RESUMEN
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.
Asunto(s)
Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP , Membrana Celular , Inhibidores de Puntos de Control Inmunológico , Melanoma , Microambiente Tumoral , Microambiente Tumoral/inmunología , Animales , Humanos , Ratones , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Melanoma/genética , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Melanoma/patología , Melanoma/inmunología , Línea Celular Tumoral , Membrana Celular/metabolismo , Receptor de Interferón gamma , Receptores de Interferón/metabolismo , Receptores de Interferón/genética , Transporte de Proteínas , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Melanoma Experimental/genética , Ratones Endogámicos C57BL , FemeninoRESUMEN
ADP ribosylation factor-like GTPase 2 (Arl2) is crucial for controlling mitochondrial fusion and microtubule assembly in various organisms. Arl2 regulates the asymmetric division of neural stem cells in Drosophila via microtubule growth. However, the function of mammalian Arl2 during cortical development was unknown. Here, we demonstrate that mouse Arl2 plays a new role in corticogenesis via regulating microtubule growth, but not mitochondria functions. Arl2 knockdown (KD) leads to impaired proliferation of neural progenitor cells (NPCs) and neuronal migration. Arl2 KD in mouse NPCs significantly diminishes centrosomal microtubule growth and delocalization of centrosomal proteins Cdk5rap2 and γ-tubulin. Moreover, Arl2 physically associates with Cdk5rap2 by in silico prediction using AlphaFold multimer, which was validated by co-immunoprecipitation and proximity ligation assay. Remarkably, Cdk5rap2 overexpression significantly rescues the neurogenesis defects caused by Arl2 KD. Therefore, Arl2 plays an important role in mouse cortical development through microtubule growth via the centrosomal protein Cdk5rap2.
Asunto(s)
Proteínas de Ciclo Celular , Centrosoma , Microtúbulos , Proteínas del Tejido Nervioso , Células-Madre Neurales , Neurogénesis , Animales , Microtúbulos/metabolismo , Ratones , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Neurogénesis/genética , Células-Madre Neurales/metabolismo , Centrosoma/metabolismo , Proliferación Celular , Movimiento Celular , Corteza Cerebral/metabolismo , Corteza Cerebral/embriología , Corteza Cerebral/crecimiento & desarrollo , Tubulina (Proteína)/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genéticaRESUMEN
Small GTPases switch between GDP- and GTP-bound states during cell signaling. The ADP-ribosylation factor (ARF) family of small GTPases is involved in vesicle trafficking. Although evolutionarily well conserved, little is known about ARF and ARF-like GTPases in plants. We characterized biochemical properties and cellular localization of the essential small ARF-like GTPase TITAN 5 (TTN5; also known as HALLIMASCH, ARL2 and ARLC1) from Arabidopsis thaliana, and two TTN5 proteins with point mutants in conserved residues, TTN5T30N and TTN5Q70L, that were expected to be unable to perform nucleotide exchange and GTP hydrolysis, respectively. TTN5 exhibited very rapid intrinsic nucleotide exchange and remarkably low GTP hydrolysis activity, functioning as a non-classical small GTPase being likely present in a GTP-loaded active form. We analyzed signals from YFP-TTN5 and HA3-TTN5 by in situ immunolocalization in Arabidopsis seedlings and through use of a transient expression system. Colocalization with endomembrane markers and pharmacological treatments suggests that TTN5 can be present at the plasma membrane and that it dynamically associates with membranes of vesicles, Golgi stacks and multivesicular bodies. Although TTN5Q70L mirrored wild-type TTN5 behavior, the TTN5T30N mutant differed in some aspects. Hence, the unusual rapid nucleotide exchange activity of TTN5 is linked with its membrane dynamics, and TTN5 likely has a role in vesicle transport within the endomembrane system.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Guanosina Trifosfato/metabolismo , Membrana Celular/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Hidrólisis , Aparato de Golgi/metabolismoRESUMEN
Targeting ferroptosis for cancer therapy has slowed because of an incomplete understanding of ferroptosis mechanisms under specific pathological contexts such as tumorigenesis and cancer treatment. Here, we identify TRPML1-mediated lysosomal exocytosis as a potential anti-ferroptotic process through genome-wide CRISPR-Cas9 activation and kinase inhibitor library screening. AKT directly phosphorylated TRPML1 at Ser343 and inhibited K552 ubiquitination and proteasome degradation of TRPML1, thereby promoting TRPML1 binding to ARL8B to trigger lysosomal exocytosis. This boosted ferroptosis defense of AKT-hyperactivated cancer cells by reducing intracellular ferrous iron and enhancing membrane repair. Correlation analysis and functional analysis revealed that TRPML1-mediated ferroptosis resistance is a previously unrecognized feature of AKT-hyperactivated cancers and is necessary for AKT-driven tumorigenesis and cancer therapeutic resistance. TRPML1 inactivation or blockade of the interaction between TRPML1 and ARL8B inhibited AKT-driven tumorigenesis and cancer therapeutic resistance in vitro and in vivo by promoting ferroptosis. A synthetic peptide targeting TRPML1 inhibited AKT-driven tumorigenesis and enhanced the sensitivity of AKT-hyperactivated tumors to ferroptosis inducers, radiotherapy, and immunotherapy by boosting ferroptosis in vivo. Together, our findings identified TRPML1 as a therapeutic target in AKT-hyperactivated cancer.
Asunto(s)
Ferroptosis , Neoplasias , Proteínas Proto-Oncogénicas c-akt , Animales , Humanos , Ratones , Factores de Ribosilacion-ADP/metabolismo , Carcinogénesis/patología , Carcinogénesis/genética , Línea Celular Tumoral , Ferroptosis/efectos de los fármacos , Lisosomas/metabolismo , Neoplasias/patología , Neoplasias/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , UbiquitinaciónRESUMEN
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.
Asunto(s)
Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP , Actinas , Proteínas Activadoras de GTPasa , Animales , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/genética , Actinas/metabolismo , Ratones , Extensiones de la Superficie Celular/metabolismo , Humanos , Línea Celular , Guanosina Trifosfato/metabolismo , HidrólisisRESUMEN
The retinal features of Bardet-Biedl syndrome (BBS) are insufficiently characterized in Arab populations. This retrospective study investigated the retinal features and genotypes of BBS in Saudi patients managed at a single tertiary eye care center. Data analysis of the identified 46 individuals from 31 families included visual acuity (VA), systemic manifestations, multimodal retinal imaging, electroretinography (ERG), family pedigrees, and genotypes. Patients were classified to have cone-rod, rod-cone, or generalized photoreceptor dystrophy based on the pattern of macular involvement on the retinal imaging. Results showed that nyctalopia and subnormal VA were the most common symptoms with 76% having VA ≤ 20/200 at the last visit (age: 5-35). Systemic features included obesity 91%, polydactyly 56.5%, and severe cognitive impairment 33%. The predominant retinal phenotype was cone-rod dystrophy 75%, 10% had rod-cone dystrophy and 15% had generalized photoreceptor dystrophy. ERGs were undetectable in 95% of patients. Among the 31 probands, 61% had biallelic variants in BBSome complex genes, 32% in chaperonin complex genes, and 6% had biallelic variants in ARL6; including six previously unreported variants. Interfamilial and intrafamilial variabilities were noted, without a clear genotype-phenotype correlation. Most BBS patients had advanced retinopathy and were legally blind by early adulthood, indicating a narrow therapeutic window for rescue strategies.
Asunto(s)
Síndrome de Bardet-Biedl , Mutación , Humanos , Síndrome de Bardet-Biedl/genética , Masculino , Arabia Saudita/epidemiología , Femenino , Niño , Adolescente , Adulto , Preescolar , Adulto Joven , Linaje , Estudios Retrospectivos , Electrorretinografía , Fenotipo , Agudeza Visual , Retina/patología , Factores de Ribosilacion-ADPRESUMEN
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.
Asunto(s)
Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP , Oocitos , Animales , Oocitos/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Porcinos , Femenino , Meiosis/fisiología , Huso Acromático/metabolismo , Envejecimiento/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Vibrio vulnificus causes life-threatening wound and gastrointestinal infections, mediated primarily by the production of a Multifunctional-Autoprocessing Repeats-In-Toxin (MARTX) toxin. The most commonly present MARTX effector domain, the Makes Caterpillars Floppy-like (MCF) toxin, is a cysteine protease stimulated by host adenosine diphosphate (ADP) ribosylation factors (ARFs) to autoprocess. Here, we show processed MCF then binds and cleaves host Ras-related proteins in brain (Rab) guanosine triphosphatases within their C-terminal tails resulting in Rab degradation. We demonstrate MCF binds Rabs at the same interface occupied by ARFs. Moreover, we show MCF preferentially binds to ARF1 prior to autoprocessing and is active to cleave Rabs only subsequent to autoprocessing. We then use structure prediction algorithms to demonstrate that structural composition, rather than sequence, determines Rab target specificity. We further determine a crystal structure of aMCF as a swapped dimer, revealing an alternative conformation we suggest represents the open, activated state of MCF with reorganized active site residues. The cleavage of Rabs results in Rab1B dispersal within cells and loss of Rab1B density in the intestinal tissue of infected mice. Collectively, our work describes an extracellular bacterial mechanism whereby MCF is activated by ARFs and subsequently induces the degradation of another small host guanosine triphosphatase (GTPase), Rabs, to drive organelle damage, cell death, and promote pathogenesis of these rapidly fatal infections.
Asunto(s)
Toxinas Bacterianas , Vibrio vulnificus , Proteínas de Unión al GTP rab , Animales , Femenino , Humanos , Ratones , Factores de Ribosilacion-ADP/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/química , Células HEK293 , Ratones Endogámicos ICR , Proteolisis , Proteínas de Unión al GTP rab/metabolismo , Vibriosis/microbiología , Vibriosis/metabolismo , Vibrio vulnificus/metabolismo , Vibrio vulnificus/patogenicidadRESUMEN
Formation of transport vesicles requires the coordinate activity of the coating machinery that selects cargo into the nascent vesicle and the membrane bending machinery that imparts curvature to the forming bud. Vesicle coating at the trans-Golgi Network (TGN) involves AP1, GGA2 and clathrin, which are recruited to membranes by activated ARF GTPases. The ARF activation at the TGN is mediated by the BIG1 and BIG2 guanine nucleotide exchange factors (GEFs). Membrane deformation at the TGN has been shown to be mediated by lipid flippases, including ATP8A1, that moves phospholipids from the inner to the outer leaflet of the TGN membrane. We probed a possible coupling between the coating and deformation machineries by testing for an interaction between BIG1, BIG2 and ATP8A1, and by assessing whether such an interaction may influence coating efficiency. Herein, we document that BIG1 and BIG2 co-localize with ATP8A1 in both, static and highly mobile TGN elements, and that BIG1 and BIG2 bind ATP8A1. We show that the interaction involves the catalytic Sec7 domain of the GEFs and the cytosolic C-terminal tail of ATP8A1. Moreover, we report that the expression of ATP8A1, but not ATP8A1 lacking the GEF-binding cytosolic tail, increases the generation of activated ARFs at the TGN and increases the selective recruitment of AP1, GGA2 and clathrin to TGN membranes. This occurs without increasing BIG1 or BIG2 levels at the TGN, suggesting that the binding of the ATP8A1 flippase tail to the Sec7 domain of BIG1/BIG2 increases their catalytic activity. Our results support a model in which a flippase component of the deformation machinery impacts the activity of the GEF component of the coating machinery.
Asunto(s)
Factores de Ribosilacion-ADP , Factores de Intercambio de Guanina Nucleótido , Red trans-Golgi , Red trans-Golgi/metabolismo , Humanos , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Adenosina Trifosfatasas/metabolismo , Células HeLa , Unión Proteica , Proteínas de la Membrana , Proteínas de Transferencia de FosfolípidosRESUMEN
Guanine nucleotide-binding proteins of the ADP ribosylation factor (Arf) family and their activating proteins (Arf-GAPs) are essential for diverse biological processes. Here, two homologous Arf-GAPs, Age1 (AoAge1) and Age2 (AoAge2), were identified in the widespread nematode-trapping fungus Arthrobotrys oligospora. Our results demonstrated that AoAge1, especially AoAge2, played crucial roles in mycelial growth, sporulation, trap production, stress response, mitochondrial activity, DNA damage, endocytosis, reactive oxygen species production, and autophagy. Notably, transcriptome data revealed that approximately 62.7% of the genes were directly or indirectly regulated by AoAge2, and dysregulated genes in Aoage2 deletion were enriched in metabolism, ribosome biogenesis, secondary metabolite biosynthesis, and autophagy. Furthermore, Aoage2 inactivation caused a substantial reduction in several compounds compared to the wild-type strain. Based on these results, a regulatory network for AoAge1 and AoAge2 was proposed and verified using a yeast two-hybrid assay. Based on our findings, AoAge1 and AoAge2 are essential for vegetative growth and mycelial development. Specifically, AoAge2 is required for sporulation and trapping morphogenesis. Our results demonstrated the critical functions of AoAge1 and AoAge2 in mycelial growth, diverse cellular processes, and pathogenicity, offering deep insights into the functions and regulatory mechanisms of Arf-GAPs in nematode-trapping fungi.
Asunto(s)
Ascomicetos , Proteínas Fúngicas , Regulación Fúngica de la Expresión Génica , Metabolismo Secundario , Esporas Fúngicas , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/genética , Esporas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ascomicetos/genética , Ascomicetos/metabolismo , Ascomicetos/crecimiento & desarrollo , Especies Reactivas de Oxígeno/metabolismo , Autofagia , Micelio/crecimiento & desarrollo , Micelio/metabolismo , Micelio/genética , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Animales , Transcriptoma , Virulencia , Daño del ADN , Perfilación de la Expresión GénicaRESUMEN
Cholesterol 25-hydroxylase (CH25H), an enzyme involved in cholesterol metabolism, regulates inflammatory responses and lipid metabolism. However, its role in kidney disease is not known. The author found that CH25H transcript is expressed mostly in glomerular and peritubular endothelial cells and that its expression increased in human and mouse diabetic kidneys. Global deletion of Ch25h in Leprdb/db mice aggravated diabetic kidney disease (DKD), which is associated with increased endothelial cell apoptosis. Treatment of 25-hydroxycholesterol (25-HC), the product of CH25H, alleviated kidney injury in Leprdb/db mice. Mechanistically, 25-HC binds to GTP-binding protein ADP-ribosylation factor 4 (ARF4), an essential protein required for maintaining protein transport in the Golgi apparatus. Interestingly, ARF4's GTPase-activating protein ASAP1 is also predominantly expressed in endothelial cells and its expression increased in DKD. Suppression of ARF4 activity by deleting ARF4 or overexpressing ASAP1 results in endothelial cell death. These results indicate that 25-HC binds ARF4 to inhibit its interaction with ASAP1, and thereby resulting in enhanced ARF4 activity to confer renoprotection. Therefore, treatment of 25-HC improves kidney injury in DKD in part by restoring ARF4 activity to maintain endothelial cell survival. This study provides a novel mechanism and a potential new therapy for DKD.
Asunto(s)
Factores de Ribosilacion-ADP , Nefropatías Diabéticas , Esteroide Hidroxilasas , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/genética , Animales , Ratones , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Humanos , Esteroide Hidroxilasas/metabolismo , Esteroide Hidroxilasas/genética , Modelos Animales de Enfermedad , Masculino , Ratones Endogámicos C57BL , HidroxicolesterolesRESUMEN
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.
Asunto(s)
Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP , Antígeno CD24 , Resistencia a Antineoplásicos , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Humanos , Neoplasias Esofágicas/patología , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/tratamiento farmacológico , Antígeno CD24/metabolismo , Carcinoma de Células Escamosas de Esófago/patología , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/metabolismo , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Animales , Línea Celular Tumoral , Masculino , Femenino , Ratones , Sistema de Señalización de MAP Quinasas , Fluorouracilo/farmacología , Regulación Neoplásica de la Expresión Génica , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Pronóstico , Persona de Mediana Edad , Ratones DesnudosRESUMEN
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.
Asunto(s)
Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP , Endosomas , Factores de Intercambio de Guanina Nucleótido , Factor de Crecimiento Nervioso , Proyección Neuronal , Receptor trkA , Animales , Ratones , Ratas , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Endosomas/metabolismo , Ganglios Espinales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Ratones Noqueados , Factor de Crecimiento Nervioso/metabolismo , Células PC12 , Transporte de Proteínas , Receptor trkA/metabolismoRESUMEN
We present a case study of a patient exhibiting acquired microcephaly along with global developmental delay and drug-resistant epilepsy. Brain magnetic resonance imaging revealed distinctive features, including a Z-shaped morphology of the brainstem, volumetric reduction of white matter, diffuse thinning of the corpus callosum, and partial fusion of the cerebellar hemispheres at their most cranial portion. Whole-exome sequencing uncovered a pathogenic variant in the ARF3 gene c.200A>T, p.(Asp67Val). The neurodevelopmental disorder associated with the ARF3 gene is exceptionally rare, with only two previously documented cases in the literature. This disorder is characterized by global developmental delay and brain malformations, particularly affecting the white matter, cerebellum, and brainstem. It can also manifest as acquired microcephaly and epilepsy. These phenotypic characteristics align with Golgipathies, underscoring the significance of considering this group of conditions in relevant clinical contexts. In cases where a Z-shaped morphology of the brainstem is observed, ARF3-associated disorder should be included in the list of differential diagnoses.
Asunto(s)
Factores de Ribosilacion-ADP , Trastornos del Neurodesarrollo , Femenino , Humanos , Factores de Ribosilacion-ADP/genética , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/patología , Secuenciación del Exoma , Predisposición Genética a la Enfermedad , Imagen por Resonancia Magnética , Microcefalia/genética , Microcefalia/patología , Microcefalia/diagnóstico , Mutación/genética , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología , Trastornos del Neurodesarrollo/diagnóstico por imagen , Fenotipo , Sustancia Blanca/patología , Sustancia Blanca/diagnóstico por imagen , PreescolarRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Activadoras de GTPasa , Cuerpos Multivesiculares , Tetraspanina 30 , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Endosomas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Células HeLa , Cuerpos Multivesiculares/metabolismo , Transporte de Proteínas , Tetraspanina 30/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Normal receptor tyrosine kinases (RTKs) need to reach the plasma membrane (PM) for ligand-induced activation, whereas its cancer-causing mutants can be activated before reaching the PM in organelles, such as the Golgi/trans-Golgi network (TGN). Inhibitors of protein export from the endoplasmic reticulum (ER), such as brefeldin A (BFA) and 2-methylcoprophilinamide (M-COPA), can suppress the activation of mutant RTKs in cancer cells, suggesting that RTK mutants cannot initiate signaling in the ER. BFA and M-COPA block the function of ADP-ribosylation factors (ARFs) that play a crucial role in ER-Golgi protein trafficking. However, among ARF family proteins, the specific ARFs inhibited by BFA or M-COPA, that is, the ARFs involved in RTKs transport from the ER, remain unclear. In this study, we showed that M-COPA blocked the export of not only KIT but also PDGFRA/EGFR/MET RTKs from the ER. ER-retained RTKs could not fully transduce anti-apoptotic signals, thereby leading to cancer cell apoptosis. Moreover, a single knockdown of ARF1, ARF3, ARF4, ARF5, or ARF6 could not block ER export of RTKs, indicating that BFA/M-COPA treatment cannot be mimicked by the knockdown of only one ARF member. Interestingly, simultaneous transfection of ARF1, ARF4, and ARF5 siRNAs mirrored the effect of BFA/M-COPA treatment. Consistent with these results, in vitro pulldown assays showed that BFA/M-COPA blocked the function of ARF1, ARF4, and ARF5. Taken together, these results suggest that BFA/M-COPA targets at least ARF1, ARF4, and ARF5; in other words, RTKs require the simultaneous activation of ARF1, ARF4, and ARF5 for their ER export.
Asunto(s)
Factor 1 de Ribosilacion-ADP , Factores de Ribosilacion-ADP , Brefeldino A , Retículo Endoplásmico , Transporte de Proteínas , Humanos , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Retículo Endoplásmico/metabolismo , Factor 1 de Ribosilacion-ADP/metabolismo , Factor 1 de Ribosilacion-ADP/genética , Brefeldino A/farmacología , Transporte de Proteínas/efectos de los fármacos , Receptores ErbB/metabolismo , Receptores ErbB/genética , Células HeLaRESUMEN
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.
Asunto(s)
Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP , Apoptosis , Células Endoteliales , Proteínas Hemolisinas , Streptococcus suis , Streptococcus suis/patogenicidad , Streptococcus suis/metabolismo , Humanos , Animales , Apoptosis/efectos de los fármacos , Ratones , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/microbiología , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/metabolismo , Virulencia , Encéfalo/metabolismoRESUMEN
The ADP-ribosylation factors (ARFs) and ARF-like (ARL) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we used proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified â¼3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely, SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.
Asunto(s)
Factores de Ribosilacion-ADP , Fosfolipasa D , Transducción de Señal , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Humanos , Fosfolipasa D/metabolismo , Fosfolipasa D/genética , Células HEK293 , Animales , Nexinas de Clasificación/metabolismo , Nexinas de Clasificación/genética , Mapeo de Interacción de ProteínasRESUMEN
The ß-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the global COVID-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release, and pathogenesis. We developed a nondisruptive tagging strategy for SARS-CoV-2 E and find that, at steady state, it localizes to the Golgi and to lysosomes. We identify sequences in E, conserved across Coronaviridae, responsible for endoplasmic reticulum-to-Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify an ADP ribosylation factor 1/adaptor protein-1 (ARFRP1/AP-1)-dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across ß-coronaviruses, and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and, by developing a trans-complementation assay for SARS-CoV-2 structural proteins, that lysosomal delivery of E and its viroporin activity is necessary for efficient viral replication and release.