RESUMO
The mammalian target of rapamycin complex 1 (mTORC1) integrates nutrients, growth factors, stress, and energy status to regulate cell growth and metabolism. Amino acids promote mTORC1 lysosomal localization and subsequent activation. However, the subcellular location or interacting proteins of mTORC1 under amino acid-deficient conditions is not completely understood. Here, we identify ADP-ribosylation factor GTPase-activating protein 1 (ArfGAP1) as a crucial regulator of mTORC1. ArfGAP1 interacts with mTORC1 in the absence of amino acids and inhibits mTORC1 lysosomal localization and activation. Mechanistically, the membrane curvature-sensing amphipathic lipid packing sensor (ALPS) motifs that bind to vesicle membranes are crucial for ArfGAP1 to interact with and regulate mTORC1 activity. Importantly, ArfGAP1 represses cell growth through mTORC1 and is an independent prognostic factor for the overall survival of pancreatic cancer patients. Our study identifies ArfGAP1 as a critical regulator of mTORC1 that functions by preventing the lysosomal transport and activation of mTORC1, with potential for cancer therapeutics.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Linhagem Celular , Feminino , Proteínas Ativadoras de GTPase/genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , PrognósticoRESUMO
Actin filament maintenance is critical for both normal cell homeostasis and events associated with malignant transformation. The ADP-ribosylation factor GTPase-activating protein ASAP1 regulates the dynamics of filamentous actin-based structures, including stress fibers, focal adhesions, and circular dorsal ruffles. Here, we have examined the molecular basis for ASAP1 association with actin. Using a combination of structural modeling, mutagenesis, and in vitro and cell-based assays, we identify a putative-binding interface between the N-Bin-Amphiphysin-Rvs (BAR) domain of ASAP1 and actin filaments. We found that neutralization of charges and charge reversal at positions 75, 76, and 79 of ASAP1 reduced the binding of ASAP1 BAR-pleckstrin homology tandem to actin filaments and abrogated actin bundle formation in vitro. In addition, overexpression of actin-binding defective ASAP1 BAR-pleckstrin homology [K75, K76, K79] mutants prevented cellular actin remodeling in U2OS cells. Exogenous expression of [K75E, K76E, K79E] mutant of full-length ASAP1 did not rescue the reduction of cellular actin fibers consequent to knockdown of endogenous ASAP1. Taken together, our results support the hypothesis that the lysine-rich cluster in the N-BAR domain of ASAP1 is important for regulating actin filament organization.
Assuntos
Citoesqueleto de Actina , Actinas , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Ativadoras de GTPase , Fatores de Ribosilação do ADP/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Lisina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
Breast cancer is a major global health burden with high morbidity and mortality rates. Previous studies have reported that increased expression of ASAP1 is associated with poor prognosis in various types of cancer. This study was conducted on 452 breast cancer patients who underwent surgery at Hanyang University Hospital, Seoul, South Korea. Data on clinicopathological characteristics including molecular pathologic markers were collected. Immunohistochemical staining of ASAP1 expression level were used to classify patients into high and low groups. In total, 452 cases low ASAP1 expression group was associated with significantly worse recurrence-free survival (p = 0.029). In ER-positive cases (n = 280), the low ASAP1 expression group was associated with significantly worse overall survival (p = 0.039) and recurrence-free survival (p = 0.029). In multivariate cox analysis, low ASAP1 expression was an independent significant predictor of poor recurrence-free survival in the overall patient group (hazard ratio = 2.566, p = 0.002) and ER-positive cases (hazard ratio = 4.046, p = 0.002). In the analysis of the TCGA dataset, the low-expression group of ASAP1 protein demonstrated a significantly poorer progression-free survival (p = 0.005). This study reports that low ASAP1 expression was associated with worse recurrence-free survival in invasive breast cancer.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/genética , Prognóstico , Hospitais Universitários , Análise Multivariada , Intervalo Livre de Progressão , Proteínas Adaptadoras de Transdução de SinalRESUMO
The Arf GTPase controls formation of the COPI vesicle coat. Recent structural models of COPI revealed the positioning of two Arf1 molecules in contrasting molecular environments. Each of these pockets for Arf1 is expected to also accommodate an Arf GTPase-activating protein (ArfGAP). Structural evidence and protein interactions observed between isolated domains indirectly suggest that each niche preferentially recruits one of the two ArfGAPs known to affect COPI, i.e. Gcs1/ArfGAP1 and Glo3/ArfGAP2/3, although only partial structures are available. The functional role of the unique non-catalytic domain of either ArfGAP has not been integrated into the current COPI structural model. Here, we delineate key differences in the consequences of triggering GTP hydrolysis through the activity of one versus the other ArfGAP. We demonstrate that Glo3/ArfGAP2/3 specifically triggers Arf1 GTP hydrolysis impinging on the stability of the COPI coat. We show that the Snf1 kinase complex, the yeast homologue of AMP-activated protein kinase (AMPK), phosphorylates the region of Glo3 that is crucial for this effect and, thereby, regulates its function in the COPI-vesicle cycle. Our results revise the model of ArfGAP function in the molecular context of COPI.This article has an associated First Person interview with the first author of the paper.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Complexo I de Proteína do Envoltório/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Complexo I de Proteína do Envoltório/genética , Proteínas Ativadoras de GTPase/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Approximately one-third of all eukaryotic proteins are delivered to their destination by trafficking within the endomembrane system. Such cargo proteins are incorporated into forming membrane vesicles on donor compartments and delivered to acceptor compartments by vesicle fusion. How cargo proteins are sorted into forming vesicles is still largely unknown. Here we review the roles of small GTPases of the ARF/SAR1 family, their regulators designated ARF guanine-nucleotide exchange factors (ARF-GEFs) and ARF GTPase-activating proteins (ARF-GAPs) as well as coat protein complexes during membrane vesicle formation. Although conserved across eukaryotes, these four functional groups of proteins display plant-specific modifications in composition, structure and function.
Assuntos
Proteínas do Capsídeo/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Transporte Proteico/fisiologia , Animais , Endocitose/fisiologia , Células Eucarióticas/metabolismo , HumanosRESUMO
Arf GAP with Src homology 3 domain, ankyrin repeat, and pleckstrin homology (PH) domain 1 (ASAP1) is a multidomain GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF)-type GTPases. ASAP1 affects integrin adhesions, the actin cytoskeleton, and invasion and metastasis of cancer cells. ASAP1's cellular function depends on its highly-regulated and robust ARF GAP activity, requiring both the PH and the ARF GAP domains of ASAP1, and is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2). The mechanistic basis of PIP2-stimulated GAP activity is incompletely understood. Here, we investigated whether PIP2 controls binding of the N-terminal extension of ARF1 to ASAP1's PH domain and thereby regulates its GAP activity. Using [Δ17]ARF1, lacking the N terminus, we found that PIP2 has little effect on ASAP1's activity. A soluble PIP2 analog, dioctanoyl-PIP2 (diC8PIP2), stimulated GAP activity on an N terminus-containing variant, [L8K]ARF1, but only marginally affected activity on [Δ17]ARF1. A peptide comprising residues 2-17 of ARF1 ([2-17]ARF1) inhibited GAP activity, and PIP2-dependently bound to a protein containing the PH domain and a 17-amino acid-long interdomain linker immediately N-terminal to the first ß-strand of the PH domain. Point mutations in either the linker or the C-terminal α-helix of the PH domain decreased [2-17]ARF1 binding and GAP activity. Mutations that reduced ARF1 N-terminal binding to the PH domain also reduced the effect of ASAP1 on cellular actin remodeling. Mutations in the ARF N terminus that reduced binding also reduced GAP activity. We conclude that PIP2 regulates binding of ASAP1's PH domain to the ARF1 N terminus, which may partially regulate GAP activity.
Assuntos
Fator 1 de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Fosfatidilinositol 4,5-Difosfato/genética , Fator 1 de Ribosilação do ADP/química , Fatores de Ribosilação do ADP/química , Actinas/química , Actinas/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Humanos , Neoplasias/genética , Fosfatidilinositol 4,5-Difosfato/química , Domínios de Homologia à Plecstrina/genética , Mutação Puntual/genética , Ligação Proteica/genéticaRESUMO
The coat protein complex I (COPI) allows the precise sorting of lipids and proteins between Golgi cisternae and retrieval from the Golgi to the ER. This essential role maintains the identity of the early secretory pathway and impinges on key cellular processes, such as protein quality control. In this Cell Science at a Glance and accompanying poster, we illustrate the different stages of COPI-coated vesicle formation and revisit decades of research in the context of recent advances in the elucidation of COPI coat structure. By calling attention to an array of questions that have remained unresolved, this review attempts to refocus the perspectives of the field.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Complexo I de Proteína do Envoltório/genética , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Complexo I de Proteína do Envoltório/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Humanos , Transporte Proteico/genéticaRESUMO
Small GTPases of the ADP-ribosylation factor (Arf) family and their activating proteins (Arf-GAPs) regulate mycelial development and pathogenicity in yeast and filamentous fungi; however, little is known about their roles in nematode-trapping (NT) fungi. In this study, an ortholog of Arf-GAP Glo3 (AoGlo3) in Saccharomyces cerevisiae was characterized in the NT fungus Arthrobotrys oligospora. Deletion of the Aoglo3 gene resulted in growth defects and an increase in hyphal septum. Meanwhile, the sporulation capacity of the ΔAoglo3 mutant was decreased by 98%, and 67.1-71.2% spores became gourd or claviform in shape (from obovoid), which was accompanied by a significant decrease in the spore germination rate. This reduced sporulation capacity correlated with the transcriptional repression of several sporulation-related genes including fluG, rodA, abaA, medA, and lreA. The ΔAoglo3 mutant was also sensitive to several chemical stressors such as Congo red, NaCl, and sorbitol. Additionally, AoGlo3 was found to be involved in endocytosis, and more myelin figures were observed in the ΔAoglo3 mutant than in the wild-type strain, which was consistent with the presence of more autophagosomes observed in the mutant. Importantly, AoGlo3 affected the production of mycelial traps and serine proteases for nematode predation. In summary, AoGlo3 is involved in the regulation of multiple cellular processes such as mycelial growth, conidiation, environmental adaption, endocytosis, and pathogenicity in A. oligospora.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Aldeído Oxirredutases/metabolismo , Ascomicetos , Nematoides/microbiologia , Animais , Ascomicetos/genética , Ascomicetos/patogenicidade , Ascomicetos/fisiologia , Ascomicetos/ultraestrutura , Autofagia , Endocitose , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Micélio/crescimento & desenvolvimento , Serina Proteases/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , VirulênciaRESUMO
The interaction of Mycobacterium tuberculosis (Mtb) with pulmonary epithelial cells is critical for early stages of bacillus colonization and during the progression of tuberculosis. Entry of Mtb into epithelial cells has been shown to depend on F-actin polymerization, though the molecular mechanisms are still unclear. Here, we demonstrate that mycobacterial uptake into epithelial cells requires rearrangements of the actin cytoskeleton, which are regulated by ADP-ribosylation factor 1 (Arf1) and phospholipase D1 (PLD1), and is dependent on the M3 muscarinic receptor (M3R). We show that this pathway is controlled by Arf GTPase-activating protein 1 (ArfGAP1), as its silencing has an impact on actin cytoskeleton reorganization leading to uncontrolled uptake and replication of Mtb. Furthermore, we provide evidence that this pathway is critical for mycobacterial entry, while the cellular infection with other pathogens, such as Shigella flexneri and Yersinia pseudotuberculosis, is not affected. Altogether, these results reveal how cortical actin plays the role of a barrier to prevent mycobacterial entry into epithelial cells and indicate a novel role for ArfGAP1 as a restriction factor of host-pathogen interactions.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/genética , Proteínas Ativadoras de GTPase/genética , Interações Hospedeiro-Patógeno , Mycobacterium tuberculosis/patogenicidade , Alvéolos Pulmonares/metabolismo , Células A549 , Fator 1 de Ribosilação do ADP/genética , Fator 1 de Ribosilação do ADP/metabolismo , Citoesqueleto de Actina/microbiologia , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica , Humanos , Mycobacterium tuberculosis/fisiologia , Fosfolipase D/genética , Fosfolipase D/metabolismo , Polimerização , Alvéolos Pulmonares/microbiologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Shigella flexneri/fisiologia , Transdução de Sinais , Especificidade da Espécie , Yersinia pseudotuberculosis/fisiologiaRESUMO
INTRODUCTION AND HYPOTHESIS: Severe mechanical injury or inadequate repair of the levator ani muscle (LAM) is a key contributor to the development of pelvic floor dysfunction (PFD). We explored the effects of mechanical stress on myoblasts and LAM at the cellular and animal level and the possible mechanism of PFD induced by mechanical trauma. METHODS: A C2C12 cell mechanical injury model was established with a four-point bending device, and a LAM injury mouse model was established via vaginal distention and distal traction, a common way of simulating the birth injury. The cells were divided into control, 1333 µ strain for 4-h cyclic mechanical strain (CMS), 1333 µ strain for 8-h CMS, and 5333 µ strain for 4-h CMS groups. Mice were divided into control and injury groups. After treatment, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS) levels, indicators of oxidative damage, cell apoptosis, muscle and cell morphology, cell differentiation, and expression of adenosine diphosphate (ADP)-ribosylation factor GTPase activating protein 3 (ArfGAP3) were detected. RESULTS: 5333 µ strain for 4-h CMS loading could induce myoblast injury with a reduction of ΔΨm, increased ROS levels, aggravation of oxidative damage-associated proteins NADPH oxidase 2 (NOX2) and xanthine oxidase (XO), and an increased apoptosis rate of C2C12 cells. At the same time, the injury CMS loading can promote the differentiation of myoblasts and increase the expression of ArfGAP3, a factor regulating intracellular transport. Mechanical trauma could also lead to the oxidative damage of LAM, indicated by 8-hydroxy-2'-deoxyguanosine(8-OHdG), NOX2 and XO protein accumulation, and increase the expression of ArfGAP3 in LAM. CONCLUSIONS: Oxidative stress caused by mechanical trauma induces dysfunction and damage repairing of LAM and C2C12 myoblast, and ArfGAP3 may promote the repairing process.
Assuntos
Proteínas Ativadoras de GTPase/genética , Distúrbios do Assoalho Pélvico , Diafragma da Pelve , Ferimentos e Lesões/patologia , Animais , Diferenciação Celular , Feminino , Camundongos , MioblastosRESUMO
The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins (inactivators) for the ADP-ribosylation factor (Arf) small GTP-binding proteins, and function to limit the activity of Arf proteins. The PIX proteins, α-PIX and ß-PIX (also known as ARHGEF6 and ARHGEF7, respectively), are guanine nucleotide exchange factors (activators) for the Rho family small GTP-binding protein family members Rac1 and Cdc42. Through their multi-domain structures, GIT and PIX proteins can also function as signaling scaffolds by binding to numerous protein partners. Importantly, the constitutive association of GIT and PIX proteins into oligomeric GIT-PIX complexes allows these two proteins to function together as subunits of a larger structure that coordinates two distinct small GTP-binding protein pathways and serves as multivalent scaffold for the partners of both constituent subunits. Studies have revealed the involvement of GIT and PIX proteins, and of the GIT-PIX complex, in numerous fundamental cellular processes through a wide variety of mechanisms, pathways and signaling partners. In this Commentary, we discuss recent findings in key physiological systems that exemplify current understanding of the function of this important regulatory complex. Further, we draw attention to gaps in crucial information that remain to be filled to allow a better understanding of the many roles of the GIT-PIX complex in health and disease.
Assuntos
Fator 1 de Ribosilação do ADP/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Proteínas Ativadoras de GTPase/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Humanos , Complexos Multiproteicos/genética , Ligação Proteica , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
In bacteria, certain shape-sensing proteins localize to differently curved membranes. During sporulation in Bacillus subtilis, the only convex (positively curved) surface in the cell is the forespore, an approximately spherical internal organelle. Previously, we demonstrated that SpoVM localizes to the forespore by preferentially adsorbing onto slightly convex membranes. Here, we used NMR and molecular dynamics simulations of SpoVM and a localization mutant (SpoVM(P9A)) to reveal that SpoVM's atypical amphipathic α-helix inserts deeply into the membrane and interacts extensively with acyl chains to sense packing differences in differently curved membranes. Based on binding to spherical supported lipid bilayers and Monte Carlo simulations, we hypothesize that SpoVM's membrane insertion, along with potential cooperative interactions with other SpoVM molecules in the lipid bilayer, drives its preferential localization onto slightly convex membranes. Such a mechanism, which is distinct from that used by high curvature-sensing proteins, may be widely conserved for the localization of proteins onto the surface of cellular organelles.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Estrutura Secundária de Proteína , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Bicamadas Lipídicas/metabolismo , Espectroscopia de Ressonância Magnética , Microscopia de Fluorescência , Simulação de Dinâmica Molecular , Método de Monte Carlo , Mutação , Ligação ProteicaRESUMO
Small GTP-binding proteins of the Ras superfamily play diverse roles in intracellular trafficking. Among them, the Rab, Arf, and Rho families function in successive steps of vesicle transport, in forming vesicles from donor membranes, directing vesicle trafficking toward target membranes and docking vesicles onto target membranes. These proteins act as molecular switches that are controlled by a cycle of GTP binding and hydrolysis regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). In this study we explored the role of GAPs in the regulation of the endocytic pathway using fluorescently labeled yeast mating pheromone α-factor. Among 25 non-essential GAP mutants, we found that deletion of the GLO3 gene, encoding Arf-GAP protein, caused defective internalization of fluorescently labeled α-factor. Quantitative analysis revealed that glo3Δ cells show defective α-factor binding to the cell surface. Interestingly, Ste2p, the α-factor receptor, was mis-localized from the plasma membrane to the vacuole in glo3Δ cells. Domain deletion mutants of Glo3p revealed that a GAP-independent function, as well as the GAP activity, of Glo3p is important for both α-factor binding and Ste2p localization at the cell surface. Additionally, we found that deletion of the GLO3 gene affects the size and number of Arf1p-residing Golgi compartments and causes a defect in transport from the TGN to the plasma membrane. Furthermore, we demonstrated that glo3Δ cells were defective in the late endosome-to-TGN transport pathway, but not in the early endosome-to-TGN transport pathway. These findings suggest novel roles for Arf-GAP Glo3p in endocytic recycling of cell surface proteins.
Assuntos
Endocitose , Proteínas Ativadoras de GTPase/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas Ativadoras de GTPase/genética , Complexo de Golgi/metabolismo , Transporte Proteico , Proteínas R-SNARE/metabolismo , Receptores de Fator de Acasalamento/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Saturated fatty acids (SFA) have been reported to alter organelle integrity and function in many cell types, including muscle and pancreatic ß-cells, adipocytes, hepatocytes and cardiomyocytes. SFA accumulation results in increased amounts of ceramides/sphingolipids and saturated phospholipids (PL). In this study, using a yeast-based model that recapitulates most of the trademarks of SFA-induced lipotoxicity in mammalian cells, we demonstrate that these lipid species act at different levels of the secretory pathway. Ceramides mostly appear to modulate the induction of the unfolded protein response and the transcription of nutrient transporters destined to the cell surface. On the other hand, saturated PL, by altering membrane properties, directly impact vesicular budding at later steps in the secretory pathway, i.e. at the trans-Golgi Network level. They appear to do so by increasing lipid order within intracellular membranes which, in turn, alters the recruitment of loose lipid packing-sensing proteins, required for optimal budding, to nascent vesicles. We propose that this latter general mechanism could account for the well-documented deleterious impacts of fatty acids on the last steps of the secretory pathway in several cell types.
Assuntos
Membrana Celular/metabolismo , Ácidos Graxos/metabolismo , Saccharomyces cerevisiae/metabolismo , Via Secretória , Ceramidas/metabolismo , Fosfolipídeos/metabolismo , Vesículas Transportadoras/metabolismo , Resposta a Proteínas não Dobradas , Rede trans-Golgi/metabolismoRESUMO
Ribbon synapses are tonically active synapses in the retina and inner ear with intense vesicle traffic. How this traffic is organized and regulated is still unknown. Synaptic ribbons, large presynaptic structures associated with numerous synaptic vesicles, appear to be essential for this process. The base of the synaptic ribbon is anchored at the active zone and is a hotspot of exocytosis. The synaptic ribbon complex is also important for vesicle replenishment. RIBEYE is a unique and major component of synaptic ribbons. It consists of a unique A-domain and an NAD(H)-binding, C-terminal B-domain. In the present study, we show that the Arf-GTPase activating protein-3 (ArfGAP3), a well characterized regulator of vesicle formation at the Golgi apparatus, is also a component of the synaptic ribbon complex in photoreceptor synapses of the mouse retina and interacts with RIBEYE as shown by multiple, independent approaches. ArfGAP3 binds to RIBEYE(B)-domain in an NAD(H)-dependent manner. The interaction is redox sensitive because NADH is more efficient than the oxidized NAD(+) in promoting ArfGAP3-RIBEYE interaction. RIBEYE competes with the GTP-binding protein Arf1 for binding to ArfGAP3. Thus, binding of RIBEYE(B) to ArfGAP3 could prevent inactivation of Arf1 by ArfGAP3 and provides the synaptic ribbon with the possibility to control Arf1 function. The interaction is relevant for endocytic vesicle trafficking because overexpression of ArfGAP3 in photoreceptors strongly inhibited endocytotic uptake of FM1-43.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endocitose , Proteínas Ativadoras de GTPase/metabolismo , NAD/metabolismo , Fosfoproteínas/metabolismo , Células Fotorreceptoras/metabolismo , Sinapses/metabolismo , Fator 1 de Ribosilação do ADP/metabolismo , Oxirredutases do Álcool , Animais , Células COS , Bovinos , Chlorocebus aethiops , Proteínas Correpressoras , Proteínas de Ligação a DNA/genética , Proteínas Ativadoras de GTPase/genética , Camundongos , Oxirredução , Fosfoproteínas/genética , Células Fotorreceptoras/fisiologia , Ligação Proteica , Sinapses/fisiologiaRESUMO
Membrane protrusions are fundamental to cellular functions like migration, adhesion, and communication and depend upon dynamic reorganization of the cytoskeleton. GAP-dependent GTP hydrolysis of Arf proteins regulates actin-dependent membrane remodeling. Here, we show that dAsap regulates membrane protrusions in S2R+ cells by a mechanism that critically relies on its ArfGAP domain and relocalization of actin regulators, SCAR, and Ena. While our data reinforce the preference of dAsap for Arf1 GTP hydrolysis in vitro, we demonstrate that induction of membrane protrusions in S2R+ cells depends on Arf6 inactivation. This study furthers our understanding of how dAsap-dependent GTP hydrolysis maintains a balance between active and inactive states of Arf6 to regulate cell shape.
Assuntos
Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Actinas , Proteínas Ativadoras de GTPase , Animais , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/genética , Actinas/metabolismo , Camundongos , Extensões da Superfície Celular/metabolismo , Humanos , Linhagem Celular , Guanosina Trifosfato/metabolismo , HidróliseRESUMO
Arf GTPase-activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors. ArfGAPs are critical for cargo sorting in the Golgi-to-ER traffic. However, the role of ArfGAPs in sorting into intralumenal vesicles (ILVs) in multivesicular bodies (MVBs) in post-Golgi traffic remains unclear. Exosomes are extracellular vesicles (EVs) of endosomal origin. CD63 is an EV marker. CD63 is enriched ILVs in MVBs of cells. However, the secretion of CD63 positive EVs has not been consistent with the data on CD63 localization in MVBs, and how CD63-containing EVs are formed is yet to be understood. To elucidate the mechanism of CD63 transport to ILVs, we focused on CD63 localization in MVBs and searched for the ArfGAPs involved in CD63 localization. We observed that ADAP1 and ARAP1 depletion inhibited CD63 localization to enlarged endosomes after Rab5Q79L overexpression. We tested epidermal growth factor (EGF) and CD9 localization in MVBs. We observed that ADAP1 and ARAP1 depletion inhibited CD9 localization in enlarged endosomes but not EGF. Our results indicate ADAP1 and ARAP1, regulate incorporation of CD63 and CD9, but not EGF, in overlapped and different MVBs. Our work will contribute to distinguish heterogenous ILVs and exosomes by ArfGAPs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Ativadoras de GTPase , Corpos Multivesiculares , Tetraspanina 30 , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Endossomos/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Células HeLa , Corpos Multivesiculares/metabolismo , Transporte Proteico , Tetraspanina 30/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismoRESUMO
Floral organ shedding is a cell separation event preceded by cell-wall loosening and generally accompanied by cell expansion. Mutations in NEVERSHED (NEV) or INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) block floral organ abscission in Arabidopsis thaliana. NEV encodes an ADP-ribosylation factor GTPase-activating protein, and cells of nev mutant flowers display membrane-trafficking defects. IDA encodes a secreted peptide that signals through the receptor-like kinases HAESA (HAE) and HAESA-LIKE2 (HSL2). Analyses of single and double mutants revealed unique features of the nev and ida phenotypes. Cell-wall loosening was delayed in ida flowers. In contrast, nev and nev ida mutants displayed ectopic enlargement of abscission zone (AZ) cells, indicating that cell expansion alone is not sufficient to trigger organ loss. These results suggest that NEV initially prevents precocious cell expansion but is later integral for cell separation. IDA is involved primarily in the final cell separation step. A mutation in KNOTTED-LIKE FROM ARABIDOPSIS THALIANA1 (KNAT1), a suppressor of the ida mutant, could not rescue the abscission defects of nev mutant flowers, indicating that NEV-dependent activity downstream of KNAT1 is required. Transcriptional profiling of mutant AZs identified gene clusters regulated by IDA-HAE/HSL2. Several genes were more strongly downregulated in nev-7 compared with ida and hae hsl2 mutants, consistent with the rapid inhibition of organ loosening in nev mutants, and the overlapping roles of NEV and IDA in cell separation. A model of the crosstalk between the IDA signalling pathway and NEV-mediated membrane traffic during floral organ abscission is presented.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica de Plantas , Transdução de Sinais , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proliferação de Células , Parede Celular/metabolismo , Regulação para Baixo , Proteínas Ativadoras de GTPase/metabolismo , Perfilação da Expressão Gênica , Inflorescência/anatomia & histologia , Inflorescência/genética , Inflorescência/fisiologia , Modelos Biológicos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/genética , Peptídeos/metabolismo , Fenótipo , Plantas Geneticamente Modificadas , Transporte ProteicoRESUMO
Skeletal muscle injuries are common, and damaged myofibers are repaired through proliferation and differentiation of muscle satellite cells. GLUT4 is enriched in GLUT4 storage vesicles (GSVs) and plays a crucial role in the maintenance of muscle function. ArfGAP3 regulates the vesicle transport especially for COPI coat assembly, but its effects on GSVs and the repair process after skeletal muscle injury remains unclear. In this study, datasets related to skeletal muscle injury and myoblast differentiation GSE469, GSE5413, GSE45577 and GSE108040 were retrieved through the GEO database and the expression of heptameric coat protein complex I (COPI) and Golgi vesicle transport-related genes in various datasets, as well as the expression correlation between ArfGAP2, ArfGAP3 and COPI-related genes COPA, COPB1, COPB2, COPE, COPZ1, COPZ2 were analyzed. The results suggested that ArfGAP3 was expressed in the process of repair after skeletal muscle injury and myoblast differentiation and that ArfGAP3 was positively correlated with COPI-related genes. In vitro experimental results showed that ArfGAP3 was colocalized with COPA, COPB, COPG protein, and GLUT4 in C2C12 myoblasts. After the downregulation of ArfGAP3 expression, intracellular vesicle transport, and glucose uptake were blocked, the proliferation of myoblasts under low glucose culture conditions was impaired, the proportion of apoptosis increased, and myotube differentiation was impaired. In summary, ArfGAP3 regulates COPI-associated vesicle and GSVs transport and affects the proliferation and differentiation ability of myoblasts by influencing glucose uptake, thereby modulating the repair process after skeletal muscle injury.
Assuntos
Complexo I de Proteína do Envoltório , Proteínas Ativadoras de GTPase , Mioblastos , Transporte Biológico , Diferenciação Celular , Complexo I de Proteína do Envoltório/genética , Complexo I de Proteína do Envoltório/metabolismo , Glucose/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Proteínas Ativadoras de GTPase/metabolismoRESUMO
Compelling evidence indicates that defects in nucleocytoplasmic transport contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS). In particular, hexanucleotide (G4C2) repeat expansions in C9orf72, the most common cause of genetic ALS, have a widespread impact on the transport machinery that regulates the nucleocytoplasmic distribution of proteins and RNAs. We previously reported that the expression of G4C2 hexanucleotide repeats in cultured human and mouse cells caused a marked accumulation of poly(A) mRNAs in the cell nuclei. To further characterize the process, we set out to systematically identify the specific mRNAs that are altered in their nucleocytoplasmic distribution in the presence of C9orf72-ALS RNA repeats. Interestingly, pathway analysis showed that the mRNAs involved in membrane trafficking are particularly enriched among the identified mRNAs. Most importantly, functional studies in cultured cells and Drosophila indicated that C9orf72 toxic species affect the membrane trafficking route regulated by ADP-Ribosylation Factor 1 GTPase Activating Protein (ArfGAP-1), which exerts its GTPase-activating function on the small GTPase ADP-ribosylation factor 1 to dissociate coat proteins from Golgi-derived vesicles. We demonstrate that the function of ArfGAP-1 is specifically affected by expanded C9orf72 RNA repeats, as well as by C9orf72-related dipeptide repeat proteins (C9-DPRs), indicating the retrograde Golgi-to-ER vesicle-mediated transport as a target of C9orf72 toxicity.