Effect of methylglyoxal modification on stress-induced aggregation of client proteins and their chaperoning by human alphaA-crystallin

alpha-Crystallin prevents protein aggregation under various stress conditions through its chaperone-like properties. Previously, we demonstrated that MGO (methylglyoxal) modification of alphaA-crystallin enhances its chaperone function and thus may affect transparency of the lens. During aging of the lens, not only alphaA-crystallin, but its client proteins are also likely to be modified by MGO. We have investigated the role of MGO modification of four model client proteins (insulin, alpha-lactalbumin, alcohol dehydrogenase and gamma-crystallin) in their aggregation and structure and the ability of human alphaA-crystallin to chaperone them. We found that MGO modification (10-1000 microM) decreased the chemical aggregation of insulin and alpha-lactalbumin and thermal aggregation of alcohol dehydrogenase and gamma-crystallin. Surface hydrophobicity in MGO-modified proteins decreased slightly relative to unmodified proteins. HPLC and MS analyses revealed argpyrimidine and hydroimidazolone in MGO-modified client proteins. The degree of chaperoning by alphaA-crystallin towards MGO-modified and unmodified client proteins was similar. Co-modification of client proteins and alphaA-crystallin by MGO completely inhibited stress-induced aggregation of client proteins. Our results indicate that minor modifications of client proteins and alphaA-crystallin by MGO might prevent protein aggregation and thus help maintain transparency of the aging lens.     

Hydroimidazolone modification of the conserved Arg12 in small heat shock proteins: studies on the structure and chaperone function using mutant mimics

Methylglyoxal (MGO) is an α-dicarbonyl compound present ubiquitously in the human body. MGO reacts with arginine residues in proteins and forms adducts such as hydroimidazolone and argpyrimidine in vivo. Previously, we showed that MGO-mediated modification of αA-crystallin increased its chaperone function. We identified MGO-modified arginine residues in αA-crystallin and found that replacing such arginine residues with alanine residues mimicked the effects of MGO on the chaperone function. Arginine 12 (R12) is a conserved amino acid residue in Hsp27 as well as αA- and αB-crystallin. When treated with MGO at or near physiological concentrations (2-10 μM), R12 was modified to hydroimidazolone in all three small heat shock proteins. In this study, we determined the effect of arginine substitution with alanine at position 12 (R12A to mimic MGO modification) on the structure and chaperone function of these proteins. Among the three proteins, the R12A mutation improved the chaperone function of only αA-crystallin. This enhancement in the chaperone function was accompanied by subtle changes in the tertiary structure, which increased the thermodynamic stability of αA-crystallin. This mutation induced the exposure of additional client protein binding sites on αA-crystallin. Altogether, our data suggest that MGO-modification of the conserved R12 in αA-crystallin to hydroimidazolone may play an important role in reducing protein aggregation in the lens during aging and cataract formation.     

The combined effect of acetylation and glycation on the chaperone and anti-apoptotic functions of human α-crystallin

N^ε-acetylation occurs on select lysine residues in α-crystallin of the human lens and alters its chaperone function. In this study, we investigated the effect of N^ε-acetylation on advanced glycation end product (AGE) formation and consequences of the combined N^ε-acetylation and AGE formation on the function of α-crystallin. Immunoprecipitation experiments revealed that N^ε-acetylation of lysine residues and AGE formation co-occurs in both αA- and αB-crystallin of the human lens. Prior acetylation of αA- and αB-crystallin with acetic anhydride (Ac₂O) before glycation with methylglyoxal (MGO) resulted in significant inhibition of the synthesis of two AGEs, hydroimidazolone (HI) and argpyrimidine. Similarly, synthesis of ascorbate-derived AGEs, pentosidine and N^ε-carboxymethyl lysine (CML), was inhibited in both proteins by prior acetylation. In all cases, inhibition of AGE synthesis was positively related to the degree of acetylation. While prior acetylation further increased the chaperone activity of MGO-glycated αA-crystallin, it inhibited the loss of chaperone activity by ascorbate-glycation in both proteins. BioPORTER-mediated transfer of αA- and αB-crystallin into CHO cells resulted in significant protection against hyperthermia-induced apoptosis. This effect was enhanced in acetylated and MGO-modified αA- and αB-crystallin. Caspase-3 activity was reduced in α-crystallin transferred cells. Glycation of acetylated proteins with either MGO or ascorbate produced no significant change in the anti-apoptotic function. Collectively, these data demonstrate that lysine acetylation and AGE formation can occur concurrently in α-crystallin of human lens, and that lysine acetylation improves anti-apoptotic function of α-crystallin and prevents ascorbate-mediated loss of chaperone function.     

Enhancement of chaperone function of alpha-crystallin by methylglyoxal modification

The molecular chaperone function of alpha-crystallin in the lens prevents the aggregation and insolubilization of lens proteins that occur during the process of aging. We found that chemical modification of alpha-crystallin by a physiological alpha-dicarbonyl compound, methylglyoxal (MG), enhances its chaperone function. Protein-modifying sugars and ascorbate have no such effect and actually reduce chaperone function. Chaperone assay after immunoprecipitation or with immunoaffinity-purified argpyrimidine-alpha-crystallin indicates that 50-60% of the increased chaperone function is due to argpyrimidine-modified protein. Incubation of alpha-crystallin with DL-glyceraldehyde and arginine-modifying agents also enhances chaperone function, and we believe that the increased chaperone activity depends on the extent of arginine modification. Far- and near-UV circular dichroism spectra indicate modest changes in secondary and tertiary structure of MG-modified alpha-crystallin. LC MS/MS analysis of MG-modified alpha-crystallin following chymotryptic digestion revealed that R21, R49, and R103 in alphaA-crystallin were converted to argpyrimidine. 1,1'-Bis(4-anilino)naphthalene-5,5'-disulfonic acid binding, an indicator of hydrophobicity of proteins, increased in alpha-crystallin modified by low concentrations of MG (2-100 microM). MG similarly enhances chaperone function of another small heat shock protein, Hsp27. Our results show that posttranslational modification by a metabolic product can enhance the chaperone function of alpha-crystallin and Hsp27 and suggest that such modification may be a protective mechanism against environmental and metabolic stresses. Augmentation of the chaperone function of alpha-crystallin might have evolved to protect the lens from deleterious protein modifications associated with aging.     

Chemical modulation of the chaperone function of human alphaA-crystallin

alphaA-crystallin is abundant in the lens of the eye and acts as a molecular chaperone by preventing aggregation of denaturing proteins. We previously found that chemical modification of the guanidino group of selected arginine residues by a metabolic alpha-dicarbonyl compound, methylglyoxal (MGO), makes human alphaA-crystallin a better chaperone. Here, we examined how the introduction of additional guanidino groups and modification by MGO influence the structure and chaperone function of alphaA-crystallin. alphaA-crystallin lysine residues were converted to homoarginine by guanidination with o-methylisourea (OMIU) and then modified with MGO. LC-ESI-mass spectrometry identified homoargpyrimidine and homohydroimidazolone adducts after OMIU and MGO treatment. Treatment with 0.25 M OMIU abolished most of the chaperone function. However, subsequent treatment with 1.0 mM MGO not only restored the chaperone function but increased it by approximately 40% and approximately 60% beyond that of unmodified alphaA-crystallin, as measured with citrate synthase and insulin aggregation assays, respectively. OMIU treatment reduced the surface hydrophobicity but after MGO treatment, it was approximately 39% higher than control. FRET analysis revealed that alphaA-crystallin subunit exchange rate was markedly retarded by OMIU modification, but was enhanced after MGO modification. These results indicate a pattern of loss and gain of chaperone function within the same protein that is associated with introduction of guanidino groups and their neutralization. These findings support our hypothesis that positively charged guanidino group on arginine residues keeps the chaperone function of alphaA-crystallin in check and that a metabolic alpha-dicarbonyl compound neutralizes this charge to restore and enhance chaperone function.     

Hydroimidazolone modification of human αA-crystallin: Effect on the chaperone function and protein refolding ability

AlphaA-crystallin is a molecular chaperone; it prevents aggregation of denaturing proteins. We have previously demonstrated that upon modification by a metabolic α-dicarbonyl compound, methylglyoxal (MGO), αA-crystallin becomes a better chaperone. AlphaA-crystallin also assists in refolding of denatured proteins. Here, we have investigated the effect of mild modification of αA-crystallin by MGO (with 20–500 µM) on the chaperone function and its ability to refold denatured proteins. Under the conditions used, mildly modified protein contained mostly hydroimidazolone modifications. The modified protein exhibited an increase in chaperone function against thermal aggregation of β_L- and γ-crystallins, citrate synthase (CS), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) and chemical aggregation of insulin. The ability of the protein to assist in refolding of chemically denatured β_L- and γ-crystallins, MDH and LDH, and to prevent thermal inactivation of CS were unchanged after mild modification by MGO. Prior binding of catalytically inactive, thermally denatured MDH or the hydrophobic probe, 2-p-toluidonaphthalene-6-sulfonate (TNS) abolished the ability of αA-crystallin to assist in the refolding of denatured MDH. However, MGO modification of chaperone-null TNS-bound αA-crystallin resulted in partial regain of the chaperone function. Taken together, these results demonstrate that: 1) hydroimidazolone modifications are sufficient to enhance the chaperone function of αA-crystallin but such modifications do not change its ability to assist in refolding of denatured proteins, 2) the sites on the αA-crystallin responsible for the chaperone function and refolding are the same in the native αA-crystallin and 3) additional hydrophobic sites exposed upon MGO modification, which are responsible for the enhanced chaperone function, do not enhance αA-crystallin's ability to refold denatured proteins.     

Effect of glycation on alpha-crystallin structure and chaperone-like function

The chaperone-like activity of alpha-crystallin is considered to play an important role in the maintenance of the transparency of the eye lens. However, in the case of aging and in diabetes, the chaperone function of alpha-crystallin is compromized, resulting in cataract formation. Several post-translational modifications, including non-enzymatic glycation, have been shown to affect the chaperone function of alpha-crystallin in aging and in diabetes. A variety of agents have been identified as the predominant sources for the formation of AGEs (advanced glycation end-products) in various tissues, including the lens. Nevertheless, glycation of alpha-crystallin with various sugars has resulted in divergent results. In the present in vitro study, we have investigated the effect of glucose, fructose, G6P (glucose 6-phosphate) and MGO (methylglyoxal), which represent the major classes of glycating agents, on the structure and chaperone function of alpha-crystallin. Modification of alpha-crystallin with all four agents resulted in the formation of glycated protein, increased AGE fluorescence, protein cross-linking and HMM (high-molecular-mass) aggregation. Interestingly, these glycation-related profiles were found to vary with different glycating agents. For instance, CML [N(epsilon)-(carboxymethyl)lysine] was the predominant AGE formed upon glycation of alpha-crystallin with these agents. Although fructose and MGO caused significant conformational changes, there were no significant structural perturbations with glucose and G6P. With the exception of MGO modification, glycation with other sugars resulted in decreased chaperone activity in aggregation assays. However, modification with all four sugars led to the loss of chaperone activity as assessed using an enzyme inactivation assay. Glycation-induced loss of alpha-crystallin chaperone activity was associated with decreased hydrophobicity. Furthermore, alpha-crystallin isolated from glycated TSP (total lens soluble protein) had also increased AGE fluorescence, CML formation and diminished chaperone activity. These results indicate the susceptibility of alpha-crystallin to non-enzymatic glycation by various sugars and their derivatives, whose levels are elevated in diabetes. We also describe the effects of glycation on the structure and chaperone-like activity of alpha-crystallin.     

The role of the cysteine residue in the chaperone and anti‐apoptotic functions of human Hsp27

The small heat shock protein Hsp27 is a molecular chaperone and an anti‐apoptotic protein. Human Hsp27 has one cysteine residue at position 137. We investigated the role of this cysteine residue in the chaperone and anti‐apoptotic functions of Hsp27 by mutating the cysteine residue to an alanine (Hsp27_C137A) and comparing it to wild‐type protein (Hsp27_WT). Both proteins were multi‐subunit oligomers, but subunits of Hsp27_WT were disulfide‐linked unlike those of Hsp27_C137A, which were monomeric. Hsp27_C137A was indistinguishable from Hsp27_WT with regard to its secondary structure, surface hydrophobicity, oligomeric size and chaperone function. S‐thiolation and reductive methylation of the cysteine residue had no apparent effect on the chaperone function of Hsp27_WT. The anti‐apoptotic function of Hsp27_C137A and Hsp27_WT was studied by overexpressing them in CHO cells. No difference in the caspase‐3 or ‐9 activity was observed in staurosporine‐treated cells. The rate of apoptosis between Hsp27_C137A and Hsp27_WT overexpressing cells was similar whether the cells were treated with staurosporine or etoposide. However, the mutant protein was less protective relative to the wild‐type protein in preventing caspase‐3 and caspase‐9 activation and apoptosis induced by 1 mM H₂O₂ in CHO and HeLa cells. These data demonstrate that in human Hsp27, disulfide formation by the lone cysteine does not affect its chaperone function and anti‐apoptotic function against chemical toxicants. However, oxidation of the lone cysteine in Hsp27 might at least partially affect the anti‐apoptotic function against oxidative stress. J. Cell. Biochem. 110: 408–419, 2010. © 2010 Wiley‐Liss, Inc.     

Effect of methylglyoxal modification on the structure and properties of human small heat shock protein HspB6 (Hsp20)

Human small heat shock protein HspB6 (Hsp20) was modified by metabolic α-dicarbonyl compound methylglyoxal (MGO). At low MGO/HspB6 molar ratio, Arg13, Arg14, Arg27, and Arg102 were the primary sites of MGO modification. At high MGO/HspB6 ratio, practically, all Arg and Lys residues of HspB6 were modified. Both mild and extensive MGO modification decreased susceptibility of HspB6 to trypsinolysis and prevented its heat-induced aggregation. Modification by MGO was accompanied by formation of small quantities of chemically crosslinked dimers and did not dramatically affect quaternary structure of HspB6. Mild modification by MGO did not affect whereas extensive modification decreased interaction of HspB6 with HspB1. Phosphorylation of HspB6 by cyclic adenosine monophosphate (cAMP)-dependent protein kinase was inhibited after mild modification and completely prevented after extensive modification by MGO. Chaperone-like activity of HspB6 measured with subfragment 1 of skeletal myosin was enhanced after MGO modifications. It is concluded that Arg residues located in the N-terminal domain of HspB6 are easily accessible to MGO modification and that even mild modification by MGO affects susceptibility to trypsinolysis, phosphorylation by cAMP-dependent protein kinase, and chaperone-like activity of HspB6.     

Transglutaminase 2 interaction with small heat shock proteins mediate cell survival upon excitotoxic stress

Transglutaminase 2 has been postulated to be involved in the pathogenesis of central nervous system neurodegenerative disorders. However, its role in neuronal cell death remains to be elucidated. Excitotoxicity is a common event underlying neurodegeneration. We aimed to evaluate the protein targets for transglutaminase 2 in cell response to NMDA-induced excitotoxic stress, using SH-SY5Y neuroblastoma cells which express high tranglutaminase 2 levels upon retinoic acid-driven differentiation toward neurons. NMDA-evoked calcium increase led to transglutaminase 2 activation that mediated cell survival, as at first suggested by the exacerbation of NMDA toxicity in the presence of R283, a synthetic competitive inhibitor of transglutaminase active site. Assays of R283-mediated transglutaminase inhibition showed the involvement of enzyme activity in NMDA-induced reduction in protein basal levels of pro-apoptotic caspase-3 and the stress protein Hsp20. However, this occurred in a way different from protein cross-linking, given that macromolecular assemblies were not observed in our experimental conditions for both proteins. Co-immunoprecipitation experiments provided evidence for the interaction, in basal conditions, between transglutaminase 2 and Hsp20, as well as between Hsp20 and Hsp27, a major anti-apoptotic protein promoting caspase-3 inactivation and degradation. NMDA treatment disrupted both these interactions that were restored upon transglutaminase 2 inhibition with R283. These results suggest that transglutaminase 2 might be protective against NMDA-evoked excitotoxic insult in neuronal-like SH-SY5Y cells in a way, independent from transamidation that likely involves its interaction with the complex Hsp20/Hsp27 playing a pro-survival role.     

Over-expression of Hsp70 in BHK-21 cells engineered to produce recombinant factor VIII promotes resistance to apoptosis and enhances secretion

Production of coagulation factor VIII (FVIII) by recombinant cell lines is limited by its failure to reach or maintain the native conformation in the endoplasmic reticulum. This results in significant cytoplasmic degradation and/or aggregation of the misfolded product. The molecular chaperone Hsp70 was overexpressed in an attempt to increase the recombinant FVIII (rFVIII) secretion. The characteristics of increased Hsp70 expression were investigated by comparing a clone of BHK-21 cells expressing rFVIII (rBHK-21(host)) to a chaperone clone derived by transfection of the host clone with human Hsp70 (rBHK-21(Hsp70)) in small-scale batch cell cultures. To aid this investigation a number of fluorescence based cellular apoptosis assays were developed and optimized. These assays demonstrated sub-populations of rBHK-21(host) cells that were apoptotic in nature and were identified prior to the loss in plasma membrane integrity. Dual staining for intracellular rFVIII and caspase-3 activation showed a reduction in intracellular rFVIII in rBHK-21(host) cells that correlated with a significant increase in active caspase-3, suggesting that apoptosis was a factor limiting rFVIII secretion. In sharp contrast there was more intracellular rFVIII and less active caspase-3 in rBHK-21(Hsp70) cell cultures. Moreover when grown in batch culture, rBHK-21(Hsp70) cells released rFVIII of higher specific activity (active FVIII protein/total FVIII protein), suggesting improved product quality. Thus, increased expression of HSP70 led to an increased yield of a secreted recombinant protein by inhibition of apoptosis and promoting proper conformational maturation of rFVIII in sub-optimal bioreactor conditions.     

Effect of site-directed mutagenesis of methylglyoxal-modifiable arginine residues on the structure and chaperone function of human alphaA-crystallin

We reported previously that chemical modification of human alphaA-crystallin by a metabolic dicarbonyl compound, methylglyoxal (MGO), enhances its chaperone-like function, a phenomenon which we attributed to formation of argpyrimidine at arginine residues (R) 21, 49, and 103. This structural change removes the positive charge on the arginine residues. To explore this mechanism further, we replaced these three R residues with a neutral alanine (A) residue one at a time or in combination and examined the impact on the structure and chaperone function. Measurement of intrinsic tryptophan fluorescence and near-UV CD spectra revealed alteration of the microenvironment of aromatic amino acid residues in mutant proteins. When compared to wild-type (wt) alphaA-crystallin, the chaperone function of R21A and R103A mutants increased 20% and 18% as measured by the insulin aggregation assay and increased it as much as 39% and 28% when measured by the citrate synthase (CS) aggregation assay. While the R49A mutant lost most of its chaperone function, R21A/R103A and R21A/R49A/R103A mutants had slightly better function (6-14% and 10-14%) than the wt protein in these assays. R21A and R103A mutants had higher surface hydrophobicity than wt alphaA-crystallin, but the R49A mutant had lower hydrophobicity. R21A and R103A mutants, but not the R49A mutant, were more efficient than wt protein in refolding guanidine hydrochloride-treated malate dehydrogenase to its native state. Our findings indicate that the positive charges on R21, R49, and R103 are important determinants of the chaperone function of alphaA-crystallin and suggest that chemical modification of arginine residues may play a role in protein aggregation during lens aging and cataract formation.     

Methylglyoxal-bovine serum albumin stimulates tumor necrosis factor alpha secretion in RAW 264.7 cells through activation of mitogen-activating protein kinase,nuclear factor kappaB and intracellular reactive oxygen species formation

Accumulating evidence suggests that the pathophysiology of diabetes is analogous to chronic inflammatory states. Circulating levels of inflammatory cytokines such as IL-6 and tumor necrosis factor alpha (TNFalpha) are increased in both type 1 and type 2 diabetes. TNFalpha plays an important role in the pathogenesis of insulin resistance in type 2 diabetes. However, the reason for this increase remains unclear. Levels of the dicarbonyl methylglyoxal (MGO) are elevated in diabetic plasma and MGO-modified bovine serum albumin (MGO-BSA) can trigger cellular uptake of TNF. Therefore we tested the hypothesis that MGO-modified proteins may cause TNFalpha secretion in macrophage-like RAW 264.7 cells. Treatment of cells with MGO-BSA induced TNFalpha release in a dose-dependent manner. MGO-modified ribonuclease A and chicken egg ovalbumin had similar effects. Cotreatment of cells with antioxidant reagent N-acetylcysteine (NAC) inhibited MGO-BSA-induced TNFalpha secretion. MGO-BSA stimulated the simultaneous activation of p44/42 and p38 mitogen-activated protein kinase. PD98059, a selective MEK inhibitor, inhibited MGO-BSA-induced TNFalpha release as well as ERK phosphorylation. Pretreatment of cells with NAC also resulted in inhibition of MGO-BSA-induced ERK phosphorylation. MGO-BSA induced dose-dependent NFkappaB activation as shown by electrophoresis mobility shift assay. The MGO-BSA-induced NFkappaB activation was prevented in the presence of PD98059, NAC, and parthenolide, a selective inhibitor of NFkappaB. Furthermore, the NFkappaB inhibitor parthenolide suppressed MGO-BSA-induced TNFalpha secretion. Confocal microscopy using dichlorofluorescein to demonstrate intracellular reactive oxygen species (ROS) showed that MGO-BSA produced more ROS compared with native BSA. MGO-BSA could also stimulate protein kinase C (PKC) translocation to the cell membrane, considered a key signaling pathway in diabetes. However, there was no evidence that PKC was involved in TNFalpha release based on inhibition by calphostin C and staurosporine. Our findings suggest that the presence of chronically elevated levels of MGO-modified bovine serum albumin may contribute to elevated levels of TNFalpha in diabetes.     

Hsp70 exerts its anti-apoptotic function downstream of caspase-3-like proteases.

The major heat shock protein, Hsp70, is an effective inhibitor of apoptosis. To study its mechanism of action, we created tumor cell lines with altered Hsp70 levels. The expression levels of Hsp70 in the cells obtained correlated well with their survival following treatments with tumor necrosis factor, staurosporine and doxorubicin. Surprisingly, the surviving Hsp70-expressing cells responded to the apoptotic stimuli by activation of stress-activated protein kinases, generation of free radicals, early disruption of mitochondrial transmembrane potential, release of cytochrome c from mitochondria and activation of caspase-3-like proteases in a manner essentially similar to that of the dying cells with low Hsp70 levels. However, Hsp70 inhibited late caspase-dependent events such as activation of cytosolic phospholipase A2 and changes in nuclear morphology. Furthermore, Hsp70 conferred significant protection against cell death induced by enforced expression of caspase-3. Thus, Hsp70 rescues cells from apoptosis later in the death signaling pathway than any known anti-apoptotic protein, making it a tempting target for therapeutic interventions.     

Redox switch of hsp33 has a novel zinc-binding motif

The chaperone activity of the heat shock protein Hsp33 is regulated by reversible disulfide bond formation. Oxidized Hsp33 is active, and reduced Hsp33 is inactive. We show that zinc binding is essential for the function of this redox switch. Our results reveal that Hps33 contains a new, high affinity (K(a) > 10(17) m(-)(1)), zinc-binding motif in the form Cys-X-Cys-X(27-32)-Cys-X-X-Cys. All four conserved cysteines within this motif act to coordinate a single zinc atom. Experiments where reduced wild type Hsp33 is reconstituted with cobalt or cadmium demonstrate that the metal-coordinating cysteines are present as highly reactive thiolate anions. This ionization may allow for the fast and successful activation of the chaperone function of Hsp33 upon incubation in oxidizing agents.     

On the role of Hsp27 in regulating apoptosis

Heat shock proteins (Hsps) comprise several different families of proteins that are induced in response to a wide variety of physiological and environmental insults. One such protein which is highly induced during the stress response is a 27-kDa protein, termed Hsp27 whose expression is seen to correlate with increased survival in response to cytotoxic stimuli. It has been shown to prevent cell death by a wide variety of agents that cause apoptosis. Hsp27 is a molecular chaperone with an ability to interact with a large number of proteins. Recent evidence has shown that Hsp27 regulates apoptosis through an ability to interact with key components of the apoptotic signalling pathway, in particular, those involved in caspase activation and apoptosis. This article will review recent advances in the field and will address some of the potential mechanisms by which Hsp27 functions as an anti-apoptotic molecule.     

The role of Hsp70 chaperone in the reaction of human leukemic cells to anticancer drugs

Most of anti-tumor factors are designed to kill selectively cancer cells; in most cases this action is related to the ability of the above substances to induce apoptosis. One of potent anti-apoptotic mechanisms is based on Hsp70 protein. Since the level of this protein is often higher in malignant tumors than in normal tissues, the aim of this study was to establish whether the elevated Hsp70 content may influence the process of apoptosis induced by anti-tumor drugs in cancer cells population. The increase of intracellular content of Hsp70 in human leukemia U-937 cells was attained by a mild heat stress or by transfection of cells with the human hsp70 gene. The elevation of Hsp70 quantity, irrespective of the way it was performed, leads to the inhibition of apoptosis in cells treated with two substances, etoposide or adriamycin. The inhibition of apoptosis was accompanied with the reducing of the share of cells with fragmented nuclei and with the delay in caspase activation. It is suggested that in addition to the previously discovered targets, whose activity is suppressed by the Hsp70 chaperone, this protein can inhibit the activity of caspase-3 and -7; this delays the onset of apoptosis in part of a cancer cell population.     

Chaperone activity with a redox switch

Hsp33, a member of a newly discovered heat shock protein family, was found to be a very potent molecular chaperone. Hsp33 is distinguished from all other known molecular chaperones by its mode of functional regulation. Its activity is redox regulated. Hsp33 is a cytoplasmically localized protein with highly reactive cysteines that respond quickly to changes in the redox environment. Oxidizing conditions like H2O2 cause disulfide bonds to form in Hsp33, a process that leads to the activation of its chaperone function. In vitro and in vivo experiments suggest that Hsp33 protects cells from oxidants, leading us to conclude that we have found a protein family that plays an important role in the bacterial defense system toward oxidative stress.