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2.
Front Immunol ; 12: 663586, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33859652

RESUMEN

As of January 2021, SARS-CoV-2 has killed over 2 million individuals across the world. As such, there is an urgent need for vaccines and therapeutics to reduce the burden of COVID-19. Several vaccines, including mRNA, vector-based vaccines, and inactivated vaccines, have been approved for emergency use in various countries. However, the slow roll-out of vaccines and insufficient global supply remains a challenge to turn the tide of the pandemic. Moreover, vaccines are important tools for preventing the disease but therapeutic tools to treat patients are also needed. As such, since the beginning of the pandemic, repurposed FDA-approved drugs have been sought as potential therapeutic options for COVID-19 due to their known safety profiles and potential anti-viral effects. One of these drugs is ivermectin (IVM), an antiparasitic drug created in the 1970s. IVM later exerted antiviral activity against various viruses including SARS-CoV-2. In this review, we delineate the story of how this antiparasitic drug was eventually identified as a potential treatment option for COVID-19. We review SARS-CoV-2 lifecycle, the role of the nucleocapsid protein, the turning points in past research that provided initial 'hints' for IVM's antiviral activity and its molecular mechanism of action- and finally, we culminate with the current clinical findings.


Asunto(s)
Transporte Activo de Núcleo Celular/efectos de los fármacos , Antivirales/uso terapéutico , Ivermectina/uso terapéutico , /efectos de los fármacos , Animales , Línea Celular , Chlorocebus aethiops , /metabolismo , Reposicionamiento de Medicamentos , Humanos , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Células Vero , Replicación Viral/efectos de los fármacos , alfa Carioferinas/antagonistas & inhibidores , beta Carioferinas/antagonistas & inhibidores
3.
Nutrients ; 13(2)2021 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-33572926

RESUMEN

Rosa canina L. is a natural polyphenol-rich medicinal plant that exhibits antioxidant and anti-inflammatory activities. Recent in vivo studies have demonstrated that a methanol extract of Rosa canina L. (RCME) has reversed an inflammatory bowel disease (IBD)-like phenotype that has been triggered by dextran sulfate sodium (DSS) in mice. In the current study, we investigated the effects of RCME on perturbations of cellular mechanisms induced by DSS-treatment of intestinal Caco-2 cells, including stress response in the endoplasmic reticulum (ER), protein trafficking and sorting as well as lipid rafts integrity and functional capacities of an intestinal enzyme. 6 days post-confluent cells were treated for 24 h with DSS (3%) or simultaneously with DSS (3%) and RCME (100 µg/mL) or exclusively with RCME (100 µg/mL) or not treated. The results obtained demonstrate the ability of RCME to counteract the substantial increase in the expression levels of several ER stress markers in DSS-treated cells. Concomitantly, the delayed trafficking of intestinal membrane glycoproteins sucrase-isomaltase (SI) and dipeptidyl peptidase 4 (DPP4) induced by DSS between the ER and the Golgi has been compromised by RCME. Furthermore, RCME restored the partially impaired polarized sorting of SI and DPP4 to the brush border membrane. An efficient sorting mechanism of SI and DPP4 is tightly associated with intact lipid rafts structures in the trans-Golgi network (TGN), which have been distorted by DSS and normalized by RCME. Finally, the enzymatic activities of SI are enhanced in the presence of RCME. Altogether, DSS treatment has triggered ER stress, impaired trafficking and function of membrane glycoproteins and distorted lipid rafts, all of which can be compromised by RCME. These findings indicate that the antioxidants in RCME act at two major sites in Caco-2 cells, the ER and the TGN and are thus capable of maintaining the membrane integrity by correcting the sorting of membrane-associated proteins.


Asunto(s)
Retículo Endoplásmico/efectos de los fármacos , Enfermedades Inflamatorias del Intestino/terapia , Metanol/farmacología , Extractos Vegetales/farmacología , Transporte de Proteínas/efectos de los fármacos , Rosa/química , Animales , Células CACO-2 , Sulfato de Dextran , Dipeptidil Peptidasa 4/metabolismo , Modelos Animales de Enfermedad , Humanos , Enfermedades Inflamatorias del Intestino/inducido químicamente , Mucosa Intestinal/efectos de los fármacos , Microdominios de Membrana/metabolismo , Ratones , Microvellosidades/metabolismo , Fenotipo , Complejo Sacarasa-Isomaltasa/metabolismo
4.
Int J Mol Med ; 47(4)2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33537817

RESUMEN

Inflammation is the most common cause of most acute and chronic debilitating diseases. Towards unveiling novel therapeutic options for patients with such complications, N­bromotaurine (TauNHBr) has emerged as a potential anti­inflammatory agent; however, its therapeutic efficacy is hindered due to its relatively poor stability. To address this challenge, the present study focused on examining the effects of a stable active bromine compound, named bromamine T (BAT). The present study examined the protective properties of BAT against lipopolysaccharide (LPS)­mediated inflammation in vitro, by using LPS­stimulated murine J774.A1 macrophages (Mφs), as well as in vivo, by using a murine LPS­mediated air­pouch model. Additionally, its efficacy was compared with that of taurine, a known potent anti­inflammatory molecule. In LPS­stimulated J774A.1 Mφs, BAT and taurine were very effective in reducing the secretion of pro­inflammatory mediators. The in vitro experiments indicated that LPS­mediated inflammation was attenuated due to the protective properties of BAT and of taurine, probably through the inhibition of phosphorylated p65 NF­κB subunit (Ser 536) nuclear translocation. The in vivo experiments also revealed that BAT and taurine inhibited LPS­mediated inflammation by reducing total cell/polymorphonuclear cell (PMN) infiltration in the air­pouch and by decreasing pouch wall thickness. The analysis of exudates obtained from pouches highlighted that the inhibitory effects of BAT and taurine on the secretion of pro­inflammatory cytokines were similar to those observed in vitro. Notably, the effect of BAT at the highest concentration tested was superior to that of taurine at the highest concentration. Taken together, the findings of the present study indicate that BAT prevents the LPS­induced inflammatory response both in vitro and in vivo.


Asunto(s)
Bromo/uso terapéutico , Inflamación/tratamiento farmacológico , Sulfonamidas/uso terapéutico , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Bromo/farmacología , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/patología , Mediadores de Inflamación/metabolismo , Lipopolisacáridos , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sulfonamidas/farmacología , Taurina/farmacología , Factor de Transcripción ReIA/metabolismo , Transcripción Genética/efectos de los fármacos
5.
Biochem J ; 478(2): 407-422, 2021 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-33393983

RESUMEN

Insulin stimulates glucose uptake in muscle cells by rapidly redistributing vesicles containing GLUT4 glucose transporters from intracellular compartments to the plasma membrane (PM). GLUT4 vesicle fusion requires the formation of SNARE complexes between vesicular VAMP and PM syntaxin4 and SNAP23. SNARE accessory proteins usually regulate vesicle fusion processes. Complexins aide in neuro-secretory vesicle-membrane fusion by stabilizing trans-SNARE complexes but their participation in GLUT4 vesicle fusion is unknown. We report that complexin-2 is expressed and homogeneously distributed in L6 rat skeletal muscle cells. Upon insulin stimulation, a cohort of complexin-2 redistributes to the PM. Complexin-2 knockdown markedly inhibited GLUT4 translocation without affecting proximal insulin signalling of Akt/PKB phosphorylation and actin fiber remodelling. Similarly, complexin-2 overexpression decreased maximal GLUT4 translocation suggesting that the concentration of complexin-2 is finely tuned to vesicle fusion. These findings reveal an insulin-dependent regulation of GLUT4 insertion into the PM involving complexin-2.


Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , Mioblastos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Transportador de Glucosa de Tipo 4/genética , Insulina/genética , Insulina/metabolismo , Músculo Esquelético/citología , Mioblastos/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismo
6.
Nat Commun ; 12(1): 61, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33397928

RESUMEN

Coat protein complex I (COP-I) mediates the retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER). Mutation of the COPA gene, encoding one of the COP-I subunits (α-COP), causes an immune dysregulatory disease known as COPA syndrome. The molecular mechanism by which the impaired retrograde transport results in autoinflammation remains poorly understood. Here we report that STING, an innate immunity protein, is a cargo of the retrograde membrane transport. In the presence of the disease-causative α-COP variants, STING cannot be retrieved back to the ER from the Golgi. The forced Golgi residency of STING results in the cGAS-independent and palmitoylation-dependent activation of the STING downstream signaling pathway. Surf4, a protein that circulates between the ER/ ER-Golgi intermediate compartment/ Golgi, binds STING and α-COP, and mediates the retrograde transport of STING to the ER. The STING/Surf4/α-COP complex is disrupted in the presence of the disease-causative α-COP variant. We also find that the STING ligand cGAMP impairs the formation of the STING/Surf4/α-COP complex. Our results suggest a homeostatic regulation of STING at the resting state by retrograde membrane traffic and provide insights into the pathogenesis of COPA syndrome.


Asunto(s)
Retículo Endoplásmico/metabolismo , Homeostasis , Proteínas de la Membrana/metabolismo , Animales , Brefeldino A/farmacología , Vesículas Cubiertas por Proteínas de Revestimiento/efectos de los fármacos , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/ultraestructura , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/ultraestructura , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Células HEK293 , Humanos , Lipoilación , Luciferasas/metabolismo , Ratones , Nucleotidiltransferasas/metabolismo , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos
7.
Biochim Biophys Acta Gen Subj ; 1865(3): 129829, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33340587

RESUMEN

BACKGROUND: Iron export via the transport protein ferroportin (Fpn) plays a critical role in the regulation of dietary iron absorption and iron recycling in macrophages. Fpn plasma membrane expression is controlled by the hepatic iron-regulated hormone hepcidin in response to high iron availability and inflammation. Hepcidin binds to the central cavity of the Fpn transporter to block iron export either directly or by inducing Fpn internalization and lysosomal degradation. Here, we investigated whether iron deficiency affects Fpn protein turnover. METHODS: We ectopically expressed Fpn in HeLa cells and used cycloheximide chase experiments to study basal and hepcidin-induced Fpn degradation under extracellular and intracellular iron deficiency. CONCLUSIONS/GENERAL SIGNIFICANCE: We show that iron deficiency does not affect basal Fpn turnover but causes a significant delay in hepcidin-induced degradation when cytosolic iron levels are low. These data have important mechanistic implications supporting the hypothesis that iron export is required for efficient targeting of Fpn by hepcidin. Additionally, we show that Fpn degradation is not involved in protecting cells from intracellular iron deficiency.


Asunto(s)
Proteínas de Transporte de Catión/genética , Hepcidinas/genética , Hierro/deficiencia , Proteínas de Transporte de Catión/metabolismo , Cicloheximida/farmacología , Deferoxamina/farmacología , Regulación de la Expresión Génica , Células HeLa , Hepcidinas/metabolismo , Humanos , Transporte Iónico/efectos de los fármacos , Quelantes del Hierro/farmacología , Unión Proteica/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Transporte de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Transducción de Señal
8.
Poult Sci ; 100(1): 26-38, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33357689

RESUMEN

Duck enteritis virus (DEV) multifunctional tegument protein UL13 is predicted to be a conserved herpesvirus protein kinase; however, little is known about its subcellular localization signal. In this study, through transfection of 2 predicted nuclear signals of DEV UL13 fused to enhanced green fluorescent protein, 2 bipartite nuclear localization signals (NLS) were identified. We found that ivermectin blocked the NLS-mediated nuclear import of DEV UL13, showing that the nuclear localization signal of DEV UL13 is a classical importin α- and ß-dependent process. We constructed a DEV UL13 mutant strain in which the NLS of DEV UL13 was deleted to explore whether deletion of the NLS affects viral replication. Amino acids 4 to 7 and 90 to 96 were predicted to be NLSs, further proving that nuclear import occurs via a classical importin α- and ß-dependent process. We also found that the NLS of pUL13 had no effect on DEV replication in cell culture. Our study enhances the understanding of DEV pUL13. Taken together, these results provide significant information regarding the biological function of pUL13 during DEV infection.


Asunto(s)
Enteritis , Mardivirus , Señales de Localización Nuclear , Proteínas Quinasas , Animales , Antiparasitarios/farmacología , Células Cultivadas , Patos , Enteritis/fisiopatología , Enteritis/veterinaria , Enteritis/virología , Espacio Intracelular/metabolismo , Espacio Intracelular/virología , Ivermectina/farmacología , Mardivirus/genética , Mardivirus/metabolismo , Mutación , Señales de Localización Nuclear/efectos de los fármacos , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética
9.
Biomed Pharmacother ; 133: 111032, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33378945

RESUMEN

Insulin resistance is associated with obesity and can lead to several metabolic disorders including type II diabetes, nonalcoholic fatty liver disease and cardiovascular problems. Search for the small molecules which can either induce or mimic the insulin action are of great interest and can be utilized to manage insulin resistance. There are several dietary phytochemicals which can potentially have insulinomimetic action. Nevertheless, high throughput screening methods to test efficiency of small molecules to act as an insulinomimetic are not fully established. In this paper we have performed chemical screen analysis based on GLUT4 translocation using a cell line CHO-HIRC-myc-GLUT4 eGFP that expresses GLUT4-GFP in association with human Insulin receptor. We have established a high content screening-based method which can track and quantify the GLUT4 translocation from perinuclear area to the cell membrane. The assay involves measuring fluorescence intensity in a defined perinuclear area and a defined area along the cell membrane; and the results are expressed as the ratio of fluorescence intensity in the perinuclear to membrane area. The assay could collect real time data of GLUT4 translocation from thousand of cells/ sample and from many such samples in one experiment. We validated the assay using Insulin, insulin mimics/sensitizers and insulin inhibitors. The agonist or antagonists were analyzed for their ability to enhance or block the GLUT4 translocation independent of insulin. The outcome of the assay was correlated by performing glucose uptake assay using differentiated 3T3L1 cells. Using this platform we further identified several plant extracts which had the insulin mimetic action. We confirmed that these plant extracts were non-toxic to the beta cells using RIN mf5cells and 3T3L1 cells. We have identified plant extracts with the potential insulinomimetic action using novel high-content screening approach; these can be further tested for their efficiency in-vivo in pre-clinical trials.


Asunto(s)
Adipocitos/metabolismo , Bioensayo , Transportador de Glucosa de Tipo 4/metabolismo , Hepatocitos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Células 3T3 , Adipocitos/efectos de los fármacos , Animales , Células CHO , Cricetulus , Transportador de Glucosa de Tipo 4/genética , Células Hep G2 , Hepatocitos/efectos de los fármacos , Humanos , Hipoglucemiantes/farmacología , Insulina/farmacología , Resistencia a la Insulina , Ratones , Transporte de Proteínas/efectos de los fármacos , Reproducibilidad de los Resultados , Factores de Tiempo
10.
Sci Adv ; 6(35): eaba7910, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32923629

RESUMEN

Targeting a universal host protein exploited by most viruses would be a game-changing strategy that offers broad-spectrum solution and rapid pandemic control including the current COVID-19. Here, we found a common YxxØ-motif of multiple viruses that exploits host AP2M1 for intracellular trafficking. A library chemical, N-(p-amylcinnamoyl)anthranilic acid (ACA), was identified to interrupt AP2M1-virus interaction and exhibit potent antiviral efficacy against a number of viruses in vitro and in vivo, including the influenza A viruses (IAVs), Zika virus (ZIKV), human immunodeficiency virus, and coronaviruses including MERS-CoV and SARS-CoV-2. YxxØ mutation, AP2M1 depletion, or disruption by ACA causes incorrect localization of viral proteins, which is exemplified by the failure of nuclear import of IAV nucleoprotein and diminished endoplasmic reticulum localization of ZIKV-NS3 and enterovirus-A71-2C proteins, thereby suppressing viral replication. Our study reveals an evolutionarily conserved mechanism of protein-protein interaction between host and virus that can serve as a broad-spectrum antiviral target.


Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Antivirales/farmacología , Cinamatos/farmacología , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por VIH/tratamiento farmacológico , Gripe Humana/tratamiento farmacológico , Neumonía Viral/tratamiento farmacológico , ortoaminobenzoatos/farmacología , Células A549 , Animales , Betacoronavirus/efectos de los fármacos , Sitios de Unión/genética , Línea Celular Tumoral , Chlorocebus aethiops , Infecciones por Coronavirus/patología , Perros , Células HEK293 , Infecciones por VIH/patología , VIH-1/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Virus de la Influenza A/efectos de los fármacos , Gripe Humana/patología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Pandemias , Neumonía Viral/patología , Unión Proteica/genética , Transporte de Proteínas/efectos de los fármacos , ARN Viral/genética , Receptor de Interferón alfa y beta/genética , Factor de Crecimiento Transformador beta1/metabolismo , Células Vero , Replicación Viral/efectos de los fármacos , Virus Zika/efectos de los fármacos , Infección por el Virus Zika/patología
11.
Life Sci ; 258: 118195, 2020 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-32781073

RESUMEN

AIMS: The estrogen-ERα axis participates in osteoblast maturation. This study was designed to further evaluated the roles of the estrogen-ERα axis in bone healing and the possible mechanisms. MAIN METHODS: Female ICR mice were created a metaphyseal bone defect in the left femurs and administered with methylpiperidinopyrazole (MPP), an inhibitor of ERα. Bone healing was evaluated using micro-computed tomography. Colocalization of ERα with alkaline phosphatase (ALP) and ERα translocation to mitochondria were determined. Levels of ERα, ERß, PECAM-1, VEGF, and ß-actin were immunodetected. Expression of chromosomal Runx2, ALP, and osteocalcin mRNAs and mitochondrial cytochrome c oxidase (COX) I and COXII mRNAs were quantified. Angiogenesis was measured with immunohistochemistry. KEY FINDINGS: Following surgery, the bone mass was time-dependently augmented in the bone-defect area. Simultaneously, levels of ERα were specifically upregulated and positively correlated with bone healing. Administration of MPP to mice consistently decreased levels of ERα and bone healing. As to the mechanisms, osteogenesis was enhanced in bone healing, but MPP attenuated osteoblast maturation. In parallel, expressions of osteogenesis-related ALP, Runx2, and osteocalcin mRNAs were induced in the injured zone. Treatment with MPP led to significant inhibition of the alp, runx2, and osteocalcin gene expressions. Remarkably, administration of MPP lessened translocation of ERα to mitochondria and expressions of mitochondrial energy production-related coxI and coxII genes. Furthermore, exposure to MPP decreased levels of PECAM-1 and VEGF in the bone-defect area. SIGNIFICANCE: The present study showed the contributions of the estrogen-ERα axis to bone healing through stimulation of energy production, osteoblast maturation, and angiogenesis.


Asunto(s)
Regeneración Ósea , Diferenciación Celular , Metabolismo Energético , Receptor alfa de Estrógeno/metabolismo , Neovascularización Fisiológica , Osteoblastos/citología , Transducción de Señal , Fosfatasa Alcalina/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Regeneración Ósea/efectos de los fármacos , Callo Óseo/efectos de los fármacos , Callo Óseo/patología , Diferenciación Celular/efectos de los fármacos , Cromosomas de los Mamíferos/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Metabolismo Energético/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Ratones Endogámicos ICR , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Tamaño de los Órganos/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteogénesis/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Pirazoles/administración & dosificación , Pirazoles/farmacología , Regulación hacia Arriba/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos
12.
PLoS One ; 15(8): e0237448, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32790800

RESUMEN

We established that Endosidin2 (ES2) affected the trafficking routes of both newly synthesized and endocytic pools of PIN-FORMED2 (PIN2) in Arabidopsis root epidermal cells. PIN2 populations accumulated in separated patches, which gradually merged into large and compact ES2 aggregates (ES2As). FM4-64 endocytic tracer labeled ES2As as well. Both PIN2 pools also appeared in vacuoles. Accelerated endocytosis of PIN2, its aggregation in the cytoplasm, and redirection of PIN2 flows to vacuoles led to a substantial reduction of the abundance of this protein in the plasma membrane. Whereas PIN-FORMED3 and PIN-FORMED4 also aggregated in the cytoplasm, SYT1 was not sensitive to ES2 treatment and did not appear either in the cytoplasmic aggregates or vacuoles. Ultrastructural analysis revealed that ES2 affects the Golgi apparatus so that stacks acquired cup-shape and even circular shape surrounded by several vesicles. Abnormally shaped Golgi stacks, stack remnants, multi-lamellar structures, separated Golgi cisterna rings, tubular structures, and vesicles formed discrete clusters.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Endocitosis/efectos de los fármacos , Limoninas/farmacología , Proteínas de Transporte de Membrana/metabolismo , Pared Celular/metabolismo , Citoplasma/metabolismo , Aparato de Golgi/metabolismo , Raíces de Plantas/citología , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Transporte de Proteínas/efectos de los fármacos , Sinaptotagmina I/metabolismo
13.
PLoS One ; 15(7): e0236185, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32730344

RESUMEN

Fluorescent markers are a powerful tool and have been widely applied in biology for different purposes. The genome sequence of Xanthomonas citri subsp. citri (X. citri) revealed that approximately 30% of the genes encoded hypothetical proteins, some of which could play an important role in the success of plant-pathogen interaction and disease triggering. Therefore, revealing their functions is an important strategy to understand the bacterium pathways and mechanisms involved in plant-host interaction. The elucidation of protein function is not a trivial task, but the identification of the subcellular localization of a protein is key to understanding its function. We have constructed an integrative vector, pMAJIIc, under the control of the arabinose promoter, which allows the inducible expression of red fluorescent protein (mCherry) fusions in X. citri, suitable for subcellular localization of target proteins. Fluorescence microscopy was used to track the localization of VrpA protein, which was visualized surrounding the bacterial outer membrane, and the GyrB protein, which showed a diffused cytoplasmic localization, sometimes with dots accumulated near the cellular poles. The integration of the vector into the amy locus of X. citri did not affect bacterial virulence. The vector could be stably maintained in X. citri, and the disruption of the α-amylase gene provided an ease screening method for the selection of the transformant colonies. The results demonstrate that the mCherry-containing vector here described is a powerful tool for bacterial protein localization in cytoplasmic and periplasmic environments.


Asunto(s)
Proteínas Bacterianas/metabolismo , Citoplasma/metabolismo , Periplasma/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Xanthomonas/metabolismo , Arabinosa/farmacología , Cromosomas Bacterianos/genética , Vectores Genéticos/metabolismo , Viabilidad Microbiana/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Almidón/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Xanthomonas/patogenicidad
14.
J Med Chem ; 63(15): 8114-8133, 2020 08 13.
Artículo en Inglés | MEDLINE | ID: mdl-32648758

RESUMEN

High-throughput screening has shown that Retro-1 inhibits ricin and Shiga toxins by diminishing their intracellular trafficking via the retrograde route, from early endosomes to the Golgi apparatus. To improve the activity of Retro-1, a structure-activity relationship (SAR) study was undertaken and yielded an analogue with a roughly 70-fold better half-maximal effective concentration (EC50) against Shiga toxin cytotoxicity measured in a cell protein synthesis assay.


Asunto(s)
Benzodiazepinonas/química , Benzodiazepinonas/farmacología , Toxinas Shiga/antagonistas & inhibidores , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Toxinas Shiga/metabolismo , Relación Estructura-Actividad
15.
Mol Cell ; 79(3): 443-458.e7, 2020 08 06.
Artículo en Inglés | MEDLINE | ID: mdl-32649883

RESUMEN

Despite the prominent role of TDP-43 in neurodegeneration, its physiological and pathological functions are not fully understood. Here, we report an unexpected role of TDP-43 in the formation of dynamic, reversible, liquid droplet-like nuclear bodies (NBs) in response to stress. Formation of NBs alleviates TDP-43-mediated cytotoxicity in mammalian cells and fly neurons. Super-resolution microscopy reveals distinct functions of the two RRMs in TDP-43 NB formation. TDP-43 NBs are partially colocalized with nuclear paraspeckles, whose scaffolding lncRNA NEAT1 is dramatically upregulated in stressed neurons. Moreover, increase of NEAT1 promotes TDP-43 liquid-liquid phase separation (LLPS) in vitro. Finally, we discover that the ALS-associated mutation D169G impairs the NEAT1-mediated TDP-43 LLPS and NB assembly, causing excessive cytoplasmic translocation of TDP-43 to form stress granules, which become phosphorylated TDP-43 cytoplasmic foci upon prolonged stress. Together, our findings suggest a stress-mitigating role and mechanism of TDP-43 NBs, whose dysfunction may be involved in ALS pathogenesis.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Cuerpos de Inclusión Intranucleares/metabolismo , Neuronas/metabolismo , ARN Largo no Codificante/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Animales Modificados Genéticamente , Arsenitos/farmacología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/ultraestructura , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Drosophila melanogaster , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Cuerpos de Inclusión Intranucleares/efectos de los fármacos , Cuerpos de Inclusión Intranucleares/ultraestructura , Ratones , Mutación , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Cultivo Primario de Células , Transporte de Proteínas/efectos de los fármacos , ARN Largo no Codificante/metabolismo , Transducción de Señal , Estrés Fisiológico
16.
Nat Chem Biol ; 16(7): 766-775, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32483376

RESUMEN

Cell surfaces are glycosylated in various ways with high heterogeneity, which usually leads to ambiguous conclusions about glycan-involved biological functions. Here, we describe a two-step chemoenzymatic approach for N-glycan-subtype-selective editing on the surface of living cells that consists of a first 'delete' step to remove heterogeneous N-glycoforms of a certain subclass and a second 'insert' step to assemble a well-defined N-glycan back onto the pretreated glyco-sites. Such glyco-edited cells, carrying more homogeneous oligosaccharide structures, could enable precise understanding of carbohydrate-mediated functions. In particular, N-glycan-subtype-selective remodeling and imaging with different monosaccharide motifs at the non-reducing end were successfully achieved. Using a combination of the expression system of the Lec4 CHO cell line and this two-step glycan-editing approach, opioid receptor delta 1 (OPRD1) was investigated to correlate its glycostructures with the biological functions of receptor dimerization, agonist-induced signaling and internalization.


Asunto(s)
Membrana Celular/química , Células Epiteliales/química , Glicoconjugados/química , Oligosacáridos/química , Receptores Opioides delta/química , Animales , Células CHO , Línea Celular Tumoral , Membrana Celular/metabolismo , Colforsina/farmacología , Cricetulus , Encefalina Leucina/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Expresión Génica , Glicoconjugados/metabolismo , Glicosilación , Células HEK293 , Humanos , Ratones , Oligosacáridos/metabolismo , Multimerización de Proteína/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismo , Transgenes
18.
Sci Rep ; 10(1): 7856, 2020 05 12.
Artículo en Inglés | MEDLINE | ID: mdl-32398691

RESUMEN

Copper (Cu) is an essential, yet potentially toxic nutrient, as illustrated by inherited diseases of copper deficiency and excess. Elevated expression of the ATP7A Cu exporter is known to confer copper tolerance, however, the contribution of metal-binding metallothioneins is less clear. In this study, we investigated the relative contributions of ATP7A and the metallothioneins MT-I and MT-II to cell viability under conditions of Cu excess or deficiency. Although the loss of ATP7A increased sensitivity to low Cu concentrations, the absence of MTs did not significantly affect Cu tolerance. However, the absence of all three proteins caused a synthetic lethal phenotype due to extreme Cu sensitivity, indicating that MTs are critical for Cu tolerance only in the absence of ATP7A. A lack of MTs resulted in the trafficking of ATP7A from the trans-Golgi complex in a Cu-dependent manner, suggesting that MTs regulate the delivery of Cu to ATP7A. Under Cu deficiency conditions, the absence of MTs and / or ATP7A enhanced cell proliferation compared to wild type cells, suggesting that these proteins compete with essential Cu-dependent pathways when Cu is scarce. These studies reveal new roles for ATP7A and metallothioneins under both Cu deficiency and excess.


Asunto(s)
ATPasas Transportadoras de Cobre/metabolismo , Cobre/farmacología , Metalotioneína/metabolismo , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , ATPasas Transportadoras de Cobre/deficiencia , ATPasas Transportadoras de Cobre/genética , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Metalotioneína/deficiencia , Metalotioneína/genética , Ratones , Mutación , Transporte de Proteínas/efectos de los fármacos
19.
Nature ; 581(7806): 83-88, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32376950

RESUMEN

Photoreceptor loss is the final common endpoint in most retinopathies that lead to irreversible blindness, and there are no effective treatments to restore vision1,2. Chemical reprogramming of fibroblasts offers an opportunity to reverse vision loss; however, the generation of sensory neuronal subtypes such as photoreceptors remains a challenge. Here we report that the administration of a set of five small molecules can chemically induce the transformation of fibroblasts into rod photoreceptor-like cells. The transplantation of these chemically induced photoreceptor-like cells (CiPCs) into the subretinal space of rod degeneration mice (homozygous for rd1, also known as Pde6b) leads to partial restoration of the pupil reflex and visual function. We show that mitonuclear communication is a key determining factor for the reprogramming of fibroblasts into CiPCs. Specifically, treatment with these five compounds leads to the translocation of AXIN2 to the mitochondria, which results in the production of reactive oxygen species, the activation of NF-κB and the upregulation of Ascl1. We anticipate that CiPCs could have therapeutic potential for restoring vision.


Asunto(s)
Reprogramación Celular/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Degeneración Retiniana/terapia , Células Fotorreceptoras Retinianas Bastones/citología , Células Fotorreceptoras Retinianas Bastones/trasplante , Visión Ocular/efectos de los fármacos , Animales , Proteína Axina/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Modelos Animales de Enfermedad , Citometría de Flujo , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Transporte de Proteínas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Visión Ocular/fisiología
20.
Cancer Sci ; 111(7): 2508-2525, 2020 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-32415868

RESUMEN

Human epidermal growth factor receptor 4 (HER4) isoforms have oncogenic or tumor suppressor functions depending on their susceptibility to proteolytic cleavage and HER4 intracellular domain (4ICD) translocation. Here, we report that the neuregulin 1 (NRG1) tumor suppressor mechanism through the HER4 JMa/CYT1 isoform can be mimicked by the agonist anti-HER4 Ab C6. Neuregulin 1 induced cleavage of poly(ADP-ribose) polymerase (PARP) and sub-G1 DNA fragmentation, and also reduced the metabolic activity of HER3- /HER4+ cervical (C-33A) and ovarian (COV318) cancer cells. This effect was confirmed in HER4 JMa/CYT1-, but not JMa/CYT2-transfected BT549 triple-negative breast cancer cells. Neuregulin 1 favored 4ICD cleavage and retention in mitochondria in JMa/CYT1-transfected BT549 cells, leading to reactive oxygen species (ROS) production through mitochondrial depolarization. Similarly, the anti-HER4 Ab C6, which binds to a conformational epitope located on a.a. 575-592 and 605-620 of HER4 domain IV, induced 4ICD cleavage and retention in mitochondria, and mimicked NRG1-mediated effects on PARP cleavage, ROS production, and mitochondrial membrane depolarization in cancer cells. In vivo, C6 reduced growth of COV434 and HCC1187 tumor cell xenografts in nude mice. Biasing 4ICD trafficking to mitochondria with anti-HER4 Abs to mimic NRG1 suppressor functions could be an alternative anticancer strategy.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Receptor ErbB-4/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Anticuerpos Monoclonales/inmunología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Mapeo Epitopo , Humanos , Espacio Intracelular/metabolismo , Ratones , Mitocondrias/metabolismo , Neurregulina-1/farmacología , Transporte de Proteínas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Receptor ErbB-4/inmunología
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