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Supplementary Material and methods and figures. Supplementary Figure 1: SHH is overexpressed in Colon, Pancreatic and NSCL cancers. Supplementary Figure 2: SHH interference lead to cell death in LoVo colon cell line. Supplementary Figure 3: SHH interference does not involve CDON-induced cell death. Supplementary Figure 4: PtcDN and interference with SHH does not affect the SHH positive signaling. Supplementary Figure 5: SHH expression in HCT8 xenograft and tumor growth inhibition in SHH siRNA treated HCT8- engrafted mice. Supplementary Figure 6: Interference with SHH in LoVo colon cell line show tumor growth inhibition in vivo.

The Sonic Hedgehog (SHH) pathway plays a key role in cancer. Alterations of SHH canonical signaling, causally linked to tumor progression, have become rational targets for cancer therapy. However, Smoothened (SMO) inhibitors have failed to show clinical benefit in patients with cancers displaying SHH autocrine/paracrine expression. We reported earlier that the SHH receptor Patched (PTCH) is a dependence receptor that triggers apoptosis in the absence of SHH through a pathway that differs from the canonical one, thus generating a state of dependence on SHH for survival. Here, we propose a dual function for SHH: its binding to PTCH not only activates the SHH canonical pathway but also blocks PTCH-induced apoptosis. Eighty percent, 64%, and 8% of human colon, pancreatic, and lung cancer cells, respectively, overexpressed SHH at transcriptional and protein levels. In addition, SHH-overexpressing cells expressed all the effectors of the PTCH-induced apoptotic pathway. Although the canonical pathway remained unchanged, autocrine SHH interference in colon, pancreatic, and lung cell lines triggered cell death through PTCH proapoptotic signaling. In vivo, SHH interference in colon cancer cell lines decreased primary tumor growth and metastasis. Therefore, the antitumor effect associated to SHH deprivation, usually thought to be a consequence of the inactivation of the canonical SHH pathway, is, at least in part, because of the engagement of PTCH proapoptotic activity. Together, these data strongly suggest that therapeutic strategies based on the disruption of SHH/PTCH interaction in SHH-overexpressing cancers should be explored.Significance:Sonic Hedgehog–overexpressing tumors express PTCH-induced cell death effectors, suggesting that this death signaling could be activated as an antitumor strategy.

The Sonic Hedgehog (SHH) pathway plays a key role in cancer. Alterations of SHH canonical signaling, causally linked to tumor progression, have become rational targets for cancer therapy. However, Smoothened (SMO) inhibitors have failed to show clinical benefit in patients with cancers displaying SHH autocrine/paracrine expression. We reported earlier that the SHH receptor Patched (PTCH) is a dependence receptor that triggers apoptosis in the absence of SHH through a pathway that differs from the canonical one, thus generating a state of dependence on SHH for survival. Here, we propose a dual function for SHH: its binding to PTCH not only activates the SHH canonical pathway but also blocks PTCH-induced apoptosis. Eighty percent, 64%, and 8% of human colon, pancreatic, and lung cancer cells, respectively, overexpressed SHH at transcriptional and protein levels. In addition, SHH-overexpressing cells expressed all the effectors of the PTCH-induced apoptotic pathway. Although the canonical pathway remained unchanged, autocrine SHH interference in colon, pancreatic, and lung cell lines triggered cell death through PTCH proapoptotic signaling. In vivo, SHH interference in colon cancer cell lines decreased primary tumor growth and metastasis. Therefore, the antitumor effect associated to SHH deprivation, usually thought to be a consequence of the inactivation of the canonical SHH pathway, is, at least in part, because of the engagement of PTCH proapoptotic activity. Together, these data strongly suggest that therapeutic strategies based on the disruption of SHH/PTCH interaction in SHH-overexpressing cancers should be explored. Significance: Sonic Hedgehog–overexpressing tumors express PTCH-induced cell death effectors, suggesting that this death signaling could be activated as an antitumor strategy.

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., 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 NFκB 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 NFκB triggers the up-regulation of BAG3 and HspB8 expression, two activators of selective autophagy, which relocalize to protein aggregates. Then NFκB-dependent autophagy allows the clearance of protein aggregates. Thus NFκB 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.

Human HspB1 (also denoted as Hsp27) belongs to the family of small (or stress) proteins (sHsps). The family, which contains ten members including αA,B-crystallin polypeptides, is characterized by a conserved C-terminal α-crystallin domain and molecular weights ranging from 20 to 40 kDa. Here, procedures are described for analyzing the dynamic oligomerization and phosphorylation patterns of HspB1 in cells exposed to different environments. Changes in the structural organization of HspB1 can reprogram its interaction with specific partner/client polypeptides. Methods are presented to analyze these interactions using tissue culture cells genetically modified to express different levels of this protein. In addition, the laboratory approaches presented here could be used to test the nine other human sHsp members as well as sHsps from other species.

Human small heat shock proteins are molecular chaperones that regulate fundamental cellular processes in normal unstressed cells as well as in many cancer cells where they are over-expressed. These proteins are characterized by cell physiology dependent changes in their oligomerization and phosphorylation status. These structural changes allow them to interact with many different client proteins that subsequently display modified activity and/or half-life. Nowdays, the protein interactomes of small Hsps are under intense investigations and will represent, when completed, key parameters to elaborate therapeutic strategies aimed at modulating the functions of these chaperones. Here, we have analyzed the potential pro-cancerous roles of several client proteins that have been described so far to interact with HspB1 (Hsp27) and its close members HspB5 (αB-crystallin) and HspB4 (αA-crystallin).

HSP90 was the first molecular target to inhibit the interaction of this heat shock protein (HSP) with client proteins in cancer cells and tissues. The HSP90 inhibition was attempted to liberate from this chaperone the oncogenic fusion proteins, mutated and activated serine/threonine protein kinases, tyrosine kinases, as well as transcription factors with oncogenic activity, in this manner, the free proteins could be recognized by the proteasome system to be degraded. We should remember here that many HSP family members are overexpressed in different kinds of cancer tissues, these molecules act as chaperones of tumorigenesis. In cancer patients, the first generation of HSP90 inhibitors showed elevated levels of toxicity, which was partially solved with the second-generation of inhibitors that could be intravenously delivered. With the arrival of the third-generation drugs that could be orally administrated, anticancer activities were achieved in clinical trials, however, the results were not as successful as expected due to: 1) limited anti-tumor efficacy, 2) acquisition of drug resistance, 3) difficulty to identify the client protein(s) specifically degraded in response to drug administration. The main problem is the redundancy of chaperones that the cancer cells have, in fact during HSP90 or HSP70 inhibition the heat shock factor (HSF1) could be liberated increasing the levels of other HSPs and in addition, HSF1 can by itself act as an inducer of the multidrug resistance MDR response and is also implicated in HER2 and hormonal responses. These difficulties, rather than decreasing the interest of having the HSPs as molecular targets, are increasing the exploration of new ways to interfere with several HSPs simultaneously and using HSP inhibitors with more “conventional” anticancer drugs. In this article we review, in addition to HSP90, HSP27, HSP70, and HSP60 as targets for anticancer therapy.

Constitutively expressed small heat shock protein HspB1 regulates many fundamental cellular processes and plays major roles in many human pathological diseases. In that regard, this chaperone has a huge number of apparently unrelated functions that appear linked to its ability to recognize many client polypeptides that are subsequently modified in their activity and/or half-life. A major parameter to understand how HspB1 is dedicated to interact with particular clients in defined cellular conditions relates to its complex oligomerization and phosphorylation properties. Indeed, HspB1 structural organization displays dynamic and complex rearrangements in response to changes in the cellular environment or when the cell physiology is modified. These structural modifications probably reflect the formation of structural platforms aimed at recognizing specific client polypeptides. Here, I have reviewed data from the literature and re-analyzed my own studies to describe and discuss these fascinating changes in HspB1 structural organization.

Human heat shock protein 27 (Hsp27, HspB1) is an anti-apoptotic protein characterized for its tumorigenic and metastatic properties, and now referenced as a major therapeutic target in many types of cancer. Hsp27 biochemical properties rely on a structural oligomeric and dynamic organization. Downregulation by small interfering RNA or inhibition with dominant-negative mutant have proven their efficiency to counteract the anti-apoptotic and protective properties of Hsp27. In this study, we report the isolation and characterization of Hsp27-targeted molecules interfering with its structural organization. Using the peptide aptamer (PA) strategy, we isolated PAs that specifically interact with Hsp27 and not with the other members of the small heat shock protein family. In mammalian cell cultures, PAs expression perturbed the dimerization and oligomerization of Hsp27, and acted as negative regulators of the anti-apoptotic and cytoprotective activities of this protein. Further studies analyzing SQ20B cell xenografts in immunocompromised mice showed that PAs strongly reduced tumor development through cell cycle arrest. Our data suggest that PAs could provide a potential tool to develop strategies for the discovery of Hsp27 chemical inhibitors.

In leukemia cells, hyperthermia enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. The phenomenon is caspase-dependent and results in membrane changes leading to an increased recognition of TRAIL death receptors by TRAIL. Because either caspase-2 or an apical proteolytic event has been recently proposed to act as an initiator of the cell death mechanism induced by heat shock, we have investigated the hierarchy of caspase activation in cells exposed to the combined heat shock plus TRAIL treatment. We report here that caspases-2, -3, and -8 were the first caspases to be activated. As expected, caspase-8 is required and indispensable during the initiation of this death signaling. Caspase-2 may also participate in the phenomenon but, in contrast to caspase-8, its presence appears dispensable because its depletion by small interfering RNA is devoid of effects. Our observations also suggest a role of caspase-3 and of a particular cleaved form of this caspase during the early signals of heat shock plus TRAIL-induced apoptosis.

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 γ-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.

In addition to being potent chaperones that protect cells against the accumulation of unfolded proteins under stress conditions, mammalian small heat shock proteins (small Hsps) regulate many vital cellular processes in normal and pathological cells. Indeed, these Hsps are constitutively expressed in many tissues and show dramatic changes in their levels of expression in most human pathologies. They are characterized by a large spectrum of activities and are particularly active in protein conformational and inflammatory diseases as well as in cancer pathologies. It is now believed that the immense cellular implications of small Hsps results from their ability to interact, through particular structural changes, with many different client proteins that are subsequently modulated in their activities or half-lifes. Here, we have integrated functionally and structurally the recent data in the literature concerning the interactions of mammalian small Hsps with specific clients. Further analysis with geneMANIA software and database confirmed the incredibly large number of functions associated with these Hsps. The consequences for human pathologies as well as putative therapeutic strategies are discussed, particularly when the expression of small Hsps is harmful (as in some cancer pathologies) or when it appears beneficial for patients.

Recently, we reported that the transient expression of huntingtin exon1 polypeptide containing polyglutamine tracts of various sizes (httEx1-polyQ) in cell models of Huntington disease generated an oxidative stress whose intensity was CAG repeat expansion-dependent. Here, we have analyzed the intracellular localization of the oxidative events generated by the httEx1-polyQ polypeptides. Analysis of live COS-7 cells as well as neuronal SK-N-SH and PC12 cells incubated with hydroethidine or dichlorofluorescein diacetate revealed oxidation of these probes at the level of the inclusion bodies formed by httEx1-polyQ polypeptides. The intensity and frequency of the oxidative events among the inclusions were CAG repeat expansion-dependent. Electron microscopic analysis of cell sections revealed the presence of oxidation-dependent morphologic alterations in the vicinity of httEx1-polyQ inclusion bodies. Moreover, a high level of oxidized proteins was recovered in partially purified inclusions. We also report that the iron chelator deferroxamine altered the structure, localization and oxidative potential of httEx1-polyQ inclusion bodies. Hence, despite the fact that the formation of inclusion bodies may represent a defense reaction of the cell to eliminate httEx1 mutant polypeptide, this phenomenon appears inherent to the generation of iron-dependent oxidative events that can be deleterious to the cell.

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.

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.

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.

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.

We show here that Hsp27 increases its level of expression during the late phase of the keratinocyte differentiation of human HaCat cells. A similar phenomenon was observed when differentiated HaCat cells underwent a dedifferentiation process. In both cases, Hsp27 accumulated in the form of large native structures, which represent the chaperone active form of the protein. Hence, the presence of Hsp27 large oligomers does not appear to be the consequence of a particular differentiation process but should be considered as a marker of endogenous stress conditions. Such conditions may arise when drastic changes in the intracellular protein organization occur, such as during differentiation, dedifferentiation and probably also during the development of the senescent phenotype.

Small stress proteins are molecular chaperones that modulate the ability of cells to respond to several types of injuries. In this regard, a protection generated by the expression of mammalian small stress proteins against the cell death induced by oxidative stress has been described. This review summarizes the current knowledge of the protective mechanism generated by the expression of the major mammalian small stress protein Hsp27 in cells exposed to oxidative stress. A possible role of this chaperone protein in the presentation of oxidized proteins to the proteasome degradation machinery is proposed.

Heat shock induces the accumulation of misfolded proteins and results in the preferential expression of heat shock proteins, which help the cell to recover from thermal damage. Heat shock is a well known transcriptional activator of the human immunodeficiency virus type 1 long terminal repeat (LTR). We report here that mutations or deletions of the LTR kappaB sites impaired the LTR transcriptional activation by heat shock. Further analysis revealed that, during heat shock recovery, the NF-kappaB p65 and p50 subunits migrated into the nucleus of HeLa cells, bound to DNA, and induced kappaB-dependent reporter gene expression. This NF-kappaB activation did not depend on new transcriptional and/or translational events and on the pro-oxidant state generated by heat shock. It was not concomitant with IkappaBalpha phosphorylation and was not abolished by the expression of IkappaB kinase or IkappaBalpha dominant-negative mutants. Moreover, NF-kappaB activation and migration into the nucleus were not concomitant with IkappaBalpha/beta or p105 degradation. However, during heat shock recovery, NF-kappaB was dissociated from its complexing partners, allowing its migration into the nucleus. Hence, we describe here a novel mechanism for activation of NF-kappaB based on the thermolability of the NF-kappaB.IkappaB complex.

The paralyse spontaneous mutation in mice involves degeneration and death of anterior horn motor neurons. Mutant mice are not viable past postnatal day 16. At present, the mechanisms involved in motor neuron death are unknown. Here, we investigate the expression of the small heat shock protein Hsp25, in the spinal cord of paralyse at two different stages during postnatal development, i.e., day 11 and day 14. Western blot analysis reveals that the level of Hsp25 was strikingly different in paralyse as compared to control littermates. Hsp25 expression level in paralyse at day 11 was much lower than in control mice. At day 14, an opposite pattern was observed. Such pattern seems to be restricted to spinal cord, since level of Hsp25 in other tissues (lung, brain, liver, and heart) was quite similar. Immunofluorescence examination of the lumbar spinal cord sections reveals that in control mice, Hsp25 was expressed at high level in motor neurons located in the ventral horn at both day 11 and day 14. By contrast, in paralyse mice, Hsp25 staining within the motor neurons was barely detectable except as a spot in the nucleolus (day 11). At the end stage of the disease (day 14), not only was Hsp25 staining even less intense in motor neurons, but also a strong Hsp25 staining was observed in reactive astrocytes within the gray matter. Taken together, these data suggest that Hsp25 expression is differently modulated in neuronal and glial cells during neurodegenerative processes leading to motor neuron death. J. Neurosci. Res. 65:247–253, 2001. © 2001 Wiley-Liss, Inc.

Treatment of mammalian cells with hydrogen peroxide induces the nuclear translocation of the transcription factor NF-kappa B and its binding to kappa B DNA sequences present in the promoter region of numerous genes. The role of selenium in NF-kappa B activation was analyzed in human T47D cells overexpressing the seleno-dependent detoxifiant enzyme glutathione peroxidase. Following exposure to H(2)O(2), these cells showed a seleno-dependent decreased accumulation of intracellular ROS and NF-kappa B activation. This phenomenon was correlated with an inhibition of the nuclear translocation of NF-kappa B (p50 subunit) and with an absence of I kappa B alpha degradation. We also report that the half-life of I kappa B alpha in untreated cells was increased two-fold by the overexpression of active glutathione peroxidase. These results suggest that selenium is a key element that through its modulation of glutathione peroxidase activity can inhibit NF-kappa B activation and can up-regulate I kappa B alpha normal half life.

Mammalian cells respond to stress by accumulating or activating a set of highly conserved proteins known as heat-shock proteins (HSPs). Several of these proteins interfere negatively with apoptosis. We show that the small HSP known as Hsp27 inhibits cytochrome-c-mediated activation of caspases in the cytosol. Hsp27 does not interfere with granzyme-B-induced activation of caspases, nor with apoptosis-inducing factor-mediated, caspase-independent, nuclear changes. Hsp27 binds to cytochrome c released from the mitochondria to the cytosol and prevents cytochrome-c-mediated interaction of Apaf-1 with procaspase-9. Thus, Hsp27 interferes specifically with the mitochondrial pathway of caspase-dependent cell death.

Simian Virus 40 Large T-antigen expressed in NIH3T3 cells increases p53 level and interacts with this tumor suppressor to form large nuclear complexes. We show here that T-antigen sensitizes NIH3T3 cells to low doses of the oxidative stress inducer menadione. This oxidant increased p53 accumulation and disrupted p53/T-antigen interaction, but not T-antigen/pRb, T-antigen/Hsc70 and p53/Hsc70 complexes; a phenomenon inhibited by the anti-oxidant N-acetyl-cysteine. Analysis of several p53 downstream gene products revealed that the level of Fas receptor, which was sharply reduced by T-antigen expression, was drastically increased in response to menadione treatment. Menadione also induced a T-antigen dependent cleavage of Fas ligand. Analysis performed with Fas receptor antagonist antibody and metalloproteinases inhibitor revealed that menadione triggers a Fas-dependent death of a fraction of T-antigen expressing cells. This Fas pathway does not activate caspase 8 or 3, probably because of the inhibition induced by T-antigen, and leads to a necrotic cell death which contributes at least in part to the hypersensitivity of T-antigen transformed cells to oxidative stress.

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.

The small stress protein Hsp27 is expressed during mammalian neural development. We have analyzed the role of this protein in immortalized rat olfactory neuroblasts. In the presence of dopamine a fraction of these cells differentiate into neurons while the remaining cells undergo apoptosis. We report here that the dopamine induced differentiation and apoptosis are associated with a transient and specific accumulation of Hsp27. Moreover, transfection experiments have shown that Hsp27 overexpression drastically decreases the fraction of cells undergoing apoptosis. In contrast, reduction of the endogenous level of Hsp27 led to abortion of differentiation and, therefore, drastically increased the number of apoptotic cells. Furthermore, in the normal cell population we show that Hsp27 accumulation takes place only in differentiating cells that were not undergoing apoptosis. We therefore conclude that Hsp27 may represent a key protein that controls the decision of olfactory precursor cells to undergo either differentiation or cell death.

The effects of thiols, ascorbic acid and thermal stress on the basal (steady-state) levels of oxidative DNA base modifications were studied. In various types of untreated cultured mammalian cells, the levels of total glutathione were found to be inversely correlated with the levels of DNA base modifications sensitive to the repair endonuclease Fpg protein, which include 8-hydroxyguanine (8-oxoG). A depletion of glutathione by treatment with buthionine sulphoximine increased the steady-state level in AS52 Chinese hamster cells by approximately 50%. However, additional thiols in the culture medium did not reduce the level of Fpg-sensitive base modifications: 0-10 mM N-acetylcysteine had no effect, whereas cysteine ethylester even increased the oxidative DNA damage at concentrations >0.1 mM. Similarly, ascorbic acid (0-20 mM) failed to reduce the steady-state levels. When AS52 cells were grown at elevated temperature (41 degrees C), the steady-state level of the oxidative DNA modifications increased by 40%, in spite of a concomitant 1.6-fold increase of the cellular level of total glutathione. Depletion of glutathione at 41 degrees C nearly doubled the already elevated level of oxidative damage. A constitutive expression of the heat-shock protein Hsp27 in L929 mouse fibrosarcoma cells at 37 degrees C increased the glutathione level by 60%, but had little effect on the level of oxidative DNA damage.

We report here that amino acid analogs, which activate hsp70 promoter, are powerful transcriptional activators of human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR), an activation which was impaired when the two κB sites present in the LTR were mutated or deleted. Amino acid analogs also stimulated the transcription of a κB-controlled reporter gene. Upon treatment with amino acid analogs, the two NF-κB subunits (p65 and p50), which are characterized by a relatively long half-life, redistributed into the nucleus where they bound to κB elements. This phenomenon, which began to be detectable after 1 h of treatment, was concomitant with the degradation of the short lived inhibitory subunit IκB-α by the proteasome. However, contrasting with other NF-κB inducers that trigger IκB-α degradation through a phosphorylation step, amino acid analogs did not change IκB-α isoform composition. Antioxidant conditions inhibited amino acid analog stimulatory action toward NF-κB. This suggests that aberrant protein conformation probably generates a pro-oxidant state that is necessary for IκB-α proteolysis by the proteasome. Moreover, this activation of NF-κB appeared different from that mediated by endoplasmic reticulum overload as it was not inhibited by calcium chelation.

The recent discoveries in the field of human small heat shock proteins (sHSPs) clearly point to the important roles played by these adenosine triphosphate (ATP)-independent chaperones in the regulation of a large spectrum of vital cellular processes and in pathological diseases. These proteins are therefore considered as very attractive therapeutic targets.To understand the functions of the stress-inducible members of the sHSP family, HspB1, HspB5 and HspB8, and be able to therapeutically modulate their activities, researchers are faced with the complex oligomerisation and phosphorylation properties of these proteins and with their ability to interact with each other and with specific protein targets. Here, we have integrated, in a functionally orientated way, the up-to-date literature data concerning HspB1, HspB5 and HspB8 protein interactions which reflect their numerous crucial cellular functions. We also present data supporting the idea that specific phospho-oligomeric domains of HspB1 are involved in the interaction with particular client proteins.More information concerning the interactions between client protein targets and sHSPs or the multiple combinatorial chimeric oligomeric complexes formed by different sHSPs are urgently required to elaborate a comprehensive sHSPs protein interactome and propose efficient and pathology-specific therapeutic approaches.

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.

Heat shock proteins (HSP) are a subset of the molecular chaperones, best known for their rapid and abundant induction by stress. HSP genes are activated at the transcriptional level by heat shock transcription factor 1 (HSF1). During the progression of many types of cancer, thisheat shock transcriptional regulon becomes co-opted by mechanisms that are currently unclear, although evidently triggered in the emerging tumor cell. Concerted activation of HSF1 and the accumulation of HSPs then participate in many of the traits that permit the malignant phenotype. Thus, cancers of many histologies exhibit activated HSF1 and increased HSP levels that may help to deter tumor suppression and evade therapy in the clinic. We review here the extensive work that has been carried out and is still in progress aimed at (1) understanding the oncogenic mechanisms by which HSP genes are switched on, (2) determining the roles of HSF1/HSP in malignant transformation and (3) discovering approaches to therapy based on disrupting the influence of the HSF1-controlled transcriptome in cancer. Fil: Ciocca, Daniel Ramon. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Arrigo, Andre Patrick. Cancer Research Center of Lyon, Apoptosis Cancer and Development; Francia. Claude Bernard University; Francia Fil: Calderwood, Stuart K.. Beth Israel Deaconess Medical Center. Department of Radiation Oncology; Estados Unidos

Bacteria and eukaryotic cells respond to cold stress by inducing and enhancing the synthesis of specific arrays of proteins. We describe here cold-induced enhancement of expression for two reporter genes; luciferase and beta-galactosidase, both under the control of HIV-1 LTR sequences, observed in mouse fibroblasts and human HeLa cells respectively. Increased expression of luciferase in fibroblasts when shifted to 25 degrees C was detectable at 30 degrees C but was not observed following cold shock at 4 degrees C. To sustain the cold-induced effect, cells had to be kept at subphysiological temperature. The observed enhancement of luciferase activity did not result from a particular site of integration of the reporter gene and was evident whether cold-stressed cells were stationary or growing. Cold-induced expression of luciferase was evidenced at the protein level, enzymatic activity and RNA level, furthermore, active transcription and translation were required for overexpression. The cold effect which has been generalized with the reporter gene beta-galactosidase appears to be a process involving, at least in part, the HIV-1 LTR sequences and might correspond to an increase in the half-life of mRNA. The cold-dependent enhanced expression of luciferase and beta-galactosidase reported here, together with data describing the activation of HIV-1 LTR by hyperthermia, point out the particular temperature sensitivity of these regulatory sequences. This potential thermal modulation may be useful in the comprehension of regulatory processes in latency and reactivation of viral expression during HIV-1 infection.

Small stress protein expression enhances the survival of mammalian cells exposed to numerous injuries that induce necrotic cell death. The cell surface receptor Fas/APO-1 and its ligand have been recently identified as important mediators of apoptosis. Here, we show that constitutive expression of human heat shock protein (hsp)27 in murine L929 cells blocks Fas/APO-1-mediated cell death. Expression of human hsp27 prevented anti-APO-1-induced DNA fragmentation and morphological changes. These results strongly suggest that human hsp27 acts as a cellular inhibitor of Fas/APO-1-induced apoptosis. We also report that the expression of small stress proteins from different species, such as human hsp27, Drosophila Dhsp27, or human αB-crystallin, confers resistance to apoptotic cell death induced by staurosporine, a protein kinase C inhibitor. Hence, small stress proteins are novel regulators that are able to block apoptosis induced by different pathways.

Expression of small stress proteins (shsp) enhances the survival of mammalian cells exposed to heat or oxidative injuries. Recently, we have shown that the expression of shsp from different species, such as human hsp27, Drosophila hsp27 or human alphaB-crystallin protected murine L929 cells against cell death induced by tumor necrosis factor (TNFalpha), hydrogen peroxide or menadione. Here, we report that, in growing L929 cell lines, the presence of these shsp decreased the intracellular level of reactive oxygen species (ROS). shsp expression also abolished the burst of intracellular ROS induced by TNFalpha. Several downstream effects resulting from the TNFalpha-mediated ROS increment, such as NF-kappaB activation, lipid peroxidation and protein oxidation, were inhibited by shsp expression. We also report that the expression of these different shsp raised the total glutathione level in both L929 cell lines and transiently transfected NIH 3T3-ras cells. This phenomenon was essential for the shsp-mediated decrease in ROS and resistance against TNFalpha. Our results therefore suggest that the protective activity shared by human hsp27, Drosophila hsp27 and human alphaB-crystallin against TNFalpha-mediated cell death and probably other types of oxidative stress results from their conserved ability to raise the intracellular concentration of glutathione.

We report here that both kappa B-dependent transactivation of a reporter gene and NF-kappa B activation in response to tumor necrosis factor (TNF alpha) or H2O2 treatments are deficient in human T47D cell transfectants that overexpress seleno-glutathione peroxidase (GSHPx). These cells feature low reactive oxygen species (ROS) levels and decreased intracellular ROS burst in response to TNF alpha treatment. Decreased ROS levels and NF-kappa B activation were likely to result from GSHPx increment since these phenomena were no longer observed when GSHPx activity was reduced by selenium depletion. The cellular contents of the two NF-kappa B subunits (p65 and p50) and of the inhibitory subunit I kappa B-alpha were unaffected by GSHPx overexpression, suggesting that increased GSHPx activity interfered with the activation, but not the synthesis or stability, of Nf-kappa B. Nuclear translocation of NF-kappa B as well as I kappa B-alpha degradation were inhabited in GSHPx-overexpressing cells exposed to oxidative stress. Moreover, in control T47D cells exposed to TNF alpha, a time correlation was observed between elevated ROS levels and I kappa B-alpha degradation. We also show that, in growing T47D cells, GSHPx overexpression altered the isoform composition of I kappa B-alpha, leading to the accumulation of the more basic isoform of this protein. GSHPx overexpression also abolished the TNF alpha-mediated transient accumulation of the acidic and highly phosphorylated I kappa B-alpha isoform. These results suggest that intracellular ROS are key elements that regulate the phosphorylation of I kappa B-alpha, a phenomenon that precedes and controls the degradation of this protein, and then NF-kappa B activation.

To investigate the role of the small 27-kDa heat-shock protein (Hsp27) in the intrinsic resistance of colon cancer cells to doxorubicin, we modified Hsp27 expression either genetically by transfection or pharmacologically by cisplatin treatment. HT-29 cells were transfected with a full-length Hsp27 construct in the sense or antisense orientation. We found a good correlation between cell survival after doxorubicin treatment and Hsp27 content. A similar correlation was found for the thermoresistance of the Hsp27-transfected cells. In contrast, the sensitivity of the different transfected cells to 5-fluorouracil was not modified. cis-Platinum(II)diammine dichloride (cisplatin) treatment of HT-29 or Caco2 cells dramatically increased their Hsp27 mRNA and protein content. Accordingly, the cells became thermoresistant. Contrary to what has been previously assumed, however, cell resistance to doxorubicin was reduced. Our data suggest that the decreased resistance of the cells to doxorubicin may be due to a concomitant increase of topoisomerase II expression, the main target of anthracyclines. In conclusion, although Hsp27 seems to participate in the natural resistance of colon cancer cells to anthracyclines, its increase after cisplatin treatment is not associated with a decreased cytotoxicity to doxorubicin.

The α-crystallin-related stress protein HSP27, which promotes cellular resistance to different types of stress, is constitutively expressed during the growth of several primate tissue culture cells. Here, we report an analysis of the cellular localization of this protein in CV-1 monkey cells. Following cell lysis and fractionation in the absence of detergent about 2 5 % of the cellular content of HSP27 was recovered in the particu late fractions while the remaining of this protein was in the soluble cytoplasmic fraction. This association of HSP27 with particulate fractions was no more observed when cells were lysed in the presence of non-ionic detergent or when cells were pretreated with drugs, such as monensin and colcemid, that disrupt cytoskeletal architecture. Immunofluorescence analysis revealed that HSP27 is concentrated in a polarized perinuclear zone of CV-1 cells from where microtubules radiate. The particular locale of HSP27 was investigated in cells exposed to drugs or treatments, such as monensin, colcemid, cold stess and serum starvation, that disrupt the cellular architecture of microtubules. A correlation was observed between HSP27 cellular locale and microtubules integrity. Our results suggest a possible interaction of a fraction of HSP27 with cytoplasmic organelles or structures, different from the Golgi apparatus, whose distribution depends upon the organization of microtubules.

The small stress protein heat-shock protein 27 (hsp27) is an oligomeric phosphoprotein, constitutively expressed in most human cells, which enhances cellular resistance to tumour necrosis factor alpha (TNF alpha). This phenomenon correlates with dramatic changes in hsp27 cellular location, structural organization and phosphorylation. To gain a better understanding of the molecular mechanisms regulating these properties of hsp27, we investigated whether they were a consequence of the intracellular production of reactive oxygen species (ROS) generated by TNF alpha. Here, we report that, in T47D carcinoma cell lines, the rapid burst of intracellular ROS production and changes in hsp27 locale, structural organization and phosphoisoform composition induced by TNF alpha were abolished by the overexpression of the antioxidant enzyme seleno-glutathione peroxidase (GSHPx). These effects were greatly diminished when GSHPx-expressing cells were grown in the absence of selenium, a cofactor that is essential for seleno-GSHPx activity, indicating that they are directly linked to the increased GSHPx activity. Moreover, in growing T47D cells, GSHPx expression induced intracellular redistribution of hsp27 and decreased the phosphorylation of this protein without altering its pattern of oligomerization. In contrast, the heat-mediated phosphorylation of hsp27 was not altered by decreased intracellular ROS levels. Hence, in growing and TNF-treated cells, several hsp27 properties appear to be modulated by fluctuations in intracellular ROS levels.

International audience; 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.

Human HspB1 (Hsp27), a molecular chaperone bearing tumorigenic and metastatic roles, is characterized by its dynamic phosphorylation and heterogenous oligomerization in response to changes in cell physiology. The phenomenon is particularly intense and specific when cells are exposed to different death inducers. This favors the hypothesis that the structural organization of HspB1 acts as a sensor which, through reversible modifications, allows cells to adapt and/or mount a protective response. A large number of HspB1 interacting partners have already been described in the literature. Specific changes in oligomerphosphorylation organization may therefore allow HspB1 to interact with the more appropriate polypeptides and to subsequently modulate their folding/activity and/or half-life. This could indirectly link HspB1 to multiple cellular functions and could explain the apparently unrelated effects associated to its over- or underexpression. In cancer, HspB1 is tumorigenic, stimulates metastasis and provide cancer cells with resistance to many anti-cancer drugs, so compounds aimed at disrupting HspB1 deleterious pro-cancer activity are actively looked for. One example, is brivudine that impairs HspB1 ability to recognize pathological protein substrates and appears as a promising anti-cancer drug. Similarly, we have observed that peptide aptamers that specifically interfere with HspB1 structural organization reduced its anti-apoptotic and tumorigenic activities. We propose that, in addition to RNA interference approaches, the tumorigenic activity of HspB1 could be inhibited by altering HspB1 structural organization and consequently its interaction with inappropriate procancerous polypeptide partners. Hence, developping HspB1 structure-based interfering strategies could lead to new anti-cancer drugs discovery.

International audience; 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.

The long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1) is activated under different conditions including heat shock. By using transient transfection assays, we have compared the thermal activation of HIV-1 LTR to that of the promoter of the gene encoding the human stress protein hsp70 which is under the control of the heat shock transcription factor HSF. In these assays, the chloramphenicol acetyl transferase (Cat) gene was used as a reporter gene. Several parameters of the heat stress were analyzed such as the temperature, the duration of heat stress and that of the recovery period. Under every condition tested, we have found that the kinetics of activation of both promoters were very similar. In addition, both showed a similar inhibition by actinomycin D. These results were compared to those obtained with a DNA construct containing the early promoter of SV-40 virus coupled to the Cat gene. In this case, no heat-mediated accumulation of CAT protein was observed, indicating that the transcriptional activation of HIV-1 LTR by heat shock is specific. HIV-1 LTR contains two NF-kappa B binding elements, involved in the activation of this promoter during oxidative stress, which are sequence related to the heat shock element HSE. However, under all the heat shock conditions tested, we have been unable to detect the binding of any protein to kappa B elements, suggesting that this site is not directly involved in the thermal activation of HIV-1 LTR. These results indicate that the thermal transcriptional activation of HIV-1 LTR and hsp70 promoters occurs through different mechanisms that are triggered by similar heat shock conditions.

Low amounts of Semliki Forest virus capsid protein transferred into target cells by electroporation-mediated delivery (10(3)-10(4) molecules incorporated/cell) confer thermal resistance resulting in enhanced survival. Furthermore, when exposed to 43 degrees C, these cells display an enhanced expression of heat-shock protein-70 and a translational thermotolerance. Similarly, low amounts of capsid protein transferred into cells in which transcription is blocked by actinomycin D, also protect the translational machinery at 43 degrees C. In a cell-free translation system, added capsid protein appears to modulate translational efficiency of endogenous mRNAs. At approximately 1 molecule/ribosome, capsid protein is able to enhance translation at 30 degrees C and at 43 degrees C. In contrast, high concentrations of capsid protein are responsible for a marked inhibition of protein synthesis at 30 degrees C, but only hamper translational thermotolerance at 43 degrees C. Our results favor the hypothesis that small amounts of capsid protein trigger a chaperone-like activity that is able to protect the translational machinery from thermal damage.

The oligomeric small heat-shock protein hsp27, also denoted hsp28, is constitutively expressed in several mammalian cells and displays a phosphorylation status that is related to cellular growth and differentiation. This protein is related to alpha-crystallin and has strong sequence similarity with an in vitro inhibitor of actin polymerization. Here, we have analyzed hsp27 phosphorylation, cellular localization and structural organization following serum stimulation of serum-starved HeLa cells. hsp27 is dephosphorylated in starved cells and quantitatively recovered in the form of small structures (200 kDa) present in the soluble phase of the cytoplasm. Immediately after the addition of serum to starved cells, a rapid phosphorylation and complex changes in the intracellular distribution and structural organization of hsp27 are observed. Phosphorylation essentially occurs at the level of small hsp27 structures (200 kDa) and is concomitant with the increased molecular mass (up to 700 kDa) of a fraction of this protein. Serum treatment also induced the detergent-sensitive association of another fraction of hsp27, still in the form of small and dephosphorylated structures, with cellular particulate fractions. Contrasting with these observations, hsp70 had the tendency to concentrate into nucleoli during serum starvation.

Human HspB1 (also denoted Hsp27) is a well-known member, together with alphaB-crystallin, of the small heat-shock (or stress) proteins (sHsps) (20-40 kDa). In this chapter, I describe procedures for testing the oligomeric and phosphorylation patterns of HspB1 as well as its interaction with specific partner/client polypeptides using tissue culture cells genetically modified to express different levels of this protein. The procedures have been developed in my laboratory and could be used in any well-established cellular laboratory. In addition, the different procedures presented here could be extended to test the nine other human sHsp members as well as sHsps from other species.

Human HspB1 (also denoted Hsp27) is a well-known member, together with alphaB-crystallin, of the small heat-shock (or stress) proteins (sHsps) (20-40 kDa). In this chapter, I describe procedures for testing the oligomeric and phosphorylation patterns of HspB1 as well as its interaction with specific partner/client polypeptides using tissue culture cells genetically modified to express different levels of this protein. The procedures have been developed in my laboratory and could be used in any well-established cellular laboratory. In addition, the different procedures presented here could be extended to test the nine other human sHsp members as well as sHsps from other species.