RESUMEN
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.
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Discapacidad Intelectual , Proteínas Activadoras de ras GTPasa , Humanos , Femenino , Proteínas Activadoras de ras GTPasa/genética , Masculino , Niño , Adolescente , Preescolar , Discapacidad Intelectual/genética , Discapacidad Intelectual/etiología , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/etiología , Reino Unido , Trastornos del Neurodesarrollo/genética , Estudios de Cohortes , Fenotipo , Epilepsia/genética , Convulsiones/genéticaRESUMEN
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.
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Actomiosina , Movimiento Celular , Polaridad 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 Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Animales , Quimiotaxis , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Actinas/metabolismo , Simulación por Computador , Ratones , Transducción de SeñalRESUMEN
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.
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Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Discapacidad Intelectual , Animales , Humanos , Ratones , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Terapia Genética/métodos , Vectores Genéticos/genética , Células Madre Hematopoyéticas/metabolismo , Discapacidad Intelectual/terapia , Discapacidad Intelectual/genética , Lentivirus/genética , Proteínas Activadoras de ras GTPasa/genética , Proteínas Activadoras de ras GTPasa/metabolismo , Transducción GenéticaRESUMEN
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.
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Carcinoma Hepatocelular , Quitosano , Histona Desacetilasas , Neoplasias Hepáticas , Nanopartículas , Proteínas Activadoras de ras GTPasa , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Células Hep G2 , Histona Desacetilasas/metabolismo , Histona Desacetilasas/genética , Quitosano/farmacología , Proteínas Activadoras de ras GTPasa/genética , Proteínas Activadoras de ras GTPasa/metabolismo , Nanopartículas/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Tamaño de la Partícula , Liposomas , Proteínas RepresorasRESUMEN
Macropinocytosis mediates the non-selective bulk uptake of extracellular fluid, enabling cells to survey the environment and obtain nutrients. A conserved set of signaling proteins orchestrates the actin dynamics that lead to membrane ruffling and macropinosome formation across various eukaryotic organisms. At the center of this signaling network are Ras GTPases, whose activation potently stimulates macropinocytosis. However, how Ras signaling is initiated and spatiotemporally regulated during macropinocytosis is not well understood. By using the model system Dictyostelium and a proteomics-based approach to identify regulators of macropinocytosis, we uncovered Leep2, consisting of Leep2A and Leep2B, as a RasGAP complex. The Leep2 complex specifically localizes to emerging macropinocytic cups and nascent macropinosomes, where it modulates macropinosome formation by regulating the activities of three Ras family small GTPases. Deletion or overexpression of the complex, as well as disruption or sustained activation of the target Ras GTPases, impairs macropinocytic activity. Our data reveal the critical role of fine-tuning Ras activity in directing macropinosome formation.
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Dictyostelium , Pinocitosis , Proteínas Activadoras de ras GTPasa , Dictyostelium/citología , Dictyostelium/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas ras/metabolismo , Transducción de SeñalRESUMEN
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.
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Proliferación Celular , Leucemia Mieloide Aguda , Proteínas Activadoras de ras GTPasa , Humanos , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Proteínas Activadoras de ras GTPasa/antagonistas & inhibidores , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/genética , Proliferación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Simulación por Computador , Antineoplásicos/farmacología , Dominios Proteicos , Animales , Proteómica/métodosRESUMEN
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.
Asunto(s)
Progresión de la Enfermedad , Neoplasias Pancreáticas , Proteómica , Factores de Transcripción SOXC , Proteínas Activadoras de ras GTPasa , Animales , Humanos , Ratones , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Ratones Desnudos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Fosfoproteínas/metabolismo , Fosforilación , Pronóstico , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Transducción de Señal , Factores de Transcripción SOXC/metabolismo , Factores de Transcripción SOXC/genéticaRESUMEN
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.
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Macrófagos , FN-kappa B , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular , Proteínas Activadoras de ras GTPasa , Animales , Ratones , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa 3/genética , Ratones Endogámicos C57BL , Infecciones por Mycobacterium no Tuberculosas/inmunología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Infecciones por Mycobacterium no Tuberculosas/metabolismo , FN-kappa B/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Células RAW 264.7 , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
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.
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Neoplasias , Proteínas Activadoras de ras GTPasa , Humanos , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Animales , Transducción de SeñalRESUMEN
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.
Asunto(s)
Resistencia a Antineoplásicos , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Ferroptosis , Paclitaxel , Proteínas Activadoras de ras GTPasa , Humanos , Ferroptosis/efectos de los fármacos , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/metabolismo , Carcinoma de Células Escamosas de Esófago/patología , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Paclitaxel/farmacología , Proteínas Señalizadoras YAP/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacosRESUMEN
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.
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Péptidos y Proteínas de Señalización Intracelular , Unión Proteica , Proteínas Activadoras de ras GTPasa , Humanos , Cristalografía por Rayos X , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/química , Modelos Moleculares , Mutación , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/química , Proteínas Activadoras de ras GTPasa/genética , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismoRESUMEN
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.
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Proteínas de Capping de la Actina , Citoesqueleto de Actina , Forminas , Proteínas Activadoras de ras GTPasa , Animales , Humanos , Proteínas de Capping de la Actina/metabolismo , Proteínas de Capping de la Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Movimiento Celular , Forminas/metabolismo , Células HeLa , Unión Proteica , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Ratones , Células 3T3 NIHRESUMEN
Breast cancer is a heterogeneous disease that remains the most common malignancy among women worldwide. During genomic analysis of breast tumours, mRNA levels of IQGAP3 were found to be upregulated in triple negative tumours. IQGAP3 was subsequently found to be expressed across a panel of triple negative breast cancer (TNBC) cell lines. Depleting expression levels of IQGAP3 delivered elongated cells, disrupted cell migration, and inhibited the ability of cells to form specialised invasive adhesion structures, termed invadopodia. The morphological changes induced by IQGAP3 depletion were found to be dependent on RhoA. Indeed, reduced expression of IQGAP3 disrupted RhoA activity and actomyosin contractility. Interestingly, IQGAP3 was also found to interact with p-21 activated kinase 6 (PAK6); a protein already associated with the regulation of cell morphology. Moreover, PAK6 depletion phenocopied IQGAP3 depletion in these cells. Whereas PAK6 overexpression rescued the IQGAP3 depletion phenotype. Our work points to an important PAK6-IQGAP3-RhoA pathway that drives the cellular contractility of breast cancer cells promoting both cell migration and adhesive invasion of these cells. As this phenotype is relevant to the process of metastasis and re-seeding of metastasis, the pharmacological targeting of PAK6 could lead to clinical benefit in TNBC patients.
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Movimiento Celular , Neoplasias de la Mama Triple Negativas , Quinasas p21 Activadas , Proteína de Unión al GTP rhoA , Humanos , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Línea Celular Tumoral , Proteína de Unión al GTP rhoA/metabolismo , Quinasas p21 Activadas/metabolismo , Quinasas p21 Activadas/genética , Femenino , Podosomas/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Adhesión Celular , Regulación Neoplásica de la Expresión Génica , Proteínas Activadoras de GTPasaRESUMEN
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.
Asunto(s)
Atención , Discapacidad Intelectual , Proteínas Activadoras de ras GTPasa , Humanos , Masculino , Femenino , Proteínas Activadoras de ras GTPasa/genética , Atención/fisiología , Discapacidad Intelectual/genética , Discapacidad Intelectual/fisiopatología , Niño , Adolescente , Adulto , Adulto Joven , Movimientos Oculares/fisiología , Percepción Visual/fisiología , Conducta SocialRESUMEN
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.
Asunto(s)
Actinas , Bleomicina , Fibroblastos , Fibrosis Pulmonar , Proteínas Activadoras de ras GTPasa , Animales , Ratones , Actinas/metabolismo , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibroblastos/patología , Pulmón/patología , Pulmón/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Polimerizacion , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/genéticaRESUMEN
This study aimed to describe the behavioral profile of individuals with SYNGAP1-ID. Parents/carers of 30 individuals aged 3-18 years old with a diagnosis of SYNGAP1-ID and 21 typically developing individuals completed the Vineland-3 Adaptive Behavior Scale and the Child Behavior Checklist. We found that those with SYNGAP1-ID showed fewer adaptive behaviors and higher levels of internalizing and externalizing behaviors across almost all domains compared to typically developing controls. There was some evidence that these differences were greatest in older children, and more apparent in those with co-occuring epilepsy. This characterization of the phenotype of SYNGAP1-ID significantly aids our understanding of the behavioral profile of this population and is a step towards the development of tailored interventions.
Asunto(s)
Discapacidad Intelectual , Proteínas Activadoras de ras GTPasa , Humanos , Niño , Masculino , Femenino , Preescolar , Proteínas Activadoras de ras GTPasa/genética , Adolescente , Adaptación Psicológica/fisiología , Conducta Infantil/fisiología , EpilepsiaRESUMEN
The activation level of RAS can be determined by GTP hydrolysis rate (khy) and GDP-GTP exchange rates (kex). Either impaired GTP hydrolysis or enhanced GDP-GTP exchange causes the aberrant activation of RAS in oncogenic mutants. Therefore, it is important to quantify the khy and kex for understanding the mechanisms of RAS oncogenesis and drug development. Conventional methods have individually measured the kex and khy of RAS. However, within the intracellular environment, GTP hydrolysis and GDP-GTP exchange reactions occur simultaneously under conditions where GTP concentration is kept constant. In addition, the intracellular activity of RAS is influenced by endogenous regulatory proteins, such as RAS GTPase activating proteins (GAPs) and the guanine-nucleotide exchange factors (GEFs). Here, we describe the in vitro and in-cell NMR methods to estimate the khy and kex simultaneously by measuring the time-dependent changes of the fraction of GTP-bound ratio under the condition of constant GTP concentration.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido , Proteínas Activadoras de ras GTPasa , Guanosina Trifosfato/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Hidrólisis , Factores de Intercambio de Guanina Nucleótido/metabolismo , Espectroscopía de Resonancia Magnética , Guanosina Difosfato/metabolismoRESUMEN
IQGAP3 (IQ Motif Containing GTPase Activating Protein 3) is member of the IQGAP family of scaffold proteins, which are essential for assembling multiprotein complexes that coordinate various intracellular signaling pathways. Previous research has shown that IQGAP3 is overexpressed in psoriatic skin lesions. Given its involvement in processes like cell proliferation and chemokine signaling, we sought to explore its molecular role in driving the psoriatic phenotype of keratinocytes. By conducting transcriptome profiling of HaCaT keratinocytes, we identified numerous psoriasis-associated pathways that were affected when IQGAP3 was knocked down. These included alterations in NFkB signaling, EGFR signaling, activation of p38/MAPK and ERK1/ERK2, lipid metabolism, cytokine production, and the response to inflammatory cytokine stimulation. Real-time analysis further revealed changes in cell growth dynamics, including proliferation and wound healing. The balance between cell proliferation and apoptosis was altered, as were skin barrier functions and the production of IL-6 and IFNγ. Despite these significant findings, the diversity of the alterations observed in the knockdown cells led us to conclude that IQGAP3 may not be the best target for the therapeutic inhibition to normalize the phenotype of keratinocytes in psoriasis.
Asunto(s)
Proliferación Celular , Proteínas Activadoras de GTPasa , Queratinocitos , Psoriasis , Humanos , Queratinocitos/metabolismo , Queratinocitos/patología , Psoriasis/metabolismo , Psoriasis/patología , Psoriasis/genética , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Transducción de Señal , Células HaCaT , Citocinas/metabolismo , Apoptosis , Piel/metabolismo , Piel/patología , Línea Celular , Perfilación de la Expresión GénicaRESUMEN
The clinical and genetic characteristics of SYNGAP1 mutations in Korean pediatric patients are not well understood. We retrospectively analyzed 13 individuals with SYNGAP1 mutations from a longitudinal aspect. Clinical data, genetic profiles, and electroencephalography (EEG) patterns were examined. Genotypic analyses included gene panels and whole-exome sequencing. All patients exhibited global developmental delay from early infancy, with motor development eventually reaching independent ambulation by 3 years of age. Language developmental delay varied significantly from nonverbal to simple sentences, which plateaued in all patients. Patients with the best language outcomes typically managed 2-3-word sentences, corresponding to a developmental age of 2-3 years. Epilepsy developed in 77% of patients, with onset consistently following developmental delays at a median age of 31 months. Longitudinal EEG data revealed a shift from occipital to frontal epileptiform discharges with age, suggesting a correlation with synaptic maturation. These findings suggest that the critical developmental plateau occurs between the ages of 2 and 5 years and is potentially influenced by epilepsy. By analyzing longitudinal data, our study contributes to a deeper understanding of SYNGAP1-related DEE, provides potential EEG biomarkers, and underlines the importance of early diagnosis and intervention to address this complex disorder.
Asunto(s)
Electroencefalografía , Epilepsia , Genotipo , Mutación , Fenotipo , Proteínas Activadoras de ras GTPasa , Preescolar , Femenino , Humanos , Masculino , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/patología , Discapacidades del Desarrollo/fisiopatología , Epilepsia/genética , Epilepsia/patología , Epilepsia/fisiopatología , Secuenciación del Exoma , Estudios de Asociación Genética , Estudios Longitudinales , Mutación/genética , Proteínas Activadoras de ras GTPasa/genética , Estudios RetrospectivosRESUMEN
Attachment of circulating tumor cells to the endothelial cells (ECs) lining blood vessels is a critical step in cancer metastatic colonization, which leads to metastatic outgrowth. Breast and prostate cancers are common malignancies in women and men, respectively. Here, we observe that ß1-integrin is required for human prostate and breast cancer cell adhesion to ECs under shear-stress conditions in vitro and to lung blood vessel ECs in vivo. We identify IQGAP1 and neural Wiskott-Aldrich syndrome protein (NWASP) as regulators of ß1-integrin transcription and protein expression in prostate and breast cancer cells. IQGAP1 and NWASP depletion in cancer cells decreases adhesion to ECs in vitro and retention in the lung vasculature and metastatic lung nodule formation in vivo. Mechanistically, NWASP and IQGAP1 act downstream of Cdc42 to increase ß1-integrin expression both via extracellular signal-regulated kinase (ERK)/focal adhesion kinase signaling at the protein level and by myocardin-related transcription factor/serum response factor (SRF) transcriptionally. Our results identify IQGAP1 and NWASP as potential therapeutic targets to reduce early metastatic dissemination.