RESUMEN
Human HSP27 (HSPB1) is a molecular chaperone sensor which, through dynamic changes in its phosphorylation and oligomerization, allows cells to adapt to changes in their physiology and/or mount a protective response to injuries. In pathological conditions, the high level of HSPB1 expression can either be beneficial, such as in diseases characterized by cellular degenerations, or be malignant in cancer cells where it promotes tumourigenesis, metastasis and anti-cancer drug resistance. Structural changes allow HSPB1 to interact with specific client protein partners in order to modulate their folding/activity and/or half-life. Therefore, the search is open for therapeutic compounds aimed at either down- or upregulating HSPB1 activity. In this respect, we have previously described two peptide aptamers (PA11 and PA50) that specifically interact with HSPB1 small oligomers and decrease its anti-apoptotic and tumourigenic activities. A novel analysis of the different HSPB1-interacting aptamers that were isolated earlier revealed that one aptamer (PA23) has the intriguing ability to stimulate the protective activity of HSPB1. We show here that this aptamer abolishes the dominant negative effect induced by the R120G mutant of αB-crystallin (HSPB5) by disrupting its interaction with HSPB1. Hence, developing structure-based interfering strategies could lead to the discovery of HSPB1-based therapeutic drugs.
Asunto(s)
Aptámeros de Péptidos/química , Aptámeros de Péptidos/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Chaperonas Moleculares/metabolismo , Regulación de la Expresión Génica , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares/genética , FosforilaciónRESUMEN
Hsp27 belongs to the heat shock protein family and displays chaperone properties in stress conditions by holding unfolded polypeptides, hence avoiding their inclination to aggregate. Hsp27 is often referenced as an anti-cancer therapeutic target, but apart from its well-described ability to interfere with different stresses and apoptotic processes, its role in non-stressed conditions is still not well defined. In the present study we report that three polypeptides (histone deacetylase HDAC6, transcription factor STAT2 and procaspase-3) were degraded in human cancerous cells displaying genetically decreased levels of Hsp27. In addition, these proteins interacted with Hsp27 complexes of different native size. Altogether, these findings suggest that HDAC6, STAT2 and procaspase-3 are client proteins of Hsp27. Hence, in non stressed cancerous cells, the structural organization of Hsp27 appears to be a key parameter in the regulation by this chaperone of the level of specific polypeptides through client-chaperone type of interactions.
Asunto(s)
Proteínas de Choque Térmico HSP27/genética , Proteínas/metabolismo , Proteolisis/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Proteínas de Choque Térmico HSP27/antagonistas & inhibidores , Proteínas de Choque Térmico HSP27/metabolismo , Células HeLa , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Transfección , Células Tumorales CultivadasRESUMEN
PURPOSE: The ability of heat shock protein 27 (Hsp27) to protect cells from stressful stimuli and its increased levels in tumors resistant to anticancer therapeutics suggest that it may represent a target for sensitization to radiotherapy. In this study, we investigate the protective role of Hsp27 against radiation-induced apoptosis and the effect of its attenuation in highly expressing radioresistant cancer cell lines. METHODS AND MATERIALS: We examined clonogenic death and the kinetics of apoptotic events in different tumor cell lines overexpressing or underexpressing Hsp27 protein irradiated with photons. The radiosensitive Jurkat cell line, which does not express Hsp27 constitutively or in response to gamma-rays, was stably transfected with Hsp27 complementary DNA. Attenuation of Hsp27 expression was accomplished by antisense or RNAi (interfering RNA) strategies in SQ20B head-and-neck squamous carcinoma, PC3 prostate cancer, and U87 glioblastoma radioresistant cells. RESULTS: We measured concentration-dependent protection against the cytotoxic effects of radiation in Jurkat-Hsp27 cells, which led to a 50% decrease in apoptotic cells at 48 hours in the highest expressing cells. Underlying mechanisms leading to radiation resistance involved a significant increase in glutathione levels associated with detoxification of reactive oxygen species, a delay in mitochondrial collapse, and caspase activation. Conversely, attenuation of Hsp27 in SQ20B cells, characterized by their resistance to apoptosis, sensitizes cells to irradiation. This was emphasized by increased apoptosis, decreased glutathione basal level, and clonogenic cell death. Sensitization to irradiation was confirmed in PC3 and U87 radioresistant cells. CONCLUSION: Hsp27 gene therapy offers a potential adjuvant to radiation-based therapy of resistant tumors.
Asunto(s)
Apoptosis/efectos de la radiación , Silenciador del Gen/fisiología , Proteínas de Choque Térmico/fisiología , Proteínas de Neoplasias/fisiología , Tolerancia a Radiación/fisiología , Apoptosis/fisiología , Caspasas/metabolismo , Línea Celular Tumoral/efectos de la radiación , Regulación hacia Abajo , Activación Enzimática/efectos de la radiación , Rayos gamma , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Glutatión/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/radioterapia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Células Jurkat/efectos de la radiación , Masculino , Mitocondrias/fisiología , Mitocondrias/efectos de la radiación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estrés Oxidativo/fisiología , Estrés Oxidativo/efectos de la radiación , Fotones , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/radioterapia , ARN sin Sentido/uso terapéutico , ARN Interferente Pequeño/uso terapéutico , Tolerancia a Radiación/genética , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , Transfección/métodos , Ensayo de Tumor de Célula MadreRESUMEN
Hsp27 and alphaB-crystallin are molecular chaperones that are constitutively expressed in several mammalian cells, particularly in pathological conditions. These proteins share functions as diverse as protection against toxicity mediated by aberrantly folded proteins or oxidative-inflammation conditions. In addition, these proteins share anti-apoptotic properties and are tumorigenic when expressed in cancer cells. This review summarizes the current knowledge about Hsp27 and alphaB-crystallin and the implications, either positive or deleterious, of these proteins in pathologies such as neurodegenerative diseases, myopathies, asthma, cataracts and cancers. Approaches towards therapeutic strategies aimed at modulating the expression and/or the activities of Hsp27 and alphaB-crystallin are presented.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Inflamación/tratamiento farmacológico , Chaperonas Moleculares/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Cadena B de alfa-Cristalina/metabolismo , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/química , Humanos , Inflamación/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Neoplasias/metabolismo , Conformación Proteica , Cadena B de alfa-Cristalina/antagonistas & inhibidores , Cadena B de alfa-Cristalina/químicaRESUMEN
BACKGROUND: Improved chemical hazard management such as REACH policy objective as well as drug ADMETOX prediction, while limiting the extent of animal testing, requires the development of increasingly high throughput as well as highly pertinent in vitro toxicity assays. METHODOLOGY: This report describes a new in vitro method for toxicity testing, combining cell-based assays in nanodrop Cell-on-Chip format with the use of a genetically engineered stress sensitive hepatic cell line. We tested the behavior of a stress inducible fluorescent HepG2 model in which Heat Shock Protein promoters controlled Enhanced-Green Fluorescent Protein expression upon exposure to Cadmium Chloride (CdCl2), Sodium Arsenate (NaAsO2) and Paraquat. In agreement with previous studies based on a micro-well format, we could observe a chemical-specific response, identified through differences in dynamics and amplitude. We especially determined IC50 values for CdCl2 and NaAsO2, in agreement with published data. Individual cell identification via image-based screening allowed us to perform multiparametric analyses. CONCLUSIONS: Using pre/sub lethal cell stress instead of cell mortality, we highlighted the high significance and the superior sensitivity of both stress promoter activation reporting and cell morphology parameters in measuring the cell response to a toxicant. These results demonstrate the first generation of high-throughput and high-content assays, capable of assessing chemical hazards in vitro within the REACH policy framework.
Asunto(s)
Bioensayo/métodos , Nanoestructuras , Pruebas de Toxicidad/métodos , Animales , Arseniatos/farmacología , Bioensayo/instrumentación , Cloruro de Cadmio/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Contaminantes Ambientales/farmacología , Contaminantes Ambientales/toxicidad , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Choque Térmico/genética , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Herbicidas/farmacología , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Análisis por Micromatrices/instrumentación , Análisis por Micromatrices/métodos , Paraquat/farmacología , Regiones Promotoras Genéticas , Pruebas de Toxicidad/instrumentaciónRESUMEN
We recently reported that the transient expression of polyglutamine tracts of various size in exon 1 of the huntingtin polypeptide (httEx1) generated abnormally high levels of intracellular reactive oxygen species that directly contributed to cell death. Here, we compared the protection generated by heat shock proteins to that provided by the antioxidant agent N-acetyl-L-cysteine. In cells expressing httEx1 with 72 glutamine repeats (httEx1-72Q), the overexpression of Hsp27 or Hsp70 plus Hdj-1(Hsp40) or treatment of the cells with N-acetyl-L-cysteine inhibited not only mitochondrial membrane potential disruption but also the increase in reactive oxygen species, nitric oxide and protein oxidation. However, only heat shock proteins and not N-acetyl-L-cysteine reduced the size of the inclusion bodies formed by httEx1-72Q. In cells expressing httEx1 polypeptide with 103 glutamine repeats (httEx1-103Q), heat shock proteins neither decreased oxidative damage nor reduced the size of the inclusions. In contrast, N-acetyl-L-cysteine still efficiently decreased the oxidative damage induced by httEx1-103Q polypeptide without altering the inclusions. N-Acetyl-L-cysteine was inactive with regard to proteasome inhibition, whereas heat shock proteins partially restored the caspase-like activity of this protease. These observations suggest some relationships between the presence of inclusion bodies and the oxidative damage induced by httEx1-polyQ.
Asunto(s)
Antioxidantes/farmacología , Proteínas de Choque Térmico/química , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Estrés Oxidativo , Acetilcisteína/química , Animales , Células COS , Chlorocebus aethiops , Exones , Glutamina/química , Proteínas de Choque Térmico HSP27 , Proteínas del Choque Térmico HSP40/química , Proteína Huntingtina , Chaperonas Moleculares , Proteínas de Neoplasias/química , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Péptido Hidrolasas/química , Péptidos/química , Especies Reactivas de OxígenoRESUMEN
Small stress proteins [small heat shock proteins (sHsps)] are molecular chaperones that modulate the ability of cells to respond to oxidative stress. The current knowledge concerning the protective mechanism generated by the expression of mammalian heat shock protein-27 (Hsp27) that allows cells to increase their resistance to oxidative stress is presented. We describe the effects mediated by Hsp27 expression toward crucial enzymes such as glucose-6-phosphate dehydrogenase and glutathione reductase that uphold glutathione in its reduced form. New data are presented showing that the expression of sHsps correlates with a drastic decrease in the intracellular level of iron, a catalyzer of hydroxyl radical (OH( . )) generation. A decreased ability of sHsps expressing cells to concentrate iron will therefore end up in a decreased level of oxidized proteins. In addition, we propose a role of Hsp27 in the presentation of oxidized proteins to the proteasome degradation machinery. We also present an analysis of several Hsp27 mutants that suggests that the C-terminal part of this stress protein is essential for its protective activity against oxidative stress.
Asunto(s)
Glutatión/metabolismo , Proteínas de Choque Térmico/fisiología , Hierro/metabolismo , Estrés Oxidativo , Animales , Regulación hacia Abajo , Proteínas de Choque Térmico/genética , Homeostasis , Ratones , Oxidación-ReducciónRESUMEN
Murine small stress protein [heat shock protein 25 (Hsp25)] expression confers thermotolerance and protection against oxidative stress. Hsp25 is an oligomeric ATP-independent phospho-chaperone that can generate a glutathione-dependent pro-reducing state in cells that are normally devoid of small stress protein constitutive expression. Hsp25 contains only one cysteine residue (position 141) that is highly susceptible to oxidation. We have explored the significance of this reactive residue by generating a mutant in which cysteine-141 was substituted by an alanine residue (C141A mutant). We report here that the C141A mutant did not form dimers when expressed in either murine L929 or human HeLa cells, hence, demonstrating that cysteine-141 regulates Hsp25 dimer formation. The C141A mutant also interfered with the dimerization of human Hsp27, a constitutively expressed small stress protein in HeLa cells. The mutated polypeptide showed a decreased ability to multimerize, but its expression was still able to induce cellular protection against oxidative stress. The C141A mutant was, however, less efficient than the wild-type protein in counteracting staurosporine-induced apoptosis, and it showed no in vivo chaperone activity. Hence, the cellular protection mediated against different stressors may require specific structural organizations of Hsp25 that are differently altered by the mutation. Of interest, when expressed concomitantly with wild-type Hsp25, the C141A polypeptide induced a dominant-negative effect, a phenomenon that may result from the ability of small stress proteins to interact and multimerize with each other.
Asunto(s)
Cisteína/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estrés Oxidativo , Sustitución de Aminoácidos , Animales , Apoptosis , Línea Celular , Dimerización , Células HeLa , Proteínas de Choque Térmico/química , Humanos , Ratones , Chaperonas Moleculares/metabolismo , Mutación , Proteínas de Neoplasias/químicaRESUMEN
There is currently great interest in the development of methods to analyze intracellular redox state and the cellular damages generated by oxidative stress. General methods for analyzing reactive oxygen species and glutathione level are presented together with more recently developed protocols to analyze the consequences of oxidative stress on the oxidation of macromolecules. Finally, techniques to study modalities of constitutive expression of Hsp27 in mammalian cells are considered as well as methods used to determine the protective activity of this small heat shock protein against the deleterious effects induced by oxidative stress.
Asunto(s)
Muerte Celular , Proteínas de Choque Térmico HSP27/fisiología , Estrés Oxidativo , Animales , Anexina A5/farmacología , Antioxidantes/química , Antioxidantes/farmacología , Células Cultivadas , Dimerización , Activación Enzimática , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Peroxidación de Lípido , Oxidación-Reducción , Especies Reactivas de OxígenoRESUMEN
Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear serine/threonine kinase of the subfamily of dual-specificity Yak1-related kinase proteins. HIPK2 was first described as a homeodomain-interacting protein kinase acting as a corepressor for homeodomain transcription factors. More recently, it was reported that HIPK2 plays a role in p53-mediated cellular apoptosis and could also participate in the regulation of the cell cycle. US11 protein of herpes simplex virus type 1 is a multifunctional protein involved in the regulation of several processes related to the survival of cells submitted to environmental stresses by mechanisms that are not fully elucidated. In an attempt to better understand the multiple functions of US11, we identified cellular binding partners of this protein by using the yeast two-hybrid system. We report that US11 interacts with HIPK2 through the PEST domain of HIPK2 and that this interaction occurs also in human cells. This interaction modifies the subcellular distribution of HIPK2 and protects the cell against the HIPK2-induced cell growth arrest.
Asunto(s)
Proteínas Portadoras/metabolismo , Herpesvirus Humano 1/patogenicidad , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Virales/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/química , Proteínas Portadoras/genética , División Celular/efectos de los fármacos , Proteínas Fluorescentes Verdes , Células HeLa/citología , Herpesvirus Humano 1/metabolismo , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Unión al ARN/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/farmacologíaRESUMEN
Neuronal loss and intraneuronal protein aggregates are characteristics of Huntington's disease (HD), which is one of 10 known neurodegenerative disorders caused by an expanded polyglutamine [poly(Q)] tract in the disease protein. N-terminal fragments of mutant huntingtin produce intracellular aggregates and cause toxicity. Several studies have shown that chaperones suppress poly(Q) aggregation and toxicity/cell death, but the mechanisms by which they prevent poly(Q)-mediated cell death remain unclear. In the present study, we identified heat shock protein 27 (HSP27) as a suppressor of poly(Q) mediated cell death, using a cellular model of HD. In contrast to HSP40/70 chaperones, we showed that HSP27 suppressed poly(Q) death without suppressing poly(Q) aggregation. We tested the hypotheses that HSP27 may reduce poly(Q)-mediated cell death either by binding cytochrome c and inhibiting the mitochondrial death pathway or by protecting against reactive oxygen species (ROS). While poly(Q)-induced cell death was reduced by inhibiting cytochrome c (cyt c) release from mitochondria, protection by HSP27 was regulated by its phosphorylation status and was independent of its ability to bind to cyt c. However, we observed that mutant huntingtin caused increased levels of ROS in neuronal and non-neuronal cells. ROS contributed to cell death because both N-acetyl-L-cysteine and glutathione in its reduced form suppressed poly(Q)-mediated cell death. HSP27 decreased ROS in cells expressing mutant huntingtin, suggesting that this chaperone protects cells against oxidative stress. We propose that a poly(Q) mutation can induce ROS that directly contribute to cell death and that HSP27 is an antagonist of this process.