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1.
Sci Adv ; 6(35): eaba7910, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32923629

RESUMO

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.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antivirais/farmacologia , Cinamatos/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por HIV/tratamento farmacológico , Influenza Humana/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , ortoaminobenzoatos/farmacologia , Células A549 , Animais , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Cães , Células HEK293 , Infecções por HIV/patologia , HIV-1/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/patologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Pandemias , Pneumonia Viral/patologia , Ligação Proteica/genética , Transporte Proteico/efeitos dos fármacos , RNA Viral/genética , Receptor de Interferon alfa e beta/genética , Fator de Crescimento Transformador beta1/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Infecção por Zika virus/patologia
2.
Sci Adv ; 6(35): eaba7910, 2020 08.
Artigo em Inglês | MEDLINE | ID: covidwho-760200

RESUMO

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.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antivirais/farmacologia , Cinamatos/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por HIV/tratamento farmacológico , Influenza Humana/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , ortoaminobenzoatos/farmacologia , Células A549 , Animais , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Cães , Células HEK293 , Infecções por HIV/patologia , HIV-1/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/patologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Pandemias , Pneumonia Viral/patologia , Ligação Proteica/genética , Transporte Proteico/efeitos dos fármacos , RNA Viral/genética , Receptor de Interferon alfa e beta/genética , Fator de Crescimento Transformador beta1/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Infecção por Zika virus/patologia
3.
PLoS One ; 15(8): e0237448, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790800

RESUMO

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.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Endocitose/efeitos dos fármacos , Limoninas/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Parede Celular/metabolismo , Citoplasma/metabolismo , Complexo de Golgi/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Transporte Proteico/efeitos dos fármacos , Sinaptotagmina I/metabolismo
4.
Life Sci ; 258: 118195, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32781073

RESUMO

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.


Assuntos
Regeneração Óssea , Diferenciação Celular , Metabolismo Energético , Receptor alfa de Estrogênio/metabolismo , Neovascularização Fisiológica , Osteoblastos/citologia , Transdução de Sinais , Fosfatase Alcalina/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Regeneração Óssea/efeitos dos fármacos , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/patologia , Diferenciação Celular/efeitos dos fármacos , Cromossomos de Mamíferos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Camundongos Endogâmicos ICR , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Regulação para Cima/efeitos dos fármacos , Cicatrização/efeitos dos fármacos
5.
Mol Cell ; 79(3): 443-458.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32649883

RESUMO

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.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteínas de Ligação a DNA/genética , Corpos de Inclusão Intranuclear/metabolismo , Neurônios/metabolismo , RNA Longo não Codificante/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Animais Geneticamente Modificados , Arsenitos/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/ultraestrutura , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Drosophila melanogaster , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Corpos de Inclusão Intranuclear/efeitos dos fármacos , Corpos de Inclusão Intranuclear/ultraestrutura , Camundongos , Mutação , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Cultura Primária de Células , Transporte Proteico/efeitos dos fármacos , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Estresse Fisiológico
6.
PLoS One ; 15(7): e0236185, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730344

RESUMO

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.


Assuntos
Proteínas de Bactérias/metabolismo , Citoplasma/metabolismo , Periplasma/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Xanthomonas/metabolismo , Arabinose/farmacologia , Cromossomos Bacterianos/genética , Vetores Genéticos/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Amido/metabolismo , Frações Subcelulares/efeitos dos fármacos , Xanthomonas/patogenicidade
7.
Nat Chem Biol ; 16(7): 766-775, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32483376

RESUMO

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.


Assuntos
Membrana Celular/química , Células Epiteliais/química , Glicoconjugados/química , Oligossacarídeos/química , Receptores Opioides delta/química , Animais , Células CHO , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Colforsina/farmacologia , Cricetulus , Encefalina Leucina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Expressão Gênica , Glicoconjugados/metabolismo , Glicosilação , Células HEK293 , Humanos , Camundongos , Oligossacarídeos/metabolismo , Multimerização Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismo , Transgenes
8.
Nature ; 581(7806): 83-88, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32376950

RESUMO

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.


Assuntos
Reprogramação Celular/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Degeneração Retiniana/terapia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/transplante , Visão Ocular/efeitos dos fármacos , Animais , Proteína Axina/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NF-kappa B/metabolismo , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Visão Ocular/fisiologia
9.
Cancer Sci ; 111(7): 2508-2525, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32415868

RESUMO

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.


Assuntos
Anticorpos Monoclonais/farmacologia , Receptor ErbB-4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Mapeamento de Epitopos , Humanos , Espaço Intracelular/metabolismo , Camundongos , Mitocôndrias/metabolismo , Neuregulina-1/farmacologia , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptor ErbB-4/imunologia
10.
Clin Sci (Lond) ; 134(10): 1167-1180, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32458968

RESUMO

In the present study, we evaluated the metabolic effects of green tea polyphenols (GTPs) in high-fat diet (HFD) fed Zucker fatty (ZF) rats, in particular the effects of GTP on skeletal muscle insulin sensitivity. Body weight, visceral fat, glucose tolerance, lipid profiles and whole-body insulin sensitivity were measured in HFD-fed ZF rats after 8-week-treatment with GTP (200 mg/kg of body weight) or saline (5 ml/kg of body weight). Zucker lean rats were studied as controls. Ex vivo insulin-mediated muscle glucose uptake was assessed. Immunoblotting was used to evaluate the expression of key insulin signalling proteins in skeletal muscle. GTP treatment attenuated weight gain (P<0.05) and visceral fat accumulation (27.6%, P<0.05), and significantly reduced fasting serum glucose (P<0.05) and insulin (P<0.01) levels. Homoeostasis model assessment of insulin resistance (HOMA-IR), a measure of insulin resistance, was lower (P<0.01) in GTP-treated animals compared with ZF controls. Moreover, insulin-stimulated glucose uptake by isolated soleus muscle was increased (P<0.05) in GTP-ZF rats compared with ZF-controls. GTP treatment attenuated the accumulation of ectopic lipids (triacyl- and diacyl-glycerols), enhanced the expression and translocation of glucose transporter-4, and decreased pSer612IRS-1 and increased pSer473Akt2 expression in skeletal muscle. These molecular changes were also associated with significantly decreased activation of the inhibitory (muscle-specific) protein kinase (PKC) isoform, PKC-θ. Taken together, the present study has shown that regular ingestion of GTP exerts a number of favourable metabolic and molecular effects in an established animal model of obesity and insulin resistance. The benefits of GTP are mediated in part by inhibiting PKC-θ and improving muscle insulin sensitivity.


Assuntos
Resistência à Insulina , Insulina/metabolismo , Músculo Esquelético/metabolismo , Polifenóis/farmacologia , Transdução de Sinais , Chá/química , Adiposidade/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Glucose/metabolismo , Teste de Tolerância a Glucose , Transportador de Glucose Tipo 4/metabolismo , Gordura Intra-Abdominal/efeitos dos fármacos , Gordura Intra-Abdominal/metabolismo , Isoenzimas/metabolismo , Masculino , Proteínas Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos Zucker
13.
PLoS One ; 15(4): e0231212, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32275682

RESUMO

Two major proteolytic systems, the proteasome and the autophagy pathway, are key components of the proteostasis network. The immunoproteasome, a proteasome subtype, and autophagy are upregulated under stress conditions, forming a coordinated unit designed to minimize the effect of cell stress. We investigated how genetic ablation of the LMP2 immunoproteasome subunit affects autophagy in retinal pigment epithelium (RPE) from WT and LMP2 knockout mice. We monitored autophagy regulation by measuring LC3, phosphorylation of AKT (S473), and phosphorylation of S6, a downstream readout of AKT (mTOR) pathway activation. We also evaluated transcription factor EB (TFEB) nuclear translocation, a transcription factor that controls expression of autophagy and lysosome genes. WT and LMP2 KO cells were monitored after treatment with EBSS to stimulate autophagy, insulin to stimulate AKT, or an AKT inhibitor (trehalose or MK-2206). Under basal conditions, we observed hyper-phosphorylation of AKT and S6, as well as lower nuclear-TFEB content in LMP2 KO RPE compared with WT. AKT inhibitors MK-2206 and trehalose significantly inhibited AKT phosphorylation and stimulated nuclear translocation of TFEB. Starvation and AKT inhibition upregulated autophagy, albeit to a lesser extent in LMP2 KO RPE. These data support the idea that AKT hyper-activation is an underlying cause of defective autophagy regulation in LMP2 KO RPE, revealing a unique link between two proteolytic systems and a previously unknown function in autophagy regulation by the immunoproteasome.


Assuntos
Autofagia , Complexo de Endopeptidases do Proteassoma/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Retina/citologia , Animais , Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Células Cultivadas , Cisteína Endopeptidases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Insulina/farmacologia , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Epitélio Pigmentado da Retina/citologia , Transdução de Sinais/efeitos dos fármacos
14.
Phytomedicine ; 68: 153178, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32126492

RESUMO

BACKGROUND: Lowering blood glucose levels by increasing glucose uptake in insulin target tissues, such as skeletal muscle and adipose tissue, is one strategy to discover and develop antidiabetic drugs from natural products used as traditional medicines. PURPOSE: Our goal was to reveal the mechanism and activity of acacetin (5,7-dihydroxy-4'-methoxyflavone), one of the major compounds in Agastache rugose, in stimulating glucose uptake in muscle cells. METHODS: To determine whether acacetin promotes GLUT4-dependent glucose uptake in cultured L6 skeletal muscle cells, we performed a [14C] 2-deoxy-D-glucose (2-DG) uptake assay after treating differentiated L6-GLUT4myc cells with acacetin. RESULTS: Acacetin dose-dependently increased 2-DG uptake by enhancing GLUT4 translocation to the plasma membrane. Our results revealed that acacetin activated the CaMKII-AMPK pathway by increasing intracellular calcium concentrations. We also found that aPKCλ/ζ phosphorylation and intracellular reactive oxygen species (ROS) production were involved in acacetin-induced GLUT4 translocation. Moreover, acacetin-activated AMPK inhibited intracellular lipid accumulation and increased 2-DG uptake in HepG2 cells. CONCLUSION: Taken together, these results suggest that acacetin might be useful as an antidiabetic functional ingredient. Subsequent experiments using disease model animals are needed to verify our results.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Flavonas/farmacologia , Glucose/metabolismo , Insulina/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Desoxiglucose/farmacocinética , Relação Dose-Resposta a Droga , Glucose/farmacocinética , Transportador de Glucose Tipo 4/metabolismo , Células Hep G2 , Humanos , Hipoglicemiantes/farmacologia , Fibras Musculares Esqueléticas/metabolismo , Fosforilação , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
15.
Biochem J ; 477(2): 359-380, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31899485

RESUMO

The lysosomal storage disorder Fabry disease is characterized by a deficiency of the lysosomal enzyme α-Galactosidase A. The observation that missense variants in the encoding GLA gene often lead to structural destabilization, endoplasmic reticulum retention and proteasomal degradation of the misfolded, but otherwise catalytically functional enzyme has resulted in the exploration of alternative therapeutic approaches. In this context, we have investigated proteostasis regulators (PRs) for their potential to increase cellular enzyme activity, and to reduce the disease-specific accumulation of the biomarker globotriaosylsphingosine in patient-derived cell culture. The PRs also acted synergistically with the clinically approved 1-deoxygalactonojirimycine, demonstrating the potential of combination treatment in a therapeutic application. Extensive characterization of the effective PRs revealed inhibition of the proteasome and elevation of GLA gene expression as paramount effects. Further analysis of transcriptional patterns of the PRs exposed a variety of genes involved in proteostasis as potential modulators. We propose that addressing proteostasis is an effective approach to discover new therapeutic targets for diseases involving folding and trafficking-deficient protein mutants.


Assuntos
Doença de Fabry/genética , Doenças por Armazenamento dos Lisossomos/genética , Proteostase/genética , alfa-Galactosidase/genética , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/uso terapêutico , Biomarcadores/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Doença de Fabry/tratamento farmacológico , Doença de Fabry/enzimologia , Doença de Fabry/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Doenças por Armazenamento dos Lisossomos/enzimologia , Doenças por Armazenamento dos Lisossomos/patologia , Lisossomos/enzimologia , Lisossomos/genética , Lisossomos/metabolismo , Mutação de Sentido Incorreto/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/metabolismo
16.
Int J Mol Sci ; 21(1)2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31947743

RESUMO

Proteasome inhibition is used therapeutically to induce proteotoxic stress and trigger apoptosis in cancer cells that are highly dependent on the proteasome. As a mechanism of resistance, inhibition of the cellular proteasome induces the synthesis of new, uninhibited proteasomes to restore proteasome activity and relieve proteotoxic stress in the cell, thus evading apoptosis. This evolutionarily conserved compensatory mechanism is referred to as the proteasome-bounce back response and is orchestrated in mammalian cells by nuclear factor erythroid derived 2-related factor 1 (NRF1), a transcription factor and master regulator of proteasome subunit genes. Upon synthesis, NRF1 is cotranslationally inserted into the endoplasmic reticulum (ER), then is rapidly retrotranslocated into the cytosol and degraded by the proteasome. In contrast, during conditions of proteasome inhibition or insufficiency, NRF1 escapes degradation, is proteolytically cleaved by the aspartyl protease DNA damage inducible 1 homolog 2 (DDI2) to its active form, and enters the nucleus as an active transcription factor. Despite these insights, the cellular compartment where the proteolytic processing step occurs remains unclear. Here we further probed this pathway and found that NRF1 can be completely retrotranslocated into the cytosol where it is then cleaved and activated by DDI2. Furthermore, using a triple-negative breast cancer cell line MDA-MB-231, we investigated the therapeutic utility of attenuating DDI2 function. We found that DDI2 depletion attenuated NRF1 activation and potentiated the cytotoxic effects of the proteasome inhibitor carfilzomib. More importantly, expression of a point-mutant of DDI2 that is protease-dead recapitulated these effects. Taken together, our results provide a strong rationale for a combinational therapy that utilizes inhibition of the proteasome and the protease function of DDI2. This approach could expand the repertoire of cancer types that can be successfully treated with proteasome inhibitors in the clinic.


Assuntos
Ácido Aspártico Proteases/metabolismo , Fator 1 Nuclear Respiratório/metabolismo , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Citosol/efeitos dos fármacos , Citosol/metabolismo , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Transporte Proteico/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos
17.
Clin Sci (Lond) ; 134(3): 331-347, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31967309

RESUMO

G protein-coupled receptor kinase 2 (GRK2), a type of cytosolic enzyme, transiently translocates to the plasma membrane upon G protein-coupled receptors (GPCRs) activation, and it also binds to extracellular signal-regulated kinase (ERK) to inhibit the activation of ERK. GRK2 deficiency in endothelial cells (ECs) leads to increased pro-inflammatory signaling and promotes recruitment of leukocytes to activated ECs. However, the role of GRK2 in regulating angiogenesis remains unclear. Here, we show that GRK2 is a novel regulatory molecule on migration and tube formation of ECs, vessel sprouting ex vivo and angiogenesis in vivo. We identify that EP4/AC/cAMP/protein kinase A (PKA)-mediated GRK2 translocation to cells membrane decreases the binding of GRK2 and ERK1/2 to inhibit ERK1/2 activation, which promotes prostaglandin E2 (PGE2)-induced angiogenesis. GRK2 small interfering RNA (siRNA) inhibits the increase in PGE2-induced HUVECs migration and tube formation. In vivo, PGE2 increases ECs sprouting from normal murine aortic segments and angiogenesis in mice, but not from GRK2-deficient ones, on Matrigel. Further research found that Lys220 and Ser685 of GRK2 play an important role in angiogenesis by regulating GRK2 translocation. Paeoniflorin-6'-O-benzene sulfonate (CP-25), as a novel ester derivative of paeoniflorin (pae), has therapeutic potential for the treatment of adjuvant arthritis (AA) and collagen-induced arthritis (CIA), but the underlying mechanism of CP-25 on angiogenesis has not been elucidated. In our study, CP-25 inhibits the migration and tube formation of HUVECs, and angiogenesis in mice by down-regulating GRK2 translocation activation without affecting GRK2 total expression. Taken together, the present results revealed that CP-25 down-regulates EP4/AC/cAMP/PKA-mediated GRK2 translocation, restoring the inhibition of GRK2 for ERK1/2, thereby inhibiting PGE2-stimulated angiogenesis.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Dinoprostona/farmacologia , Regulação para Baixo/efeitos dos fármacos , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Glucosídeos/farmacologia , Monoterpenos/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Adenilil Ciclases/metabolismo , Animais , Artrite Experimental/patologia , Artrite Reumatoide/patologia , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Fenótipo , Transporte Proteico/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/patologia
18.
Biochem Pharmacol ; 174: 113813, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31954717

RESUMO

P-glycoprotein (Pgp) is an ATP-dependent efflux transporter and plays a major role in anti-cancer drug resistance by pumping a chemically diverse range of cytotoxic drugs from cancerous tumors. Despite numerous studies with the transporter, the molecular features that drive anti-cancer drug efflux are not well understood. Even subtle differences in the anti-cancer drug molecular structure can lead to dramatic differences in their transport rates. To unmask these structural differences, this study focused on two closely-related anthracycline drugs, daunorubicin (DNR), and doxorubicin (DOX), with mouse Pgp. While only differing by a single hydroxyl functional group, DNR has a 4 to 5-fold higher transport rate than DOX. They both non-competitively inhibited Pgp-mediated ATP hydrolysis below basal levels. The Km of Pgp-mediated ATP hydrolysis extracted from the kinetics curves was lower for DOX than DNR. However, the dissociation constants (KDs) for these drugs determined by fluorescence quenching were virtually identical. Acrylamide quenching of Pgp tryptophan fluorescence to probe the tertiary structure of Pgp suggested that DNR shifts Pgp to a "closed" conformation, while DOX shifts Pgp to an "intermediate" conformation. The effects of these drugs on the Pgp conformational distributions in a lipid bilayer were also examined by atomic force microscopy (AFM). Analysis of AFM images revealed that DNR and DOX cause distinct and significant shifts in the conformational distribution of Pgp. The results were combined to build a conformational distribution model for anthracycline transport by Pgp.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antraciclinas/farmacologia , Antibióticos Antineoplásicos/farmacologia , Daunorrubicina/farmacologia , Doxorrubicina/farmacologia , Animais , Relação Dose-Resposta a Droga , Camundongos , Conformação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia
19.
Neuron ; 105(4): 663-677.e8, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-31837915

RESUMO

A major function of GPCRs is to inhibit presynaptic neurotransmitter release, requiring ligand-activated receptors to couple locally to effectors at terminals. The current understanding of how this is achieved is through receptor immobilization on the terminal surface. Here, we show that opioid peptide receptors, GPCRs that mediate highly sensitive presynaptic inhibition, are instead dynamic in axons. Opioid receptors diffuse rapidly throughout the axon surface and internalize after ligand-induced activation specifically at presynaptic terminals. We delineate a parallel regulated endocytic cycle for GPCRs operating at the presynapse, separately from the synaptic vesicle cycle, which clears activated receptors from the surface of terminals and locally reinserts them to maintain the diffusible surface pool. We propose an alternate strategy for achieving local control of presynaptic effectors that, opposite to using receptor immobilization and enforced proximity, is based on lateral mobility of receptors and leverages the inherent allostery of GPCR-effector coupling.


Assuntos
Endocitose/fisiologia , Terminações Pré-Sinápticas/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Vesículas Sinápticas/metabolismo , Analgésicos Opioides/farmacologia , Animais , Células Cultivadas , Endocitose/efeitos dos fármacos , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas-G/agonistas , Receptores de Neurotransmissores/agonistas , Receptores de Neurotransmissores/metabolismo , Vesículas Sinápticas/efeitos dos fármacos
20.
Biochim Biophys Acta Mol Cell Res ; 1867(1): 118566, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31672613

RESUMO

Nuclear-cytoplasmic trafficking of proteins is a highly regulated process that modulates multiple biological processes in eukaryotic cells. In Giardia lamblia, shuttling has been described from the cytoplasm to nuclei of proteins during the biological cell cycle of the parasite. This suggests that a mechanism of nucleocytoplasmic transport is present and functional in G. lamblia. By means of computational biology analyses, we found that there are only two genes for nuclear transport in this parasite, named Importin α and Importin ß. When these transporters were overexpressed, both localized close to the nuclear envelope, and no change was observed in trophozoite growth rate. However, during the encystation process, both transporters induced an increase in the number of cysts produced. Importazole and Ivermectin, two known specific inhibitors of importins, separately influenced the encysting process by inducing an arrest in the trophozoite stage that prevents the production of cysts. This effect was more noticeable when Ivermectin, an anti-parasitic drug, was used. Finally, we tested whether the enzyme arginine deiminase, which shuttles from the cytoplasm to the nuclei during encystation, was influenced by these transporters. We found that treatment with each of the inhibitors abrogates arginine deiminase nuclear translocation and favors perinuclear localization. This suggests that Importin α and Importin ß are key transporters during the encystation process and are involved, at least, in the transport of arginine deiminase into the nuclei. Considering the effect produced by Ivermectin during growth and encystation, we postulate that this drug could be used to treat giardiasis.


Assuntos
Núcleo Celular/metabolismo , Giardia lamblia/metabolismo , Proteínas de Protozoários/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Antiparasitários/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Biologia Computacional , Giardia lamblia/efeitos dos fármacos , Giardia lamblia/genética , Giardia lamblia/crescimento & desenvolvimento , Hidrolases/metabolismo , Ivermectina/farmacologia , Encistamento de Parasitas/efeitos dos fármacos , Encistamento de Parasitas/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Proteínas de Protozoários/genética , Quinazolinas/farmacologia , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismo
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