RESUMEN

ER network formation depends on membrane fusion by the atlastin (ATL) GTPase. In humans, three paralogs are differentially expressed with divergent N- and C-terminal extensions, but their respective roles remain unknown. This is partly because, unlike Drosophila ATL, the fusion activity of human ATLs has not been reconstituted. Here, we report successful reconstitution of fusion activity by the human ATLs. Unexpectedly, the major splice isoforms of ATL1 and ATL2 are each autoinhibited, albeit to differing degrees. For the more strongly inhibited ATL2, autoinhibition mapped to a C-terminal α-helix is predicted to be continuous with an amphipathic helix required for fusion. Charge reversal of residues in the inhibitory domain strongly activated its fusion activity, and overexpression of this disinhibited version caused ER collapse. Neurons express an ATL2 splice isoform whose sequence differs in the inhibitory domain, and this form showed full fusion activity. These findings reveal autoinhibition and alternate splicing as regulators of atlastin-mediated ER fusion.

Asunto(s)

Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Fusión de Membrana , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Animales , Células COS , Chlorocebus aethiops , Retículo Endoplásmico/metabolismo , Proteínas de Unión al GTP/antagonistas & inhibidores , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Mutación/genética , Estructura Secundaria de Proteína

RESUMEN

Solidagenone (SOL) is a labdane-type diterpenoid found in Solidago chilensis, a plant traditionally used to treat skin diseases, kidney pain and ovarian inflammation. In this study, the topical anti-inflammatory activity of SOL was evaluated using in vivo and in silico assays. Croton oil-, arachidonic acid (AA)- and phenol-induced ear oedema mouse models were applied in the in vivo studies. Myeloperoxidase (MPO) and N-acetyl-ß-D-glucosaminidase (NAG) activities and tumour necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and nitric oxide (NO) levels were determined, as well as histopathological analyses were conducted. Interaction profiles between SOL and cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), glucocorticoid receptor, estradiol-17-ß-dehydrogenase and prostaglandin-E(2)-9-reductase were established using molecular docking. SOL significantly inhibited croton oil-, AA- and phenol-induced ear oedema (P < .001) at doses of 0.1, 0.5 and 1.0 mg/ear. The MPO and NAG activities and TNF-α, IL-6 and NO levels were decreased (P < .001). The histopathological data revealed that inflammatory parameters (oedema thickness, leucocyte infiltration and vasodilatation) were reduced by treatment with SOL at doses of 0.1, 0.5 and 1.0 mg/ear. The docking study showed that SOL interacts with COX-1 and prostaglandin-E(2)-9-reductase through hydrogen bonding, inhibiting these enzymes. These results indicate that SOL may be a promising compound for the treatment of cutaneous inflammatory disorders and has potential as a topical anti-inflammatory agent.

Asunto(s)

Inhibidores de la Ciclooxigenasa/farmacología , Dermatitis/prevención & control , Edema/prevención & control , Furanos/farmacología , Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , Proteínas de la Membrana/antagonistas & inhibidores , Naftalenos/farmacología , Extractos Vegetales/farmacología , Piel/efectos de los fármacos , Solidago , Acetilglucosaminidasa/metabolismo , Animales , Ciclooxigenasa 1/metabolismo , Inhibidores de la Ciclooxigenasa/aislamiento & purificación , Inhibidores de la Ciclooxigenasa/metabolismo , Dermatitis/metabolismo , Dermatitis/patología , Modelos Animales de Enfermedad , Edema/inducido químicamente , Edema/metabolismo , Edema/patología , Furanos/aislamiento & purificación , Furanos/metabolismo , Enlace de Hidrógeno , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Interleucina-6/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Simulación del Acoplamiento Molecular , Naftalenos/aislamiento & purificación , Naftalenos/metabolismo , Óxido Nítrico/metabolismo , Peroxidasa/metabolismo , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/metabolismo , Unión Proteica , Transducción de Señal , Piel/metabolismo , Piel/patología , Solidago/química , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Protein trafficking is altered when normal cells acquire a tumor phenotype. A key subcellular compartment in regulating protein trafficking is the Golgi apparatus, but its role in carcinogenesis is still not well defined. Golgi phosphoprotein 3 (GOLPH3), a peripheral membrane protein mostly localized at the trans-Golgi network, is overexpressed in several tumor types including glioblastoma multiforme (GBM), the most lethal primary brain tumor. Moreover, GOLPH3 is currently considered an oncoprotein, however its precise function in GBM is not fully understood. Here, we analyzed in T98G cells of GBM, which express high levels of epidermal growth factor receptor (EGFR), the effect of stable RNAi-mediated knockdown of GOLPH3. We found that silencing GOLPH3 caused a significant reduction in the proliferation of T98G cells and an unexpected increase in total EGFR levels, even at the cell surface, which was however less prone to ligand-induced autophosphorylation. Furthermore, silencing GOLPH3 decreased EGFR sialylation and fucosylation, which correlated with delayed ligand-induced EGFR downregulation and its accumulation at endo-lysosomal compartments. Finally, we found that EGF failed at promoting EGFR ubiquitylation when the levels of GOLPH3 were reduced. Altogether, our results show that GOLPH3 in T98G cells regulates the endocytic trafficking and activation of EGFR likely by affecting its extent of glycosylation and ubiquitylation.

Asunto(s)

Carcinogénesis/genética , Glioblastoma/genética , Proteínas de la Membrana/genética , Línea Celular Tumoral , Proliferación Celular/genética , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica/genética , Glioblastoma/patología , Glicosilación , Aparato de Golgi/genética , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Transporte de Proteínas/genética , Ubiquitinación/genética , Red trans-Golgi/genética

RESUMEN

Here, we tested the hypothesis that TNAP (tissue nonspecific alkaline phosphatase) modulates vascular responsiveness to norepinephrine. In the isolated, Tyrode's-perfused rat mesentery, 50 µmol/L of L-p-bromotetramisole (L-p-BT; selective TNAP inhibitor, Ki=56 µmol/L) significantly reduced TNAP activity and caused a significant 9.0-fold rightward-shift in the norepinephrine concentration versus vasoconstriction relationship. At 100 µmol/L, L-p-BT further reduced mesenteric TNAP activity and caused an additional significant right-shift of the norepinephrine concentration versus vasoconstriction relationship. A higher concentration (200 µmol/L) of L-p-BT had no further effect on either mesenteric TNAP activity or norepinephrine-induced vasoconstriction. L-p-BT did not alter vascular responses to vasopressin, thus ruling-out nonspecific suppression of vascular reactivity. Since in the rat mesenteric vasculature α1-adrenoceptors mediate norepinephrine-induced vasoconstriction, these finding indicate that TNAP inhibition selectively interferes with α1-adrenoceptor signaling. Additional experiments showed that the effects of TNAP inhibition on norepinephrine-induced vasoconstriction were not mediated by accumulation of pyrophosphate or ATP (TNAP substrates) nor by reduced adenosine levels (TNAP product). TNAP inhibition significantly reduced the Hillslope of the norepinephrine concentration versus vasoconstriction relationship from 1.8±0.2 (consistent with positive cooperativity of α1-adrenoceptor signaling) to 1.0±0.1 (no cooperativity). Selective activation of A1-adenosine receptors, which are known to participate in coincident signaling with α1-adrenoceptors, reversed the suppressive effects of L-p-BT on norepinephrine-induced vasoconstriction. In vivo, L-p-BT administration achieved plasma levels of ≈60 µmol/L and inhibited mesenteric vascular responses to exogenous norepinephrine and sympathetic nerve stimulation. TNAP modulates vascular responses to norepinephrine likely by affecting positive cooperativity of α1-adrenoceptor signaling via a mechanism involving A1 receptor signaling.

Asunto(s)

Fosfatasa Alcalina/metabolismo , Proteínas de la Membrana/metabolismo , Mesenterio/efectos de los fármacos , Norepinefrina/farmacología , Tetramisol/análogos & derivados , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Antagonistas del Receptor de Adenosina A1/farmacología , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/genética , Animales , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Mesenterio/metabolismo , Ratas , Tetramisol/farmacología , Xantinas/farmacología

RESUMEN

ß-Thalassemia intermedia is a disorder characterized by ineffective erythropoiesis (IE), anemia, splenomegaly, and systemic iron overload. Novel approaches are being explored based on the modulation of pathways that reduce iron absorption (ie, using hepcidin activators like Tmprss6-antisense oligonucleotides [ASOs]) or increase erythropoiesis (by erythropoietin [EPO] administration or modulating the ability of transferrin receptor 2 [Tfr2] to control red blood cell [RBC] synthesis). Targeting Tmprss6 messenger RNA by Tmprss6-ASO was proven to be effective in improving IE and splenomegaly by inducing iron restriction. However, we postulated that combinatorial strategies might be superior to single therapies. Here, we combined Tmprss6-ASO with EPO administration or removal of a single Tfr2 allele in the bone marrow of animals affected by ß-thalassemia intermedia (Hbbth3/+). EPO administration alone or removal of a single Tfr2 allele increased hemoglobin levels and RBCs. However, EPO or Tfr2 single-allele deletion alone, respectively, exacerbated or did not improve splenomegaly in ß-thalassemic mice. To overcome this issue, we postulated that some level of iron restriction (by targeting Tmprss6) would improve splenomegaly while preserving the beneficial effects on RBC production mediated by EPO or Tfr2 deletion. While administration of Tmprss6-ASO alone improved the anemia, the combination of Tmprss6-ASO + EPO or Tmprss6-ASO + Tfr2 single-allele deletion produced significantly higher hemoglobin levels and reduced splenomegaly. In conclusion, our results clearly indicate that these combinatorial approaches are superior to single treatments in ameliorating IE and anemia in ß-thalassemia and could provide guidance to translate some of these approaches into viable therapies.

Asunto(s)

Eritropoyetina/administración & dosificación , Eritropoyetina/genética , Terapia Genética/métodos , Proteínas de la Membrana/antagonistas & inhibidores , Oligonucleótidos Antisentido/administración & dosificación , Talasemia beta/terapia , Animales , Células Cultivadas , Eritropoyesis/efectos de los fármacos , Eritropoyesis/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hierro/metabolismo , Sobrecarga de Hierro/genética , Sobrecarga de Hierro/prevención & control , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oligonucleótidos Antisentido/farmacología , Receptores de Transferrina/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Talasemia beta/metabolismo

RESUMEN

We recently reported, for the first time, the expression and regulation of the PDZ polarity proteins Scrib and Dlg1 in human APCs, and also described the viral targeting of these proteins by NS1 of influenza A virus in human dendritic cells (DCs). Scrib plays an important role in reactive oxygen species (ROS) production in Mϕs and uropod formation and migration in T cells, while Dlg1 is important for T cell downstream activation after Ag recognition. Nevertheless, the functions of these proteins in human DCs remain unknown. Here, we knocked-down the expression of both Scrib and Dlg1 in human DCs and then evaluated the expression of co-stimulatory molecules and cytokine production during maturation. We demonstrated that Scrib is necessary for adequate CD86 expression, while Dlg1 is important for CD83 up-regulation and IL-6 production upon maturation, suggesting that Scrib and Dlg1 participate in separate pathways in DCs. Additionally, both proteins are required for adequate IL-12 production after maturation. Furthermore, we showed that the inefficient maturation of DCs induced by Scrib or Dlg1 depletion leads to impaired T cell activation. Our results revealed the previously unknown contribution of Scrib and Dlg1 in human DCs pivotal functions, which may be able to impact innate and adaptive immune response.

Asunto(s)

Presentación de Antígeno , Células Dendríticas/inmunología , Homólogo 1 de la Proteína Discs Large/fisiología , Proteínas de la Membrana/fisiología , Proteínas Supresoras de Tumor/fisiología , Inmunidad Adaptativa , Antígenos CD/biosíntesis , Antígenos CD/genética , Antígeno B7-2/biosíntesis , Antígeno B7-2/genética , Células Cultivadas , Citocinas/biosíntesis , Citocinas/genética , Homólogo 1 de la Proteína Discs Large/antagonistas & inhibidores , Homólogo 1 de la Proteína Discs Large/genética , Técnicas de Silenciamiento del Gen , Humanos , Inmunidad Innata , Inmunoglobulinas/biosíntesis , Inmunoglobulinas/genética , Interleucina-12/metabolismo , Interleucina-6/biosíntesis , Interleucina-6/genética , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Densidad Postsináptica/fisiología , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Regulación hacia Arriba , Antígeno CD83

RESUMEN

Immune checkpoint blockers improve the overall survival of a limited number of patients among different cancers. Identifying pathways that influence the immunological and clinical response to treatment is critical to improve the therapeutic efficacy and predict clinical responses. Recently, a key role has been assigned to innate immune mechanisms in checkpoint blockade-driven anti-tumor responses. However, inflammatory pathways can both improve and impair anti-tumor immunity. In this review, we discuss how different inflammatory pathways, particularly inflammasome activation, can influence the clinical outcome of immune checkpoint blockers. Inflammasome activation may reinforce anti-tumor immunity by boosting CD8+ T cell priming as well as by enhancing T helper type 17 (Th17) responses. In particular, we focus on the modulation of the cation channel transmembrane protein 176B (TMEM176B) and the ectonucleotidase CD39 as potential targets to unleash inflammasome activation leading to reinforced anti-tumor immunity and improved efficacy of immune checkpoint blockers. Future studies should be aimed at investigating the mechanisms and cell subsets involved in inflammasome-driven anti-tumor responses.

Asunto(s)

Antineoplásicos Inmunológicos/uso terapéutico , Apirasa , Inflamasomas/inmunología , Proteínas de la Membrana , Proteínas de Neoplasias , Neoplasias , Animales , Apirasa/antagonistas & inhibidores , Apirasa/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/inmunología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Neoplasias/patología , Células Th17/inmunología , Células Th17/patología

RESUMEN

This study in patients with relapsed, refractory, or high-risk multiple myeloma (MM) evaluated the safety and activity of autologous T cells engineered to express an affinity-enhanced T-cell receptor (TCR) that recognizes a peptide shared by cancer antigens New York esophageal squamous cell carcinoma-1 (NY-ESO-1) and L-antigen family member 1 (LAGE-1) and presented by HLA-A*02:01. T cells collected from 25 HLA-A*02:01-positive patients with MM expressing NY-ESO-1 and/or LAGE-1 were activated, transduced with self-inactivating lentiviral vector encoding the NY-ESO-1c259TCR, and expanded in culture. After myeloablation and autologous stem cell transplant (ASCT), all 25 patients received an infusion of up to 1 × 1010 NY-ESO-1 specific peptide enhanced affinity receptor (SPEAR) T cells. Objective response rate (International Myeloma Working Group consensus criteria) was 80% at day 42 (95% confidence interval [CI], 0.59-0.93), 76% at day 100 (95% CI, 0.55-0.91), and 44% at 1 year (95% CI, 0.24-0.65). At year 1, 13/25 patients were disease progression-free (52%); 11 were responders (1 stringent complete response, 1 complete response, 8 very good partial response, 1 partial response). Three patients remained disease progression-free at 38.6, 59.2, and 60.6 months post-NY-ESO-1 SPEAR T-cell infusion. Median progression-free survival was 13.5 months (range, 3.2-60.6 months); median overall survival was 35.1 months (range, 6.4-66.7 months). Infusions were well tolerated; cytokine release syndrome was not reported. No fatal serious adverse events occurred during study conduct. NY-ESO-1 SPEAR T cells expanded in vivo, trafficked to bone marrow, demonstrated persistence, and exhibited tumor antigen-directed functionality. In this MM patient population, NY-ESO-1 SPEAR T-cell therapy in the context of ASCT was associated with antitumor activity. This trial was registered at www.clinicaltrials.gov as #NCT01352286.

Asunto(s)

Antígenos de Neoplasias/inmunología , Inmunoterapia Adoptiva , Proteínas de la Membrana/inmunología , Mieloma Múltiple/inmunología , Mieloma Múltiple/terapia , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Terapia Combinada , Citocinas/metabolismo , Femenino , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T/genética , Receptores Quiméricos de Antígenos/genética , Trasplante Autólogo , Resultado del Tratamiento , Adulto Joven

RESUMEN

Golgi phosphoprotein 3 (GOLPH3) is a conserved protein of the Golgi apparatus that in humans has been implicated in tumorigenesis. However, the precise function of GOLPH3 in malignant transformation is still unknown. Nevertheless, clinicopathological data shows that in more than a dozen kinds of cancer, including gliomas, GOLPH3 could be found overexpressed, which correlates with poor prognosis. Experimental data shows that overexpression of GOLPH3 leads to transformation of primary cells and to tumor growth enhancement. Conversely, the knocking down of GOLPH3 in GOLPH3-overexpressing tumor cells reduces tumorigenic features, such as cell proliferation and cell migration and invasion. The cumulative evidence indicate that GOLPH3 is an oncoprotein that promotes tumorigenicity by a mechanism that impact at different levels in different types of cells, including the sorting of Golgi glycosyltransferases, signaling pathways, and the actin cytoskeleton. How GOLPH3 connects mechanistically these processes has not been determined yet. Further studies are important to have a more complete understanding of the role of GOLPH3 as oncoprotein. Given the genetic diversity in cancer, a still outstanding aspect is how in this inherent heterogeneity GOLPH3 could possibly exert its oncogenic function. We have aimed to evaluate the contribution of GOLPH3 overexpression in the malignant phenotype of different types of tumor cells. Here, we analyzed the effect on cell migration that resulted from stable, RNAi-mediated knocking down of GOLPH3 in T98G cells of glioblastoma multiforme, a human glioma cell line with unique features. We found that the reduction of GOLPH3 levels produced dramatic changes in cell morphology, involving rearrangements of the actin cytoskeleton and reduction in the number and dynamics of focal adhesions. These effects correlated with decreased cell migration and invasion due to affected persistence and directionality of cell motility. Moreover, the knocking down of GOLPH3 also caused a reduction in autoactivation of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase that regulates focal adhesions. Our data support a model in which GOLPH3 in T98G cells promotes cell migration by stimulating the activity of FAK.

Asunto(s)

Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Adhesiones Focales/fisiología , Proteínas de la Membrana/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/metabolismo

RESUMEN

We previously demonstrated that beta II protein kinase C (ßIIPKC) activity is elevated in failing hearts and contributes to this pathology. Here we report that ßIIPKC accumulates on the mitochondrial outer membrane and phosphorylates mitofusin 1 (Mfn1) at serine 86. Mfn1 phosphorylation results in partial loss of its GTPase activity and in a buildup of fragmented and dysfunctional mitochondria in heart failure. ßIIPKC siRNA or a ßIIPKC inhibitor mitigates mitochondrial fragmentation and cell death. We confirm that Mfn1-ßIIPKC interaction alone is critical in inhibiting mitochondrial function and cardiac myocyte viability using SAMßA, a rationally-designed peptide that selectively antagonizes Mfn1-ßIIPKC association. SAMßA treatment protects cultured neonatal and adult cardiac myocytes, but not Mfn1 knockout cells, from stress-induced death. Importantly, SAMßA treatment re-establishes mitochondrial morphology and function and improves cardiac contractility in rats with heart failure, suggesting that SAMßA may be a potential treatment for patients with heart failure.

Asunto(s)

Insuficiencia Cardíaca/tratamiento farmacológico , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas Mitocondriales/antagonistas & inhibidores , Péptidos/farmacología , Proteína Quinasa C beta/antagonistas & inhibidores , Animales , GTP Fosfohidrolasas/metabolismo , Técnicas de Inactivación de Genes , Insuficiencia Cardíaca/metabolismo , Masculino , Membranas Mitocondriales/metabolismo , Contracción Miocárdica , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Fosforilación , ARN Interferente Pequeño , Ratas Wistar

RESUMEN

Saccharomyces cerevisiae, the budding yeast, must remodel initial cell shape and cell wall integrity during vegetative growth and pheromone-induced morphogenesis. The cell wall remodeling is monitored and regulated by the cell wall integrity (CWI) signaling pathway. Wsc1p, together with Wsc2p and Wsc3p, belongs to a family of highly O-glycosylated cell surface proteins that function as stress sensors of the cell wall in S. cerevisiae. These cell surface proteins have the main role of activating the CWI signaling pathway by stimulating the small G-protein Rho1p, which subsequently activates protein kinase C (Pkc1p) and a mitogen activated protein (MAP) kinase cascade that activates downstream transcription factors of stress-response genes. Wsc1p, Wsc2p, and Wsc3p possess a cytoplasmic domain where two conserved regions of the sequence have been assessed to be important for Rom2p interaction. Meanwhile, other research groups have also proposed that these transmembrane proteins could support protein-protein interactions with Ras2p. Molecular structures of the cytoplasmic domain of Wsc1p, Wsc2p and Wsc3p were generated using the standard and fully-automated ORCHESTAR procedures provided by the Sybyl-X 2.1.1 program. The tridimensional structure of full length Ras2p was also generated with Phyre2. These protein models were validated with Procheck-PDBsum and ProSA-web tools and subsequently used in docking-based modeling of protein-protein and protein-compound interfaces for extensive structural and functional characterization of their interaction. The results retrieved from STRING 10.5 suggest that the Wsc-family is involved in protein-protein interactions with each other and with Ras2p. Docking-based studies also validated the existence of protein-protein interactions mainly between Motif I (Wsc3p > Wsc1p > Wsc2p) and Ras2p, in agreement with the data provided by STRING 10.5. Additionally, it has shown that Calcofluor White preferably binds to Wsc1p (-9.5 kcal/mol), meanwhile Caspofungin binds to Wsc3p (-9.1 kcal/mol), Wsc1p (-9.1 kcal/mol) and more weakly Wsc2p (-6.9 kcal/mol). Thus, these data suggests Caspofungin as a common inhibitor for the Wsc-family. MTiOpenScreen database has provided a list of new compounds with energy scores higher than those compounds used in our docking studies, thus suggesting these new compounds have a better affinity towards the cytoplasmic domains and Ras2p. Based on these data, there are new and possibly more effective compounds that should be considered as therapeutic agents against yeast infection.

Asunto(s)

Antifúngicos/farmacología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas de la Membrana/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas ras/antagonistas & inhibidores , Antifúngicos/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas ras/metabolismo

RESUMEN

Redox impairment and mitochondrial dysfunction have been seen in inflammation. Thus, there is interest in studies aiming to find molecules that would exert mitochondrial protection in mammalian tissues undergoing inflammation. Sesamol (SES) is an antioxidant and anti-inflammatory molecule as demonstrated in both in vitro and in vivo experimental models. Nonetheless, it was not previously demonstrated whether and how SES would cause mitochondrial protection during inflammation. Thus, we investigated here whether a pretreatment (for 1 h) with SES (1-100 µM) would prevent mitochondrial impairment in lipopolysaccharide (LPS)-treated RAW 264.7 cells. It was also evaluated whether the heme oxigenase-1 (HO-1) would be involved in the effects on mitochondria induced by SES. We found that SES reduced the levels of lipid peroxidation and protein nitration in the membranes of mitochondria obtained from LPS-treated RAW 264.7 cells. SES also attenuated the production of superoxide anion radical (O2-•) and nitric oxide (NO•) in this experimental model. SES suppressed the LPS-elicited mitochondrial dysfunction, as assessed through the analyses of the activities of the mitochondrial complexes I and V. SES also abrogated the LPS-induced decrease in the levels of adenosine triphosphate (ATP) and in the mitochondrial membrane potential (MMP). SES induced mitochondria-related anti-apoptotic effects in LPS-treated cells. Besides, SES pretreatment abrogated the LPS-triggered inflammation by decreasing the levels of pro-inflammatory proteins. The SES-induced mitochondria-associated protection was blocked by the specific inhibitor of HO-1, ZnPP IX (20 µM). Therefore, SES induced mitochondrial protection in LPS-treated cells by a mechanism involving HO-1.

Asunto(s)

Benzodioxoles/farmacología , Hemo-Oxigenasa 1/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Proteínas de la Membrana/antagonistas & inhibidores , Mitocondrias/efectos de los fármacos , Fenoles/farmacología , Animales , Benzodioxoles/administración & dosificación , Benzodioxoles/antagonistas & inhibidores , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Hemo-Oxigenasa 1/metabolismo , Lipopolisacáridos/antagonistas & inhibidores , Macrófagos/citología , Macrófagos/enzimología , Proteínas de la Membrana/metabolismo , Ratones , Mitocondrias/metabolismo , Fenoles/administración & dosificación , Fenoles/antagonistas & inhibidores , Protoporfirinas/farmacología , Células RAW 264.7 , Relación Estructura-Actividad

RESUMEN

The herbicide atrazine (ATZ) is used worldwide in the control of annual grasses and broad-leaved weeds. The present study evaluated sperm quality parameters in zebrafish Danio rerio after 11-day exposure to nominal ATZ concentrations of 2, 10, and 100 µg L-1. All ATZ concentrations caused a decrease in motility, mitochondrial functionality, and membrane integrity, as measured using conventional microscopy or fluorescence microscopy with specific probes. The DNA integrity of sperm was not affected. The levels of expression of genes related to spermatogenesis, antioxidant defenses, and DNA repair were also investigated using RT-qPCR. The ATZ caused transcriptional repression of the spermatogenesis-related genes SRD5A2 and CFTR, the antioxidant defense genes SOD2 and GPX4B, and the DNA repair gene XPC. This is the first study to show that environmentally relevant concentrations of ATZ significantly affect the sperm quality in fish, possibly resulting in reduced fertility rates. In addition, we showed that the repression of genes related to spermatogenesis and cellular defense could be part of the mechanisms involved in the ATZ toxicity in the testes of male fish.

Asunto(s)

Atrazina/toxicidad , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Herbicidas/toxicidad , Espermatogénesis/efectos de los fármacos , Testículo/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Pez Cebra/fisiología , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/química , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Animales , Proteínas de Ciclo Celular , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Glutatión Peroxidasa/antagonistas & inhibidores , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/metabolismo , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/enzimología , Membranas Mitocondriales/metabolismo , Concentración Osmolar , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Distribución Aleatoria , Proteínas de Saccharomyces cerevisiae , Motilidad Espermática/efectos de los fármacos , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Testículo/citología , Testículo/metabolismo , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/metabolismo , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo

RESUMEN

Hepatitis C virus can lead to chronic infection, cirrhosis and hepatocellular carcinoma. With more than 170 million people infected worldwide, eradication remains a challenge even with the revolutionary current direct antiviral agents (DAAs). The risk of resistance, the safety profile in some populations, the genotype specificity and the high price of current DAAs explain why there is still interest in developing host targeting agents (HTA) that may help overcome some of these difficulties. Specifically, targeting the entry of HCV to the cell seems like a promising strategy. Recently it has been shown that the cholesterol transporter NPC1L1, a protein located in the small bowel epithelium and in the canalicular membrane of the hepatocyte is also an HCV receptor. Just as this protein is key in the entero-hepatic cycle of cholesterol, we hypothesize that there is an entero-hepatic cycle of HCV that could be disrupted by blocking NPC1L1 with ezetimibe, an already approved and readily available safe drug. Ezetimibe, either alone or in combination with DAAs, could decrease relapse rates, reduce resistance and even make treatments cheaper.

Asunto(s)

Colesterol/metabolismo , Ezetimiba/administración & dosificación , Hepacivirus/fisiología , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Proteínas de la Membrana/antagonistas & inhibidores , Internalización del Virus/efectos de los fármacos , Fármacos Anti-VIH/administración & dosificación , Anticolesterolemiantes/administración & dosificación , Hepacivirus/efectos de los fármacos , Humanos , Intestinos/efectos de los fármacos , Intestinos/virología , Hígado/efectos de los fármacos , Hígado/virología , Proteínas de Transporte de Membrana , Modelos Biológicos

RESUMEN

The human growth hormone receptor antagonist G120R-hGH precludes dimerization of GH and prolactin receptors and consequently JAK/STAT signaling. Some modifications in this antagonist resulted in a drug specific for the GH receptor, called Pegvisomant (Somavert®). However, the original G120R-hGH is usually synthesized in bacterial cytoplasm as inclusion bodies, not being a commercial product. The present work describes the synthesis and characterization of G120R-hGH secreted into bacterial periplasm and obtained with a vector based on a constitutive lambda-PL promoter. This antagonist can be useful for studies aiming at investigating the effects of a simultaneous inhibition of GH and prolactin signaling, as a potential anti-tumoral or anti-diabetic compound. G120R-hGH, synthesized using the W3110 E. coli strain, showed a yield of 1.34 ± 0.24 µg/ml/A600 (∼0.79 mg G120R-hGH/g of wet weight cells) after cultivation at 30 °C up to 3 A600 units and induction at 37 °C, for 6 h, with final 4.3 ± 0.3 A600. A laboratory scale purification was carried out using three chromatographic steps with a total yield of 32%, reaching 98% purity. The obtained protein was characterized by SDS-PAGE, Western Blotting, Mass spectrometry, RP-HPLC, HPSEC and in vitro proliferation bioassay. The proliferation assay, based on Ba/F3-LLP cells, shows that G120R-hGH (100 ng/ml) significantly inhibited (64%) the proliferative action of hGH (1 ng/ml). This is the first time that G120R-hGH is synthesized in bacterial periplasmic space and therefore correctly folded, without the initial methionine. The reasons for a divergent efficacy for antagonizing hGH versus hPRL is currently unknown and deserves further investigation.

Asunto(s)

Sustitución de Aminoácidos , Escherichia coli/metabolismo , Hormona de Crecimiento Humana , Proteínas de la Membrana/antagonistas & inhibidores , Periplasma/metabolismo , Animales , Línea Celular , Escherichia coli/química , Escherichia coli/genética , Hormona de Crecimiento Humana/biosíntesis , Hormona de Crecimiento Humana/química , Hormona de Crecimiento Humana/genética , Hormona de Crecimiento Humana/aislamiento & purificación , Humanos , Ratones , Mutación Missense , Periplasma/química , Periplasma/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación

RESUMEN

BACKGROUND: Breast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes to invasiveness. Although proteolytically activated receptors (PARs) are activated by proteases, the direct interaction of cysteine cathepsins with PARs is poorly understood. In human platelets, PAR-1 and -4 are highly expressed, but PAR-3 shows low expression and unclear functions. METHODS: Platelet aggregation was monitored by measuring changes in turbidity. Platelets were immunoblotted with anti-phospho and total p38, Src-Tyr-416, FAK-Tyr-397, and TGFß monoclonal antibody. Activation was measured in a flow cytometer and calcium mobilization in a confocal microscope. Mammary epithelial cells were prepared from the primary breast cancer samples of 15 women with Luminal-B subtype to produce primary cells. RESULTS: We demonstrate that platelets are aggregated by cathepsin K in a dose-dependent manner, but not by other cysteine cathepsins. PARs-3 and -4 were confirmed as the cathepsin K target by immunodetection and specific antagonists using a fibroblast cell line derived from PARs deficient mice. Moreover, through co-culture experiments, we show that platelets activated by cathepsin K mediated the up-regulation of SHH, PTHrP, OPN, and TGFß in epithelial-mesenchymal-like cells from patients with Luminal B breast cancer. CONCLUSIONS: Cathepsin K induces platelet dysfunction and affects signaling in breast cancer cells.

Asunto(s)

Plaquetas/metabolismo , Neoplasias de la Mama/metabolismo , Catepsina K/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Animales , Plaquetas/efectos de los fármacos , Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , Calcio/metabolismo , Catepsina K/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Proteínas Hedgehog/metabolismo , Humanos , Hidrólisis , Ligandos , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Fosforilación , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Proteolisis , Receptores de Trombina/antagonistas & inhibidores , Trombina/metabolismo , Trombina/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Familia-src Quinasas/metabolismo

Asunto(s)

Eritropoyesis/efectos de los fármacos , Quelantes del Hierro/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Piridonas/farmacología , Talasemia beta/tratamiento farmacológico , Animales , Deferiprona , Modelos Animales de Enfermedad , Eritropoyesis/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Talasemia beta/genética , Talasemia beta/metabolismo

RESUMEN

BACKGROUND: Malignant melanoma is an aggressive type of skin cancer, and despite recent advances in treatment, the survival rate of the metastatic form remains low. Nifuroxazide analogues are drugs based on the substitution of the nitrofuran group by benzofuroxan, in view of the pharmacophore similarity of the nitro group, improving bioavailability, with higher intrinsic activity and less toxicity. Benzofuroxan activity involves the intracellular production of free-radical species. In the present work, we evaluated the antitumor effects of different benzofuroxan derivatives in a murine melanoma model. METHODS: B16F10-Nex2 melanoma cells were used to investigate the antitumor effects of Benzofuroxan derivatives in vitro and in a syngeneic melanoma model in C57Bl/6 mice. Cytotoxicity, morphological changes and reactive oxygen species (ROS) were assessed by a diphenyltetrasolium reagent, optical and fluorescence microscopy, respectively. Annexin-V binding and mitochondrial integrity were analyzed by flow cytometry. Western blotting and colorimetry identified cell signaling proteins. RESULTS: Benzofuroxan N-Br and N-I derivatives were active against murine and human tumor cell lines, exerting significant protection against metastatic melanoma in a syngeneic model. N-Br and N-I induce apoptosis in melanoma cells, evidenced by specific morphological changes, DNA condensation and degradation, and phosphatidylserine translocation in the plasma membrane. The intrinsic mitochondrial pathway in B16F10-Nex2 cells is suggested owing to reduced outer membrane potential in mitochondria, followed by caspase -9, -3 activation and cleavage of PARP. The cytotoxicity of N-Br and N-I in B16F10-Nex2 cells is mediated by the generation of ROS, inhibited by pre-incubation of the cells with N-acetylcysteine (NAC). The induction of ROS by N-Br and N-I resulted in the inhibition of AKT activation, an important molecule related to tumor cell survival, followed by upregulation of BIM. CONCLUSION: We conclude that N-Br and N-I are promising agents aiming at cancer treatment. They may be useful in melanoma therapy as inducers of intrinsic apoptosis and by exerting significant antitumor activity against metastatic melanoma, as presently shown in syngeneic mice.

Asunto(s)

Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/efectos de los fármacos , Benzoxazoles/química , Benzoxazoles/uso terapéutico , Melanoma Experimental/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Oncogénica v-akt/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteína 11 Similar a Bcl2 , Benzoxazoles/farmacología , Línea Celular Tumoral , Células HeLa , Humanos , Masculino , Melanoma Experimental/tratamiento farmacológico , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Proteína Oncogénica v-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología

RESUMEN

This work aimed to investigate mechanisms underlying the inflammatory response caused by Potamotrygon motoro stingray venom (PmV) in mouse paws. Pre-treatment of animals with a mast cell degranulation inhibitor (cromolyn) diminished edema (62% of inhibition) and leukocyte influx into the site of PmV injection. Promethazine (histamine type 1 receptor antagonist) or thioperamide (histamine type 3 and 4 receptor antagonist) also decreased edema (up to 30%) and leukocyte numbers, mainly neutrophils (40-50 %). Cimetidine (histamine type 2 receptor antagonist) had no effect on PmV-induced inflammation. In the RBL-2H3 lineage of mast cells, PmV caused proper cell activation, in a dose-dependent manner, with release of PGD2 and PGE2. In addition, the role of COXs products on PmV inflammatory response was evaluated. Indomethacin (COX-1/COX-2 inhibitor) or etoricoxib (COX-2 inhibitor) partially diminished edema (around 20%) in PmV-injected mice. Indomethacin, but not etoricoxib, modulated neutrophil influx into the site of venom injection. In conclusion, mast cell degranulation and histamine, besides COXs products, play an important role in PmV-induced reaction. Since PmV mechanism of action remains unknown, hindering accurate treatment, clinical studies can be performed to validate the prescription of antihistaminic drugs, besides NSAIDs, to patients injured by freshwater stingrays.

Asunto(s)

Edema/patología , Elasmobranquios/metabolismo , Venenos de los Peces/toxicidad , Histamina/toxicidad , Leucocitos/efectos de los fármacos , Mastocitos/efectos de los fármacos , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 1/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Dinoprostona/metabolismo , Edema/inducido químicamente , Etoricoxib , Antagonistas de los Receptores Histamínicos H1/farmacología , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Ratones , Prometazina/farmacología , Prostaglandina D2/metabolismo , Piridinas/farmacología , Ratas , Sulfonas/farmacología

RESUMEN

PURPOSE: Human Apo2-Ligand/TRAIL secreted by natural killer cells and cytotoxic T lymphocytes plays an important role immunosurveillance controlling tumor growth and metastasis. Moreover, the fact that Apo2L/TRAIL is capable of inducing cell death in tumor cells but not in normal cells makes this death ligand a promising anti-tumor agent. Previous data from our group demonstrated that Apo2L/TRAIL was physiologically released as transmembrane protein inserted in lipid vesicles, called exosomes. Recently, we demonstrated that artificial lipid nanoparticles coated with bioactive Apo2L/TRAIL (LUV-TRAIL) resembling the natural exosomes, greatly improved Apo2L/TRAIL activity and were able to induce apoptosis in hematological malignancies. In this study, we have deepened in the underlying mechanism of action of LUV-TRAIL in hematologic cells. METHODS/PATIENTS: Cytotoxic ability of LUV-TRAIL was assessed on Jurkat cells either over-expressing the anti-apoptotic protein Mcl1 or down-regulating the pro-apoptotic protein Bim previously generated in our laboratory. We also tested LUV-TRAIL cytotoxic ability against primary human leukemic cells from T-cell ALL patient. RESULTS: Silencing Bim but not Mcl-1 over-expression partially protects Jurkat cells from apoptosis induced by sTRAIL. LUV-TRAIL induced caspase-8 and caspase-3 activation and killed Jurkat-Mcl1 and Jurkat-shBim more efficiently than sTRAIL independently of the mitochondrial pathway. On the other hand, LUV-TRAIL were clearly more cytotoxic against primary leukemic cells from a T-cell ALL patient than sTRAIL. CONCLUSION: Tethering Apo2L/TRAIL to the surface of lipid nanoparticles greatly increases its bioactivity and could be of potential use in anti-tumor therapeutics.

Asunto(s)

Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Apoptosis , Inmunoterapia , Leucemia/patología , Leucemia/terapia , Liposomas , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Western Blotting , Proliferación Celular , Células Cultivadas , Regulación hacia Abajo , Humanos , Técnicas para Inmunoenzimas , Leucemia/inmunología , Leucemia/metabolismo , Linfocitos/citología , Linfocitos/inmunología , Linfocitos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal