RESUMEN
In this study, we determined whether binase, a ribonuclease from Bacillus pumilus, increases interferon sensitivity and apoptosis in SiHa cervical cancer cells infected with high-risk human papilloma virus (HPV) strain 16. Binase treatment increased SiHa cell apoptosis in a time- and concentration-dependent manner, as determined by flow cytometry, WST tests and real time xCelligence cell index analysis. Binase-treated SiHa cells showed reduced expression of E6 and E7 viral oncoproteins and increased expression of their intracellular targets, p53 and pRb. Combined treatment with binase and IFNα2b enhanced the interferon sensitivity of HPV-positive SiHa cells. By contrast, combined treatment with binase and IFNα2b in HPV-negative C33A cervical cancer cells, which do no expess E6 and E7, elicited no changes in interferon sensitivity or p53 and pRb expression. These findings suggest binase enhances interferon sensitivity and apoptosis in HPV-positive SiHa cervical cancer cells by suppressing E6 and E7 viral protein expression.
RESUMEN
Many viruses induce oxidative stress and cause S-glutathionylation of Cys residues of the host and viral proteins. Changes in cell functioning during viral infection may be associated with glutathionylation of a number of key proteins including Na,K-ATPase which creates a gradient of sodium and potassium ions. It was found that Na,K-ATPase α-subunit has a basal glutathionylation which is not abrogated by reducing agent. We have shown that acute hypoxia leads to increase of total glutathionylation level of Na,K-ATPase α-subunit; however, basal glutathionylation of α-subunit increases under prolonged hypoxia only. The role of basal glutathionylation in Na,K-ATPase function remains unclear. Understanding significance of basal glutathionylation is complicated by the fact that there are no X-ray structures of Na,K-ATPase with the identified glutathione molecules. We have analyzed all X-ray structures of the Na,K-ATPase α-subunit from pig kidney and found that there are a number of isolated cavities with unresolved electron density close to the relevant cysteine residues. Analysis of the structures showed that this unresolved density in the structure can be occupied by glutathione associated with cysteine residues. Here, we discuss the role of basal glutathionylation of Na,K-ATPase α-subunit and provide evidence supporting the view that this modification is cotranslational.
Asunto(s)
Riñón/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Sitios de Unión , Hipoxia de la Célula , Línea Celular , Cristalografía por Rayos X , Cisteína/química , Cisteína/metabolismo , Glutatión/química , Glutatión/metabolismo , Inmunoprecipitación , Riñón/metabolismo , Ratones , Simulación de Dinámica Molecular , Oxidación-Reducción , Fosfinas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/química , PorcinosRESUMEN
By maintaining the Na(+) and K(+) transmembrane gradient mammalian Na,K-ATPase acts as a key regulator of neuronal electrotonic properties. Na,K-ATPase has an important role in synaptic transmission and memory formation. Accumulation of beta-amyloid (Aß) at the early stages of Alzheimer's disease is accompanied by reduction of Na,K-ATPase functional activity. The molecular mechanism behind this phenomenon is not known. Here we show that the monomeric Aß(1-42) forms a tight (Kd of 3 µM), enthalpy-driven equimolar complex with α1ß1 Na,K-ATPase. The complex formation results in dose-dependent inhibition of the enzyme hydrolytic activity. The binding site of Aß(1-42) is localized in the "gap" between the alpha- and beta-subunits of Na,K-ATPase, disrupting the enzyme functionality by preventing the subunits from shifting towards each other. Interaction of Na,K-ATPase with exogenous Aß(1-42) leads to a pronounced decrease of the enzyme transport and hydrolytic activity and Src-kinase activation in neuroblastoma cells SH-SY5Y. This interaction allows regulation of Na,K-ATPase activity by short-term increase of the Aß(1-42) level. However prolonged increase of Aß(1-42) level under pathological conditions could lead to chronical inhibition of Na,K-ATPase and disruption of neuronal function. Taken together, our data suggest the role of beta-amyloid as a novel physiological regulator of Na,K-ATPase.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Activación Enzimática , Fluoresceína/metabolismo , Humanos , Hidrólisis , Modelos Moleculares , Neuroblastoma/metabolismo , Neuroblastoma/patología , Unión Proteica , Transporte de Proteínas , ATPasa Intercambiadora de Sodio-Potasio/química , Soluciones , Familia-src Quinasas/metabolismoRESUMEN
Accumulation of amyloid-ß (Aß) in neurons accompanies Alzheimer's disease progression. In the cytoplasm Aß influences activity of proteasomes, the multisubunit protein complexes that hydrolyze the majority of intracellular proteins. However, the manner in which Aß affects the proteolytic activity of proteasomes has not been established. In this study the effect of Aß42 and Aß42 with isomerized Asp7 on activity of different forms of proteasomes has been analyzed. It has been shown that Aß peptides efficiently reduce activity of the 20S proteasomes, but increase activity of the 20S proteasomes capped with the 19S and/or 11S regulators. Modulation of proteasome activity is mainly determined by the C-terminal segment of Aß (amino acids 17-42). This study demonstrated an important role of proteasome regulators in the interplay between Aß and the proteasomes.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Fragmentos de Péptidos/farmacología , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Humanos , Complejo de la Endopetidasa Proteasomal/químicaRESUMEN
The majority of known cytotoxic RNases are basic proteins which destroy intracellular RNA. Cationization of RNases is considered to be an effective strategy for strengthening their antitumor properties. We constructed a set of RNase Sa variants consisting of charge reversal mutants, charge neutralization mutants, and variants with positively charged cluster at the N-terminus. All constructs retain a high level of catalytic activity and differ in net charge. Using acute myeloid leukemia cells Kasumi-1 we have shown that (i) cytotoxicity of RNase Sa mutants is linearly enhanced by cationization, (ii) the ability of cytotoxic mutants to induce cell death is caused by induction of apoptosis and (iii) localization of positive charge on N-terminus does not contribute to RNase Sa cytotoxicity. Capacity to induce apoptosis in malignant cells and the absence of necrotic effects make the RNase Sa mutants with high positive charge a suitable anti-cancer agent.
RESUMEN
Cytotoxic exogenous RNases triggering apoptotic response in malignant cells have potential as anticancer drugs; surprisingly, detailed characterization of the RNase-induced apoptosis has not been conducted so far. Here we show that a cytotoxic RNase from Bacillus intermedius (binase) induces extrinsic and intrinsic apoptotic pathways in leukemic Kasumi-1 cells. The experiments were performed using TaqMan Array Human Apoptosis 96-well Plate for gene expression analysis, and flow cytometry. Cytometric studies demonstrated dissipation of the mitochondrial membrane potential, opening of mitochondrial permeability transition pores, activation of caspases, increase of intracellular Ca(2+) and decrease of reactive oxygen species levels. We found that expression of 62 apoptotic genes is up-regulated, including 16 genes that are highly up-regulated, and only one gene was found to be down-regulated. The highest, 16 fold increase of the expression level was observed for TNF gene. Highly up-regulated genes also include the non-canonical NF-κB signaling pathway and inflammatory caspases 1,4. The obtained results suggest that binase induces evolutionary acquired cellular response to a microbial agent and triggers unusual apoptosis pathway.