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

L’autophagie dépendante du facteur de transcription NFκB : un mécanisme de réponse à l’hyperthermie et à l’agrégation protéique

The heat shock response is a widely described defense mechanism during which the preferential expression of heat shock proteins (Hsps) helps the cell to recover from thermal damages such as protein denaturation/aggregation. We have previously reported that NFκB transcription factor is activated during the recovery period after heat shock. Thus, we aimed to analyze the consequences of NFκB activation during heat shock recovery, by comparing the heat shock response of NFκB competent and incompetent cells. We demonstrated that NFκB plays a major and crucial role during the heat shock response by activating autophagy, which increases the survival of heat-treated cells. Indeed, we observed that autophagy is not activated during heat shock recovery leading to an increased level of necrotic cell death in NFκB incompetent cells. Moreover, when autophagy is artificially induced in these cells, the heat shock cytotoxicity is turned back to normal. We showed that the key regulators of autophagy (mTOR complex, and class III PI3Kinase complex) are not regulated by NFκB after heat shock. In contrast, we observed that aberrantly folded/aggregated proteins accumulation is a prime event in the activation of NFκB -mediated autophagy. Moreover, NFκB -depleted cells accumulate higher levels of protein aggregates induced by either heat shock treatment or mutated form of HspB5, indicating that the protein quality control process seem to be altered in these cells. This alteration could be caused by a defect in BAG3-HspB8 complex formation in NFκB -depleted cells. We demonstrated that heat shock treatment induces a NFB-dependent overexpression of the bag3 and hspb8 genes. Moreover, the accumulation of BAG3-HspB8 complex in heat shocked NFκB -competent cells is inhibited by NFκB depletion. Our findings how / prove / highlight revealed that NFκB -induced autophagy during heat shock recovery is an additional response to protein denaturation/aggregation induced by heat shock. This process depends on the BAG3-HspB8 complex formation and increases cell survival, probably through clearance of aggregated proteinsLa réponse au choc thermique est un mécanisme de défense largement décrit au cours duquel l’expression préférentielle des protéines de choc thermique Hsp aide la cellule à récupérer des dommages causés par l’hyperthermie, comme la dénaturation/agrégation des protéines. Une des conséquences du choc thermique mise en évidence au laboratoire, est l’activation du facteur de transcription NFκB. Cette activation a lieu pendant la période de récupération suivant ce stress. Par comparaison de la réponse au choc thermique de cellules témoins ou déficientes en NFκB, nous avons cherché à étudier les conséquences de l’activation de NFκB par le choc thermique. Nous avons montré que NFκB active un mécanisme augmentant la survie des cellules soumises à une hyperthermie : l’autophagie. L’absence d’induction de ce mécanisme conduit à la mort par nécrose des cellules déplétées en NFκB. Dans ces cellules, l’induction artificielle de l’autophagie restaure une survie normale au stress thermique. Nous avons montré que les principaux régulateurs de l’autophagie (complexes mTOR et PI3Kinase de Classe III) ne sont pas des cibles modulées par NFκB, en réponse à une hyperthermie. En revanche, l’accumulation de protéines dénaturées voire agrégées est un élément primordial pour l’activation de l’autophagie-dépendante de NFκB. En effet dans les cellules déficientes pour NFκB, contrairement aux cellules témoins, l’accumulation de protéines agrégées induite par le traitement hyperthermique, mais aussi par l’expression de formes mutées d’HspB5, n’est pas résorbée ; ceci indique que le contrôle qualité des protéines est altéré dans ces cellules. Cette altération pourrait provenir d’un défaut de formation du complexe BAG3-HspB8 en absence de NFκB. En effet, nous avons montré que la forte expression des gènes bag3 et hspb8, induite suite au stress thermique, est dépendante de NFκB et que l’accumulation du complexe BAG3-HspB8, observé dans les cellules témoins soumises au choc thermique, est inhibée dans les cellules déficientes pour NFκB. Nos résultats démontrent que NFκB induit un processus autophagique en réponse à l’agrégation protéique induite par l’hyperthermie. Ce mécanisme, nécessitant la formation du complexe BAG3-HspB8, augmente la survie des cellules probablement par l’élimination des protéines agrégées générées au cours du stress thermiqu

NFκB-dependent autophagy : a response mechanism to hypothermia and protein aggregation

La réponse au choc thermique est un mécanisme de défense largement décrit au cours duquel l’expression préférentielle des protéines de choc thermique Hsp aide la cellule à récupérer des dommages causés par l’hyperthermie, comme la dénaturation/agrégation des protéines. Une des conséquences du choc thermique mise en évidence au laboratoire, est l’activation du facteur de transcription NFκB. Cette activation a lieu pendant la période de récupération suivant ce stress. Par comparaison de la réponse au choc thermique de cellules témoins ou déficientes en NFκB, nous avons cherché à étudier les conséquences de l’activation de NFκB par le choc thermique. Nous avons montré que NFκB active un mécanisme augmentant la survie des cellules soumises à une hyperthermie : l’autophagie. L’absence d’induction de ce mécanisme conduit à la mort par nécrose des cellules déplétées en NFκB. Dans ces cellules, l’induction artificielle de l’autophagie restaure une survie normale au stress thermique. Nous avons montré que les principaux régulateurs de l’autophagie (complexes mTOR et PI3Kinase de Classe III) ne sont pas des cibles modulées par NFκB, en réponse à une hyperthermie. En revanche, l’accumulation de protéines dénaturées voire agrégées est un élément primordial pour l’activation de l’autophagie-dépendante de NFκB. En effet dans les cellules déficientes pour NFκB, contrairement aux cellules témoins, l’accumulation de protéines agrégées induite par le traitement hyperthermique, mais aussi par l’expression de formes mutées d’HspB5, n’est pas résorbée ; ceci indique que le contrôle qualité des protéines est altéré dans ces cellules. Cette altération pourrait provenir d’un défaut de formation du complexe BAG3-HspB8 en absence de NFκB. En effet, nous avons montré que la forte expression des gènes bag3 et hspb8, induite suite au stress thermique, est dépendante de NFκB et que l’accumulation du complexe BAG3-HspB8, observé dans les cellules témoins soumises au choc thermique, est inhibée dans les cellules déficientes pour NFκB. Nos résultats démontrent que NFκB induit un processus autophagique en réponse à l’agrégation protéique induite par l’hyperthermie. Ce mécanisme, nécessitant la formation du complexe BAG3-HspB8, augmente la survie des cellules probablement par l’élimination des protéines agrégées générées au cours du stress thermiqueThe heat shock response is a widely described defense mechanism during which the preferential expression of heat shock proteins (Hsps) helps the cell to recover from thermal damages such as protein denaturation/aggregation. We have previously reported that NFκB transcription factor is activated during the recovery period after heat shock. Thus, we aimed to analyze the consequences of NFκB activation during heat shock recovery, by comparing the heat shock response of NFκB competent and incompetent cells. We demonstrated that NFκB plays a major and crucial role during the heat shock response by activating autophagy, which increases the survival of heat-treated cells. Indeed, we observed that autophagy is not activated during heat shock recovery leading to an increased level of necrotic cell death in NFκB incompetent cells. Moreover, when autophagy is artificially induced in these cells, the heat shock cytotoxicity is turned back to normal. We showed that the key regulators of autophagy (mTOR complex, and class III PI3Kinase complex) are not regulated by NFκB after heat shock. In contrast, we observed that aberrantly folded/aggregated proteins accumulation is a prime event in the activation of NFκB -mediated autophagy. Moreover, NFκB -depleted cells accumulate higher levels of protein aggregates induced by either heat shock treatment or mutated form of HspB5, indicating that the protein quality control process seem to be altered in these cells. This alteration could be caused by a defect in BAG3-HspB8 complex formation in NFκB -depleted cells. We demonstrated that heat shock treatment induces a NFB-dependent overexpression of the bag3 and hspb8 genes. Moreover, the accumulation of BAG3-HspB8 complex in heat shocked NFκB -competent cells is inhibited by NFκB depletion. Our findings how / prove / highlight revealed that NFκB -induced autophagy during heat shock recovery is an additional response to protein denaturation/aggregation induced by heat shock. This process depends on the BAG3-HspB8 complex formation and increases cell survival, probably through clearance of aggregated protein

NF-κB regulates protein quality control after heat stress through modulation of the BAG3-HspB8 complex.

We previously found that the NF-κB transcription factor is activated during the recovery period after heat shock; moreover, we demonstrated that NF-κB is essential for cell survival after heat shock by activating autophagy, a mechanism that probably helps the cell to cope with hyperthermic stress through clearance of damaged proteins. In this study, we analyze the involvement of NF-κB in basal and heat-stress-induced protein quality control, by comparing the level of multiubiquitylated and/or aggregated proteins, and proteasome and autophagic activity in NF-κB-competent and NF-κB-incompetent cells. We show that NF-κB has only a minor role in basal protein quality control, where it modulates autophagosome maturation. By contrast, NF-κB is shown to be a key player in protein quality control after hyperthermia. Indeed, NF-κB-incompetent cells show highly increased levels of multiubiquitylated and/or aggregated proteins and aggresome clearance defects; a phenotype that disappears when NF-κB activity is restored to normal. We demonstrate that during heat shock recovery NF-κB activates selective removal of misfolded or aggregated proteins--a process also called 'aggrephagy'--by controlling the expression of BAG3 and HSPB8 and by modulating the level of the BAG3-HspB8 complex. Thus NF-κB-mediated increase in the level of the BAG3-HspB8 complex leads to upregulation of aggrephagy and clearance of irreversibly damaged proteins and might increase cell survival in conditions of hyperthermia

NF kappa B is a central regulator of protein quality control in response to protein aggregation stresses via autophagy modulation

During cell life, proteins often misfold, depending on particular mutations or environmental changes, which may lead to protein aggregates that are toxic for the cell. Such protein aggregates are the root cause of numerous diseases called "protein conformational diseases," such as myofibrillar myopathy and familial amyotrophic lateral sclerosis. To fight against aggregates, cells are equipped with protein quality control mechanisms. Here we report that NF kappa B transcription factor is activated by misincorporation of amino acid analogues into proteins, inhibition of proteasomal activity, expression of the R120G mutated form of HspB5 (associated with myofibrillar myopathy), or expression of the G985R and G93A mutated forms of superoxide dismutase 1 (linked to familial amyotrophic lateral sclerosis). This noncanonical stimulation of NF kappa B triggers the up-regulation of BAG3 and HspB8 expression, two activators of selective autophagy, which relocalize to protein aggregates. Then NF kappa B-dependent autophagy allows the clearance of protein aggregates. Thus NF kappa B appears as a central and major regulator of protein aggregate clearance by modulating autophagic activity. In this context, the pharmacological stimulation of this quality control pathway might represent a valuable strategy for therapies against protein conformational diseases.Ligue contre le Cancer, comite du Rhone Ligue contre le Cancer, comite de Savoie Bonus Qualite Recherche from Universite Claude Bernard Lyon 1 Centre National de la Recherche Scientifique Millennium Institute P09-015-F FONDAP Program 15150012 French Department of research Fondation pour la Recherche Medicale Association Francaise contre les Myopathies/Teletho

Analysis of the Dominant Effects Mediated by Wild Type or R120G Mutant of αB-crystallin (HspB5) towards Hsp27 (HspB1)

<div><p>Several human small heat shock proteins (sHsps) are phosphorylated oligomeric chaperones that enhance stress resistance. They are characterized by their ability to interact and form polydispersed hetero-oligomeric complexes. We have analyzed the cellular consequences of the stable expression of either wild type HspB5 or its cataracts and myopathies inducing R120G mutant in growing and oxidative stress treated HeLa cells that originally express only HspB1. Here, we describe that wild type and mutant HspB5 induce drastic and opposite effects on cell morphology and oxidative stress resistance. The cellular distribution and phosphorylation of these polypeptides as well as the oligomerization profile of the resulting hetero-oligomeric complexes formed by HspB1 with the two types of exogenous polypeptides revealed the dominant effects induced by HspB5 polypeptides towards HspB1. The R120G mutation enhanced the native size and salt resistance of HspB1-HspB5 complex. However, in oxidative conditions the interaction between HspB1 and mutant HspB5 was drastically modified resulting in the aggregation of both partners. The mutation also induced the redistribution of HspB1 phosphorylated at serine 15, originally observed at the level of the small oligomers that do not interact with wild type HspB5, to the large oligomeric complex formed with mutant HspB5. This phosphorylation stabilized the interaction of HspB1 with mutant HspB5. A dominant negative effect towards HspB1 appears therefore as an important event in the cellular sensitivity to oxidative stress mediated by mutated HspB5 expression. These observations provide novel data that describe how a mutated sHsp can alter the protective activity of another member of this family of chaperones.</p></div

Quantitative analysis of the cellular distribution of phosphorylated HspB1 and HspB5.

<p>The data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#pone-0070545-g002" target="_blank">Fig. 2A</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#pone-0070545-g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#pone-0070545-g005" target="_blank">5</a> were used to calculate the percentage in regard to the total cellular content of HspB1, HspB5 and their different phosphorylated isoforms (HspB1: Ser15, Ser78 and Ser82/HspB5: Ser19, Ser45 and Ser59) in the different size populations (S10,000×<i>g</i>, gel filtration) and pellet fractions (P10,000×<i>g</i>). Size populations from gel filtration columns and color codes are the same as those described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#pone-0070545-g005" target="_blank">Fig. 5</a>. Standard deviations are indicated from three independent experiments. <i>**P</i><0.01.</p

Characterization of HspB1, HspB5 and mutant HspB5 in HeLa cell clones.

<p>A) Cellular distribution of HspB1, HspB5 (wild type and mutant) and Hsp70 upon cell lysis. Neo, WT and R120G cells were lysed in the presence of 0.1% Triton X-100 and spun at 10,000×<i>g</i> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#s4" target="_blank">Materials and Methods</a>. The levels of HspB1, HspB5 and Hsp70 present in the supernatant and pellet fractions were detected in immunoblots probed with the corresponding antibodies (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#s4" target="_blank">Materials and Methods</a>). Autoradiographs of ECL-revealed immunoblots are presented. Quantitative analysis of three independent experiments is presented in the adjacent figure. B) Effect of shRNA-mediated depletion of HspB1. WT and R120G cells were transiently transfected with control mismatch pSuperNeo-MsRNA27 (Mismatch: Ms) or pSuperNeo-ShRNA27 (ShB1) vector targeting HspB1 mRNA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#s4" target="_blank">Materials and Methods</a>). Two days after transfection, cells were analyzed in immunoblots probed with HspB1, HspB5 and actin antibodies. ShB1 transfected cells were also treated for the last 20 h before being analyzed with 0.5 µmol/l of MG132. Quantitative analysis of one particular experiment where the RNAi-mediated transient depletion of HspB1 was almost complete is presented in the adjacent figure. C) Analysis of HspB1 and HspB5 native sizes in Neo, WT and R120G cells. Cells were lysed as above and the 10,000×<i>g</i> cytosolic supernatant fractions were applied to Sepharose 6B gel filtration columns (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#s4" target="_blank">Materials and Methods</a>). The presence of HspB1 and HspB5 in pooled fractions eluted from the columns was detected in immunoblots probed with the corresponding antibodies. Autoradiographs of ECL-revealed immunoblots are presented. 29, 66, 150, 200, 443, 669 kDa are gel filtration markers. Exclusion size of the column is 2000 kDa. Brackets indicate fractions that were pooled for further immunoprecipitation analysis. Size population I is from WT cells and size population II is from R120G cells. D) Co-immunoprecipitation studies. a) Size population I from WT cells was immunoprecipitated with either anti-HspB1 (IPαB1) or anti-HspB5 antibody (IPαB5). Immunoprecipitated proteins-bound to proteinG-sepharose were washed in IPP150 buffer containing 150 mM NaCl before being processed for gel electrophoresis. After migration in SDS-PAGE, proteins were revealed in immunoblots probed with either anti-HspB1 or anti-HspB5 antibody. T: aliquot of cytosolic supernatant fractions before immunoprecipitation, IP: immunoprecipitated proteins, S: aliquot from supernatant after immunoprecipitation. b) Same as a) except that washes of the immunoprecipitated proteins were performed in IPP300 buffer containing 300 mM NaCl. c–d) same as a–b) but in this case analysis was performed with size population II from R120G cells. Autoradiographs of ECL-revealed immunoblots are presented.</p

Enhanced oxidoresistance induced by wild type HspB5 and sensitivity mediated by the R120G mutation.

<p>Neo, WT or R120G cells were treated or not for different time periods with several concentrations of menadione. A) Crystal violet staining. The percentage of cell survival corresponded to the ratio of the relative absorbance of the different samples to that of untreated cells. Values are means ± SDM of three independent experiments. 2-way ANOVA indicates statistically significant differences in the survival to menadione treatment between Neo, WT and R120G cell lines, <i>*P</i><0.05, <i>**P</i><0.01. B) Clonogenic colony formation assay. The number of colonies was visually estimated. All experiments were performed in triplicate. C) Phase-contrast analysis of cell morphology. Before and after treatments, phase contrast analysis of the morphology of live cells was performed and photographs are presented. Bar: 10 µm. Black arrows: perinuclear granules; black arrowheads: filamentous bridges between cells; white arrowheads: membranous ruffles; white arrows: vacuoles. D) Immunoblot analysis of the level of HspB1, HspB5, Hsp70 and Hsp90 in menadione-treated Neo, WT and R120G cells.</p

Characterization of Neo, WT and R120G cells.

<p>A) Immunoblot analysis of total cellular extracts of Neo, WT and R120G HeLa cells. The levels of HspB1, HspB5, Hsp70, Hsp90, HspB6 and Actin were detected in immunoblots probed with the corresponding antibodies (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#s4" target="_blank">Materials and Methods</a>). B) Phase contrast analysis of Neo, WT and R120G. bar: 10 µm. Black arrows: perinuclear granules; white arrowheads: membranous ruffles. Analysis of the biggest dimension of cells (cell length) is presented in the adjacent figure. Mean, SD (standard deviation) and SEM (standard error of mean) of twenty different measurements are presented. C) Analysis of the number of cells in the cultures was from day 0 to days 1 and 2 (d1, d2). Values are means ± SEM of three independent experiments. One-way ANOVA within a time point analysis indicates statistically significant growth differences between Neo and WT and R120G cell lines, <i>*P</i><0.05. D) Immunofluorescence analysis. Neo, WT and R120G cells were processed for the immunofluorescence detection of HspB5, HspB1 and nuclei as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#s4" target="_blank">Materials and Methods</a>. Bar: 10 µm. Cells were stained for HspB5 (red fluorescence), HspB1 (green fluorescence), nuclei (blue fluorescence) and processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070545#s4" target="_blank">Materials and Methods</a>. The fusion images (Merge) of WT and R20G cells are shown. Overlap and Pearson's coefficients are indicated. E) The graphs represent the fluorescence distribution of HspB1 (green; Ch1-1), wild type or mutant HspB5 (red; Ch1-2) and nucleus (blue; Ch1-3) of the section of WT or R120G cells shown in the green/red fusion images (Merge). #: areas where the co-localization of HspB1 and HspB5 (wild type or mutant) may not occur.</p