities. The high-resolution crystal structure of AIDA-1 PTB domain in complex with the SynGAP NPxF-motif revealed the molecular mechanism governing the specific interaction between AIDA-1 and SynGAP. Our study not only explains why patients with ANKS1B or SYNGAP1 mutations share overlapping clinical phenotypes, but also allows identification of new AIDA-1 binding targets such as Ras and Rab interactors.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Ligação Proteica , Proteínas Ativadoras de ras GTPase , Humanos , Cristalografia por Raios X , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/química , Modelos Moleculares , Mutação , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Ativadoras de ras GTPase/química , Proteínas Ativadoras de ras GTPase/genética , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
Cell processes require precise regulation of actin polymerization that is mediated by plus-end regulatory proteins. Detailed mechanisms that explain plus-end dynamics involve regulators with opposing roles, including factors that enhance assembly, e.g., the formin mDia1, and others that stop growth (capping protein, CP). We explore IQGAP1's roles in regulating actin filament plus-ends and the consequences of perturbing its activity in cells. We confirm that IQGAP1 pauses elongation and interacts with plus ends through two residues (C756 and C781). We directly visualize the dynamic interplay between IQGAP1 and mDia1, revealing that IQGAP1 displaces the formin to influence actin assembly. Using four-color TIRF, we show that IQGAP1's displacement activity extends to formin-CP "decision complexes," promoting end-binding protein turnover at plus-ends. Loss of IQGAP1 or its plus-end activities disrupts morphology and migration, emphasizing its essential role. These results reveal a new role for IQGAP1 in promoting protein turnover on filament ends and provide new insights into how plus-end actin assembly is regulated in cells.