Chaperons moléculaires et repliement des protéines : L'exemple de certaines protéines de choc thermique

Des conditions ou agents déstabilisant l'environnement cellulaire altèrent souvent le repliement des protéines. Suivant son intensité, ce phénomène peut induire une agrégation irréversible des protéines et entraîner la mort des cellules. Un mécanisme cellulaire de défense contre cette atteinte à l'intégrité des protéines existe, qui est conservé au cours de l'évolution. En effet, la cellule réagit aux stress altérant le repliement des protéines en activant l'expression d'un petit nombre de gènes codant pour des protéines spécialisées, les Hsp (heat shock proteins). Certaines de ces protéines ont des activités de chaperons moléculaires aidant au repliement des polypeptides ayant une structure altérée. Mais la cellule contient également des homologues de Hsp constitutifs, non induits par un stress, qui participent au contrôle de qualité des protéines. Ces Hsp constitutives sont impliquées dans le repliement des protéines après leur synthèse, dans l'assemblage de structures multiprotéiques dans le réticulum endoplasmique, dans le dépliement des polypeptides lors de leur passage à travers les membranes ou dans le masquage de certaines mutations altérant le repliement des protéines. Les pathologies neurodégénératives et cancéreuses sont données en exemple pour souligner le fait qu'une concentration élevée en Hsp peut, selon la maladie concernée, être bénéfique ou délétère pour la cellule.Exposure to different conditions or agents that destabilize cell homeostasis often alters protein folding. Depending on stress intensity irreversible protein aggregation and cell death can occur. Cells have developed a conserved defense mechanism aimed at reducing the deleterious effects induced by protein folding alteration. This mechanism is characterized by the expression of a small number of genes encoding specific proteins, named Hsps. Several of these proteins act as molecular chaperones through their ability to refold polypeptides with an altered conformation. Moreover, constitutive Hsps homologues have been characterized that participate in the folding of newly made polypeptides, in the assembly of protein complexes in the endoplasmic reticulum, in the translocation of polypeptides through membranes or in masking mutations that alter protein folding. Neurodegeneratives and cancereous diseases are discussed as examples where high levels of Hsp expression can be either beneficial or deleterious to the cells

Evaluation of an in vitro hsp 70 induction test for toxicity assessment of complex mixtures : comparaison with chemical analyses and ecotoxicity tests

International audienceThe aim of this study was to assess the potential of a human cell line containing the hsp70 promoter linked to the chloramphenicol acetyltransferase reporter gene in evaluating the toxic potential of complex mixtures. Cells were exposed to eluates of industrial wastes and the cellular responses were compared with the metal contents of the samples and with standardized aquatic (microalgal growth inhibition, daphnia Immobilization, bacterial luminescence inhibition, Ceriodaphnia dubia reproduction inhibition) and terrestrial (earthworm lethality, plant growth inhibition) tests. The hsp70 promoter was significantly induced by 11 of 14 samples, with different dose-response patterns. Significant correlations of in vitro induction potency with aquatic ecotoxicity, especially with chronic tests, and with the metal contents of the samples were observed. Our study provides new information on the relevance of hsp70 gene induction as a criterion of toxicity and suggests its usefulness for the detection of toxicity associated with metallic pollution in complex mixtures

Induction of the HSP70 gene promoter by various anticancer drugs

International audienceHeLa cells containing the chloramphenicol acetyl transferase (CAT) gene under the control of the hsp70 promoter have been exposed in vitro to various anticancer drugs. Cisplatin induced CAT production with a dose-effect relationship at a non-cytotoxic dose, whereas no induction was detected with carboplatin. Etoposid induced a significant response at a cytotoxic concentration. The limited positive response with doxorubicin, daunomycin and mitoxantrone was not statistically significant. These chemicals are known to produce reactive oxygen species and induce apoptosis. No induction of the hsp70 promoter could be detected with the other cytostatic compounds that have been tested such as base analogues (5-fluorouracil, cytosine arabinoside 3'-MP), inhibitors of DNA synthesis (amethopterin, aminopterin), antimitotics (vinblastine, colchicine), and alkylating (streptozotocine, carboplatin, melphalan) or intercalating agents (bleomycin). In addition, the role of the transcription inhibitory activity of doxorubicin in this model is evidenced and the consequent question of the suitability of the reporter gene system is discussed. Our results suggest that specific genotoxic compounds are not able to induce the hsp70 promoter, and are in agreement with the concept that stimulation of HSP70 synthesis occurs through a biochemical process involving proteotoxicity

Hsp27 (HspB1) and αB-crystallin (HspB5) as therapeutic targets

AbstractHsp27 and αB-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 αB-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 αB-crystallin are presented

Toxicity Assays in Nanodrops Combining Bioassay and Morphometric Endpoints

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 (CdCl(2)), Sodium Arsenate (NaAsO(2)) 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 CdCl(2) and NaAsO(2), 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

Knock Down of Heat Shock Protein 27 (HspB1) Induces Degradation of Several Putative Client Proteins

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

Cellular Localization of HSP23 during <i>Drosophila</i> Development and following Subsequent Heat Shock

The low-molecular-weight heat-shock protein HSP23 is synthesized in the absence of heat shock during Drosophila development. Here, I present a quantitative analysis of this phenomenon and describe the cellular localization of this protein during normal development and after a subsequent heat shock. HSP23 is first detected in the late third instar larvae and continues to accumulate reaching a maximum level in late pupae. In a 1-week-old adult, HSP23 can no longer be detected. Following lysis of whole pupae, HSP23 is found in the soluble lysate fraction in a form which sediments between 10 and 20 S. Exposure of larvae, pupae, and the adult fly to heat stress (37°C) results in an increased amount of HSP23 which, however, is recovered in an insoluble particulate form following insect lysis. During recovery from heat shock, HSP23 is again found in the soluble 10- to 20-S lysate fraction. In pupae which are exposed to a severe heat stress (41°C) HSP23 remains in the pellet fraction after the heat stress and no pupae are able to emerge as adult flies. However, when pupae are first exposed to a mild heat-shock treatment prior to the 41°C stress, the thermotolerance process is induced and HSP23 is again rapidly found in the soluble lysate fraction during the recovery from heat shock. These observations suggest a possible correlation between the survival of pupae after heat shock and the recovery of HSP23 in the soluble lysate fraction as 10- to 20-S structures after the heat shock