RESUMEN

Style penetration by pollen tubes is essential for reproductive success, a process requiring canonical Rab5s in Arabidopsis. However, functional loss of Arabidopsis Vps9a, the gene encoding for guanine nucleotide exchange factor (GEF) of Rab5s, did not affect male transmission, implying the presence of a compensation program or redundancy. By combining genetic, cytological, and molecular approaches, we report that Arabidopsis Vps9b is a pollen-preferential gene, redundantly mediating pollen tube penetration of style with Vps9a. Vps9b is functionally interchangeable with Vps9a, whose functional distinction results from distinct expression profiles. Functional loss of Vps9a and Vps9b results in the mis-targeting of Rab5-dependent tonoplast proteins, defective vacuolar biogenesis, disturbed distribution of post-Golgi vesicles, increased cellular turgor, cytosolic acidification, and disrupted organization of actin microfilaments (MF) in pollen tubes, which collectively lead to the failure of pollen tubes to grow through style.

RESUMEN

RhoA and its effectors, the transcriptional coactivators myocardin-related transcription factor (MRTF) and serum response factor (SRF), control epithelial phenotype and are indispensable for profibrotic epithelial reprogramming during fibrogenesis. Context-dependent control of RhoA and fibrosis-associated changes in its regulators, however, remain incompletely characterized. We previously identified the guanine nucleotide exchange factor GEF-H1 as a central mediator of RhoA activation in renal tubular cells exposed to inflammatory or fibrotic stimuli. Here we found that GEF-H1 expression and phosphorylation were strongly elevated in two animal models of fibrosis. In the Unilateral Ureteral Obstruction mouse kidney fibrosis model, GEF-H1 was upregulated predominantly in the tubular compartment. GEF-H1 was also elevated and phosphorylated in a rat pulmonary artery banding (PAB) model of right ventricular fibrosis. Prolonged stimulation of LLC-PK1 tubular cells with tumor necrosis factor (TNF)-α or transforming growth factor (TGF)-ß1 increased GEF-H1 expression and activated a luciferase-coupled GEF-H1 promoter. Knockdown and overexpression studies revealed that these effects were mediated by RhoA, cytoskeleton remodeling, and MRTF, indicative of a positive feedback cycle. Indeed, silencing endogenous GEF-H1 attenuated activation of the GEF-H1 promoter. Of importance, inhibition of MRTF using CCG-1423 prevented GEF-H1 upregulation in both animal models. MRTF-dependent increase in GEF-H1 was prevented by inhibition of the transcription factor Sp1, and mutating putative Sp1 binding sites in the GEF-H1 promoter eliminated its MRTF-dependent activation. As the GEF-H1/RhoA axis is key for fibrogenesis, this novel MRTF/Sp1-dependent regulation of GEF-H1 abundance represents a potential target for reducing renal and cardiac fibrosis.NEW & NOTEWORTHY We show that expression of the RhoA regulator GEF-H1 is upregulated in tubular cells exposed to fibrogenic cytokines and in animal models of kidney and heart fibrosis. We identify a pathway wherein GEF-H1/RhoA-dependent MRTF activation through its noncanonical partner Sp1 upregulates GEF-H1. Our data reveal the existence of a positive feedback cycle that enhances Rho signaling through control of both GEF-H1 activation and expression. This feedback loop may play an important role in organ fibrosis.

Asunto(s)

Fibrosis , Factores de Intercambio de Guanina Nucleótido Rho , Factor de Transcripción Sp1 , Transactivadores , Proteína de Unión al GTP rhoA , Animales , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp1/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Transactivadores/metabolismo , Transactivadores/genética , Ratones , Ratas , Retroalimentación Fisiológica , Masculino , Ratones Endogámicos C57BL , Humanos , Transducción de Señal , Porcinos , Fosforilación , Modelos Animales de Enfermedad , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patología , Obstrucción Ureteral/genética , Ratas Sprague-Dawley , Línea Celular , Factores de Transcripción

RESUMEN

INTRODUCTION: Guanine nucleotide exchange factors (GEFs) regulate the activation of small GTPases (G proteins) of the Ras superfamily proteins controlling cellular functions. Ras superfamily proteins act as 'molecular switches' that are turned 'ON' by guanine exchange. There are five major groups of Ras family GTPases: Ras, Ran, Rho, Rab and Arf, with a variety of different GEFs regulating their GTP loading. GEFs have been implicated in various diseases including cancer. This makes GEFs attractive targets to modulate signaling networks controlled by small GTPases. AREAS COVERED: In this review, the roles and mechanisms of GEFs in malignancy are outlined. The mechanism of guanine exchange activity by GEFs on a small GTPase is illustrated. Then, some examples of GEFs that are significant in cancer are presented with a discussion on recent progress in therapeutic targeting efforts using a variety of approaches. EXPERT OPINION: Recently, GEFs have emerged as potential therapeutic targets for novel cancer drug development. Targeting small GTPases is challenging; thus, targeting their activation by GEFs is a promising strategy. Most GEF-targeted drugs are still in preclinical development. A deeper biological understanding of the underlying mechanisms of GEF activity and utilizing advanced technology are necessary to enhance drug discovery for GEFs in cancer.

Asunto(s)

Antineoplásicos , Desarrollo de Medicamentos , Descubrimiento de Drogas , Factores de Intercambio de Guanina Nucleótido , Terapia Molecular Dirigida , Neoplasias , Transducción de Señal , Humanos , Neoplasias/tratamiento farmacológico , Factores de Intercambio de Guanina Nucleótido/metabolismo , Descubrimiento de Drogas/métodos , Antineoplásicos/farmacología , Animales , Desarrollo de Medicamentos/métodos

RESUMEN

Small Ras homologous guanosine triphosphatase (Rho GTPase) family proteins are highly associated with tumorigenesis and development. As intrinsic exchange activity regulators of Rho GTPases, Rho guanine nucleotide exchange factors (RhoGEFs) have been demonstrated to be closely involved in tumor development and received increasing attention. They mainly contain two families: the diffuse B-cell lymphoma (Dbl) family and the dedicator of cytokinesis (Dock) family. More and more emphasis has been paid to the Dbl family members for their abnormally high expression in various cancers and their correlation to poor prognosis. In this review, the common and distinctive structures of Dbl family members are discussed, and their roles in cancer are summarized with a focus on Ect2, Tiam1/2, P-Rex1/2, Vav1/2/3, Trio, KALRN, and LARG. Significantly, the strategies targeting Dbl family RhoGEFs are highlighted as novel therapeutic opportunities for cancer.

Asunto(s)

Linfoma de Células B , Neoplasias , Humanos , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Carcinogénesis

RESUMEN

The Rab family of guanosine triphosphatases (GTPases) includes key regulators of intracellular transport and membrane trafficking targeting specific steps in exocytic, endocytic, and recycling pathways. DENND5B (Rab6-interacting Protein 1B-like protein, R6IP1B) is the longest isoform of DENND5, an evolutionarily conserved DENN domain-containing guanine nucleotide exchange factor (GEF) that is highly expressed in the brain. Through exome sequencing and international matchmaking platforms, we identified five de novo variants in DENND5B in a cohort of five unrelated individuals with neurodevelopmental phenotypes featuring cognitive impairment, dysmorphism, abnormal behavior, variable epilepsy, white matter abnormalities, and cortical gyration defects. We used biochemical assays and confocal microscopy to assess the impact of DENND5B variants on protein accumulation and distribution. Then, exploiting fluorescent lipid cargoes coupled to high-content imaging and analysis in living cells, we investigated whether DENND5B variants affected the dynamics of vesicle-mediated intracellular transport of specific cargoes. We further generated an in silico model to investigate the consequences of DENND5B variants on the DENND5B-RAB39A interaction. Biochemical analysis showed decreased protein levels of DENND5B mutants in various cell types. Functional investigation of DENND5B variants revealed defective intracellular vesicle trafficking, with significant impairment of lipid uptake and distribution. Although none of the variants affected the DENND5B-RAB39A interface, all were predicted to disrupt protein folding. Overall, our findings indicate that DENND5B variants perturb intracellular membrane trafficking pathways and cause a complex neurodevelopmental syndrome with variable epilepsy and white matter involvement.

Asunto(s)

Epilepsia , Discapacidad Intelectual , Trastornos del Neurodesarrollo , Humanos , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Encéfalo/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Lípidos , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Proteínas de Unión al GTP rab/metabolismo

RESUMEN

Introduction: Rac-GTPases and their Rac-GEF activators play important roles in neutrophil-mediated host defence. These proteins control the adhesion molecules and cytoskeletal dynamics required for neutrophil recruitment to inflamed and infected organs, and the neutrophil effector responses that kill pathogens. Methods: Here, we used live cell TIRF-FRET imaging in neutrophils from Rac-FRET reporter mice with deficiencies in the Rac-GEFs Dock2, Tiam1 or Prex1/Vav1 to evaluate if these proteins activate spatiotemporally distinct pools of Rac, and to correlate patterns of Rac activity with the neutrophil responses they control. Results: All the GEFs were required for neutrophil adhesion, and Prex1/Vav1 were important during spreading and for the velocity of migration during chemotaxis. However, Dock2 emerged as the prominent regulator of neutrophil responses, as this GEF was required for neutrophil polarisation and random migration, for migration velocity during chemokinesis, for the likelihood to migrate and for the speed of migration and of turning during chemotaxis, as well as for rapid particle engulfment during phagocytosis. We identified characteristic spatiotemporal patterns of Rac activity generated by Dock2 which correlate with the importance of the Rac-GEF in these neutrophil responses. We also demonstrate a requirement for Dock2 in neutrophil recruitment during aseptic peritonitis. Discussion: Collectively, our data provide a first direct comparison of the pools of Rac activity generated by different types of Rac-GEFs, and identify Dock2 as a key regulator of polarisation, migration and phagocytosis in primary neutrophils.

Asunto(s)

Proteínas Activadoras de GTPasa , Factores de Intercambio de Guanina Nucleótido , Neutrófilos , Fagocitosis , Animales , Ratones , Quimiotaxis , Citoesqueleto , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Activadoras de GTPasa/metabolismo

RESUMEN

Metastatic cancer cells dynamically adjust their shape to adhere, invade, migrate, and expand to generate secondary tumors. Inherent to these processes is the constant assembly and disassembly of cytoskeletal supramolecular structures. The subcellular places where cytoskeletal polymers are built and reorganized are defined by the activation of Rho GTPases. These molecular switches directly respond to signaling cascades integrated by Rho guanine nucleotide exchange factors (RhoGEFs), which are sophisticated multidomain proteins that control morphological behavior of cancer and stromal cells in response to cell-cell interactions, tumor-secreted factors and actions of oncogenic proteins within the tumor microenvironment. Stromal cells, including fibroblasts, immune and endothelial cells, and even projections of neuronal cells, adjust their shapes and move into growing tumoral masses, building tumor-induced structures that eventually serve as metastatic routes. Here we review the role of RhoGEFs in metastatic cancer. They are highly diverse proteins with common catalytic modules that select among a variety of homologous Rho GTPases enabling them to load GTP, acquiring an active conformation that stimulates effectors controlling actin cytoskeleton remodeling. Therefore, due to their strategic position in oncogenic signaling cascades, and their structural diversity flanking common catalytic modules, RhoGEFs possess unique characteristics that make them conceptual targets of antimetastatic precision therapies. Preclinical proof of concept, demonstrating the antimetastatic effect of inhibiting either expression or activity of ßPix (ARHGEF7), P-Rex1, Vav1, ARHGEF17, and Dock1, among others, is emerging.

Asunto(s)

Neoplasias , Proteínas de Unión al GTP rho , Humanos , Proteínas de Unión al GTP rho/metabolismo , Células Endoteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal/fisiología , Neoplasias/metabolismo , Microambiente Tumoral

RESUMEN

Rho GTPases family are considered to be molecular switches that regulate various cellular processes, including cytoskeleton remodeling, cell polarity, synaptic development and maintenance. Accumulating evidence shows that Rho GTPases are involved in neuronal development and brain diseases, including substance dependence. However, the functions of Rho GTPases in substance dependence are divergent and cerebral nuclei-dependent. Thereby, comprehensive integration of their roles and correlated mechanisms are urgently needed. In this review, the molecular functions and regulatory mechanisms of Rho GTPases and their regulators such as GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) in substance dependence have been reviewed, and this is of great significance for understanding their spatiotemporal roles in addictions induced by different addictive substances and in different stages of substance dependence.

RESUMEN

Rho family GTPases serve as molecular switches in numerous cellular processes, and their overexpression is involved in disease conditions. RhoG is one of the less explored Rho GTPases with significant sequential and structural homology with Rac1. Experimental mutations in RhoG (i.e., RhoGG12V and RhoGQ61L) are shown to dysregulate cell migration. Thus, targeting upstream activators of RhoG, such as guanine nucleotide exchange factors (GEFs), maybe an important strategy for inhibiting RhoG activation. In the current study, we have modelled the 3D structure of RhoG with greater accuracy as confirmed through PROCHECK, ProSA, and Verify3D. Our results indicate that 90.4% of residues are in the Ramachandran plots favoured region, with the Z-score of -6.46, and 87.96% of residues had an averaged 3D-1D score ≥0.2. Further, we have evaluated and binding dynamics of ten Rac1 inhibitors to investigate their potential to inhibit RhoG by targeting GEFs binding grooves. To this end, the binding energy of the docked complexes of the wild-type (WT) RhoG and its mutant proteins with inhibitor molecules was calculated using the MM/PBSA method. Our results from docking studies showed that macrolide1 binds efficiently with the GEF site of WT RhoG and the mutants mentioned above. However, an extensive analysis using MD simulations (200 ns) showed that the Rac1 based inhibitor, EHop-016, and NSC23766 might bind with greater affinity to GEF sites of mutants and WT RhoG. Thus, the results from the study indicate that Rac1 inhibitors have the potential for use as therapeutics in conditions involving dysregulation of RhoG.Communicated by Ramaswamy H. Sarma.

Asunto(s)

Proteína de Unión al GTP cdc42 , Proteína de Unión al GTP rac1 , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/química , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Transducción de Señal , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP rho

RESUMEN

DOCK proteins are a family of multi-domain guanine nucleotide exchange factors (GEFs) that activate the RHO GTPases CDC42 and RAC1, thereby regulating several RHO GTPase-dependent cellular processes. DOCK proteins are characterized by the catalytic DHR2 domain (DOCKDHR2 ), and a phosphatidylinositol(3,4,5)P3 -binding DHR1 domain (DOCKDHR1 ) that targets DOCK proteins to plasma membranes. DOCK-family GEFs are divided into four subfamilies (A to D) differing in their specificities for CDC42 and RAC1, and the composition of accessory signalling domains. Additionally, the DOCK-A and DOCK-B subfamilies are constitutively associated with ELMO proteins that auto-inhibit DOCK GEF activity. We review structural studies that have provided mechanistic insights into DOCK-protein functions. These studies revealed how a conserved nucleotide sensor in DOCKDHR2 catalyses nucleotide exchange, the basis for how different DOCK proteins activate specifically CDC42 and RAC1, and sometimes both, and how up-stream regulators relieve the ELMO-mediated auto-inhibition. We conclude by presenting a model for full-length DOCK9 of the DOCK-D subfamily. The involvement of DOCK GEFs in a range of diseases highlights the importance of gaining structural insights into these proteins to better understand and specifically target them.

Asunto(s)

Factores de Intercambio de Guanina Nucleótido , Proteína de Unión al GTP cdc42 , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rho/genética , Transducción de Señal , Nucleótidos/metabolismo , Biología , Proteína de Unión al GTP rac1/metabolismo

RESUMEN

Small GTPases act as molecular switches in regulating a myriad of cellular signaling, cytoskeletal dynamics, vesicular trafficking, and membrane/organelle transport processes. Here, I provide an editorial overview of papers collected in this Special Issue on the "Regulation and Function of Small GTPases 2.0".

Asunto(s)

Proteínas de Unión al GTP Monoméricas , Proteínas de Unión al GTP Monoméricas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Transducción de Señal , Citoesqueleto/metabolismo

RESUMEN

Chemotaxis plays an essential role in recruitment of leukocytes to sites of inflammation. Eukaryotic cells sense chemoattractant with G protein-coupled receptors (GPCRs) and chemotax toward gradients with an enormous concentration range through adaptation. Cells in adaptation no longer respond to the present stimulus but remain sensitive to stronger stimuli. Thus, adaptation provides a fundamental strategy for eukaryotic cells to chemotax through a gradient. Ras activation is the first step in the chemosensing GPCR signaling pathways that displays a transient activation behavior in both model organism Dictyostelium discoideum and mammalian neutrophils. Recently, it has been revealed that C2GAP1 and CAPRI control the GPCR-mediated adaptation in D. discoideum and human neutrophils, respectively. More importantly, both Ras inhibitors regulate the sensitivity of the cells. These findings suggest an evolutionarily conserved molecular mechanism by which eukaryotic cells gate concentration range of chemoattractants for chemotaxis.

Asunto(s)

Quimiotaxis , Dictyostelium , Animales , Humanos , Quimiotaxis/fisiología , Dictyostelium/metabolismo , Factores Quimiotácticos/farmacología , Factores Quimiotácticos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Mamíferos/metabolismo

RESUMEN

ADP-ribosylation factor (Arf)-GTPase-activating protein (GAP) with coiled-coil, ankyrin repeat and PH domains 1 (ACAP1) has been reported to serve as an adaptor for clathrin coat complex playing a role in endocytic recycling and cellular migration. The potential role of ACAP1 in lung adenocarcinoma (LUAD) has not been yet completely defined. We performed the comprehensive analyses, including gene expression, survival analysis, genetic alteration, function enrichment, and immune characteristics. ACAP1 was remarkably downregulated in tumor tissues, and linked with the clinicopathologic features in LUAD patients. Prognostic analysis demonstrated that low ACAP1 expression was correlated with unsatisfactory overall survival (OS) and disease specific survival (DSS) in LUAD patients. Moreover, ACAP1 could be determined as a prognostic biomarker according to Cox proportional hazard model and nomogram model. We also confirmed that ACAP1 was downregulated in two LUAD cell lines, comparing to normal lung cell. Overexpression of ACAP1 caused a profound attenuation in cell proliferation, migration, invasion, and promoted cell apoptosis. Additionally, functional enrichment analyses confirmed that ACAP1 was highly correlated with T cell activation and immune response. Then, we further conducted immune landscape analyses, including single cell RNA sequencing, immune cells infiltration, and immune checkpoints. ACAP1 expression was positively associated with the infiltrating level of immune cells in TME and the expression of immune checkpoint molecules. This study first comprehensively analyzed molecular expression, clinical implication, and immune landscape features of ACAP1 in LUAD, suggesting that ACAP1 was predictive of prognosis and could serve as a potential biomarker predicting immunotherapy response for LUAD patients.

Asunto(s)

Lisofosfolípidos , Esfingosina , Linfocitos/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1

RESUMEN

BACKGROUND: Guanine nucleotide exchange factors (GEFs) play pivotal roles in neuronal cell functions by exchanging GDP to GTP nucleotide and activation of GTPases. We aimed to determine the genotype and phenotype spectrum of GEF mutations by collecting data from a large Iranian cohort with intellectual disability (ID) and/or developmental delay (DD). METHODS: We collected data from nine families with 20 patients extracted from Iranian cohort of 640 families with ID and/or DD. Next-generation sequencing (NGS) was used to identify the causing variants in recruited families. We also compared our clinical and molecular findings with previously reported patients carrying mutations in these GEF genes in the literature published until mid-2021. RESULTS: We identified disease-causing variants in eight GEF genes including ALS2, IQSEC2, MADD, RAB3GAP1, RAB3GAP2, TRIO, ITSN1, and DENND2A. The major clinical manifestations in 203 previously reported cases along with our 20 patients with disease causing variants in eight GEF genes were as follow; speech disorder (85.2%), ID (81.6%), DD (81.1%), inability to walk (71.3%), facial dysmorphisms features (52.4%), abnormalities in skull morphology (55.6%), hypotonia and muscle weakness (47%), and brain MRI abnormalities (43.4%). CONCLUSION: Our study provides new insights into the genotype and phenotype spectrum of mutations in GEF genes.

Asunto(s)

Factores de Intercambio de Guanina Nucleótido , Discapacidad Intelectual , Genotipo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Discapacidad Intelectual/genética , Irán , Fenotipo , Proteínas de Unión al GTP rab3/genética

RESUMEN

GTP binding proteins known as small GTPases make up one of the largest groups of regulatory proteins and control almost all functions of living cells. Their activity is under, respectively, positive and negative regulation by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which together with their upstream regulators and the downstream targets of the small GTPases form formidable signalling networks. While genomics has revealed the large size of the GTPase, GEF and GAP repertoires, only a small fraction of their interactions and functions have yet been experimentally explored. Dictyostelid social amoebas have been particularly useful in unravelling the roles of many proteins in the Rac-Rho and Ras-Rap families of GTPases in directional cell migration and regulation of the actin cytoskeleton. Genomes and cell-type specific and developmental transcriptomes are available for Dictyostelium species that span the 0.5 billion years of evolution of the group from their unicellular ancestors. In this work, we identified all GTPases, GEFs and GAPs from genomes representative of the four major taxon groups and investigated their phylogenetic relationships and evolutionary conservation and changes in their functional domain architecture and in their developmental and cell-type specific expression. We performed a hierarchical cluster analysis of the expression profiles of the ~2000 analysed genes to identify putative interacting sets of GTPases, GEFs and GAPs, which highlight sets known to interact experimentally and many novel combinations. This work represents a valuable resource for research into all fields of cellular regulation.

Asunto(s)

Dictyostelium , Proteínas de Unión al GTP Monoméricas , Dictyostelium/genética , Dictyostelium/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Filogenia

RESUMEN

RAS, a member of the small GTPase family, functions as a binary switch by shifting between inactive GDP-loaded and active GTP-loaded state. RAS gain-of-function mutations are one of the leading causes in human oncogenesis, accounting for ∼19% of the global cancer burden. As a well-recognized target in malignancy, RAS has been intensively studied in the past decades. Despite the sustained efforts, many failures occurred in the earlier exploration and resulted in an 'undruggable' feature of RAS proteins. Phosphorylation at several residues has been recently determined as regulators for wild-type and mutated RAS proteins. Therefore, the development of RAS inhibitors directly targeting the RAS mutants or towards upstream regulatory kinases supplies a novel direction for tackling the anti-RAS difficulties. A better understanding of RAS phosphorylation can contribute to future therapeutic strategies. In this review, we comprehensively summarized the current advances in RAS phosphorylation and provided mechanistic insights into the signaling transduction of associated pathways. Importantly, the preclinical and clinical success in developing anti-RAS drugs targeting the upstream kinases and potential directions of harnessing allostery to target RAS phosphorylation sites were also discussed.

RESUMEN

Dysfunction of vascular barriers is a critical step in inflammatory diseases. Endothelial tight junctions (TJs) control barrier function, and the cytoplasmic adaptor protein cingulin connects TJs to signalling pathways. However, local events at TJs during inflammation are largely unknown. In this study, we investigate the local response of TJ adaptor protein cingulin and its interaction with Rho guanine nucleotide exchange factor H1 (GEF-H1, also known as ARHGEF2) upon vascular barrier disruption to find a new approach to counteract vascular leak. Based on transendothelial-electrical-resistance (TEER) measurements, cingulin strengthened barrier integrity upon stimulation with histamine, thrombin and VEGF. Cingulin also attenuated myosin light chain 2 (MLC2; also known as MYL2) phosphorylation by localising GEF-H1 to cell junctions. By using cingulin phosphomutants, we verified that the phosphorylation of the cingulin head domain is required for its protective effect. Increased colocalisation of GEF-H1 and cingulin was observed in the vessels of vasculitis patients compared to those in healthy skin. Our findings demonstrate that cingulin can counteract vascular leak at TJs, suggesting the existence of a novel mechanism in blood endothelial cells that protects barrier function during disease.

Asunto(s)

Células Endoteliales , Uniones Estrechas , Permeabilidad Capilar , Células Endoteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal , Uniones Estrechas/metabolismo

RESUMEN

Several small guanosine triphosphatases (GTPases) from the Ras protein superfamily regulate glucose-stimulated insulin secretion in the pancreatic islet ß-cell. The Rho family GTPases Cdc42 and Rac1 are primarily involved in relaying key signals in several cellular functions, including vesicle trafficking, plasma membrane homeostasis, and cytoskeletal dynamics. They orchestrate specific changes at each spatiotemporal region within the ß-cell by coordinating with signal transducers, guanine nucleotide exchange factors (GEFs), GTPase-activating factors (GAPs), and their effectors. The Arf family of small GTPases is involved in vesicular trafficking (exocytosis and endocytosis) and actin cytoskeletal dynamics. Rab-GTPases regulate pre-exocytotic and late endocytic membrane trafficking events in ß-cells. Several additional functions for small GTPases include regulating transcription factor activity and mitochondrial dynamics. Importantly, defects in several of these GTPases have been found associated with type 2 diabetes (T2D) etiology. The purpose of this review is to systematically denote the identities and molecular mechanistic steps in the glucose-stimulated insulin secretion pathway that leads to the normal release of insulin. We will also note newly identified defects in these GTPases and their corresponding regulatory factors (e.g., GDP dissociation inhibitors (GDIs), GEFs, and GAPs) in the pancreatic ß-cells, which contribute to the dysregulation of metabolism and the development of T2D.

Asunto(s)

Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Endocitosis , Exocitosis , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/patología , Islotes Pancreáticos/patología , Proteínas de Unión al GTP Monoméricas/fisiología , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas ras/metabolismo , Proteínas ras/fisiología , Proteínas de Unión al GTP rho/metabolismo

RESUMEN

Guanine nucleotide exchange factors (GEFs) activate GTPases by stimulating the release of guanosine diphosphate to permit the binding of guanosine triphosphate. ARHGEF3 or XPLN (exchange factor found in platelets, leukemic, and neuronal tissues) is a selective guanine nucleotide exchange factor for Rho GTPases (RhoGEFs) that activates RhoA and RhoB but not RhoC, RhoG, Rac1, or Cdc42. ARHGEF3 contains the diffuse B-cell lymphoma homology and pleckstrin homology domains but lacks similarity with other known functional domains. ARHGEF3 also binds the mammalian target of rapamycin complex 2 (mTORC2) and subsequently inhibits mTORC2 and Akt. In vivo investigation has also indicated the communication between ARHGEF3 and autophagy-related muscle pathologies. Moreover, studies on genetic variation in ARHGEF3 and genome-wide association studies have predicted exciting novel roles of ARHGEF3 in controlling bone mineral density, platelet formation and differentiation, and Hirschsprung disease. In conclusion, we hypothesized that additional biochemical and functional studies are required to elucidate the detailed mechanism of ARHGEF3-related pathologies and therapeutics.