RESUMO
Tumour-host immune interactions lead to complex changes in the tumour microenvironment (TME), impacting progression, metastasis and response to therapy. While it is clear that cancer cells can have the capacity to alter immune landscapes, our understanding of this process is incomplete. Herein we show that endocytic trafficking at the plasma membrane, mediated by the small GTPase ARF6, enables melanoma cells to impose an immunosuppressive TME that accelerates tumour development. This ARF6-dependent TME is vulnerable to immune checkpoint blockade therapy (ICB) but in murine melanoma, loss of Arf6 causes resistance to ICB. Likewise, downregulation of ARF6 in patient tumours correlates with inferior overall survival after ICB. Mechanistically, these phenotypes are at least partially explained by ARF6-dependent recycling, which controls plasma membrane density of the interferon-gamma receptor. Collectively, our findings reveal the importance of endomembrane trafficking in outfitting tumour cells with the ability to shape their immune microenvironment and respond to immunotherapy.
Assuntos
Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Membrana Celular , Inibidores de Checkpoint Imunológico , Melanoma , Microambiente Tumoral , Microambiente Tumoral/imunologia , Animais , Humanos , Camundongos , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Melanoma/genética , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma/patologia , Melanoma/imunologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Receptor de Interferon gama , Receptores de Interferon/metabolismo , Receptores de Interferon/genética , Transporte Proteico , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Melanoma Experimental/genética , Camundongos Endogâmicos C57BL , FemininoRESUMO
Arf6 is a member of ADP-ribosylation factor (Arf) family, which is widely implicated in the regulation of multiple physiological processes including endocytic recycling, cytoskeletal organization, and membrane trafficking during mitosis. In this study, we investigated the potential relationship between Arf6 and aging-related oocyte quality, and its roles on organelle rearrangement and cytoskeleton dynamics in porcine oocytes. Arf6 expressed in porcine oocytes throughout meiotic maturation, and it decreased in aged oocytes. Disruption of Arf6 led to the failure of cumulus expansion and polar body extrusion. Further analysis indicated that Arf6 modulated ac-tubulin for meiotic spindle organization and microtubule stability. Besides, Arf6 regulated cofilin phosphorylation and fascin for actin assembly, which further affected spindle migration, indicating the roles of Arf6 on cytoskeleton dynamics. Moreover, the lack of Arf6 activity caused the dysfunction of Golgi and ER for protein synthesis and signal transduction. Mitochondrial dysfunction was also observed in Arf6-deficient porcine oocytes, which was supported by the increased ROS level and abnormal membrane potential. In conclusion, our results reported that insufficient Arf6 was related to aging-induced oocyte quality decline through spindle organization, actin assembly, and organelle rearrangement in porcine oocytes.
Assuntos
Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Oócitos , Animais , Oócitos/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Suínos , Feminino , Meiose/fisiologia , Fuso Acromático/metabolismo , Envelhecimento/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
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
Endosomal trafficking of TrkA is a critical process for nerve growth factor (NGF)-dependent neuronal cell survival and differentiation. The small GTPase ADP-ribosylation factor 6 (Arf6) is implicated in NGF-dependent processes in PC12 cells through endosomal trafficking and actin cytoskeleton reorganization. However, the regulatory mechanism for Arf6 in NGF signaling is largely unknown. In this study, we demonstrated that EFA6A, an Arf6-specific guanine nucleotide exchange factor, was abundantly expressed in PC12 cells and that knockdown of EFA6A significantly inhibited NGF-dependent Arf6 activation, TrkA recycling from early endosomes to the cell surface, prolonged ERK1/2 phosphorylation, and neurite outgrowth. We also demonstrated that EFA6A forms a protein complex with TrkA through its N-terminal region, thereby enhancing its catalytic activity for Arf6. Similarly, we demonstrated that EFA6A forms a protein complex with TrkA in cultured dorsal root ganglion (DRG) neurons. Furthermore, cultured DRG neurons from EFA6A knockout mice exhibited disturbed NGF-dependent TrkA trafficking compared with wild-type neurons. These findings provide the first evidence for EFA6A as a key regulator of NGF-dependent TrkA trafficking and signaling.
Assuntos
Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Endossomos , Fatores de Troca do Nucleotídeo Guanina , Fator de Crescimento Neural , Crescimento Neuronal , Receptor trkA , Animais , Camundongos , Ratos , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Endossomos/metabolismo , Gânglios Espinais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Camundongos Knockout , Fator de Crescimento Neural/metabolismo , Células PC12 , Transporte Proteico , Receptor trkA/metabolismoRESUMO
Increasing evidence suggests the importance of CD24 in tumor progression, but its role and mechanism in esophageal squamous cell carcinoma (ESCC) remain unclear. The present study aims to explore the potential of CD24 as a novel predictive biomarker in ESCC, as well as its mechanism and therapeutic implications in metastasis and 5-FU chemoresistance. By using tissue microarray and immunohistochemistry, we found that CD24 expression was higher in ESCC tumor tissues than paired non-tumor tissues, further indicating that CD24 was markedly associated with poor prognosis. CD24 significantly promoted metastasis and 5-FU chemoresistance in vitro and in vivo. Mechanistically, CD24 competes with GIT2 to bind to Arf6, and stabilizes Arf6-GTP to activate the subsequent ERK pathway, thus promoting cancer progression. In addition, a significant positive correlation between CD24 and p-ERK was observed in clinical ESCC tissues. In summary, this study not only reveals CD24 as a regulatory factor for Arf6 activity, but also uncovers CD24-Arf6-ERK signaling axis as a novel mechanism of ESCC progression. Our findings suggest CD24 as a promising biomarker and therapeutic target in ESCC.
Assuntos
Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Antígeno CD24 , Resistencia a Medicamentos Antineoplásicos , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Humanos , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/tratamento farmacológico , Antígeno CD24/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Animais , Linhagem Celular Tumoral , Masculino , Feminino , Camundongos , Sistema de Sinalização das MAP Quinases , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Prognóstico , Pessoa de Meia-Idade , Camundongos NusRESUMO
Streptococcus suis (S. suis) is a significant zoonotic microorganism that causes a severe illness in both pigs and humans and is characterized by severe meningitis and septicemia. Suilysin (SLY), which is secreted by S. suis, plays a crucial role as a virulence factor in the disease. To date, the interaction between SLY and host cells is not fully understood. In this study, we identified the interacting proteins between SLY and human brain microvascular endothelial cells (HBMECs) using the TurboID-mediated proximity labeling method. 251 unique proteins were identified in TurboID-SLY treated group, of which six plasma membrane proteins including ARF6, GRK6, EPB41L5, DSC1, TJP2, and PNN were identified. We found that the proteins capable of interacting with SLY are ARF6 and PNN. Subsequent investigations revealed that ARF6 substantially increased the invasive ability of S. suis in HBMECs. Furthermore, ARF6 promoted SLY-induced the activation of p38 MAPK signaling pathway in HBMECs. Moreover, ARF6 promoted the apoptosis in HBMECs through the activation of p38 MAPK signaling pathway induced by SLY. Finally, we confirmed that ARF6 could increase the virulence of SLY in C57BL/6 mice. These findings offer valuable insights that contribute to a deeper understanding of the pathogenic mechanism of SLY.
Assuntos
Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Apoptose , Células Endoteliais , Proteínas Hemolisinas , Streptococcus suis , Streptococcus suis/patogenicidade , Streptococcus suis/metabolismo , Humanos , Animais , Apoptose/efeitos dos fármacos , Camundongos , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/microbiologia , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/metabolismo , Virulência , Encéfalo/metabolismoRESUMO
BACKGROUND: Much of what we know about insulin resistance is based on studies from metabolically active tissues such as the liver, adipose tissue, and skeletal muscle. Emerging evidence suggests that the vascular endothelium plays a crucial role in systemic insulin resistance; however, the underlying mechanisms remain incompletely understood. Arf6 (ADP ribosylation factor 6) is a small GTPase that plays a critical role in endothelial cell function. Here, we tested the hypothesis that the deletion of endothelial Arf6 will result in systemic insulin resistance. METHODS: We used mouse models of constitutive endothelial cell-specific Arf6 deletion (Arf6f/- Tie2Cre+) and tamoxifen-inducible Arf6 knockout (Arf6f/f Cdh5CreER+). Endothelium-dependent vasodilation was assessed using pressure myography. Metabolic function was assessed using a battery of metabolic assessments including glucose and insulin tolerance tests and hyperinsulinemic-euglycemic clamps. We used a fluorescence microsphere-based technique to measure tissue blood flow. Skeletal muscle capillary density was assessed using intravital microscopy. RESULTS: Endothelial Arf6 deletion impaired insulin-stimulated vasodilation in white adipose tissue and skeletal muscle feed arteries. The impairment in vasodilation was primarily due to attenuated insulin-stimulated nitric oxide bioavailability but independent of altered acetylcholine-mediated or sodium nitroprusside-mediated vasodilation. Endothelial cell-specific deletion of Arf6 also resulted in systematic insulin resistance in normal chow-fed mice and glucose intolerance in high-fat diet-fed obese mice. The underlying mechanisms of glucose intolerance were reductions in insulin-stimulated blood flow and glucose uptake in the skeletal muscle and were independent of changes in capillary density or vascular permeability. CONCLUSIONS: Results from this study support the conclusion that endothelial Arf6 signaling is essential for maintaining insulin sensitivity. Reduced expression of endothelial Arf6 impairs insulin-mediated vasodilation and results in systemic insulin resistance. These results have therapeutic implications for diseases that are associated with endothelial cell dysfunction and insulin resistance such as diabetes.
Assuntos
Fator 6 de Ribosilação do ADP , Endotélio , Resistência à Insulina , Músculo Esquelético , Camundongos , Fator 6 de Ribosilação do ADP/genética , Fator 6 de Ribosilação do ADP/metabolismo , Endotélio/metabolismo , Camundongos Endogâmicos C57BL , Intolerância à Glucose , Tamoxifeno , Camundongos Knockout , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Obesidade/metabolismo , Obesidade/patologia , Glucose/metabolismo , Dieta Hiperlipídica , Camundongos Obesos , VasodilataçãoRESUMO
Rab35 (Ras-associated binding protein) is a small GTPase that regulates endosomal membrane trafficking and functions in cell polarity, cytokinesis, and growth factor signaling. Altered Rab35 function contributes to progression of glioblastoma, defects in primary cilia formation, and altered cytokinesis. Here, we report a pediatric patient with global developmental delay, hydrocephalus, a Dandy-Walker malformation, axial hypotonia with peripheral hypertonia, visual problems, and conductive hearing impairment. Exome sequencing identified a homozygous missense variant in the GTPase fold of RAB35 (c.80G>A; p.R27H) as the most likely candidate. Functional analysis of the R27H-Rab35 variant protein revealed enhanced interaction with its guanine-nucleotide exchange factor, DENND1A and decreased interaction with a known effector, MICAL1, indicating that the protein is in an inactive conformation. Cellular expression of the variant drives the activation of Arf6, a small GTPase under negative regulatory control of Rab35. Importantly, variant expression leads to delayed cytokinesis and altered length, number, and Arl13b composition of primary cilia, known factors in neurodevelopmental disease. Our findings provide evidence of altered Rab35 function as a causative factor of a neurodevelopmental disorder.
Assuntos
Mutação de Sentido Incorreto , Transtornos do Neurodesenvolvimento , Proteínas rab de Ligação ao GTP , Feminino , Humanos , Masculino , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Linhagem Celular , Cílios/metabolismo , Cílios/genética , Cílios/patologia , Citocinese/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mutação com Perda de Função , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/patologia , Linhagem , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Murine leukemia viruses (MuLVs) are simple retroviruses that cause several diseases in mice. Retroviruses encode three basic genes: gag, pol, and env. Gag is translated as a polyprotein and moves to assembly sites where viral particles are shaped by cleavage of poly-Gag. Viral release depends on the intracellular trafficking of viral proteins, which is determined by both viral and cellular factors. ADP-ribosylation factor 6 (Arf6) is a small GTPase that regulates vesicular trafficking and recycling of different types of cargo in cells. Arf6 also activates phospholipase D (PLD) and phosphatidylinositol-4-phosphate 5-kinase (PIP5K) and produces phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). We investigated how Arf6 affected MuLV release with a constitutively active form of Arf6, Arf6Q67L. Expression of Arf6Q67L impaired Gag release by accumulating Gag at PI(4,5)P2-enriched compartments in the cytoplasm. Treatment of the inhibitors for PLD and PIP5K impaired or recovered MuLV Gag release in the cells expressing GFP (control) and Arf6Q67L, implying that regulation of PI(4,5)P2 through PLD and PIP5K affected MuLV release. Interference with the phosphoinositide 3-kinases, mammalian target of rapamycin (mTOR) pathway, and vacuolar-type ATPase activities showed further impairment of Gag release from the cells expressing Arf6Q67L. In contrast, mTOR inhibition increased Gag release in the control cells. The proteasome inhibitors reduced viral release in the cells regardless of Arf6Q67L expression. These data outline the differences in MuLV release under the controlled and overactivated Arf6 conditions and provide new insight into pathways for MuLV release.
Assuntos
Fator 6 de Ribosilação do ADP , Vírus da Leucemia Murina , Proteínas Virais , Animais , Camundongos , Vírus da Leucemia Murina/fisiologia , Serina-Treonina Quinases TORRESUMO
Male germ cells are connected by intercellular bridges (ICBs) in a syncytium due to incomplete cytokinesis. Syncytium is thought to be important for synchronized germ cell development by interchange of cytoplasmic factors via ICBs. Mammalian ADP-ribosylation factor 6 (ARF6) is a small GTPase that is involved in many cellular mechanisms including but not limited to regulating cellular structure, motility, vesicle trafficking and cytokinesis. ARF6 localizes to ICBs in spermatogonia and spermatocytes in mice. Here we report that mice with global depletion of ARF6 in adulthood using Ubc-CreERT2 display no observable phenotypes but are male sterile. ARF6-deficient males display a progressive loss of germ cells, including LIN28A-expressing spermatogonia, and ultimately develop Sertoli-cell-only syndrome. Specifically, intercellular bridges are lost in ARF6-deficient testis. Furthermore, germ cell-specific inactivation using the Ddx4-CreERT2 results in the same testicular morphological phenotype, showing the germ cell-intrinsic requirement of ARF6. Therefore, ARF6 is essential for spermatogenesis in mice and this function is conserved from Drosophila to mammals.
Assuntos
Fator 6 de Ribosilação do ADP , Espermatogênese , Animais , Feminino , Masculino , Camundongos , Drosophila , Mamíferos , Espermatócitos , Espermatogênese/genética , Espermatogônias , TestículoRESUMO
ADP-ribosylation factor 6 (ARF6) is a small G protein with extensive functions, including regulation of cellular membrane transport and viral infection. Infectious bursal disease (IBD) is caused by infectious bursal disease virus (IBDV), which mainly invades the bursa of Fabricius and causes low immunity in poultry. Our study demonstrated that IBDV infection could promote the expression of ARF6; however, the underlying mechanism remains unclear. Herein, the function of ARF6 in IBDV infection was explored, and it was revealed that viral replication was significantly promoted by ARF6 overexpression and hampered by siRNA-mediated inhibition of ARF6. Using two site mutants of ARF6 (ARF6-T27N and ARF6-Q67L), we found that IBDV replication was repressed by ARF6-T27N, indicating that ARF6 promotes IBDV replication. Further exploration of its mechanism revealed that ARF6 affects the copy number of IBDVs entering cells. A clathrin inhibitor (pitstop 2) impeded the early replication of IBDV, even when ARF6 was overexpressed. These results indicated that ARF6 promotes viral replication by affecting the internalization of IBDV, which may involve clathrin-dependent endocytosis. Our findings improve the understanding of the processes governing IBDV infection and provide insights into its prevention and control.
Assuntos
Infecções por Birnaviridae , Vírus da Doença Infecciosa da Bursa , Doenças das Aves Domésticas , Animais , Clatrina/metabolismo , Fator 6 de Ribosilação do ADP , Internalização do Vírus , Endocitose , Replicação Viral , Galinhas , Infecções por Birnaviridae/veterinária , Bolsa de FabriciusRESUMO
BACKGROUND: Severe burns are devastating injuries with significant immune dysfunction and result in substantial mortality and morbidity due to sepsis induced organ failure. Acute lung injury is the most common type of organ injury in sepsis, however, the mechanisms of which are poorly understood and effective therapeutic measures are limited. This study is aimed to investigate the effect of a small Guanosine triphosphatase (GTPase), Adenosine diphosphate ribosylation factor 6 (ARF6), on burn sepsis induced lung injury, and discuss the possible mechanisms. METHODS: Burn sepsis was established in male C57BL/6 mice. Mice were anesthetised by intramuscular injection of ketamine and xylazine hydrochloride, then 30% TBSA full thickness burn followed by sub-eschar injection of lipopolysaccharide. Animals were treated with intraperitoneal injection of a small molecule inhibitor of ARF6: NAV-2729, or vehicle, right after the burn and sepsis stimuli were inflicted. Lung tissues were harvested for histopathological observation and the acute lung injury scores were calculated. Organ permeability, Vascular Endothelial Cadherin (VE-cadherin) expression, inflammatory cytokine levels and myeloperoxidase activity in lung tissues were detected. Rat pulmonary microvascular endothelial cells (PMVECs) were stimulated by burn sepsis serum with or without 10 µM NAV-2729. The ARF6 activation, VE-cadherin expression, inflammasome activity, adapter protein apoptosis speck-like protein containing a caspase recruiting domain (ASC) specks and cytokines secretion were determined. Student's t test was used for comparison between two groups. Multiple comparisons among groups were performed by using analysis of variance, with Tukey's test for the post hoc test. RESULTS: NAV-2729 treatment attenuated burn sepsis induced lung injury and promoted survival of burn septic mice by preserving VE-cadherin expression in endothelial cell adherent junction and limited vascular hyperpermeability in lung tissues. Moreover, inflammatory cytokine expression and inflammatory injury in lung tissues were alleviated. Mechanistically, NAV-2729 enhanced vascular integrity by inhibiting ARF6 activation and restoring VE-cadherin expression in PMVECs. In addition, NAV-2729 inhibited ARF6-dependent phagocytosis of ASC specks, thus preventing inflammation propagation mediated by cell-to-cell transmission of ASC specks. CONCLUSIONS: ARF6 inhibition preserved vascular integrity by restoring expression of VE-cadherin and suppressed the spread of inflammation by affecting phagocytosis of ASC specks, thus protected against sepsis induced lung injury and improve survival of burn septic animals. The findings of this study implied potential therapeutics by which ARF6 inhibition can protect lung function from septic induced lung injury and improve outcomes in burn sepsis.
Assuntos
Fator 6 de Ribosilação do ADP , Lesão Pulmonar Aguda , Queimaduras , Caderinas , Inflamassomos , Camundongos Endogâmicos C57BL , Sepse , Animais , Queimaduras/complicações , Queimaduras/metabolismo , Sepse/complicações , Sepse/metabolismo , Camundongos , Inflamassomos/metabolismo , Inflamassomos/efeitos dos fármacos , Caderinas/metabolismo , Masculino , Lesão Pulmonar Aguda/prevenção & controle , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/etiologia , Antígenos CD/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Peroxidase/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Ratos , Modelos Animais de Doenças , Citocinas/metabolismoRESUMO
IQSEC2 gene mutations are associated with epilepsy, autism, and intellectual disability. The primary function IQSEC2, mediated via its Sec 7 domain, is to act as a guanine nucleotide exchange factor for ARF6. We sought to develop a molecular model, which may explain the aberrant Sec 7 activity on ARF6 of different human IQSEC2 mutations. We integrated experimental data of IQSEC2 mutants with protein structure prediction by the RaptorX server combined with molecular modeling and molecular dynamics simulations. Normally, apocalmodulin (apoCM) binds to IQSEC2 resulting in its N-terminal fragment inhibiting access of its Sec 7 domain to ARF6. An increase in Ca2+ concentration destabilizes the interaction of IQSEC2 with apoCM and removes steric hindrance of Sec 7 binding with ARF6. Mutations at amino acid residue 350 of IQSEC2 result in loss of steric hindrance of Sec 7 binding with ARF6 leading to constitutive activation of ARF6 by Sec 7. On the other hand, a mutation at amino acid residue 359 of IQSEC2 results in constitutive hindrance of Sec 7 binding to ARF6 leading to the loss of the ability of IQSEC2 to activate ARF6. These studies provide a model for dysregulation of IQSEC2 Sec 7 activity by mutant IQSEC2 proteins.Communicated by Ramaswamy H. Sarma.
Assuntos
Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Humanos , Fatores de Ribosilação do ADP/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mutação , Modelos Moleculares , Aminoácidos/genéticaRESUMO
Circulating plasma extracellular vesicles (EVs) mostly originate from platelets and may promote organ dysfunction in sepsis. However, the role of platelet-derived EVs in sepsis-induced acute kidney injury (AKI) remains poorly understood. The present study extracted EVs from the supernatant of human platelets treated with phosphate buffer saline (PBS) or lipopolysaccharide (LPS). Then, we subjected PBS-EVs or LPS-EVs to cecal ligation and puncture (CLP) mice in vivo or LPS-stimulated renal tubular epithelial cells (RTECs) in vitro. Our results indicated that LPS-EVs aggravate septic AKI via promoting apoptosis, inflammation and oxidative stress. Further, ADP-ribosylation factor 6 (ARF6) was identified as a differential protein between PBS-EVs and LPS-EVs by quantitative proteomics analysis. Mechanistically, ARF6 activated ERK/Smad3/p53 signaling to exacerbate sepsis-induced AKI. LPS upregulated ARF6 in RTECs was dependent on TLR4/MyD88 pathway. Both genetically and pharmacologically inhibition of ARF6 attenuated septic AKI. Moreover, platelets were activated by TLR4 and its downstream mediator IKK controlled platelet secretion during sepsis. Inhibition of platelet secretion alleviated septic AKI. Collectively, our study demonstrated that platelet-derived EVs may be a therapeutic target in septic AKI.
Assuntos
Injúria Renal Aguda , Vesículas Extracelulares , Sepse , Camundongos , Humanos , Animais , Lipopolissacarídeos/toxicidade , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator 6 de Ribosilação do ADP , Injúria Renal Aguda/induzido quimicamente , Vesículas Extracelulares/metabolismo , Sepse/metabolismoRESUMO
Despite the "big data" on cancer from recent breakthroughs in high-throughput technology and the development of new therapeutic modalities, it remains unclear as to how intra-tumor heterogeneity and phenotypic plasticity created by various somatic abnormalities and epigenetic and metabolic adaptations orchestrate therapy resistance, immune evasiveness, and metastatic ability. Tumors are formed by various cells, including immune cells, cancer-associated fibroblasts, and endothelial cells, and their tumor microenvironment (TME) plays a crucial role in malignant tumor progression and responses to therapy. ADP-ribosylation factor 6 (ARF6) and AMAP1 are often overexpressed in cancers, which statistically correlates with poor outcomes. The ARF6-AMAP1 pathway promotes the intracellular dynamics and cell-surface expression of various proteins. This pathway is also a major target for KRAS/TP53 mutations to cooperatively promote malignancy in pancreatic ductal adenocarcinoma (PDAC), and is closely associated with immune evasion. Additionally, this pathway is important in angiogenesis, acidosis, and fibrosis associated with tumor malignancy in the TME, and its inhibition in PDAC cells results in therapeutic synergy with an anti-PD-1 antibody in vivo. Thus, the ARF6-based pathway affects the TME and the intrinsic function of tumors, leading to malignancy. Here, we discuss the potential mechanisms of this ARF6-based pathway in tumorigenesis, and novel therapeutic strategies.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Células Endoteliais/metabolismo , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/genética , Microambiente Tumoral , Neoplasias PancreáticasRESUMO
OBJECTIVE: Endocytosis is a process vital to angiogenesis and vascular homeostasis. In pathologies where supraphysiological growth factor signaling underlies disease etiology, such as in diabetic retinopathy and solid tumors, strategies to limit chronic growth factor signaling by way of blunting endocytic processes have been shown to have tremendous clinical value. ADP ribosylation factor 6 (Arf6) is a small GTPase that promotes the assembly of actin necessary for clathrin-mediated and clathrin-independent endocytosis. In its absence, growth factor signaling is greatly diminished, which has been shown to ameliorate pathological signaling input in diseased vasculature. However, it is less clear if there are bystander effects related to loss of Arf6 on angiogenic behaviors. Our goal was to provide an analysis of Arf6's function in angiogenic endothelium, focusing on its role in actin and endocytosis as well as sprouting morphogenesis. METHODS: Primary endothelial cells were cultured in both 2D and 3D environments. Here, endothelial cells were fixed and stained for various proteins or transfected with fluorescently-tagged constructs for live-cell imaging. RESULTS: We found that Arf6 localized to both filamentous actin and sites of endocytosis in two-dimensional culture. Loss of Arf6 distorted both apicobasal polarity and reduced the total cellular filamentous actin content, which may be the primary driver underlying gross sprouting dysmorphogenesis in its absence. CONCLUSIONS: Our findings highlight that endothelial Arf6 is a potent mediator of both actin regulation and endocytosis and is required for proper sprout formation.
Assuntos
Fator 6 de Ribosilação do ADP , Actinas , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Células Endoteliais/metabolismo , Endocitose/fisiologia , Clatrina/metabolismo , Peptídeos e Proteínas de Sinalização IntercelularRESUMO
Infection of the central nervous system caused by enterovirus 71 (EV71) remains the main cause of death in hand-foot-and-mouth disease. However, the mechanism responsible for how EV71 breaks through the blood-brain barrier to infect brain cells has yet to be elucidated. By performing a high-throughput small interfering RNA (siRNA) screening and validation, we found that the infection of human brain microvascular endothelial cells (HBMECs) by EV71 was independent of the endocytosis pathways mediated by caveolin, clathrin, and macropinocytosis but dependent on ADP-ribosylation factor 6 (ARF6), a small guanosinetriphosphate (GTP)-binding protein of the Ras superfamily. The specific siRNA targeting ARF6 markedly inhibited HBMECs susceptibility to EV71. EV71 infectivity was inhibited by NAV-2729, a specific inhibitor of ARF6, in a dose-dependent manner. The subcellular analysis demonstrated the co-localization of the endocytosed EV71 and ARF6, while knockdown of ARF6 with siRNA remarkably influenced EV71 endocytosis. By immunoprecipitation assays, we found a direct interaction of ARF6 with EV71 viral protein. Furthermore, ARF1, another small GTP-binding protein, was also found to participate in ARF6-mediated EV71 endocytosis. Murine experiments demonstrated that NAV-2729 significantly alleviated mortality caused by EV71 infection. Our study revealed a new pathway by which EV71 enters the HBMECs and provides new targets for drug development.
Assuntos
Fator 6 de Ribosilação do ADP , Enterovirus Humano A , Infecções por Enterovirus , Animais , Humanos , Camundongos , Fator 6 de Ribosilação do ADP/metabolismo , Encéfalo/metabolismo , Células Endoteliais , Enterovirus Humano A/genética , RNA Interferente Pequeno/genéticaRESUMO
BACKGROUND: Childhood asthma is a major global health concern. ADP-ribosylation factor 6 (ARF6) is a low-molecular-weight GTPase; however, its role in childhood asthma remains unclear. METHODS: Ovalbumin (OVA)-challenged neonatal mice and transforming growth factor-ß1 (TGF-ß1)-induced BEAS-2B cells were used as in vivo and in vitro models of childhood asthma, respectively. RESULTS: Upon OVA stimulation, ARF6 expression was upregulated in the lung tissue. Neonatal mice administered SehinH3 (an ARF6 inhibitor) exhibited improved pulmonary pathological injury, along with reduced inflammatory cell infiltration in the lungs and cytokine release in bronchial alveolar lavage fluid and serum (interleukin [IL]-3, IL-5, IL-13, IgE, and OVA-specific IgE). SehinH3 treatment restrained epithelial - mesenchymal transition (EMT) in the lungs of asthmatic mice, as evidenced by increased E-cadherin and decreased N-cadherin and α-smooth muscle actin expression. Different TGF-ß1 exposures to BEAS-2B cells induced a time- and dose-dependent increase in ARF6 expression in vitro. Upon TGF-ß1 stimulation, ARF6 knockdown repressed EMT and SehinH3 treatment caused similar results in BEAS-2B cells. The transcription factor E2F8 is involved in diverse biological functions and its increased expression was confirmed in vivo and in vitro. Dual-luciferase assays confirmed that E2F8 binds to the ARF6 promoter and promotes its transcriptional activity. In vitro results revealed that E2F8 silencing suppressed EMT, whereas rescue experiments showed that ARF6 overexpression partly reversed these phenomena. CONCLUSION: Our study showed that ARF6 is associated with childhood asthma progression and may be positively regulated by E2F8. These results provide insight into the pathogenesis and treatment of childhood asthma.
Assuntos
Asma , Fator de Crescimento Transformador beta1 , Camundongos , Animais , Fator de Crescimento Transformador beta1/metabolismo , Ovalbumina , Fator 6 de Ribosilação do ADP , Transição Epitelial-Mesenquimal , Asma/metabolismo , Inflamação , Imunoglobulina E , Fatores de Transcrição E2F/metabolismo , Camundongos Endogâmicos BALB C , Modelos Animais de DoençasRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerged beta-coronavirus that enter cells via two routes, direct fusion at the plasma membrane or endocytosis followed by fusion with the late endosome/lysosome. While the viral receptor, ACE2, multiple entry factors and the mechanism of fusion of the virus at the plasma membrane have been investigated extensively, viral entry via the endocytic pathway is less understood. By using a human hepatocarcinoma cell line, Huh-7, which is resistant to the antiviral action of the TMPRSS2 inhibitor camostat, we discovered that SARS-CoV-2 entry is not dependent on dynamin but on cholesterol. ADP-ribosylation factor 6 (ARF6) has been described as a host factor for SARS-CoV-2 replication and is involved in the entry and infection of several pathogenic viruses. Using CRISPR/Cas9 genetic deletion, a modest reduction in SARS-CoV-2 uptake and infection in Huh-7 was observed. In addition, pharmacological inhibition of ARF6 with the small molecule NAV-2729 showed a dose-dependent reduction of viral infection. Importantly, NAV-2729 also reduced SARS-CoV-2 viral loads in more physiological models of infection: Calu-3 cells and kidney organoids. This highlighted a role for ARF6 in multiple cell contexts. Together, these experiments point to ARF6 as a putative target to develop antiviral strategies against SARS-CoV-2.