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1.
Cell ; 176(1-2): 113-126.e15, 2019 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-30633902

RESUMEN

Here, we describe a novel pathogenic entity, the activated PMN (polymorphonuclear leukocyte, i.e., neutrophil)-derived exosome. These CD63+/CD66b+ nanovesicles acquire surface-bound neutrophil elastase (NE) during PMN degranulation, NE being oriented in a configuration resistant to α1-antitrypsin (α1AT). These exosomes bind and degrade extracellular matrix (ECM) via the integrin Mac-1 and NE, respectively, causing the hallmarks of chronic obstructive pulmonary disease (COPD). Due to both ECM targeting and α1AT resistance, exosomal NE is far more potent than free NE. Importantly, such PMN-derived exosomes exist in clinical specimens from subjects with COPD but not healthy controls and are capable of transferring a COPD-like phenotype from humans to mice in an NE-driven manner. Similar findings were observed for another neutrophil-driven disease of ECM remodeling (bronchopulmonary dysplasia [BPD]). These findings reveal an unappreciated role for exosomes in the pathogenesis of disorders of ECM homeostasis such as COPD and BPD, providing a critical mechanism for proteolytic damage.


Asunto(s)
Exosomas/fisiología , Neutrófilos/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/citología , Células Cultivadas , Matriz Extracelular/metabolismo , Femenino , Humanos , Inflamación , Integrinas , Elastasa de Leucocito/metabolismo , Pulmón/metabolismo , Pulmón/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/fisiología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , alfa 1-Antitripsina/metabolismo
2.
J Virol ; 95(2)2020 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-33087467

RESUMEN

Enterovirus replication requires the cellular protein GBF1, a guanine nucleotide exchange factor for small Arf GTPases. When activated, Arfs associate with membranes, where they regulate numerous steps of membrane homeostasis. The requirement for GBF1 implies that Arfs are important for replication, but which of the different Arfs function(s) during replication remains poorly understood. Here, we established cell lines expressing each of the human Arfs fused to a fluorescent tag and investigated their behavior during enterovirus infection. Arf1 was the first to be recruited to the replication organelles, where it strongly colocalized with the viral antigen 2B and mature virions but not double-stranded RNA. By the end of the infectious cycle, Arf3, Arf4, Arf5, and Arf6 were also concentrated on the replication organelles. Once on the replication membranes, all Arfs except Arf3 were no longer sensitive to inhibition of GBF1, suggesting that in infected cells they do not actively cycle between GTP- and GDP-bound states. Only the depletion of Arf1, but not other class 1 and 2 Arfs, significantly increased the sensitivity of replication to GBF1 inhibition. Surprisingly, depletion of Arf6, a class 3 Arf, normally implicated in plasma membrane events, also increased the sensitivity to GBF1 inhibition. Together, our results suggest that GBF1-dependent Arf1 activation directly supports the development and/or functioning of the replication complexes and that Arf6 plays a previously unappreciated role in viral replication. Our data reveal a complex pattern of Arf activation in enterovirus-infected cells that may contribute to the resilience of viral replication in different cellular environments.IMPORTANCE Enteroviruses include many known and emerging pathogens, such as poliovirus, enteroviruses 71 and D68, and others. However, licensed vaccines are available only against poliovirus and enterovirus 71, and specific anti-enterovirus therapeutics are lacking. Enterovirus infection induces the massive remodeling of intracellular membranes and the development of specialized domains harboring viral replication complexes, replication organelles. Here, we investigated the roles of small Arf GTPases during enterovirus infection. Arfs control distinct steps in intracellular membrane traffic, and one of the Arf-activating proteins, GBF1, is a cellular factor required for enterovirus replication. We found that all Arfs expressed in human cells, including Arf6, normally associated with the plasma membrane, are recruited to the replication organelles and that Arf1 appears to be the most important Arf for enterovirus replication. These results document the rewiring of the cellular membrane pathways in infected cells and may provide new ways of controlling enterovirus infections.


Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Infecciones por Enterovirus/metabolismo , Enterovirus/fisiología , Compartimentos de Replicación Viral/metabolismo , Factores de Ribosilacion-ADP/genética , Antígenos Virales/metabolismo , Enterovirus/clasificación , Infecciones por Enterovirus/virología , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Viral/metabolismo , Replicación Viral
3.
Gynecol Oncol ; 157(3): 765-774, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32192732

RESUMEN

OBJECTIVES: Wnt pathway mutations are a hallmark of endometrioid and clear cell subtypes of epithelial ovarian carcinoma (EOC). However, no drugs targeting the Wnt pathway in EOC are FDA-approved. Dickkopf-related protein 1 (DKK1), a modulator of the Wnt pathway, has emerged as a promising therapeutic target. We aimed to examine the role of DKK1 and the effects of a monoclonal antibody against DKK1 (DKN-01) in vivo and in a murine model of ovarian cancer. METHODS: We examined in vitro the role of DKK1 and the effects of DKK1 inhibition in EOC cell lines. We then studied in vivo the role of DKN-01 and DKK1 overexpression on tumor burden and anti-tumor immune cell populations using the ID8 syngeneic mouse model. RESULTS: DKN-01 did not phenotypically alter ES2 cells in vitro; however, DKK1 inhibition promoted Wnt signaling. Tumor burden and immune populations were unchanged in ID8 challenged mice treated with mDKN01. Mice challenged with ID8 cells overexpressing DKK1 had tumor burden similar to controls (p = 0.175). However, the overexpression of DKK1 decreased CD45+ leukocyte infiltration into the peritoneum (p = 0.008) and omentum (p = 0.032), reducing both natural killer (NK) and CD8 T cells, and reducing interferon-gamma (IFNγ) expression on activated CD8 T cells. CONCLUSIONS: Our results suggest that DKK1 inhibition does not affect tumor growth in the ID8 ovarian cancer model. DKK1 overexpression alters anti-tumor immune populations within the tumor microenvironment. Thus, our findings confirm DKK1 as a new therapeutic target in EOC and suggest that DKK1 inhibition may function best in a combinatorial, immune-modulatory therapy.

4.
Am J Respir Cell Mol Biol ; 61(5): 560-566, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-30958968

RESUMEN

Chronic obstructive pulmonary disease (COPD) is a major cause of mortality worldwide and is characterized by an excessive airway neutrophilic response. The neutrophil chemoattractant proline-glycine-proline (PGP) and its more potent acetylated form (acPGP) have been found to be elevated in patients with COPD and act via CXCR2. Here, we investigated the impact of neutralizing PGP peptides in a murine model for emphysema. The PGP-neutralizing peptide l-arginine-threonine-arginine (RTR) was used first in a 6-week model of cigarette smoke exposure, where it attenuated lung inflammation. Then, in a model of chronic smoke exposure, mice were exposed to cigarette smoke and RTR treatment was initiated after 10 weeks of smoke exposure. This treatment was continued together with smoke exposure for another 13 weeks, for a total of 23 weeks of smoke exposure. RTR significantly inhibited neutrophil and macrophage influx into the lungs in the 6-week model of exposure. RTR also attenuated the development of emphysema, normalized lung volumes, and reduced right ventricular hypertrophy in the chronic exposure model. Murine epithelia expressed CXCR2, and this expression was increased after smoke exposure. In vitro, human bronchial epithelial cells also demonstrated robust expression of CXCR2, and stimulation of primary human bronchial epithelial cells with acPGP led to increased release of MMP-9 and IL-8. Overall, these results provide evidence that acPGP plays a critical role during the development of emphysema in cigarette smoke-induced injury, and highlight a new epithelial mechanism by which acPGP augments neutrophilic inflammation.


Asunto(s)
Inflamación/metabolismo , Neutrófilos/metabolismo , Enfisema Pulmonar/etiología , Animales , Células Cultivadas , Humanos , Pulmón/metabolismo , Pulmón/patología , Ratones , Oligopéptidos/metabolismo , Prolina/análogos & derivados , Prolina/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/etiología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfisema Pulmonar/metabolismo , Humo/efectos adversos
5.
Cancer ; 125 Suppl 24: 4563-4572, 2019 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-31967683

RESUMEN

Biomarkers are becoming increasingly important in the treatment of epithelial ovarian cancer. Recent work from many laboratories has begun to provide clinically meaningful biomarkers. This review summarizes the state of the science regarding biomarkers for stratifying early-stage patients into those who benefit from adjuvant treatment, primary debulking versus interval debulking, and specific targeted therapy. In addition, new molecular imaging technologies have been developed to allow the surgeon to resect subvisible tumor deposits. These efforts should increase clinical effectiveness while minimizing toxicities for patients.


Asunto(s)
Biomarcadores/metabolismo , Neoplasias Ováricas/diagnóstico , Femenino , Humanos , Neoplasias Ováricas/patología
6.
Am J Respir Crit Care Med ; 197(5): 632-643, 2018 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-29232160

RESUMEN

RATIONALE: MicroRNAs (miRNAs) destabilize mRNA transcripts and inhibit protein translation. miR-145 is of particular interest in cystic fibrosis (CF) as it has a direct binding site in the 3'-untranslated region of CFTR (cystic fibrosis transmembrane conductance regulator) and is upregulated by the CF genetic modifier TGF (transforming growth factor)-ß. OBJECTIVES: To demonstrate that miR-145 mediates TGF-ß inhibition of CFTR synthesis and function in airway epithelia. METHODS: Primary human CF (F508del homozygous) and non-CF airway epithelial cells were grown to terminal differentiation at the air-liquid interface on permeable supports. TGF-ß (5 ng/ml), a miR-145 mimic (20 nM), and a miR-145 antagonist (20 nM) were used to manipulate CFTR function. In CF cells, lumacaftor (3 µM) and ivacaftor (10 µM) corrected mutant F508del CFTR. Quantification of CFTR mRNA, protein, and function was done by standard techniques. MEASUREMENTS AND MAIN RESULTS: miR-145 is increased fourfold in CF BAL fluid compared with non-CF (P < 0.01) and increased 10-fold in CF primary airway epithelial cells (P < 0.01). Exogenous TGF-ß doubles miR-145 expression (P < 0.05), halves wild-type CFTR mRNA and protein levels (P < 0.01), and nullifies lumacaftor/ivacaftor F508del CFTR correction. miR-145 overexpression similarly decreases wild-type CFTR protein synthesis (P < 0.01) and function (P < 0.05), and eliminates F508del corrector benefit. miR-145 antagonism blocks TGF-ß suppression of CFTR and enhances lumacaftor correction of F508del CFTR. CONCLUSIONS: miR-145 mediates TGF-ß inhibition of CFTR synthesis and function in airway epithelia. Specific antagonists to miR-145 interrupt TGF-ß signaling to restore F508del CFTR modulation. miR-145 antagonism may offer a novel therapeutic opportunity to enhance therapeutic benefit of F508del CFTR correction in CF epithelia.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Epitelio/metabolismo , MicroARNs/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , MicroARNs/genética , Factor de Crecimiento Transformador beta/genética
7.
Am J Respir Cell Mol Biol ; 54(3): 359-69, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26222144

RESUMEN

Proteases are important regulators of pulmonary remodeling and airway inflammation. Recently, we have characterized the enzyme prolyl endopeptidase (PE), a serine peptidase, as a critical protease in the generation of the neutrophil chemoattractant tripeptide Pro-Gly-Pro (PGP) from collagen. However, PE has been characterized as a cytosolic enzyme, and the mechanism mediating PE release extracellularly remains unknown. We examined the role of exosomes derived from airway epithelia as a mechanism for PE release and the potential extracellular signals that regulate the release of these exosomes. We demonstrate a specific regulatory pathway of exosome release from airway epithelia and identify PE as novel exosome cargo. LPS stimulation of airway epithelial cells induces release of PE-containing exosomes, which is significantly attenuated by small interfering RNA depletion of Toll-like receptor 4 (TLR4). These differences were recapitulated upon intratracheal LPS administration in mice competent versus deficient for TLR4 signaling. Finally, sputum samples from subjects with cystic fibrosis colonized with Pseudomonas aeruginosa demonstrate elevated exosome content and increased PE levels. This TLR4-based mechanism highlights the first report of nonstochastic release of exosomes in the lung and couples TLR4 activation with matrikine generation. The increased quantity of these proteolytic exosomes in the airways of subjects with chronic lung disease highlights a new mechanism of injury and inflammation in the pathogenesis of pulmonary disorders.


Asunto(s)
Bronquios/enzimología , Fibrosis Quística/enzimología , Células Epiteliales/enzimología , Exosomas/enzimología , Proteínas Mitocondriales/metabolismo , Serina Endopeptidasas/metabolismo , Receptor Toll-Like 4/metabolismo , Adulto , Animales , Bronquios/efectos de los fármacos , Bronquios/microbiología , Estudios de Casos y Controles , Línea Celular , Fibrosis Quística/genética , Fibrosis Quística/microbiología , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Exosomas/efectos de los fármacos , Exosomas/microbiología , Femenino , Humanos , Lipopolisacáridos/farmacología , Masculino , Ratones Endogámicos C3H , Ratones Noqueados , Prolil Oligopeptidasas , Pseudomonas aeruginosa/aislamiento & purificación , Interferencia de ARN , Transducción de Señal , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/genética , Transfección , Adulto Joven
9.
Am J Respir Crit Care Med ; 190(1): 51-61, 2014 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-24874071

RESUMEN

RATIONALE: Chronic neutrophilic inflammation is a hallmark in the pathogenesis of chronic obstructive pulmonary disease (COPD) and persists after cigarette smoking has stopped. Mechanisms involved in this ongoing inflammatory response have not been delineated. OBJECTIVES: We investigated changes to the leukotriene A4 hydrolase (LTA4H)-proline-glycine-proline (PGP) pathway and chronic inflammation in the development of COPD. METHODS: A/J mice were exposed to air or cigarette smoke for 22 weeks followed by bronchoalveolar lavage and lung and cardiac tissue analysis. Two human cohorts were used to analyze changes to the LTA4H-PGP pathway in never smokers, control smokers, COPD smokers, and COPD former smokers. PGP/AcPGP and LTA4H aminopeptidase activity were detected by mass spectroscopy, LTA4H amounts were detected by ELISA, and acrolein was detected by Western blot. MEASUREMENTS AND MAIN RESULTS: Mice exposed to cigarette smoke developed emphysema with increased PGP, neutrophilic inflammation, and selective inhibition of LTA4H aminopeptidase, which ordinarily degrades PGP. We recapitulated these findings in smokers with and without COPD. PGP and AcPGP are closely associated with cigarette smoke use. Once chronic inflammation is established, changes to LTA4H aminopeptidase remain, even in the absence of ongoing cigarette use. Acrolein modifies LTA4H and inhibits aminopeptidase activity to the same extent as cigarette smoke. CONCLUSIONS: These results demonstrate a novel pathway of aberrant regulation of PGP/AcPGP, suggesting this inflammatory pathway may be intimately involved in disease progression in the absence of ongoing cigarette smoke exposure. We highlight a mechanism by which acrolein potentiates neutrophilic inflammation through selective inhibition of LTA4H aminopeptidase activity. Clinical trial registered with www.clinicaltrials.gov (NCT 00292552).


Asunto(s)
Epóxido Hidrolasas/inmunología , Inflamación/fisiopatología , Neutrófilos/inmunología , Enfermedad Pulmonar Obstructiva Crónica/etiología , Fumar/efectos adversos , Anciano , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Estudios de Cohortes , Modelos Animales de Enfermedad , Enfisema/etiología , Enfisema/inmunología , Femenino , Glicina/metabolismo , Humanos , Inflamación/complicaciones , Pulmón/inmunología , Masculino , Ratones , Persona de Mediana Edad , Miocardio/inmunología , Prolina/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Fumar/inmunología
10.
J Biol Chem ; 288(16): 11532-45, 2013 Apr 19.
Artículo en Inglés | MEDLINE | ID: mdl-23386609

RESUMEN

Three Sec7 guanine nucleotide exchange factors (GEFs) activate ADP-ribosylation factors (ARFs) to facilitate coating of transport vesicles within the secretory and endosomal pathways. GBF1 recruits COPI to pre-Golgi and Golgi compartments, whereas BIG1 and BIG2 recruit AP1 and GGA clathrin adaptors to the trans-Golgi network (TGN) and endosomes. Here, we report a functional cascade between these GEFs by showing that GBF1-activated ARFs (ARF4 and ARF5, but not ARF3) facilitate BIG1 and BIG2 recruitment to the TGN. We localize GBF1 ultrastructurally to the pre-Golgi, the Golgi, and also the TGN. Our findings suggest a model in which GBF1 localized within pre-Golgi and Golgi compartments mediates ARF activation to facilitate recruitment of COPI to membranes, whereas GBF1 localized at the TGN mediates ARF activation that leads to the recruitment of BIG1 and BIG2 to the TGN. Membrane-associated BIG1/2 then activates ARFs that recruit clathrin adaptors. In this cascade, an early acting GEF (GBF1) activates ARFs that mediate recruitment of late acting GEFs (BIG1/2) to coordinate coating events within the pre-Golgi/Golgi/TGN continuum. Such coordination may optimize the efficiency and/or selectivity of cargo trafficking through the compartments of the secretory pathway.


Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Membranas Intracelulares/metabolismo , Vías Secretoras/fisiología , Red trans-Golgi/metabolismo , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Animales , Proteína Coat de Complejo I/genética , Proteína Coat de Complejo I/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Células HeLa , Humanos , Ratones , Red trans-Golgi/genética
11.
J Cell Sci ; 125(Pt 8): 1896-909, 2012 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-22328511

RESUMEN

The tethering factor p115 (known as Uso1p in yeast) has been shown to facilitate Golgi biogenesis and membrane traffic in cells in culture. However, the role of p115 within an intact animal is largely unknown. Here, we document that depletion of p115 by using RNA interference (RNAi) in C. elegans causes accumulation of the 170 kD soluble yolk protein (YP170) in the body cavity and retention of the yolk receptor RME-2 in the ER and the Golgi within oocytes. Structure-function analyses of p115 have identified two homology regions (H1 and H2) within the N-terminal globular head and the coiled-coil 1 (CC1) domain as essential for p115 function. We identify a new C-terminal domain of p115 as necessary for Golgi ribbon formation and cargo trafficking. We show that p115 mutants that lack the fourth CC domain (CC4) act in a dominant-negative manner to disrupt Golgi and prevent cargo trafficking in cells containing endogenous p115. Furthermore, using RNAi of p115 and the subsequent transfection with p115 deletion mutants, we show that CC4 is necessary for Golgi ribbon formation and membrane trafficking in cells depleted of endogenous p115. p115 has been shown to bind a subset of ER-Golgi SNAREs through CC1 and CC4 domains (Shorter et al., 2002). Our findings show that CC4 is required for p115 function, and suggest that both the CC1 and the CC4 SNARE-binding motifs participate in p115-mediated membrane tethering.


Asunto(s)
Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Animales , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Membrana Celular/genética , Aparato de Golgi/química , Aparato de Golgi/genética , Proteínas de la Matriz de Golgi , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas de Transporte Vesicular/genética
12.
Eur Respir J ; 43(4): 1086-96, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24311764

RESUMEN

Respiratory syncytial virus (RSV) infection is a potent stimulus for airway epithelial expression of matrix metalloproteinase (MMP)-9. MMP-9 activity in vivo is a predictor of disease severity in children with RSV-induced respiratory failure. Human airway epithelial cells were infected with RSV A2 strain and analysed for MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 (a natural inhibitor of MMP-9) release. In addition, endotracheal samples from children with RSV-RF and controls (non-RSV pneumonia and nonlung disease controls) were analysed for MMP-9, TIMP-1, human neutrophil elastase and myeloperoxidase activity. RSV infection of airway epithelia was sufficient to rapidly induce MMP-9 transcription and protein release. Pulmonary MMP-9 activity peaked at 48 h in infants with RSV-induced respiratory failure. In the RSV group, MMP-9 activity and MMP-9/TIMP-1 ratio imbalance predicted higher oxygen requirement and worse paediatric risk of mortality scores. The highest levels of human neutrophil elastase and myeloperoxidase activity were measured in the RSV cohort; however, unlike MMP-9, these neutrophil markers failed to predict disease severity. These results support the hypothesis that RSV is a potent stimulus for MMP-9 expression and release from human airway epithelium, and that MMP-9 is an important biomarker of disease severity in mechanically ventilated children with RSV lung infection.


Asunto(s)
Regulación Enzimológica de la Expresión Génica , Pulmón/enzimología , Metaloproteinasa 9 de la Matriz/metabolismo , Respiración Artificial/métodos , Infecciones por Virus Sincitial Respiratorio/enzimología , Virus Sincitial Respiratorio Humano , Biomarcadores/metabolismo , Células Cultivadas , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Intubación , Elastasa de Leucocito/metabolismo , Masculino , Oxígeno/uso terapéutico , Peroxidasa/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo
13.
Front Cell Dev Biol ; 11: 1233272, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37745300

RESUMEN

Golgi homeostasis require the activation of Arf GTPases by the guanine-nucleotide exchange factor requires GBF1, whose recruitment to the Golgi represents a rate limiting step in the process. GBF1 contains a conserved, catalytic, Sec7 domain (Sec7d) and five additional (DCB, HUS, HDS1-3) domains. Herein, we identify the HDS3 domain as essential for GBF1 membrane association in mammalian cells and document the critical role of HDS3 during the development of Drosophila melanogaster. We show that upon binding to Golgi membranes, GBF1 undergoes conformational changes in regions bracketing the catalytic Sec7d. We illuminate GBF1 interdomain arrangements by negative staining electron microscopy of full-length human GBF1 to show that GBF1 forms an anti-parallel dimer held together by the paired central DCB-HUS core, with two sets of HDS1-3 arms extending outward in opposite directions. The catalytic Sec7d protrudes from the central core as a largely independent domain, but is closely opposed to a previously unassigned α-helix from the HDS1 domain. Based on our data, we propose models of GBF1 engagement on the membrane to provide a paradigm for understanding GBF1-mediated Arf activation required for cellular and organismal function.

14.
J Biol Chem ; 286(42): 36898-906, 2011 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-21828055

RESUMEN

ADP-ribosylation factors (ARFs) and their activating guanine nucleotide exchange factors (GEFs) play key roles in membrane traffic and signaling. All ARF GEFs share a ∼200-residue Sec7 domain (Sec7d) that alone catalyzes the GDP to GTP exchange that activates ARF. We determined the crystal structure of human BIG2 Sec7d. A C-terminal loop immediately following helix J (loop>J) was predicted to form contacts with helix H and the switch I region of the cognate ARF, suggesting that loop>J may participate in the catalytic reaction. Indeed, we identified multiple alanine substitutions within loop>J of the full length and/or Sec7d of two large brefeldin A-sensitive GEFs (GBF1 and BIG2) and one small brefeldin A-resistant GEF (ARNO) that abrogated binding of ARF and a single alanine substitution that allowed ARF binding but inhibited GDP to GTP exchange. Loop>J sequences are highly conserved, suggesting that loop>J plays a crucial role in the catalytic activity of all ARF GEFs. Using GEF mutants unable to bind ARF, we showed that GEFs associate with membranes independently of ARF and catalyze ARF activation in vivo only when membrane-associated. Our structural, cell biological, and biochemical findings identify loop>J as a key regulatory motif essential for ARF binding and GDP to GTP exchange by GEFs and provide evidence for the requirement of membrane association during GEF activity.


Asunto(s)
Factores de Ribosilacion-ADP/química , Proteínas Activadoras de GTPasa/química , Factores de Intercambio de Guanina Nucleótido/química , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Catálisis , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Difosfato/química , Guanosina Difosfato/genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Mutación Missense , Estructura Terciaria de Proteína
15.
Physiology (Bethesda) ; 26(5): 348-64, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22013193

RESUMEN

Protein traffic is necessary to maintain homeostasis in all eukaryotic organisms. All newly synthesized secretory proteins destined to the secretory and endolysosmal systems are transported from the endoplasmic reticulum to the Golgi before delivery to their final destinations. Here, we describe the COPII and COPI coating machineries that generate carrier vesicles and the tethers and SNAREs that mediate COPII and COPI vesicle fusion at the ER-Golgi interface.


Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Animales , Humanos , Transporte de Proteínas
16.
Mol Biol Cell ; 32(5): 446-459, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33405949

RESUMEN

The components and subprocesses underlying the formation of COPI-coated vesicles at the Golgi are well understood. The coating cascade is initiated after the small GTPase Arf1 is activated by the Sec7 domain-containing guanine nucleotide exchange factor GBF1 (Golgi brefeldin A resistant guanine nucleotide exchange factor 1). This causes a conformational shift within Arf1 that facilitates stable association of Arf1 with the membrane, a process required for subsequent recruitment of the COPI coat. Although we have atomic-level knowledge of Arf1 activation by Sec7 domain-containing GEFs, our understanding of the biophysical processes regulating Arf1 and GBF1 dynamics is limited. We used fluorescence recovery after photobleaching data and kinetic Monte Carlo simulation to assess the behavior of Arf1 and GBF1 during COPI vesicle formation in live cells. Our analyses suggest that Arf1 and GBF1 associate with Golgi membranes independently, with an excess of GBF1 relative to Arf1. Furthermore, the GBF1-mediated Arf1 activation is much faster than GBF1 cycling on/off the membrane, suggesting that GBF1 is regulated by processes other than its interactions Arf1. Interestingly, modeling the behavior of the catalytically inactive GBF1/E794K mutant stabilized on the membrane is inconsistent with the formation of a stable complex between it and an endogenous Arf1 and suggests that GBF1/E794K is stabilized on the membrane independently of complex formation.


Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factor 1 de Ribosilacion-ADP/fisiología , Factores de Ribosilacion-ADP/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/fisiología , Proteína Coat de Complejo I/metabolismo , Endocitosis , Retículo Endoplásmico/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Aparato de Golgi/metabolismo , Factores de Intercambio de Guanina Nucleótido/fisiología , Células HeLa , Humanos , Cinética , Proteínas de Unión al GTP Monoméricas/metabolismo , Método de Montecarlo , Unión Proteica , Transporte de Proteínas
17.
Mol Biol Cell ; 17(7): 2996-3008, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16624868

RESUMEN

The mechanisms regulating membrane recruitment of the p115 tethering factor in vivo are unknown. Here, we describe cycling of p115 between membranes and cytosol and document the effects of Golgi matrix proteins, Rab1, and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) on this process. Rapid membrane/cytosol exchange is shown by swift (t1/2 approximately 20 s) loss of Golgi-localized p115-green fluorescent protein (GFP) after repeated photobleaching of cell periphery and rapid (t1/2 approximately 13 s) fluorescence recovery after photobleaching Golgi-localized p115-GFP. p115 mutant missing the GM130/giantin binding site exhibits analogous fluorescence recovery after photobleaching (FRAP) (t1/2 approximately 13 s), suggesting that GM130 and giantin are not major determinants of p115 membrane dynamics. In contrast, p115-GFP exchanges more rapidly (t1/2 approximately 8 s) in cells expressing the inactive Rab1/N121I mutant, indicating that p115 cycling is influenced by Rab1. p115-GFP dynamics is also influenced by the assembly status of SNAREs. In cells expressing an ATPase-deficient NSF/E329Q mutant that inhibits SNARE complex disassembly, the cycling kinetics of p115-GFP are significantly slower (t1/2 approximately 21 s). In contrast, in cells incubated at reduced temperature (10 degrees C) that inhibits vesicular traffic, the cycling kinetics of p115-GFP are faster (t1/2 approximately 7 s). These data suggest that p115-binding sites on the membrane are provided by unassembled SNAREs. In agreement, biochemical studies show increased p115 recruitment to membranes in the presence of NSF and alpha-SNAP. Our data support a model in which recruitment of tethers is directly regulated by the assembly status of SNAREs.


Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Autoantígenos , Citosol/química , Citosol/metabolismo , Retículo Endoplásmico/química , Proteínas de la Matriz de Golgi , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Humanos , Membranas Intracelulares/química , Cinética , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Proteínas Sensibles a N-Etilmaleimida/metabolismo , Transporte de Proteínas , Ratas , Proteínas de Transporte Vesicular/análisis , Proteínas de Transporte Vesicular/genética , Proteínas de Unión al GTP rab1/genética , Proteínas de Unión al GTP rab1/metabolismo
18.
JCI Insight ; 3(5)2018 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-29515035

RESUMEN

Premature infants are at high risk for developing bronchopulmonary dysplasia (BPD), characterized by chronic inflammation and inhibition of lung development, which we have recently identified as being modulated by microRNAs (miRNAs) and alterations in the airway microbiome. Exosomes and exosomal miRNAs may regulate cell differentiation and tissue and organ development. We discovered that tracheal aspirates from infants with severe BPD had increased numbers of, but smaller, exosomes compared with term controls. Similarly, bronchoalveolar lavage fluid from hyperoxia-exposed mice (an animal model of BPD) and supernatants from hyperoxia-exposed human bronchial epithelial cells (in vitro model of BPD) had increased exosomes compared with air controls. Next, in a prospective cohort study of tracheal aspirates obtained at birth from extremely preterm infants, utilizing independent discovery and validation cohorts, we identified unbiased exosomal miRNA signatures predictive of severe BPD. The strongest signal of reduced miR-876-3p in BPD-susceptible compared with BPD-resistant infants was confirmed in the animal model and in vitro models of BPD. In addition, based on our recent discovery of increased Proteobacteria in the airway microbiome being associated with BPD, we developed potentially novel in vivo and in vitro models for BPD combining Proteobacterial LPS and hyperoxia exposure. Addition of LPS led to a larger reduction in exosomal miR 876-3p in both hyperoxia and normoxia compared with hyperoxia alone, thus indicating a potential mechanism by which alterations in microbiota can suppress miR 876-3p. Gain of function of miR 876-3p improved the alveolar architecture in the in vivo BPD model, demonstrating a causal link between miR 876-3p and BPD. In summary, we provide evidence for the strong predictive biomarker potential of miR 876-3p in severe BPD. We also provide insights on the pathogenesis of neonatal lung disease, as modulated by hyperoxia and microbial product-induced changes in exosomal miRNA 876-3p, which could be targeted for future therapeutic development.


Asunto(s)
Células Epiteliales Alveolares/inmunología , Displasia Broncopulmonar/diagnóstico , Exosomas/metabolismo , Recien Nacido Extremadamente Prematuro/inmunología , MicroARNs/metabolismo , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/microbiología , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar/citología , Displasia Broncopulmonar/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular , Modelos Animales de Enfermedad , Exosomas/genética , Exosomas/inmunología , Femenino , Humanos , Hiperoxia/inmunología , Recien Nacido con Peso al Nacer Extremadamente Bajo/inmunología , Recién Nacido , Lipopolisacáridos/inmunología , Masculino , Ratones , MicroARNs/genética , MicroARNs/inmunología , Microbiota/inmunología , Pronóstico , Estudios Prospectivos , Proteobacteria/inmunología , Índice de Severidad de la Enfermedad
19.
Mol Biol Cell ; 14(6): 2250-61, 2003 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12808027

RESUMEN

ADP-ribosylation factor (ARF) mediated recruitment of COPI to membranes plays a central role in transport between the endoplasmic reticulum (ER) and the Golgi. The activation of ARFs is mediated by guanine nucleotide exchange factors (GEFs). Although several ARF-GEFs have been identified, the transport steps in which they function are still poorly understood. Here we report that GBF1, a member of the Sec7-domain family of GEFs, is responsible for the regulation of COPI-mediated events at the ER-Golgi interface. We show that GBF1 is essential for the formation, differentiation, and translocation of pre-Golgi intermediates and for the maintenance of Golgi integrity. We also show that the formation of transport-competent ER-to-Golgi intermediates proceeds in two stages: first, a COPI-independent event leads to the formation of an unstable compartment, which is rapidly reabsorbed in the absence of GBF1 activity. Second, the association of GBF1 with this compartment allows COPI recruitment and leads to its maturation into transport intermediates. The recruitment of GBF1 to this compartment is specifically inhibited by brefeldin A. Our findings imply that the continuous recruitment of GBF1 to spatially differentiated membrane domains is required for sustained membrane remodeling that underlies membrane traffic and Golgi biogenesis.


Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Proteína Coat de Complejo I/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Genes Reporteros , Factores de Intercambio de Guanina Nucleótido/genética , Humanos
20.
ERJ Open Res ; 2(1)2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-27730172

RESUMEN

In chronic inflammatory lung disorders such as chronic obstructive pulmonary disease (COPD), the concurrent organ-specific and systemic inflammatory responses lead to airway remodelling and vascular dysfunction. Although a major common risk factor for COPD, cigarette smoke alone cannot explain the progression of this disease; there is increasing evidence that genetic predisposition also plays a role in COPD susceptibility and progression. A key enzyme in chronic lung inflammation is leukotriene A4 hydrolase (LTA4H). With its aminopeptidase activity, LTA4H degrades the neutrophil chemoattractant tripeptide PGP. In this study, we used the luciferase reporter gene analysis system and quantitative trait locus analysis to explore the impact of single-nucleotide polymorphisms (SNPs) in the putative promoter region of LTA4H on LTA4H expression. We show that not only is the putative promoter of LTA4H larger than previously reported but also that SNPs in the expanded promoter region regulate expression of LTA4H both in cell-based systems and in peripheral blood samples from human subjects. These findings provide significant evidence for an active region upstream of the previously reported LTA4H promoter, which may have implications related to ongoing inflammatory processes in chronic lung disease.

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