RESUMEN
Glucocorticoids are the drugs most commonly used to manage inflammatory diseases. However, they are prone to inducing muscle atrophy by increasing muscle proteolysis and decreasing protein synthesis. Various studies have demonstrated that antioxidants can mitigate glucocorticoid-induced skeletal muscle atrophy. Here, we investigated the effect of a potent antioxidative natural flavonoid, morin, on the muscle atrophy and oxidative stress induced by dexamethasone (Dex) using mouse C2C12 skeletal myotubes. Dex (10 µM) enhanced the production of reactive oxygen species (ROS) in C2C12 myotubes via glucocorticoid receptor. Moreover, Dex administration reduced the diameter and expression levels of the myosin heavy chain protein in C2C12 myotubes, together with the upregulation of muscle atrophy-associated ubiquitin ligases, such as muscle atrophy F-box protein 1/atrogin-1, muscle ring finger protein-1, and casitas B-lineage lymphoma proto-oncogene-b. Dex also significantly decreased phosphorylated Foxo3a and increased total Foxo3a expression. Interestingly, Dex-induced ROS accumulation and Foxo3a expression were inhibited by morin (10 µM) pretreatment. Morin also prevented the Dex-induced reduction of myotube thickness, together with muscle protein degradation and suppression of the upregulation of atrophy-associated ubiquitin ligases. In conclusion, our results suggest that morin effectively prevents glucocorticoid-induced muscle atrophy by reducing oxidative stress.
Asunto(s)
Dexametasona , Flavonoides/farmacología , Fibras Musculares Esqueléticas , Proteínas Musculares/metabolismo , Atrofia Muscular , Estrés Oxidativo/efectos de los fármacos , Animales , Línea Celular , Dexametasona/efectos adversos , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Ratones , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Atrofia Muscular/inducido químicamente , Atrofia Muscular/tratamiento farmacológico , Atrofia Muscular/metabolismo , Atrofia Muscular/patologíaRESUMEN
Unloading-mediated muscle atrophy is associated with increased reactive oxygen species (ROS) production. We previously demonstrated that elevated ubiquitin ligase casitas B-lineage lymphoma-b (Cbl-b) resulted in the loss of muscle volume (Nakao R, Hirasaka K, Goto J, Ishidoh K, Yamada C, Ohno A, Okumura Y, Nonaka I, Yasutomo K, Baldwin KM, Kominami E, Higashibata A, Nagano K, Tanaka K, Yasui N, Mills EM, Takeda S, Nikawa T. Mol Cell Biol 29: 4798-4811, 2009). However, the pathological role of ROS production associated with unloading-mediated muscle atrophy still remains unknown. Here, we showed that the ROS-mediated signal transduction caused by microgravity or its simulation contributes to Cbl-b expression. In L6 myotubes, the assessment of redox status revealed that oxidized glutathione was increased under microgravity conditions, and simulated microgravity caused a burst of ROS, implicating ROS as a critical upstream mediator linking to downstream atrophic signaling. ROS generation activated the ERK1/2 early-growth response protein (Egr)1/2-Cbl-b signaling pathway, an established contributing pathway to muscle volume loss. Interestingly, antioxidant treatments such as N-acetylcysteine and TEMPOL, but not catalase, blocked the clinorotation-mediated activation of ERK1/2. The increased ROS induced transcriptional activity of Egr1 and/or Egr2 to stimulate Cbl-b expression through the ERK1/2 pathway in L6 myoblasts, since treatment with Egr1/2 siRNA and an ERK1/2 inhibitor significantly suppressed clinorotation-induced Cbl-b and Egr expression, respectively. Promoter and gel mobility shift assays revealed that Cbl-b was upregulated via an Egr consensus oxidative responsive element at -110 to -60 bp of the Cbl-b promoter. Together, this indicates that under microgravity conditions, elevated ROS may be a crucial mechanotransducer in skeletal muscle cells, regulating muscle mass through Cbl-b expression activated by the ERK-Egr signaling pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Atrofia Muscular/enzimología , Mioblastos Esqueléticos/enzimología , Estrés Oxidativo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ingravidez , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antioxidantes/farmacología , Células COS , Chlorocebus aethiops , Factores de Transcripción de la Respuesta de Crecimiento Precoz/genética , Factores de Transcripción de la Respuesta de Crecimiento Precoz/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glutatión/metabolismo , Mecanotransducción Celular , Atrofia Muscular/genética , Atrofia Muscular/patología , Atrofia Muscular/prevención & control , Mioblastos Esqueléticos/efectos de los fármacos , Mioblastos Esqueléticos/patología , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Fosforilación , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-cbl/genética , Ratas , Vuelo Espacial , Factores de Tiempo , Regulación hacia Arriba , Simulación de IngravidezRESUMEN
Skeletal muscle atrophy occurs when the rate of protein degradation exceeds that of protein synthesis in various catabolic conditions, such as fasting, disuse, aging, and chronic diseases. Insulin-like growth factor-1 (IGF-1) signaling stimulates muscle growth and suppresses muscle protein breakdown. In atrophied muscles, ubiquitin ligase, Cbl-b, increases and stimulates the ubiquitination and degradation of IRS-1, an intermediate in IGF-1 signaling pathway, resulting in IGF-1 resistance. In this study, we evaluated the efficacy of atelocollagen (ATCOL)-transported anti-ubiquitination oligopeptide (Cblin: Cbl-b inhibitor) (consisting of tyrosine phosphorylation domain of IRS-1) in starved C2C12 myotubes. The amount of IRS-1 protein was lower in starved versus unstarved myotubes. The Cblin-ATCOL complex inhibited IRS-1 degradation in a concentration-dependent manner. Myotubes incubated with Cblin-ATCOL complex showed significant resistance to starvation-induced atrophy (p<0.01). Furthermore, the Cblin-ATCOL complex significantly inhibited any decrease in Akt phosphorylation (p<0.01) and localization of FOXO3a to the nucleus in starved myotubes. These results suggest that Cblin prevented starvation-induced C2C12 myotube atrophy by maintaining the IGF-1/Akt/FOXO signaling. Therefore, attachment of anti-ubiquitination oligopeptide, Cblin, to ATCOL enhances its delivery to myotubes and could be a potentially effective strategy in the treatment of atrophic myopathies.
Asunto(s)
Colágeno/farmacología , Músculo Esquelético/patología , Atrofia Muscular/prevención & control , Oligopéptidos/farmacología , Ubiquitinación/efectos de los fármacos , Animales , Medio de Cultivo Libre de Suero , Proteínas Sustrato del Receptor de Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-cbl , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
Uncoupling protein 3 (UCP3) is known to regulate energy dissipation, proton leakage, fatty acid oxidation, and oxidative stress. To identify the putative protein regulators of UCP3, we performed yeast two-hybrid screens. Here we report that UCP3 interacted with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that was localized in the mitochondria, and is involved in cellular responses to Ca(2+). The hydrophilic sequences within loop 2, and the matrix-localized hydrophilic domain of mouse UCP3, were necessary for binding to Hax-1 at the C-terminal domain, adjacent to the mitochondrial inner membrane. Interestingly, interaction of these proteins occurred in a calcium-dependent manner. Moreover, the NMR spectrum of the C-terminal domain of Hax-1 was dramatically changed by removal of Ca(2+), suggesting that the C-terminal domain of Hax-1 underwent a Ca(2+)-induced conformational change. In the Ca(2+)-free state, the C-terminal Hax-1 tended to unfold, suggesting that Ca(2+) binding may induce protein folding of the Hax-1 C-terminus. These results suggested that the UCP3-Hax-1 complex may regulate mitochondrial functional changes caused by mitochondrial Ca(2+).
Asunto(s)
Calcio/metabolismo , Canales Iónicos/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas/metabolismo , Animales , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Canales Iónicos/química , Canales Iónicos/genética , Ratones , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Técnicas del Sistema de Dos Híbridos , Proteína Desacopladora 3RESUMEN
Cbl-b is a RING-type ubiquitin ligase. Previously, we showed that Cbl-b-mediated ubiquitination and proteosomal degradation of IRS-1 contribute to muscle atrophy caused by unloading stress. The phospho-pentapeptide DGpYMP (Cblin) mimics Tyr612-phosphorylated IRS-1 and inhibits the Cbl-b-mediated ubiquitination and degradation of IRS-1 in vitro and in vivo. In this study, we confirmed the direct interaction between Cblin and the TKB domain of Cbl-b using NMR. Moreover, we showed that the shortened tripeptide GpYM also binds to the TKB domain. To elucidate the inhibitory mechanism of Cblin, we solved the crystal structure of the TKB-Cblin complex at a resolution of 2.5 Å. The pY in Cblin inserts into a positively charged pocket in the TKB domain via hydrogen-bond networks and hydrophobic interactions. Within this complex, the Cblin structure closely resembles the TKB-bound form of another substrate-derived phosphopeptide, Zap-70-derived phosphopeptide. These peptides lack the conserved intrapeptidyl hydrogen bond between pY and a conserved residue involved in TKB-domain binding. Instead of the conserved interaction, these peptides specifically interact with the TKB domain. Based on this binding mode of Cblin to the TKB domain, we can design drugs against unloading-mediated muscle atrophy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Oligopéptidos/metabolismo , Proteínas Proto-Oncogénicas c-cbl/química , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Células HEK293 , Humanos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Modelos Moleculares , Oligopéptidos/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-cbl/antagonistas & inhibidores , Ubiquitinación/efectos de los fármacosRESUMEN
BACKGROUND: There is evidence that several messenger RNAs (mRNAs) are bifunctional RNAs, i.e. RNA transcript carrying both protein-coding capacity and activity as functional non-coding RNA via 5' and 3' untranslated regions (UTRs). RESULTS: In this study, we identified a novel bifunctional RNA that is transcribed from insulin receptor substrate-1 (Irs-1) gene with full-length 5'UTR sequence (FL-Irs-1 mRNA). FL-Irs-1 mRNA was highly expressed only in skeletal muscle tissue. In cultured skeletal muscle C2C12 cells, the FL-Irs-1 transcript functioned as a bifunctional mRNA. The FL-Irs-1 transcript produced IRS-1 protein during differentiation of myoblasts into myotubes; however, this transcript functioned as a regulatory RNA in proliferating myoblasts. The FL-Irs-1 5'UTR contains a partial complementary sequence to Rb mRNA, which is a critical factor for myogenic differentiation. The overexpression of the 5'UTR markedly reduced Rb mRNA expression, and this reduction was fully dependent on the complementary element and was not compensated by IRS-1 protein. Conversely, knockdown of FL-Irs-1 mRNA increased Rb mRNA expression and enhanced myoblast differentiation into myotubes. CONCLUSIONS: Our findings suggest that the FL-Irs-1 transcript regulates myogenic differentiation as a regulatory RNA in myoblasts.
Asunto(s)
Proteínas Sustrato del Receptor de Insulina/genética , Regiones no Traducidas 5' , Animales , Secuencia de Bases , Diferenciación Celular , Línea Celular , Proteínas Sustrato del Receptor de Insulina/antagonistas & inhibidores , Proteínas Sustrato del Receptor de Insulina/metabolismo , Ratones , Músculo Esquelético/metabolismo , Mioblastos/citología , Mioblastos/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Alineación de SecuenciaRESUMEN
A DGpYMP peptide mimetic of tyrosine(608)-phosphorylated insulin receptor substrate-1 (IRS-1), named Cblin, was previously shown to significantly inhibit Cbl-b-mediated IRS-1 ubiquitination. In the present study, we developed N-myristoylated Cblin and investigated whether it was effective in preventing glucocorticoid-induced muscle atrophy. Using HEK293 cells overexpressing Cbl-b, IRS-1 and ubiquitin, we showed that the 50% inhibitory concentrations of Cbl-b-mediated IRS-1 ubiquitination by N-myristoylated Cblin and Cblin were 30 and 120 µM, respectively. Regarding the DEX-induced atrophy of C2C12 myotubes, N-myristoylated Cblin was more effective than Cblin for inhibiting the DEX-induced decreases in C2C12 myotube diameter and IRS-1 degradation. The inhibitory efficacy of N-myristoylated Cblin on IRS-1 ubiquitination in C2C12 myotubes was approximately fourfold larger than that of Cblin. Furthermore, N-myristoylation increased the incorporation of Cblin into HEK293 cells approximately 10-folds. Finally, we demonstrated that N-myristoylated Cblin prevented the wet weight loss, IRS-1 degradation, and MAFbx/atrogin-1 and MuRF-1 expression in gastrocnemius muscle of DEX-treated mice approximately fourfold more effectively than Cblin. Taken together, these results suggest that N-myristoylated Cblin prevents DEX-induced skeletal muscle atrophy in vitro and in vivo, and that N-myristoylated Cblin more effectively prevents muscle atrophy than unmodified Cblin.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Glucocorticoides/efectos adversos , Músculo Esquelético/metabolismo , Péptidos/química , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Animales , Sistema Libre de Células , Femenino , Células HEK293 , Humanos , Proteínas Sustrato del Receptor de Insulina/química , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efectos de los fármacos , Atrofia Muscular/inducido químicamente , Ácido Mirístico/química , Proteínas Proto-Oncogénicas c-cbl/antagonistas & inhibidores , Ubiquitina/químicaRESUMEN
In-cell NMR is an isotope-aided multi-dimensional NMR technique that enables observations of conformations and functions of proteins in living cells at the atomic level. This method has been successfully applied to proteins overexpressed in bacteria, providing information on protein-ligand interactions and conformations. However, the application of in-cell NMR to eukaryotic cells has been limited to Xenopus laevis oocytes. Wider application of the technique is hampered by inefficient delivery of isotope-labelled proteins into eukaryote somatic cells. Here we describe a method to obtain high-resolution two-dimensional (2D) heteronuclear NMR spectra of proteins inside living human cells. Proteins were delivered to the cytosol by the pyrenebutyrate-mediated action of cell-penetrating peptides linked covalently to the proteins. The proteins were subsequently released from cell-penetrating peptides by endogenous enzymatic activity or by autonomous reductive cleavage. The heteronuclear 2D spectra of three different proteins inside human cells demonstrate the broad application of this technique to studying interactions and protein processing. The in-cell NMR spectra of FKBP12 (also known as FKBP1A) show the formation of specific complexes between the protein and extracellularly administered immunosuppressants, demonstrating the utility of this technique in drug screening programs. Moreover, in-cell NMR spectroscopy demonstrates that ubiquitin has much higher hydrogen exchange rates in the intracellular environment, possibly due to multiple interactions with endogenous proteins.
Asunto(s)
Espacio Intracelular/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas Recombinantes de Fusión/química , Animales , Permeabilidad de la Membrana Celular , Supervivencia Celular/efectos de los fármacos , Medición de Intercambio de Deuterio , Evaluación Preclínica de Medicamentos/métodos , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , Células HeLa , Humanos , Inmunosupresores/química , Inmunosupresores/metabolismo , Inmunosupresores/farmacología , Unión Proteica , Pirenos/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína 1A de Unión a Tacrolimus/química , Proteína 1A de Unión a Tacrolimus/genética , Proteína 1A de Unión a Tacrolimus/metabolismo , Transfección , Ubiquitina/genética , Ubiquitina/metabolismoRESUMEN
Obesity causes type 2 diabetes, atherosclerosis and cardiovascular diseases by inducing systemic insulin resistance. It is now recognized that obesity is related to chronic low-grade inflammation in adipose tissue. Specifically, activated immune cells infiltrate adipose tissue and cause inflammation. There is increasing evidence that activated macrophages accumulate in the hypertrophied adipose tissue of rodents and humans and induce systemic insulin resistance by secreting inflammatory cytokines. Accordingly, a better understanding of the molecular mechanisms underlying macrophage activation in adipose tissue will facilitate the development of new therapeutic strategies. Currently, little is known about the regulation of macrophage activation, although E3 ubiquitin ligase Casitas B-lineage lymphoma (Cbl)-b was identified recently as a novel negative regulator of macrophage activation in adipose tissue. Cbl-b, which is a suppressor of T- and B-cell activation, inhibits intracellular signal transduction by targeting some tyrosine kinases. Notably, preventing Cbl-b-mediated macrophage activation improves obesity-induced insulin resistance in mice. c-Cbl is another member of the Cbl family that is associated with insulin resistance in obesity. These reports suggest that Cbl-b and c-Cbl are potential therapeutic targets for treating obesity-induced insulin resistance. In this review, we focus on the importance of Cbl-b in macrophage activation in aging-induced and high-fat diet-induced obesity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Resistencia a la Insulina/genética , Obesidad/metabolismo , Proteínas Proto-Oncogénicas c-cbl/fisiología , Envejecimiento/fisiología , Animales , Dieta Alta en Grasa , Humanos , Sistema Inmunológico/enzimología , Sistema Inmunológico/metabolismo , Activación de Macrófagos/genética , Ratones , Obesidad/complicaciones , Obesidad/genéticaRESUMEN
Osteoactivin is a type I transmembrane protein upregulated by unloading stresses, including denervation, prolonged bed rest, and space flight, but the regulatory mechanisms of its expression and activation under these conditions remain undefined. Here we report that osteoactivin protein exists in two forms: an intact transmembrane form and a secreted form. The secreted form, the extracellular fragment of osteoactivin, was produced by ectodomain shedding and was released into a culture medium. Amino acid sequence analysis of the carboxy-terminal fragment of osteoactivin (OA-CTF) revealed that cleavage of osteoactivin by proteases occurred both at the cell surface and within the cell membrane. Localization analysis demonstrated translocalization of OA-CTF to the nucleus and the endoplasmic reticulum. Moreover, RNA binding proteins, which regulate pre-mRNA splicing, were identified as OA-CTF binding proteins. These results suggest that OA-CTF formed by ectodomain shedding is involved in the regulation of pre-mRNA splicing.
Asunto(s)
Núcleo Celular/metabolismo , Espacio Intracelular/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , ProteolisisRESUMEN
Deficiency of the Cbl-b ubiquitin ligase gene activates macrophages in mice. This study aimed to elucidate the pathophysiological roles of macrophages in muscle degeneration/regeneration in Cbl-b-deficient mice. We examined immune cell infiltration and cytokine expression in cardiotoxin-injected tibialis anterior muscle of Cbl-b-deficient mice. Ablation of the Cbl-b gene expression delayed regeneration of cardiotoxin-induced skeletal muscle damage compared with wild-type mice. CD8-positive T cells were still present in the damaged muscle on day 14 after cardiotoxin injection in Cbl-b-deficient mice, but there was dispersal of the same cells over that time-frame in wild-type mice. Infiltrating macrophages in Cbl-b-deficient mice showed strong expression of RANTES (regulated-on-activation, normal T cell expressed and secreted), a chemokine for CD8-positive T cells. In turn, a neutralizing antibody against RANTES significantly suppressed the infiltration of CD8-positive T cells into the muscle, resulting in restoration of the disturbed muscle regeneration. Cbl-b is an important regulatory factor for cytotoxic T-cell infiltration via RANTES production in macrophages.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/deficiencia , Cardiotoxinas/farmacología , Quimiocina CCL5/metabolismo , Macrófagos/efectos de los fármacos , Músculo Esquelético/patología , Enfermedades Musculares/patología , Proteínas Proto-Oncogénicas c-cbl/deficiencia , Regeneración/efectos de los fármacos , Análisis de Varianza , Animales , Anticuerpos/farmacología , Antígenos CD/metabolismo , Quimiocina CCL5/genética , Quimiocina CCL5/inmunología , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Enfermedades Musculares/genética , Enfermedades Musculares/fisiopatología , Factores Reguladores Miogénicos , ARN Mensajero/metabolismo , Regeneración/genética , Factores de TiempoRESUMEN
In heterologous protein production by filamentous fungi, target proteins are expressed as fusions with homologous secretory proteins, called carriers, for higher production yields. Although carrier fusion is thought to overcome the bottleneck in transcriptional and (post)translational processes during heterologous protein production, there is limited knowledge of its physiological effects on the host strain. In this study, we performed DNA microarray analysis by comparing gene expression patterns of two Aspergillus oryzae strains expressing either carrier- or non-carrier-fused bovine chymosin (CHY). When CHY was expressed as a fusion with α-amylase (AmyB), the production level increased by approximately 2-fold as compared with the non-carrier-fused CHY. DNA microarray analysis revealed that the carrier fusion significantly up-regulated many genes involved in endoplasmic reticulum (ER) protein-folding and secretion. Consistently, hacA transcripts were efficiently spliced in the strain expressing the carrier-fused CHY, indicating an unfolded protein response (UPR). The carrier-fused CHY was detected intracellularly without processing at the Kex2 cleavage site, which is likely recognized in the Golgi, and the carrier fusion delayed extracellular CHY production in the early growth phase as compared with the non-carrier-fused expression. Taken together, our data suggest a proposal that the carrier fusion temporarily accumulates the carrier-fused CHY in the ER and significantly induces UPR.
Asunto(s)
Aspergillus oryzae/metabolismo , Quimosina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Respuesta de Proteína Desplegada , Animales , Aspergillus oryzae/genética , Bovinos , Quimosina/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Expresión Génica , Regulación Fúngica de la Expresión Génica , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genéticaRESUMEN
BACKGROUND: Tobacco-related lung diseases, including chronic obstructive pulmonary disease (COPD), are major causes of lung-related disability and death worldwide. Acute exacerbation of COPD (AE-COPD) is commonly associated with upper and lower respiratory tract viral infections and can result in respiratory failure in those with advanced lung disease. OBJECTIVE: We sought to determine the mechanism underlying COPD exacerbation and host response to pathogen-derived factors. METHODS: Over a 24-month period, we assessed the viral causes for upper and lower respiratory tract infections in patients with COPD (n = 155) and control subjects (n = 103). We collected nasal and bronchoalveolar lavage fluid and peripheral blood under baseline and exacerbated conditions. We determined the effect of human rhinovirus (HRV) proteinases on T-cell activation in human subjects and mice. RESULTS: HRVs are isolated from nasal and lung fluid from subjects with AE-COPD. Bronchoalveolar lavage fluid and CD4 T cells from patients with COPD exhibited a T(H)1 and T(H)2 cell cytokine phenotype during acute infection. HRV-encoded proteinase 2A activated monocyte-derived dendritic cells in vitro and induced strong T(H)1 and T(H)2 immune responses from CD4 T cells. Intranasal administration of recombinant rhinovirus proteinase 2A in mice resulted in an increase in airway hyperreactivity, lung inflammation, and IL-4 and IFN-gamma production from CD4 T cells. CONCLUSION: Our findings suggest that patients with severe COPD show T(H)1- and T(H)2-biased responses during AE-COPD. HRV-encoded proteinase 2A, like other microbial proteinases, could provide a T(H)1- and T(H)2-biasing adjuvant factor during upper and lower respiratory tract infection in patients with severe COPD. Alteration of the immune response to secreted viral proteinases might contribute to worsening of dyspnea and respiratory failure in patients with COPD.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Cisteína Endopeptidasas/administración & dosificación , Infecciones por Picornaviridae/inmunología , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Rhinovirus/inmunología , Proteínas Virales/administración & dosificación , Adulto , Anciano , Animales , Hiperreactividad Bronquial , Líquido del Lavado Bronquioalveolar , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Células Cultivadas , Cisteína Endopeptidasas/farmacología , Células Dendríticas/efectos de los fármacos , Femenino , Estudios de Seguimiento , Humanos , Interferón gamma/metabolismo , Interleucina-4/metabolismo , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Infecciones por Picornaviridae/complicaciones , Infecciones por Picornaviridae/virología , Neumonía , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Enfermedad Pulmonar Obstructiva Crónica/virología , Rhinovirus/enzimología , Rhinovirus/patogenicidad , Células TH1/inmunología , Células Th2/inmunología , Proteínas Virales/farmacologíaRESUMEN
We previously reported that intramuscular injections of ubiquitin ligase CBLB inhibitory pentapeptide (Cblin; Asp-Gly-pTyr-Met-Pro) restored lost muscle mass caused by sciatic denervation. Here, we detected Cblin on the basolateral side of Caco-2 cells after being placed on the apical side, and found that cytochalasin D, a tight junction opener, enhanced Cblin transport. Orally administered Cblin was found in rat plasma, indicating that intact Cblin was absorbed in vitro and in vivo. Furthermore, transgenic Cblin peptide-enriched rice (CbR) prevented the denervation-induced loss of muscle mass and the upregulation of muscle atrophy-related ubiquitin ligases in mice. These findings indicated that CbR could serve as an alternative treatment for muscle atrophy.
RESUMEN
Infection of certain influenza viruses is triggered when its HA is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, whereas the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R↓ sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal α-helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a homology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19.
Asunto(s)
Gripe Aviar/virología , Proteínas de la Membrana/química , Orthomyxoviridae/patogenicidad , Serina Endopeptidasas/química , Animales , Aves , Cristalografía por Rayos X , Humanos , Conformación ProteicaRESUMEN
OBJECTIVE: This study aimed to: 1) obtain data about occurrence of childhood domestic injuries in Gunma prefecture according to children's age; 2) ascertain parental awareness of injury prevention; and 3) develop ideas for creating concrete strategies of childhood injury prevention. METHODS: The participants were 551 parents of children living in 14 cities/towns in Gunma prefecture that showed interest in cooperating with this survey. A self-reported questionnaire was handed to parents when they took their children to health check-ups provided by the cities/towns either during the child's first year, at 18 months, or at 3 years. Parents completed the questionnaire asking whether their child had been injured at home during the past year, and if so, they were asked about the type of injury, the cause of injury, and what action they took. We also asked whether the parents took specific injury prevention measures at home. Data were analyzed statistically. RESULTS: The injury experienced most frequently during the first year of life was "fall" (30.8%), followed by "ingestion of a foreign body" (22.7%), and then "choking" (11.5%). For children around the age of 18 months, the most frequently experienced injury was "fall" (41.0%), followed by "burn" (20.3%), and "ingestion of a foreign body" (19.3%). At 3 years, "burn" was reported most frequently (32.3%), followed by "fall" (31.0%), and "choking" (14.5%). Chi2-test revealed significant correlations among the three age groups concerning the rate of burn injury, foreign body ingestion, and drowning. The rate of burn injury was higher at 3 years than at 18 months, and also higher at 18 months as compared to under the age of one. In contrast, the rate of foreign body ingestion was higher under the age of one than at 18 months, and also higher at 18 months as compared to the age of 3 years. Drowning was more common at 18 months and 3 years than under the age of one. As for prevention of domestic injury, investigation of preventive means taken according to type of injury revealed that parents taking any of the measures to prevent an injury were significantly more likely to also take other means to prevent that injury. CONCLUSION: Characteristics of injury differed according to children's age. Furthermore, it was clarified that parents' performance of injury prevention depends on their awareness of preventive measures.
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Accidentes Domésticos/estadística & datos numéricos , Heridas y Lesiones/epidemiología , Accidentes por Caídas , Accidentes Domésticos/prevención & control , Factores de Edad , Obstrucción de las Vías Aéreas/epidemiología , Quemaduras/epidemiología , Preescolar , Humanos , Lactante , Japón/epidemiología , Heridas y Lesiones/prevención & controlRESUMEN
Anti-angiogenic therapies targeting vascular endothelial growth factor (VEGF) and its receptor (VEGF-R) are important treatments for a number of human malignancies, including colorectal cancers. However, there is increasing evidence that VEGF/VEGF-R inhibitors promote the adaptive and evasive resistance of tumor cells to the therapies. The mechanism by which the cancer cells become resistant remains unclear. One potential mechanism is that VEGF/VEGF-R blockers directly act on tumor cells independently of anti-angiogenic effects. In this study, the direct effects of an anti-VEGF antibody (bevacizumab) and a VEGF-R tyrosine kinase inhibitor (sunitinib) on the evasive adaptation of colon cancer cells were compared. HCT116 and RKO human colon cancer cell lines were chronically exposed (3 months) to bevacizumab or sunitinib in vitro to establish bevacizumab- and sunitinib-adapted cells, respectively. Transwell migration and invasion assays, western blotting, reverse transcription-quantitative polymerase chain reaction, co-immunoprecipitation analysis, cell survival assays and ELISAs were conducted to analyze the adapted cells. Compared with the control vehicle-treated cells, the two cell models exhibited increased migration and invasion activities to different degrees and through different mechanisms. The bevacizumab-adapted cells, but not in the sunitinib-adapted cells, exhibited redundantly increased expression levels of VEGF/VEGF-R family members, including VEGF-A, placental growth factor, VEGF-C, VEGF-R1 and VEGF-R3. In addition, the phosphorylation levels of VEGF-R1 and VEGF-R3 were increased in the bevacizumab-adapted cells compared with the control cells. Thus, the inhibition of VEGF-R1 and VEGF-R3 decreased the evasive activities of the cells, suggesting that they remained dependent on redundant VEGF/VEGF-R signaling. By contrast, the sunitinib-adapted cells exhibited increased neuropilin-1 (NRP1) expression levels compared with the control cells. In the sunitinib-adapted cells, NRP1 interacted with phosphorylated cMet, and the cMet activation was dependent on NRP1. Thus, NRP1 or cMet blockade suppressed the evasive activation of the sunitinib-adapted cells. These results suggest that the sunitinib-adapted cells switched from a VEGF-R-dependent pathway to an alternative NRP1/cMet-dependent one. The findings of the present study indicate that VEGF/VEGF-R inhibitors directly act on colon cancer cells and activate their evasive adaptation via different mechanisms.
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Bevacizumab/farmacología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Indoles/farmacología , Neuropilina-1/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Pirroles/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Movimiento Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Células HCT116 , Humanos , Invasividad Neoplásica , Transducción de Señal/efectos de los fármacos , SunitinibRESUMEN
The ubiquitin-associated (UBA) domain is one of the most frequently occurring motifs that recognize ubiquitin tags. Dsk2p, a UBA-containing protein from Saccharomyces cerevisiae, is involved in the ubiquitin-proteasome proteolytic pathway and has been implicated in spindle pole duplication. Here we present the solution structure of the UBA domain of Dsk2p (Dsk2(UBA)) in complex with ubiquitin. The structure reveals that the UBA domain uses a mode of ubiquitin recognition that is similar to that of the CUE domain, another ubiquitin binding motif that shares low sequence homology but high structural similarity with UBA domains. These two domains, as well as the structurally unrelated ubiquitin binding motif UIM, provide a common, crucial recognition site for ubiquitin, comprising a hydrogen-bonding acceptor for the amide group of Gly-47, and a methyl group that packs against the hydrophobic pocket of ubiquitin formed by Leu-8, Ile-44, His-68, and Val-70.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Saccharomyces cerevisiae/química , Ubiquitina/química , Ubiquitinas/química , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Ubiquitina/metabolismo , Ubiquitinas/metabolismoRESUMEN
Although vascular endothelial growth factor receptor (VEGF-R)-targeted antiangiogenic agents are important treatment for a number of human malignancies, there is accumulating evidence that the therapies may promote disease progression, such as invasion and metastasis. How tumors become to promote their evasiveness remains fully uncertain. One of possible mechanisms for the adaptation may be a direct effect of VEGF-R inhibitors on tumor cells expressing VEGF-R. To elucidate a direct effect of VEGF-R-targeting drug (sunitinib), we established a human colorectal cancer cell model adapted to sunitinib. The sunitinib-conditioned cells showed a significant increase in cellular motility and migration activities, compared to the vehicle-treated control cells. Consistent with the phenotype, the sunitinib-conditioned cells decreased the expression levels of E-cadherin (an epithelial marker), while significantly increased the levels of Slug and Zeb1 (mesenchymal markers). Expression profiles of VEGF-R in the sunitinib-conditioned cells showed that only neuropilin-1 (NRP1) expression was significantly increased among all VEGF-R tested. Blockade of NRP1 using its antagonist clearly repressed the migration activation in sunitinib-conditioned cells, but not in the control cells. These results suggest that inhibition of VEGF-R on colorectal cancer cells can drive the epithelial-mesenchymal transition, leading to activation of cell motility in an NRP1-dependent manner. J. Med. Invest. 64: 250-254, August, 2017.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Transición Epitelial-Mesenquimal/efectos de los fármacos , Indoles/farmacología , Pirroles/farmacología , Movimiento Celular/efectos de los fármacos , Neoplasias Colorrectales/patología , Células HCT116 , Humanos , Neuropilina-1/análisis , Receptores de Factores de Crecimiento Endotelial Vascular/análisis , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Sunitinib , TranscriptomaRESUMEN
Recently, inhibition of tumor angiogenesis has become an important anti-cancer therapy. Tumor angiogenesis is regulated by multiple signaling pathways, including VEGF and VEGF receptor (VEGF-R), FGF and FGF receptor (FGF-R), and PDGF and PDGF receptor (PDGF-R) pathways. Thus, the antiangiogenic agents, such as regorafenib, simultaneously target those receptors on vascular endothelial cells. In addition to endothelial cells, cancer cells express the three receptors, suggesting that the antiangiogenic inhibitors affect tumor cells. In fact, we previously demonstrated that regorafenib directly acted on human colorectal cancer cells and accelerated their apoptosis resistance and migration capability. Thus, we here elucidated how regorafenib induced the malignant phenotypes in colorectal cancer cells. To identify the responsible receptor among the regorafenib-targeting proangiogenic receptors, we examined the effects of a potent selective inhibitor for VEGF-R, FGF-R or PDGF-R on apoptosis resistance and migration capability. We clarified that blockade of VEGF-R, but not FGF-R and PDGF-R, induced the malignant phenotypes. We confirmed that blocking of VEGF ligands derived from colorectal cancer cells also induced the phenotypes. These results suggest that regorafenib progressed the malignancy via prevention of autocrine and paracrine VEGF signaling in colorectal cancer cells. J. Med. Invest. 64: 262-265, August, 2017.