Carole Kretz-Remy

Hsp27 as a Negative Regulator of Cytochrome c Release

ABSTRACT We previously showed that Hsp27 protects against apoptosis through its interaction with cytosolic cytochrome c. We have revisited this protective activity in murine cell lines expressing different levels of Hsp27. We report that Hsp27 also interferes, in a manner dependent on level of expression, with the release of cytochrome c from mitochondria. Moreover, a decreased level of endogenous Hsp27, which sensitized HeLa cells to apoptosis, reduced the delay required for cytochrome c release and procaspase 3 activation. The molecular mechanism regulating this function of Hsp27 is unknown. In our cell systems, Hsp27 is mainly cytosolic and only a small fraction of this protein colocalized with mitochondria. Moreover, we show that only a very small fraction of cytochrome c interacts with Hsp27, hence excluding a role of this interaction in the retention of cytochrome c in mitochondria. We also report that Bid intracellular relocalization was altered by changes in Hsp27 level of expression, suggesting that Hsp27 interferes with apoptotic signals upstream of mitochondria. We therefore investigated if the ability of Hsp27 to act as an expression-dependent modulator of F-actin microfilaments integrity was linked to the retention of cytochrome c in mitochondria. We show here that the F-actin depolymerizing agent cytochalasin D rapidly induced the release of cytochrome c from mitochondria and caspase activation. This phenomenon was delayed in cells pretreated with the F-actin stabilizer phalloidin and in cells expressing a high level of Hsp27. This suggests the existence of an apoptotic signaling pathway linking cytoskeleton damages to mitochondria. This pathway, which induces Bid intracellular redistribution, is negatively regulated by the ability of Hsp27 to protect F-actin network integrity. However, this upstream pathway is probably not the only one to be regulated by Hsp27 since, in staurosporine-treated cells, phalloidin only partially inhibited cytochrome c release and caspase activation. Moreover, in etoposide-treated cells, Hsp27 still delayed the release of cytochrome c from mitochondria and Bid intracellular redistribution in conditions where F-actin was not altered.

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

Hsp27 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.

Autophagy activation by NFκB is essential for cell survival after heat shock

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. In this study, we analyze the consequences of NFκB activation during heat shock recovery, by comparing the heat shock response of NFκB competent and incompetent (p65/RelA-depleted) cells. We demonstrate for the first time that NFκB plays a major and crucial role during the heat shock response by activating autophagy, which increases survival of heat-treated cells. Indeed, we observed that autophagy is not activated during heat shock recovery and cell death is strongly increased in NFκB incompetent cells. Moreover, if autophagy is artificially induced in these cells, the cytotoxicity of heat shock is turned back to normal. We show that despite a post-heat shock increase of Beclin 1 level in NFκB competent cells, neither Beclin 1/class III PI3K complex, Bcl2/BclXL nor mTOR kinase are NFκB targets whose modulation of expression could be responsible for NFκB activation of autophagy during heat shock recovery. In contrast, we demonstrate that aberrantly folded/aggregated proteins are prime events in the signaling pathway leading to NFκB mediated autophagy after heat shock. Hence, our findings demonstrate that NFκB-induced autophagy during heat shock recovery is an additional cell response to HS-induced protein denaturation/aggregation; this mechanism increases cell survival, probably through clearance of irreversibly damaged proteins.

Small stress proteins: novel negative modulators of apoptosis induced independently of reactive oxygen species.

The execution phase of the apoptotic cell death process occurs throughout the proteolytic activation of proteolytic enzymes called caspases (Nicholson and Thornberry 1997; Thornberry and Lazebnik 1998). Several different pathways can lead to the activation of caspases, among them, one can cite the death receptors (i.e. Fas) (Scaffidi et al. 1998) and mitochondria pathways (Reed 1997; Green and Reed 1998). When activated by ligand binding, death receptors (i.e. Fas) recruit adapter polypeptides (i.e. FADD) that interact with and subsequently activate pro-caspases (i.e. pro-caspase 8) or trigger a signal transduction pathway that activates specific genes (i.e. the DAXX/ASK1/JNK pathway). In contrast, in the mitochondria pathway, different inducers have the ability to induce the release in the cytoplasm of different proteins present in mitochondria, such as cytochrome c, apoptosis-inducing factor (AIP), Hsp60, HsplO, adenylate kinase, Smac/Diablo as well as the fraction of pro-caspase 2,3,8, and 9 present in mitochondria (Kluck et al. 1997; Reed 1997; Yang et al. 1997; Kohler et al. 1999; Samali et al. 1999a; Susin et al. 1999a,1999b; Xanthoudakis et al. 1999; Du et al. 2000; Verhagen et al. 2000). Once it has been released from the mitochondria, cytochrome c interacts with Apaf-1 in the presence of ATP/dATP. This results in the formation of the apoptosome complex which recruits and activates pro-caspase 9 which subsequently activates pro-caspase 3 (Li et al. 1997; Saleh et al. 1999). The release of cytochrome c from the mitochondria is a caspase-independent early phenomenon that precedes mitochondrial membrane potential loss (Bossy-Wetzel et al. 1998). This phenomenon may be induced by conformational changes of Bax (Desagher et al. 1999) or by BAK oligomerization induced by tBID, a membrane-targeted death ligand (Wei et al. 2000). Stress-induced apoptosis usually occurs through the activation of the mitochondria pathway. This is particularly the case when mild oxidative or heat stresses are considered (Samali et al. 2000).

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.

Gene expression and thiol redox state.

The intracellular redox status is a tightly regulated parameter which provides the cell with an optimal ability to counteract the highly oxidizing extracellular environment. Intracellular redox homeostasis is regulated by thiol-containing molecules, such as glutathione and thioredoxin. Essential cellular functions, such as gene expression, are influenced by the balance between pro- and antioxidant conditions. The mechanism by which the transcription of specific eukaryotic genes is redox regulated is complex, however, recent findings suggest that redox-sensitive transcription factors play an essential role in this process. This review is focused on the recent knowledge concerning some eukaryotic transcription factors, whose activation and DNA binding is controlled by the thiol redox status of the cell.

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.

Modulation of the chymotrypsin-like activity of the 20S proteasome by intracellular redox status: Effects of glutathione peroxidase-1 overexpression and antioxidant drugs

Abstract ATP- and ubiquitin-independent proteolysis by the 20S proteasome is responsible for the selective degradation of oxidized proteins. In vitro, the 20S proteasome shows an increased proteolytic activity toward oxidized polypeptides and the suc-LLVY-MCA peptide specific for its chymotrypsinlike activity. We have analyzed the effect of the intracellular redox status on the chymotrypsin-like activity of the 20S proteasome in human T47D cells overexpressing the detoxifiant enzyme seleno-glutathione peroxidase-1 (GPx-1). We report a 30% decreased activity of the chymotrypsin-like activity in cells overexpressing GPx-1. This phenomenon correlated with a 2-fold increase in IκBα half-life, a protein whose basal turnover is 20S proteasome-dependent. Following exposure to H2O2, these cells showed a seleno-dependently decreased accumulation of intracellular reactive oxygen species and 20S proteasome chymotrypsin like activity. Similar results were obtained in HeLa cells transiently overexpressing human GPx-1. Moreover, exposure of HeLa cells to antioxidant compounds reduced the proteasome 20S chymotrypsin like activity. In contrast, no effects were observed when HeLa cell extracts used to determine proteasome activity were incubated with either reduced or oxidized glutathione. These results suggest that GPx-1 activity or proreducing conditions can downregulate basal 20S proteasome activity. Hence, the intracellular redox status, probably through the level of oxidized proteins, is an important element that can either activate or down-regulate the 20S proteasome chymotrypsin-like activity in living cells.

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

NFκB is a master regulator of protein quality control. It helps the cells to survive proteotoxicity by modulating autophagy via up-regulation of BAG3 and HspB8 expression, a molecular mechanism relevant to protein conformational diseases.

Modulation of Protein Quality Control and Proteasome to Autophagy Switch in Immortalized Myoblasts from Duchenne Muscular Dystrophy Patients

The maintenance of proteome integrity is of primary importance in post-mitotic tissues such as muscle cells; thus, protein quality control mechanisms must be carefully regulated to ensure their optimal efficiency, a failure of these processes being associated with various muscular disorders. Duchenne muscular dystrophy (DMD) is one of the most common and severe forms of muscular dystrophies and is caused by mutations in the dystrophin gene. Protein quality control modulations have been diversely observed in degenerating muscles of patients suffering from DMD or in animal models of the disease. In this study, we investigated whether modulations of protein quality control mechanisms already pre-exist in undifferentiated myoblasts originating from DMD patients. We report for the first time that the absence of dystrophin in human myoblasts is associated with protein aggregation stress characterized by an increase of protein aggregates. This stress is combined with BAG1 to BAG3 switch, NFκB activation and up-regulation of BAG3/HSPB8 complexes that ensure preferential routing of misfolded/aggregated proteins to autophagy rather than to deficient 26S proteasome. In this context, restoration of pre-existing alterations of protein quality control processes might represent an alternative strategy for DMD therapies.