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1.
FASEB J ; 38(13): e23772, 2024 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-38963337

RESUMEN

Ovarian cancer is one of the most common gynecologic malignancies that has a poor prognosis. THUMPD3-AS1 is an oncogenic long noncoding RNA (lncRNA) in several cancers. Moreover, miR-320d is downregulated and inhibited proliferation in ovarian cancer cells, whereas ARF1 was upregulated and promoted the malignant progression in epithelial ovarian cancer. Nevertheless, the role of THUMPD3-AS1 in ovarian cancer and the underlying mechanism has yet to be elucidated. Human normal ovarian epithelial cells (IOSE80) and ovarian cancer cell lines (CAVO3, A2780, SKOV3, OVCAR3, and HEY) were adopted for in vitro experiments. The functional roles of THUMPD3-AS1 in cell viability and apoptosis were determined using CCK-8, flow cytometry, and TUNEL assays. Western blot was performed to assess the protein levels of ARF1, Bax, Bcl-2, and caspase 3, whereas RT-qPCR was applied to measure ARF1 mRNA, THUMPD3-AS1, and miR-320d levels. The targeting relationship between miR-320d and THUMPD3-AS1 or ARF1 was validated with dual luciferase assay. THUMPD3-AS1 and ARF1 were highly expressed in ovarian cancer cells, whereas miR-320d level was lowly expressed. THUMPD3-AS1 knockdown was able to repress cell viability and accelerate apoptosis of OVCAR3 and SKOV3 cells. Also, THUMPD3-AS1 acted as a sponge of miR-320d, preventing the degradation of ARF1. MiR-320d downregulation reversed the tumor suppressive function induced by THUMPD3-AS1 depletion. Additionally, miR-320d overexpression inhibited ovarian cancer cell viability and accelerated apoptosis, which was overturned by overexpression of ARF1. THUMPD3-AS1 inhibited ovarian cancer cell apoptosis by modulation of miR-320d/ARF1 axis. The discoveries might provide a prospective target for ovarian cancer treatment.


Asunto(s)
Factor 1 de Ribosilacion-ADP , Apoptosis , Regulación Neoplásica de la Expresión Génica , MicroARNs , Neoplasias Ováricas , ARN Largo no Codificante , Femenino , Humanos , Factor 1 de Ribosilacion-ADP/metabolismo , Factor 1 de Ribosilacion-ADP/genética , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN sin Sentido/genética
2.
Plant Sci ; 347: 112194, 2024 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-39009307

RESUMEN

Revealing the effector-host molecular interactions is crucial for understanding the host immunity against Plasmopara viticola and devising innovative disease management strategies. As a pathogenic oomycete causing grapevine downy mildew, Plasmopara viticola employs various effectors to manipulate the defense systems of host plants. One of these P. viticola derived effectors is necrosis- and ethylene-inducing peptide 1 (Nep1) -like protein (PvNLP7), which has been known to elicit cell death and immune responses in plants. However, the underlying molecular mechanisms remain obscure, prompting the focus of this study. Through yeast two-hybrid screening, we have identified the Vitis rotundifolia ADP-ribosylation factor (VrARF1) as a host interactor of PvNLP7. This interaction is corroborated through bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays. Heterologous expression of VrARF1 in Nicotiana benthamiana verifies its accumulation in both the cytoplasm and nucleus, and induction of cell death. Moreover, the VrARF1 gene is strongly induced during early P. viticola infection and upon PvNLP7 transient expression. Overexpression of the VrARF1 gene in grapevine and N. benthamiana enhances resistance to P. viticola and Phytophthora capsici, respectively, via induction of defense related genes PR1 and PR2. Conversely, virus-induced gene silencing (VIGS) of NbARF1 in N. benthamiana, homologous to VrARF1, markedly attenuates PvNLP7-triggered cell death and reduces the expression of four PTI marker genes (PTI5, Acre31, WRKY7 and Cyp71D20) and two defense related genes (PR1 and PR2), rendering plants transiently transformed with PvNLP7 more susceptible to oomycete P. capsici. These findings highlight the role of ARF1 in mediating PvNLP7-induced immunity and indicate its potential as a target for engineering disease-resistant transgenic plants against oomycete pathogens.


Asunto(s)
Factor 1 de Ribosilacion-ADP , Nicotiana , Oomicetos , Enfermedades de las Plantas , Inmunidad de la Planta , Proteínas de Plantas , Vitis , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/inmunología , Nicotiana/genética , Nicotiana/microbiología , Nicotiana/inmunología , Nicotiana/metabolismo , Oomicetos/fisiología , Vitis/genética , Vitis/microbiología , Vitis/metabolismo , Vitis/inmunología , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Factor 1 de Ribosilacion-ADP/genética , Regulación de la Expresión Génica de las Plantas , Interacciones Huésped-Patógeno
3.
J Virol ; 98(7): e0081324, 2024 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-38904364

RESUMEN

Enteroviruses are single-stranded, positive-sense RNA viruses causing endoplasmic reticulum (ER) stress to induce or modulate downstream signaling pathways known as the unfolded protein responses (UPR). However, viral and host factors involved in the UPR related to viral pathogenesis remain unclear. In the present study, we aimed to identify the major regulator of enterovirus-induced UPR and elucidate the underlying molecular mechanisms. We showed that host Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1), which supports enteroviruses replication, was a major regulator of the UPR caused by infection with enteroviruses. In addition, we found that severe UPR was induced by the expression of 3A proteins encoded in human pathogenic enteroviruses, such as enterovirus A71, coxsackievirus B3, poliovirus, and enterovirus D68. The N-terminal-conserved residues of 3A protein interact with the GBF1 and induce UPR through inhibition of ADP-ribosylation factor 1 (ARF1) activation via GBF1 sequestration. Remodeling and expansion of ER and accumulation of ER-resident proteins were observed in cells infected with enteroviruses. Finally, 3A induced apoptosis in cells infected with enteroviruses via activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/C/EBP homologous protein (CHOP) pathway of UPR. Pharmaceutical inhibition of PERK suppressed the cell death caused by infection with enteroviruses, suggesting the UPR pathway is a therapeutic target for treating diseases caused by infection with enteroviruses.IMPORTANCEInfection caused by several plus-stranded RNA viruses leads to dysregulated ER homeostasis in the host cells. The mechanisms underlying the disruption and impairment of ER homeostasis and its significance in pathogenesis upon enteroviral infection remain unclear. Our findings suggested that the 3A protein encoded in human pathogenic enteroviruses disrupts ER homeostasis by interacting with GBF1, a major regulator of UPR. Enterovirus-mediated infections drive ER into pathogenic conditions, where ER-resident proteins are accumulated. Furthermore, in such scenarios, the PERK/CHOP signaling pathway induced by an unresolved imbalance of ER homeostasis essentially drives apoptosis. Therefore, elucidating the mechanisms underlying the virus-induced disruption of ER homeostasis might be a potential target to mitigate the pathogenesis of enteroviruses.


Asunto(s)
Estrés del Retículo Endoplásmico , Retículo Endoplásmico , Factores de Intercambio de Guanina Nucleótido , Homeostasis , Respuesta de Proteína Desplegada , Humanos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Infecciones por Enterovirus/virología , Infecciones por Enterovirus/metabolismo , Apoptosis , Enterovirus/fisiología , Enterovirus/metabolismo , Células HeLa , Replicación Viral , Factor 1 de Ribosilacion-ADP/metabolismo , Factor 1 de Ribosilacion-ADP/genética , Células HEK293 , Interacciones Huésped-Patógeno , Transducción de Señal , eIF-2 Quinasa/metabolismo
4.
PLoS One ; 19(4): e0295103, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38574162

RESUMEN

The ADP-ribosylation factors (Arfs) constitute a family of small GTPases within the Ras superfamily, with a distinguishing structural feature of a hypervariable N-terminal extension of the G domain modified with myristate. Arf proteins, including Arf1, have roles in membrane trafficking and cytoskeletal dynamics. While screening for Arf1:small molecule co-crystals, we serendipitously solved the crystal structure of the non-myristoylated engineered mutation [L8K]Arf1 in complex with a GDP analogue. Like wild-type (WT) non-myristoylated Arf1•GDP, we observed that [L8K]Arf1 exhibited an N-terminal helix that occludes the hydrophobic cavity that is occupied by the myristoyl group in the GDP-bound state of the native protein. However, the helices were offset from one another due to the L8K mutation, with a significant change in position of the hinge region connecting the N-terminus to the G domain. Hypothesizing that the observed effects on behavior of the N-terminus affects interaction with regulatory proteins, we mutated two hydrophobic residues to examine the role of the N-terminal extension for interaction with guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs. Different than previous studies, all mutations were examined in the context of myristoylated Arf. Mutations had little or no effect on spontaneous or GEF-catalyzed guanine nucleotide exchange but did affect interaction with GAPs. [F13A]myrArf1 was less than 1/2500, 1/1500, and 1/200 efficient as substrate for the GAPs ASAP1, ARAP1 and AGAP1; however, [L8A/F13A]myrArf1 was similar to WT myrArf1. Using molecular dynamics simulations, the effect of the mutations on forming alpha helices adjacent to a membrane surface was examined, yet no differences were detected. The results indicate that lipid modifications of GTPases and consequent anchoring to a membrane influences protein function beyond simple membrane localization. Hypothetical mechanisms are discussed.


Asunto(s)
Proteínas Activadoras de GTPasa , Miristatos , Proteínas Activadoras de GTPasa/metabolismo , Mutación Puntual , Ácido Mirístico , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo
5.
J Biol Chem ; 300(6): 107327, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38679330

RESUMEN

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 HeLa
6.
Adv Sci (Weinh) ; 11(4): e2303009, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38014604

RESUMEN

ADP-ribosylation factor 1 (Arf1) is a small GTPase belonging to the Arf family. As a molecular switch, Arf1 is found to regulate retrograde and intra-Golgi transport, plasma membrane signaling, and organelle function during mitosis. This study aimed to explore the noncanonical roles of Arf1 in cell cycle regulation and cytoskeleton dynamics in meiosis with a mouse oocyte model. Arf1 accumulated in microtubules during oocyte meiosis, and the depletion of Arf1 led to the failure of polar body extrusion. Unlike mitosis, it finds that Arf1 affected Myt1 activity for cyclin B1/CDK1-based G2/M transition, which disturbed oocyte meiotic resumption. Besides, Arf1 modulated GM130 for the dynamic changes in the Golgi apparatus and Rab35-based vesicle transport during meiosis. Moreover, Arf1 is associated with Ran GTPase for TPX2 expression, further regulating the Aurora A-polo-like kinase 1 pathway for meiotic spindle assembly and microtubule stability in oocytes. Further, exogenous Arf1 mRNA supplementation can significantly rescue these defects. In conclusion, results reported the noncanonical functions of Arf1 in G2/M transition and meiotic spindle organization in mouse oocytes.


Asunto(s)
Factor 1 de Ribosilacion-ADP , Huso Acromático , Ratones , Animales , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Huso Acromático/metabolismo , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Meiosis , Oocitos/metabolismo , Aparato de Golgi/metabolismo
7.
Sci Adv ; 9(49): eadi5545, 2023 12 08.
Artículo en Inglés | MEDLINE | ID: mdl-38055815

RESUMEN

Infection response and other immunity-linked genes (ILGs) were first named in Caenorhabditis elegans-based expression after pathogen challenge, but many are also up-regulated when lipid metabolism is perturbed. Why pathogen attack and metabolic changes both increase ILGs is unclear. We find that ILGs are activated when phosphatidylcholine (PC) levels change in membranes of secretory organelles in C. elegans. RNAi targeting of the ADP-ribosylation factor arf-1, which disrupts the Golgi and secretory function, also activates ILGs. Low PC limits ARF-1 function, suggesting a mechanism for ILG activation via lipid metabolism, as part of a membrane stress response acting outside the ER. RNAi of selected ILGs uncovered defects in the secretion of two GFP reporters and the accumulation of a pathogen-responsive complement C1r/C1s, Uegf, Bmp1 (CUB) domain fusion protein. Our data argue that up-regulation of some ILGs is a coordinated response to changes in trafficking and may act to counteract stress on secretory function.


Asunto(s)
Caenorhabditis elegans , GTP Fosfohidrolasas , Animales , GTP Fosfohidrolasas/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Aparato de Golgi/metabolismo , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Transporte Biológico
8.
Nat Commun ; 14(1): 6770, 2023 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-37914730

RESUMEN

Type I interferon (IFN) signalling is tightly controlled. Upon recognition of DNA by cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING) translocates along the endoplasmic reticulum (ER)-Golgi axis to induce IFN signalling. Termination is achieved through autophagic degradation or recycling of STING by retrograde Golgi-to-ER transport. Here, we identify the GTPase ADP-ribosylation factor 1 (ARF1) as a crucial negative regulator of cGAS-STING signalling. Heterozygous ARF1 missense mutations cause a previously unrecognized type I interferonopathy associated with enhanced IFN-stimulated gene expression. Disease-associated, GTPase-defective ARF1 increases cGAS-STING dependent type I IFN signalling in cell lines and primary patient cells. Mechanistically, mutated ARF1 perturbs mitochondrial morphology, causing cGAS activation by aberrant mitochondrial DNA release, and leads to accumulation of active STING at the Golgi/ERGIC due to defective retrograde transport. Our data show an unexpected dual role of ARF1 in maintaining cGAS-STING homeostasis, through promotion of mitochondrial integrity and STING recycling.


Asunto(s)
Interferón Tipo I , Humanos , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Interferón Tipo I/metabolismo , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Transducción de Señal
9.
Nat Commun ; 14(1): 7570, 2023 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-37989735

RESUMEN

ADP-ribosylation factor 1 (Arf1) interacts with multiple cellular partners and membranes to regulate intracellular traffic, organelle structure and actin dynamics. Defining the dynamic conformational landscape of Arf1 in its active form, when bound to the membrane, is of high functional relevance and key to understanding how Arf1 can alter diverse cellular processes. Through concerted application of nuclear magnetic resonance (NMR), neutron reflectometry (NR) and molecular dynamics (MD) simulations, we show that, while Arf1 is anchored to the membrane through its N-terminal myristoylated amphipathic helix, the G domain explores a large conformational space, existing in a dynamic equilibrium between membrane-associated and membrane-distal conformations. These configurational dynamics expose different interfaces for interaction with effectors. Interaction with the Pleckstrin homology domain of ASAP1, an Arf-GTPase activating protein (ArfGAP), restricts motions of the G domain to lock it in what seems to be a conformation exposing functionally relevant regions.


Asunto(s)
Factor 1 de Ribosilacion-ADP , Factores de Ribosilacion-ADP , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Membranas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Actinas/metabolismo
10.
PLoS Pathog ; 19(9): e1011673, 2023 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-37721955

RESUMEN

The cellular protein GBF1, an activator of Arf GTPases (ArfGEF: Arf guanine nucleotide exchange factor), is recruited to the replication organelles of enteroviruses through interaction with the viral protein 3A, and its ArfGEF activity is required for viral replication, however how GBF1-dependent Arf activation supports the infection remains enigmatic. Here, we investigated the development of resistance of poliovirus, a prototype enterovirus, to increasing concentrations of brefeldin A (BFA), an inhibitor of GBF1. High level of resistance required a gradual accumulation of multiple mutations in the viral protein 2C. The 2C mutations conferred BFA resistance even in the context of a 3A mutant previously shown to be defective in the recruitment of GBF1 to replication organelles, and in cells depleted of GBF1, suggesting a GBF1-independent replication mechanism. Still, activated Arfs accumulated on the replication organelles of this mutant even in the presence of BFA, its replication was inhibited by a pan-ArfGEF inhibitor LM11, and the BFA-resistant phenotype was compromised in Arf1-knockout cells. Importantly, the mutations strongly increased the interaction of 2C with the activated form of Arf1. Analysis of other enteroviruses revealed a particularly strong interaction of 2C of human rhinovirus 1A with activated Arf1. Accordingly, the replication of this virus was significantly less sensitive to BFA than that of poliovirus. Thus, our data demonstrate that enterovirus 2Cs may behave like Arf1 effector proteins and that GBF1 but not Arf activation can be dispensable for enterovirus replication. These findings have important implications for the development of host-targeted anti-viral therapeutics.


Asunto(s)
Infecciones por Enterovirus , Enterovirus , Proteínas de Unión al GTP Monoméricas , Poliovirus , Humanos , Enterovirus/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Células HeLa , Poliovirus/genética , Proteínas Virales/metabolismo , Antígenos Virales/metabolismo , Brefeldino A/farmacología , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo
11.
J Agric Food Chem ; 71(39): 14251-14262, 2023 Oct 04.
Artículo en Inglés | MEDLINE | ID: mdl-37738360

RESUMEN

Glutamine (Gln) is the major energy source of intestinal porcine epithelial cells (IPEC-J2 cells) and plays a critical role in the nutritional physiological function of the intestine. However, the underlying mechanism requires further investigation. Here, the Gln-sensing pathway in IPEC-J2 cells was investigated. The results showed that Gln increased the cell proliferation. Subsequently, an analysis of the phosphorylated proteome revealed that Gln markedly upregulated ribosomal protein S6 (RPS6) phosphorylation at serine 235/236, suggesting that Gln activated the mTORC1 pathway. mTOR inhibition revealed that Gln promotes cell proliferation through the mTORC1 pathway. Similarly, blocking ADP-ribosylation factor 1 (Arf1) activity indicated that Gln-induced mTORC1 activation promoted cell proliferation in an Arf1-dependent manner. Additionally, the RagA/B pathway did not participate in Gln-induced mTORC1 activation. Collectively, these findings suggest that Gln-induced mTORC1 activation promotes IPEC-J2 cell proliferation via Arf1, not Rag GTPases. These results broaden our understanding of functional-cell-sensing amino acids, particularly Gln, that are regulated by mTORC1.


Asunto(s)
Factor 1 de Ribosilacion-ADP , Glutamina , Animales , Porcinos , Glutamina/farmacología , Glutamina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Factor 1 de Ribosilacion-ADP/metabolismo , Intestinos , Proliferación Celular
12.
Mol Biol Cell ; 34(12): ar119, 2023 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-37672345

RESUMEN

Arf GTPases are central regulators of the Golgi complex, which serves as the nexus of membrane-trafficking pathways in eukaryotic cells. Arf proteins recruit dozens of effectors to modify membranes, sort cargos, and create and tether transport vesicles, and are therefore essential for orchestrating Golgi trafficking. The regulation of Arf activity is controlled by the action of Arf-GEFs which activate via nucleotide exchange, and Arf-GAPs which inactivate via nucleotide hydrolysis. The localization dynamics of Arf GTPases and their Arf-GAPs during Golgi maturation have not been reported. Here we use the budding yeast model to examine the temporal localization of the Golgi Arf-GAPs. We also determine the mechanisms used by the Arf-GAP Age2 to localize to the Golgi. We find that the catalytic activity of Age2 and a conserved sequence in the unstructured C-terminal domain of Age2 are both required for Golgi localization. This sequence is predicted to form an amphipathic helix and mediates direct binding of Age2 to membranes in vitro. We also report the development of a probe for sensing active Arf1 in living cells and use this probe to characterize the temporal dynamics of Arf1 during Golgi maturation.


Asunto(s)
Aparato de Golgi , Factores de Intercambio de Guanina Nucleótido , Factores de Intercambio de Guanina Nucleótido/metabolismo , Aparato de Golgi/metabolismo , Factor 1 de Ribosilacion-ADP/metabolismo , GTP Fosfohidrolasas/metabolismo , Nucleótidos/metabolismo , Factores de Ribosilacion-ADP/metabolismo
13.
Cells ; 12(15)2023 08 05.
Artículo en Inglés | MEDLINE | ID: mdl-37566088

RESUMEN

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.


Asunto(s)
Esclerosis Amiotrófica Lateral , Proteína C9orf72 , Proteínas Activadoras de GTPasa , Animales , Humanos , Ratones , Factor 1 de Ribosilacion-ADP/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Drosophila/genética , Drosophila/metabolismo , ARN/metabolismo , ARN Mensajero/genética , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo
14.
Life Sci ; 328: 121902, 2023 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-37392777

RESUMEN

AIMS: The small GTPase protein ARF1 has been shown to be involved in the lipolysis pathway and to selectively kill stem cells in Drosophila melanogaster. However, the role of ARF1 in mammalian intestinal homeostasis remains elusive. This study aimed to explore the role of ARF1 in intestinal epithelial cells (IECs) and reveal the possible mechanism. MATERIALS AND METHODS: IEC-specific ARF1 deletion mouse model was used to evaluate the role of ARF1 in intestine. Immunohistochemistry and immunofluorescence analyses were performed to detect specific cell type markers, and intestinal organoids were cultured to assess intestinal stem cell (ISC) proliferation and differentiation. Fluorescence in situ hybridization, 16S rRNA-seq analysis, and antibiotic treatments were conducted to elucidate the role of gut microbes in ARF1-mediated intestinal function and the underlying mechanism. Colitis was induced in control and ARF1-deficient mice by dextran sulfate sodium (DSS). RNA-seq was performed to elucidate the transcriptomic changes after ARF1 deletion. KEY FINDINGS: ARF1 was essential for ISC proliferation and differentiation. Loss of ARF1 increased susceptibility to DSS-induced colitis and gut microbial dysbiosis. Gut microbiota depletion by antibiotics could rescue the intestinal abnormalities to a certain extent. Furthermore, RNA-seq analysis revealed alterations in multiple metabolic pathways. SIGNIFICANCE: This work is the first to elucidate the essential role of ARF1 in regulating gut homeostasis, and provides novel insights into the pathogenesis of intestinal diseases and potential therapeutic targets.


Asunto(s)
Factor 1 de Ribosilacion-ADP , Células Madre Adultas , Microbioma Gastrointestinal , Intestino Delgado , Animales , Ratones , Ratones Noqueados , Intestino Delgado/citología , Intestino Delgado/metabolismo , Intestino Delgado/microbiología , Factor 1 de Ribosilacion-ADP/metabolismo , Células Madre Adultas/metabolismo , Disbiosis/metabolismo , Antibacterianos/administración & dosificación , Transcripción Genética , Homeostasis , Redes y Vías Metabólicas
15.
J Cell Biol ; 222(9)2023 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-37289133

RESUMEN

Formation and fission of tubules from autolysosomes, endolysosomes, or phagolysosomes are required for lysosome reformation. However, the mechanisms governing these processes in these different lysosomal organelles are poorly understood. Thus, the role of phosphatidylinositol-4-phosphate (PI(4)P) is unclear as it was shown to promote the formation of tubules from phagolysosomes but was proposed to inhibit tubule formation on autolysosomes because the loss of PI4KIIIß causes extensive lysosomal tubulation. Using super-resolution live-cell imaging, we show that Arf1-PI4KIIIß positive vesicles are recruited to tubule fission sites from autolysosomes, endolysosomes, and phagolysosomes. Moreover, we show that PI(4)P is required to form autolysosomal tubules and that increased lysosomal tubulation caused by loss of PI4KIIIß represents impaired tubule fission. At the site of fission, we propose that Arf1-PI4KIIIß positive vesicles mediate a PI(3)P signal on lysosomes in a process requiring the lipid transfer protein SEC14L2. Our findings indicate that Arf1-PI4KIIIß positive vesicles and their regulation of PI(3)P are critical components of the lysosomal tubule fission machinery.


Asunto(s)
Factor 1 de Ribosilacion-ADP , Lisosomas , Fosfotransferasas (Aceptor de Grupo Alcohol) , Transducción de Señal , Lisosomas/metabolismo , Factor 1 de Ribosilacion-ADP/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo
16.
J Cell Biol ; 222(7)2023 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-37102998

RESUMEN

ADP-ribosylation factor (ARF) GTPases are major regulators of cellular membrane homeostasis. High sequence similarity and multiple, possibly redundant functions of the five human ARFs make investigating their function a challenging task. To shed light on the roles of the different Golgi-localized ARF members in membrane trafficking, we generated CRISPR-Cas9 knockins (KIs) of type I (ARF1 and ARF3) and type II ARFs (ARF4 and ARF5) and mapped their nanoscale localization with stimulated emission depletion (STED) super-resolution microscopy. We find ARF1, ARF4, and ARF5 on segregated nanodomains on the cis-Golgi and ER-Golgi intermediate compartments (ERGIC), revealing distinct roles in COPI recruitment on early secretory membranes. Interestingly, ARF4 and ARF5 define Golgi-tethered ERGIC elements decorated by COPI and devoid of ARF1. Differential localization of ARF1 and ARF4 on peripheral ERGICs suggests the presence of functionally different classes of intermediate compartments that could regulate bi-directional transport between the ER and the Golgi. Furthermore, ARF1 and ARF3 localize to segregated nanodomains on the trans-Golgi network (TGN) and are found on TGN-derived post-Golgi tubules, strengthening the idea of distinct roles in post-Golgi sorting. This work provides the first map of the nanoscale organization of human ARF GTPases on cellular membranes and sets the stage to dissect their numerous cellular roles.


Asunto(s)
Factores de Ribosilacion-ADP , Aparato de Golgi , Humanos , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Aparato de Golgi/metabolismo , Red trans-Golgi/metabolismo , Transporte de Proteínas , Transporte Biológico , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo
17.
Biol Open ; 12(4)2023 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-36946871

RESUMEN

Arf1 belongs to the Arf family of small GTPases that localise at the Golgi and plasma membrane. Active Arf1 plays a crucial role in regulating Golgi organisation and function. In mouse fibroblasts, loss of adhesion triggers a consistent drop (∼50%) in Arf1 activation that causes the Golgi to disorganise but not fragment. In suspended cells, the trans-Golgi (GalTase) disperses more prominently than cis-Golgi (Man II), accompanied by increased active Arf1 (detected using GFP-ABD: ARHGAP10 Arf1 binding domain) associated with the cis-Golgi compartment. Re-adhesion restores Arf1 activation at the trans-Golgi as it reorganises. Arf1 activation at the Golgi is regulated by Arf1 Guanine nucleotide exchange factors (GEFs), GBF1, and BIG1/2. In non-adherent fibroblasts, the cis-medial Golgi provides a unique setting to test and understand the role GEF-mediated Arf1 activation has in regulating Golgi organisation. Labelled with Man II-GFP, non-adherent fibroblasts treated with increasing concentrations of Brefeldin-A (BFA) (which inhibits BIG1/2 and GBF1) or Golgicide A (GCA) (which inhibits GBF1 only) comparably decrease active Arf1 levels. They, however, cause a concentration-dependent increase in cis-medial Golgi fragmentation and fusion with the endoplasmic reticulum (ER). Using selected BFA and GCA concentrations, we find a change in the kinetics of Arf1 inactivation could mediate this by regulating cis-medial Golgi localisation of GBF1. On loss of adhesion, a ∼50% drop in Arf1 activation over 120 min causes the Golgi to disorganise. The kinetics of this drop, when altered by BFA or GCA treatment causes a similar decline in Arf1 activation but over 10 min. This causes the Golgi to now fragment which affects cell surface glycosylation and re-adherent cell spreading. Using non-adherent fibroblasts this study reveals the kinetics of Arf1 inactivation, with active Arf1 levels, to be vital for Golgi organisation and function.


Asunto(s)
Factor 1 de Ribosilacion-ADP , Aparato de Golgi , Ratones , Animales , Aparato de Golgi/metabolismo , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Membrana Celular/metabolismo , Fibroblastos/metabolismo
18.
J Biol Chem ; 299(3): 102992, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36758799

RESUMEN

The ADP-ribosylation factor (Arf) GTPases and their regulatory proteins are implicated in cancer progression. NAV-2729 was previously identified as a specific inhibitor of Arf6 that reduced progression of uveal melanoma in an orthotopic xenograft. Here, our goal was to assess the inhibitory effects of NAV-2729 on the proliferation of additional cell types. We found NAV-2729 inhibited proliferation of multiple cell lines, but Arf6 expression did not correlate with NAV-2729 sensitivity, and knockdown of Arf6 affected neither cell viability nor sensitivity to NAV-2729. Furthermore, binding to native Arf6 was not detected; however, we determined that NAV-2729 inhibited both Arf exchange factors and Arf GTPase-activating proteins. ASAP1, a GTPase-activating protein linked to cancer progression, was further investigated. We demonstrated that NAV-2729 bound to the PH domain of ASAP1 and changed ASAP1 cellular distribution. However, ASAP1 knockdown did not fully recapitulate the cytoskeletal effects of NAV-2729 nor affect cell proliferation. Finally, our screens identified 48 other possible targets of NAV-2729. These results illustrate the complexities of defining targets of small molecules and identify NAV-2729 as a model PH domain-binding inhibitor.


Asunto(s)
Factores de Ribosilacion-ADP , Neoplasias , Humanos , Factores de Ribosilacion-ADP/metabolismo , Clorobencenos , Pirazoles , Proteínas Activadoras de GTPasa/metabolismo , Factor 1 de Ribosilacion-ADP/metabolismo
19.
J Cell Biol ; 222(4)2023 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-36811888

RESUMEN

The essential COPI coat mediates retrieval of transmembrane proteins at the Golgi and endosomes following recruitment by the small GTPase, Arf1. ArfGAP proteins regulate COPI coats, but molecular details for COPI recognition by ArfGAPs remain elusive. Biochemical and biophysical data reveal how ß'-COP propeller domains directly engage the yeast ArfGAP, Glo3, with a low micromolar binding affinity. Calorimetry data demonstrate that both ß'-COP propeller domains are required to bind Glo3. An acidic patch on ß'-COP (D437/D450) interacts with Glo3 lysine residues located within the BoCCS (binding of coatomer, cargo, and SNAREs) region. Targeted point mutations in either Glo3 BoCCS or ß'-COP abrogate the interaction in vitro, and loss of the ß'-COP/Glo3 interaction drives Ste2 missorting to the vacuole and aberrant Golgi morphology in budding yeast. These data suggest that cells require the ß'-COP/Glo3 interaction for cargo recycling via endosomes and the TGN, where ß'-COP serves as a molecular platform to coordinate binding to multiple proteins, including Glo3, Arf1, and the COPI F-subcomplex.


Asunto(s)
Proteína Coatómero , Proteínas Activadoras de GTPasa , Proteínas de Saccharomyces cerevisiae , Proteína Coat de Complejo I/metabolismo , Proteína Coatómero/metabolismo , Aparato de Golgi/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas SNARE/metabolismo , Factor 1 de Ribosilacion-ADP/metabolismo
20.
Hum Mol Genet ; 32(7): 1162-1174, 2023 03 20.
Artículo en Inglés | MEDLINE | ID: mdl-36345169

RESUMEN

ADP-ribosylation factor 1 (ARF1) is a small GTPase that regulates membrane traffic at the Golgi apparatus and endosomes through recruitment of several coat proteins and lipid-modifying enzymes. Here, we report a pediatric patient with an ARF1-related disorder because of a monoallelic de novo missense variant (c.296 G > A; p.R99H) in the ARF1 gene, associated with developmental delay, hypotonia, intellectual disability and motor stereotypies. Neuroimaging revealed a hypoplastic corpus callosum and subcortical white matter abnormalities. Notably, this patient did not exhibit periventricular heterotopias previously observed in other patients with ARF1 variants (including p.R99H). Functional analysis of the R99H-ARF1 variant protein revealed that it was expressed at normal levels and properly localized to the Golgi apparatus; however, the expression of this variant caused swelling of the Golgi apparatus, increased the recruitment of coat proteins such as coat protein complex I, adaptor protein complex 1 and GGA3 and altered the morphology of recycling endosomes. In addition, we observed that the expression of R99H-ARF1 prevented dispersal of the Golgi apparatus by the ARF1-inhibitor brefeldin A. Finally, protein interaction analyses showed that R99H-ARF1 bound more tightly to the ARF1-effector GGA3 relative to wild-type ARF1. These properties were similar to those of the well-characterized constitutively active Q71L-ARF1 mutant, indicating that the pathogenetic mechanism of the R99H-ARF1 variant involves constitutive activation with resultant Golgi and endosomal alterations. The absence of periventricular nodular heterotopias in this R99H-ARF1 subject also indicates that this finding may not be a consistent phenotypic expression of all ARF1-related disorders.


Asunto(s)
Factor 1 de Ribosilacion-ADP , Trastornos del Neurodesarrollo , Humanos , Animales , Ratones , Factor 1 de Ribosilacion-ADP/química , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Mutación Missense , Femenino , Niño , Aparato de Golgi/patología , Endosomas/patología , Trastornos del Neurodesarrollo/diagnóstico , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología
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