Adenine nucleotide translocase 3 (ANT3) overexpression induces apoptosis in cultured cells

Cne1p, a calnexin homologue from Saccharomyces cerevisiae, has been shown to possess a conserved P-domain and lectin site as mammalian calnexin. The effect of P-domain and lectin site on the function of Cne1p was investigated in vitro using recombinant P-domain, P-domain deletion mutant of Cne1p, and lectin site mutant of Cne1ps (E181A and E398A) The binding of monoglucosylated oligosaccharide (G1M9) with Cne1p was clearly demonstrated using lectin site mutants. The P-domain deletion mutant and the letcin site mutants partially decreased the ability to suppress the aggregation of citrate synthase (CS) and chicken egg yolk immunoglobulin at levels different from Cne1p. Furthermore, the P-domain deletion mutant and the lectin site mutants decreased the ability to enhance the refolding of CS. These results suggest that the cooperation between the P-domain and the lectin site are important for the complete function of Cne1p. Thus, we conclude that P-domain in cooperation with the lectin site of Cne1p functions as a chaperone.     

Interactions among yeast protein-disulfide isomerase proteins and endoplasmic reticulum chaperone proteins influence their activities

We previously reported that the reductive activities of yeast protein-disulfide isomerase (PDI) family proteins did not completely explain their contribution to the viability of Saccharomyces cerevisiae (Kimura, T., Hosoda, Y., Kitamura, Y., Nakamura, H., Horibe, T., and Kikuchi, M. (2004) Biochem. Biophys. Res. Commun. 320, 359-365). In this study, we examined oxidative refolding activities and found that Mpd1p, Mpd2, and Eug1p exhibit activities of 13.8, 16.0, and 2.16%, respectively, compared with Pdi1p and that activity for Eps1p is undetectable. In analyses of interactions between yeast PDI proteins and endoplasmic reticulum molecular chaperones, we found that Mpd1p alone does not have chaperone activity but that it interacts with and inhibits the chaperone activity of Cne1p, a homologue of mammalian calnexin, and that Cne1p increases the reductive activity of Mpd1p. These results suggest that the interface between Mpd1p and Cne1p is near the peptide-binding site of Cne1p. In addition, Eps1p interacts with Pdi1p, Eug1p, Mpd1p, and Kar2p with dissociation constants (KD) in the range of 10(-7) to 10(-6). Interestingly, co-chaperone activities were completely suppressed in Eps1p-Pdi1p and Eps1p-Mpd1p complexes, although only Eps1p and Pdi1p have chaperone activity. The in vivo consequences of these results are discussed.     

Connexin45 directly binds to ZO-1 and localizes to the tight junction region in epithelial MDCK cells

The molecular chaperone protein Hsp78, a member of the Clp/Hsp100 family localized in the mitochondria of Saccharomyces cerevisiae, is required for maintenance of mitochondrial functions under heat stress. To characterize the biochemical mechanisms of Hsp78 function, Hsp78 was purified to homogeneity and its role in the reactivation of chemically and heat-denatured substrate protein was analyzed in vitro. Hsp78 alone was not able to mediate reactivation of firefly luciferase. Rather, efficient refolding was dependent on the simultaneous presence of Hsp78 and the mitochondrial Hsp70 machinery, composed of Ssc1p/Mdj1p/Mge1p. Bacterial DnaK/DnaJ/GrpE, which cooperates with the Hsp78 homolog, ClpB in Escherichia coli, could not substitute for the mitochondrial Hsp70 system. However, efficient Hsp78-dependent refolding of luciferase was observed if DnaK was replaced by Ssc1p in these experiments, suggesting a specific functional interaction of both chaperone proteins. These findings establish the cooperation of Hsp78 with the Hsp70 machinery in the refolding of heat-inactivated proteins and demonstrate a conserved mode of action of ClpB homologs.     

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.     

The microtubule-associated protein, NUD-1, exhibits chaperone activity in vitro

Regulation of cell division requires the concerted function of proteins and protein complexes that properly mediate cytoskeletal dynamics. NudC is an evolutionarily conserved protein of undetermined function that associates with microtubules and interacts with several key regulators of mitosis, such as polo-kinase 1 (Plk1) and dynein. NudC is essential for proper mitotic progression, and homologs have been identified in species ranging from fungi to humans. In this paper, we report the characterization of the Caenorhabditis elegans NudC homolog, NUD-1, as a protein exhibiting molecular chaperone activity. All NudC/NUD-1 proteins share a conserved p23/HSP20 domain predicted by three-dimensional modeling [Garcia-Ranea, Mirey, Camonis, Valencia, FEBS Lett 529(2–3):162–167, 2002]. We demonstrate that nematode NUD-1 is able to prevent the aggregation of two substrate proteins, citrate synthase (CS) and luciferase, at stoichiometric concentrations. Further, NUD-1 also protects the native state of CS from thermal inactivation by significantly reducing the inactivation rate of this enzyme. To further determine if NUD-1/substrate complexes were productive or simply “dead-end” unfolding intermediates, a luciferase refolding assay was utilized. Following thermal denaturation, rabbit reticulocyte lysate and ATP were added and luciferase activity measured. In the presence of NUD-1, nearly all of the luciferase activity was regained, indicating that unfolded intermediates complexed with NUD-1 could be refolded. These studies represent the first functional evidence for a member of this mitotically essential protein family as having chaperone activity and facilitates elucidation of the role such proteins play in chaperone complexes utilized in cell division. C. elegans NUD-1 is a member of an evolutionary conserved protein family of unknown function involved in the regulation of cytoskeletal dynamics. NUD-1 and its mammalian homolog, NudC, function with the dynein motor complex to ensure proper cell division, and knockdown or overexpression of these proteins leads to disruption of mitosis. In this paper, we show that NUD-1 possesses ATP-independent chaperone activity comparable to that of small heat shock proteins and cochaperones and that changes in phosphorylation state functionally alter chaperone activity in a phosphomimetic NUD-1 mutant.     

The cytoplasmic chaperone hsp104 is required for conformational repair of heat-denatured proteins in the yeast endoplasmic reticulum

Severe heat stress causes protein denaturation in various cellular compartments. If Saccharomyces cerevisiae cells grown at 24 degrees C are preconditioned at 37 degrees C, proteins denatured by subsequent exposure to 48-50 degrees C can be renatured when the cells are allowed to recover at 24 degrees C. Conformational repair of vital proteins is essential for survival, because gene expression is transiently blocked after the thermal insult. Refolding of cytoplasmic proteins requires the Hsp104 chaperone, and refolding of lumenal endoplasmic reticulum (ER) proteins requires the Hsp70 homologue Lhs1p. We show here that conformational repair of heat-damaged glycoproteins in the ER of living yeast cells required functional Hsp104. A heterologous enzyme and a number of natural yeast proteins, previously translocated and folded in the ER and thereafter denatured by severe heat stress, failed to be refolded to active and secretion-competent structures in the absence of Hsp104 or when an ATP-binding site of Hsp104 was mutated. During recovery at 24 degrees C, the misfolded proteins persisted in the ER, although the secretory apparatus was fully functional. Hsp104 appears to control conformational repair of heat-damaged proteins even beyond the ER membrane.     

Regulation and recovery of functions of Saccharomyces cerevisiae chaperone BiP/Kar2p after thermal insult

We described earlier a novel mode of regulation of Hsp104, a cytosolic chaperone directly involved in the refolding of heat-denatured proteins, and designated it delayed upregulation, or DUR. When Saccharomyces cerevisiae cells grown at the physiological temperature of 24 degrees C, preconditioned at 37 degrees C, and treated briefly at 50 degrees C were shifted back to 24 degrees C, Hsp104 expression was strongly induced after 2.5 h of recovery and returned back to normal after 5 h. Here we show that the endoplasmic reticulum (ER) chaperones BiP/Kar2p and Lhs1p and the mitochondrial chaperone Hsp78 were also upregulated at the physiological temperature during recovery from thermal insult. The heat shock element (HSE) in the KAR2 promoter was found to be sufficient to drive DUR. The unfolded protein element could also evoke DUR, albeit weakly, in the absence of a functional HSE. BiP/Kar2p functions in ER translocation and assists protein folding. Here we found that the synthesis of new BiP/Kar2p molecules was negligible for more than an hour after the shift of the cells from 50 degrees C to 24 degrees C. Concomitantly, ER translocation was blocked, suggesting that preexisting BiP/Kar2p molecules or other necessary proteins were not functioning. Translocation resumed concomitantly with enhanced synthesis of BiP/Kar2p after 3 h of recovery, after which ER exit and protein secretion also resumed. For a unicellular organism like S. cerevisiae, conformational repair of denatured proteins is the sole survival strategy. Chaperones that refold proteins in the cytosol, ER, and mitochondria of S. cerevisiae appear to be subject to DUR to ensure survival after thermal insults.     

Temperature-induced denaturation and renaturation of triosephosphate isomerase from Saccharomyces cerevisiae: evidence of dimerization coupled to refolding of the thermally unfolded protein

The thermal denaturation of the dimeric enzyme triosephosphate isomerase (TIM) from Saccharomyces cerevisiae was studied by spectroscopic and calorimetric methods. At low protein concentration the structural transition proved to be reversible in thermal scannings conducted at a rate greater than 1.0 degrees C min(-1). Under these conditions, however, the denaturation-renaturation cycle exhibited marked hysteresis. The use of lower scanning rates lead to pronounced irreversibility. Kinetic studies indicated that denaturation of the enzyme likely consists of an initial first-order reaction that forms thermally unfolded (U) TIM, followed by irreversibility-inducing reactions which are probably linked to aggregation of the unfolded protein. As judged from CD measurements, U possesses residual secondary structure but lacks most of the tertiary interactions present in native TIM. Furthermore, the large increment in heat capacity upon denaturation suggests that extensive exposure of surface area occurs when U is formed. Above 63 degrees C, reactions leading to irreversibility were much slower than the unfolding process; as a result, U was sufficiently long-lived as to allow an investigation of its refolding kinetics. We found that U transforms into nativelike TIM through a second-order reaction in which association is coupled to the regain of secondary structure. The rate constants for unfolding and refolding of TIM displayed temperature dependences resembling those reported for monomeric proteins but with considerably larger activation enthalpies. Such large temperature dependences seem to be determinant for the occurrence of kinetically controlled transitions and thus constitute a simple explanation for the hysteresis observed in thermal scannings.     

Interaction of the Hsp110 Molecular Chaperones from S. cerevisiae with Substrate Protein

Hsp110 proteins act as nucleotide exchange factors of the molecular chaperone Hsp70 in eukaryotes. In addition, they have been reported to stabilize unfolded proteins for subsequent refolding. Hsp110 proteins belong to the Hsp70 superfamily and, in analogy to Hsp70, the substrate-binding site was proposed to be located at the interface of the β-sandwich domain and the three-helix-bundle domain. Saccharomyces cerevisiae has two closely related cytosolic isoforms of Hsp110, Sse1p and Sse2p. Under normal growth conditions, Sse1p is the predominant form. Sse2p is induced under stress conditions, such as heat shock. Consistent with these findings, we find that Sse2p has increased temperature stability. Both Sse1p and Sse2p accelerate nucleotide exchange on the yeast Hsp70 Ssa1p. Furthermore, Sse1p and Sse2p effectively compete for binding of unfolded luciferase. In contrast to Sse1p, however, Sse2p fails to stabilize this model substrate under thermal stress for subsequent Hsp70-mediated refolding. Using a domain shuffling approach, we show that both the nucleotide-binding domain and the β-sandwich domain of Sse1p are required to preserve nonnative luciferase in a folding-competent state. Our findings suggest that Sse1p must undergo partial unfolding for efficient protection of luciferase, and that the β-sandwich domain of Sse1p acts as an intramolecular chaperone for refolding of the nucleotide-binding domain. Under extreme stress conditions, Sse2p appears to take over the nucleotide exchange factor function of Sse1p and might promote the controlled aggregation of stress-denatured proteins.     

Glycosylation inhibits the interaction of invertase with the chaperone GroEL.

During refolding and reassociation of chemically denatured non-glycosylated invertase from Saccharomyces cerevisiae, aggregation competes with correct folding, leading to low yields of reactivation (Kern et al. (1992) Protein Sci. 1, 120-131). In the presence of the chaperone GroEL, refolding is completely arrested. This suggests the formation of a stable complex between GroEL and non-native non-glycosylated invertase. Addition of MgATP results in a slow release of active invertase from the chaperone complex. When GroEL/ES and MgATP are present during refolding, the final reactivation yield increases from 14% to 36%. In contrast, refolding of the core-glycosylated and the high-mannose glycosylated forms of invertase is not arrested by GroEL. Only a short lag phase at the beginning of reactivation and a slightly increased reactivation yield (64% to 86% for core-glycosylated and 62% to 76% for external invertase) indicate a weak interaction of the glycosylated forms with the chaperone.     

Characterization of recombinant forms of the yeast Gas1 protein and identification of residues essential for glucanosyltransferase activity and folding.

Gas1p is a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae and is a representative of Family GH72 of glycosidases/transglycosidases, which also includes proteins from human fungal pathogens. Gas1p, Phr1-2p from Candida albicans and Gel1p from Aspergillus fumigatus have been shown to be beta-(1,3)-glucanosyltransferases required for proper cell wall assembly and morphogenesis. Gas1p is organized into three modules: a catalytic domain; a cys-rich domain; and a highly O-glycosylated serine-rich region. In order to provide an experimental system for the biochemical and structural analysis of Gas1p, we expressed soluble forms in the methylotrophic yeast Pichia pastoris. Here we report that 48 h after induction with methanol, soluble Gas1p was produced at a yield of approximately 10 mg x L(-1) of medium, and this value was unaffected by the further removal of the serine-rich region or by fusion to a 6 x His tag. Purified soluble Gas1 protein showed beta-(1,3)-glucanosyltransferase activity that was abolished by replacement of the putative catalytic residues, E161 and E262, with glutamine. Spectral studies confirmed that the recombinant soluble Gas1 protein assumed a stable conformation in P. pastoris. Interestingly, thermal denaturation studies demonstrated that Gas1p is highly resistant to heat denaturation, and a complete refolding of the protein following heat treatment was observed. We also showed that Gas1p contains five intrachain disulphide bonds. The effects of the C74S, C103S and C265S substitutions in the membrane-bound Gas1p were analyzed in S. cerevisiae. The Gas1-C74S protein was totally unable to complement the phenotype of the gas1 null mutant. We found that C74 is an essential residue for the proper folding and maturation of Gas1p.     

Structural organization of double-stranded RNA from killer yeasts Saccharomyces cerevisiae by the thermal denaturation method

The thermal denaturation method for studying the structural organization of double-stranded RNA (dsRNA) from virus-like particles of killer yeasts Saccharomyces cerevisiae was used. High resolution derivative denaturation profiles of total dsRNA and its L- and M-types were obtained. Comparative analysis of these data with those on phage DNA denaturation demonstrated that the processes of denaturation of dsRNA and phage DNA were identical in quality. Increase of thermostability, interval of thermal denaturation and width of local helix-to-coil transitions in dsRNA as compared with phage DNA are caused by the differences of corresponding thermodynamic parameters. Derivative denaturation profiles of L- and M-types of yeasts dsRNA were shown to have certain identical local transitions. Low melting transition, consisting of three local thermalites, is due to the denaturation of AU-rich region (about 200 n.b.p.) in M-dsRNA.     

Effect of Methylglyoxal Modification of Human α-Crystallin on the Structure,Stability and Chaperone Function

α-Crystallin functions as a molecular chaperone and maintains transparency of eye lens by protecting other lens-proteins. Non-enzymatic glycation of α-crystallin by methylglyoxal, plays a crucial role on its chaperone function and structural stability. Our studies showed that methylglyoxal modification even in lower concentration caused significant decrease in chaperone function of α-crystallin as reflected both in thermal aggregation assay and enzyme refolding assay. Thermal denaturation studies showed drastic reduction of denaturation temperature with increase in the degree of modification. Thermodynamic stability studies by urea denaturation assay reflected a decrease of transition midpoint. Quantitatively we found that ΔG° of native α-crystallin decreased from 21.6 kJ/mol to 10.4 kJ/mol due to 72 h modification by 10 mM methylglyoxal. The surface hydrophobicity of α-crystallin after MG modification, was found to be decreased. Circular dichroism spectroscopy revealed conversion of β-sheet structure to random coil structure. Significant cross-linking was also observed due to methylglyoxal modification of human α-crystallin.     

Influence of trehalose on the molecular chaperone activity of p26, a small heat shock/alpha-crystallin protein

Encysted embryos of the primitive crustacean Artemia franciscana are among the most resistant of all multicellular eukaryotes to environmental stress, in part due to massive amounts of a small heat shock/alpha-crystallin protein (p26) that acts as a molecular chaperone. These embryos also contain very large amounts of the disaccharide trehalose, well known for its ability to protect macromolecules and membranes against damage due to water removal and temperature extremes. Therefore, we looked for potential interactions between trehalose and p26 in the protection of a model substrate, citrate synthase (CS), against heat denaturation and aggregation and in the restoration of activity after heating in vitro. Both trehalose and p26 decreased the aggregation and irreversible inactivation of CS at 43 degrees C. At approximate physiological concentrations (0.4 M), trehalose did not interfere with the ability of p26 to assist in the reactivation of CS after heating, but higher concentrations (0.8 M) were inhibitory. We also showed that CS and p26 interact physically during heating and that trehalose interferes with complex formation and disrupts CS-p26 complexes that form at high temperatures. We suggest from these results that trehalose may act as a "release factor," freeing folding intermediates of CS that p26 can chaperone to the native state. Trehalose and p26 can act synergistically in vitro, during and after thermal stress, suggesting that these interactions also occur in vivo.     

Atp11p and Atp12p are chaperones for F(1)-ATPase biogenesis in mitochondria

The bioenergetic needs of aerobic cells are met principally through the action of the F(1)F(0) ATP synthase, which catalyzes ATP synthesis during oxidative phosphorylation. The catalytic unit of the enzyme (F(1)) is a multimeric protein of the subunit composition alpha(3)beta(3)(gamma)(delta) epsilon. Our work, which employs the yeast Saccharomyces cerevisiae as a model system for studies of mitochondrial function, has provided evidence that assembly of the mitochondrial alpha and beta subunits into the F(1) oligomer requires two molecular chaperone proteins called Atp11p and Atp12p. Comprehensive knowledge of Atp11p and Atp12p activities in mitochondria bears relevance to human physiology and disease as these chaperone actions are now known to exist in mitochondria of human cells.     

The Drosophila gene 2A5 complements the defect in mitochondrial F1-ATPase assembly in yeast lacking the molecular chaperone Atp11p.

Assembly of mitochondrial F1-ATPase in Saccharomyces cerevisiae requires the molecular chaperone, Atp11p. Database searches have identified protein sequences from Schizosaccharomyces pombe and two species of Drosophila that are homologous to S. cerevisiae Atp11p. A cDNA encoding the putative Atp11p from Drosophila yakuba was shown to complement the respiratory deficient phenotype of yeast harboring an atp11::HIS3 disruption allele. Furthermore, the product of this Drosophila gene was shown to interact with the S. cerevisiae F1 beta subunit in the yeast two-hybrid assay. These results indicate that Atp11p function is conserved in higher eukaryotes.     

Trehalose is required for conformational repair of heat-denatured proteins in the yeast endoplasmic reticulum but not for maintenance of membrane traffic functions after severe heat stress

Saccharomyces cerevisiae cells grown at physiological temperature 24 degrees C require preconditioning at 37 degrees C to acquire tolerance towards brief exposure to 48-50 degrees C. During preconditioning, the cytosolic trehalose content increases remarkably and in the absence of trehalose synthesis yeast cannot acquire thermotolerance. It has been speculated that trehalose protects proteins and membranes under environmental stress conditions, but recently it was shown to assist the Hsp104 chaperone in refolding of heat-damaged proteins in the yeast cytosol. We have demonstrated that heat-denatured proteins residing in the endoplasmic reticulum (ER) also can be refolded once the cells are returned to physiological temperature. Unexpectedly, not only ER chaperones but also the cytosolic Hsp104 chaperone is required for conformational repair events in the ER lumen. Here we show that trehalose facilitates refolding of glycoproteins in the ER after severe heat stress. In the absence of Tps1p, a subunit of trehalose synthase, refolding of heat-damaged glycoproteins to bioactive and secretion-competent forms failed or was retarded. In contrast, membrane traffic operated many hours after severe heat stress even in the absence of the TPS1 gene, demonstrating that trehalose had no role in thermoprotection of membranes engaged in vesicular traffic. However, cytosolic proteins were aggregated and protein synthesis abolished, resulting finally in cell death.     

The molecular chaperone, Atp12p, from Homo sapiens. In vitro studies with purified wild type and mutant (E240K) proteins

Work in Saccharomyces cerevisiae has shown that Atp12p binds to unassembled alpha subunits of F(1) and in so doing prevents the alpha subunit from associating with itself in non-productive complexes during assembly of the F(1) moiety of the mitochondrial ATP synthase. We have developed a method to prepare recombinant Atp12p after expression of its human cDNA in bacterial cells. The molecular chaperone activity of HuAtp12p was studied using citrate synthase as a model substrate. Wild type HuAtp12p suppresses the aggregation of thermally inactivated citrate synthase. In contrast, the mutant protein HuAtp12p(E240K), which harbors a lysine at the position of the highly conserved Glu-240, fails to prevent citrate synthase aggregation at 43 degrees C. No significant differences were observed between the wild type and the mutant proteins as judged by sedimentation analysis, cysteine titration, tryptophan emission spectra, or limited proteolysis, which suggests that the E240K mutation alters the activity of HuAtp12p with minimal effects on the physical integrity of the protein. An additional important finding of this work is that the equilibrium chemical denaturation curve of HuAtp12p shows two components, the first of which is associated with protein aggregation. This result is consistent with a model for Atp12p structure in which there is a hydrophobic chaperone domain that is buried within the protein interior.     

The chaperonin-related protein Tcm62p ensures mitochondrial gene expression under heat stress

Tcm62p, distantly related to chaperonins, is required for the assembly of succinate dehydrogenase in mitochondria of Saccharomyces cerevisiae and was proposed to exert chaperone activity. We demonstrate here crucial functions of Tcm62p under heat stress. It ensures mitochondrial gene expression at elevated temperatures and prevents heat-aggregation of the ribosomal subunit Var1p. Similar to chaperonins, Tcm62p forms a high molecular mass protein complex of approximately 850 kDa in the mitochondrial matrix space. These results suggest a more general chaperone function of Tcm62p in mitochondria.     

Saccharomyces cerevisiae Hsp104 enhances the chaperone capacity of human cells and inhibits heat stress-induced proapoptotic signaling

Hsp104, the most potent thermotolerance factor in Saccharomyces cerevisiae, is an unusual molecular chaperone that is associated with the dispersal of aggregated, non-native proteins in vivo and in vitro. The close cooperation between Hsp100 oligomeric disaggregases and specific Hsp70 chaperone/cochaperone systems to refold and reactivate heat-damaged proteins has been dubbed a "bichaperone network". Interestingly, animal genomes do not encode a Hsp104 ortholog. To investigate the biochemical and biological consequences of introducing into human cells a stress tolerance factor that has protein refolding capabilities distinct from those already present, Hsp104 was expressed as a transgene in a human leukemic T-cell line (PEER). Hsp104 inhibited heat-shock-induced loss of viability in PEER cells, and this action correlated with reduced procaspase-3 cleavage but not with reduced c-Jun N-terminal kinase phosphorylation. Hsp104 cooperated with endogenous human Hsp70 and Hsc70 molecular chaperones and their J-domain-containing cochaperones Hdj1 and Hdj2 to produce a functional hybrid bichaperone network capable of refolding aggregated luciferase. We also established that Hsp104 shuttles across the nuclear envelope and enhances the chaperoning capacity of both the cytoplasm and nucleoplasm of intact cells. Our results establish the fundamental properties of protein disaggregase function in human cells with implications for the use of Hsp104 or related proteins as therapeutic agents in diseases associated with protein aggregation.