Nucleolar protein B23 has molecular chaperone activities

Protein B23 is an abundant, multifunctional nucleolar phosphoprotein whose activities are proposed to play a role in ribosome assembly. Szebeni et al. (1997) showed stimulation of nuclear import in vitro by protein B23 and suggested that this effect was due to a molecular chaperone-like activity. Protein B23 was tested for chaperone activities using several protein substrates. The temperature-dependent and -independent aggregation of the HIV-1 Rev protein was measured using a zero angle light scattering (turbidity) assay. Protein B23 inhibited the aggregation of the Rev protein, with the amount of inhibition proportional to the concentration of B23 added. This activity was saturable with nearly complete inhibition when the molar ratio of B23:Rev was slightly above one. Protein B23 also protected liver alcohol dehydrogenase (LADH), carboxypeptidase A, citrate synthase, and rhodanese from aggregation during thermal denaturation and preserved the enzyme activity of LADH under these conditions. In addition, protein B23 was able to promote the restoration of activity of LADH previously denatured with guanidine-HCl. Protein B23 preferentially bound denatured substrates and exposed hydrophobic regions when complexed with denatured proteins. Thus, by several criteria, protein B23 behaves like a molecular chaperone; these activities may be related to its role in ribosome biogenesis.     

The p23 co-chaperone protein is a novel substrate of CK2 in Arabidopsis

The ubiquitous Ser/Thr protein kinase CK2, which phosphorylates hundreds of substrates and is essential for cell life, plays important roles also in plants; however, only few plant substrates have been identified so far. During a study aimed at identifying proteins targeted by CK2 in plant response to salicylic acid (SA), we found that the Arabidopsis co-chaperone protein p23 is a CK2 target, readily phosphorylated in vitro by human and maize CK2, being also a substrate for an endogenous casein kinase activity present in Arabidopsis extracts, which displays distinctive characteristics of protein kinase CK2. We also demonstrated that p23 and the catalytic subunit of CK2 interact in vitro and possibly in Arabidopsis mesophyll protoplasts, where they colocalize in the cytosol and in the nucleus. Although its exact function is presently unknown, p23 is considered a co-chaperone because of its ability to associate to the chaperone protein Hsp90; therefore, an involvement of p23 in plant signal transduction pathways, such as SA signaling, is highly conceivable, and its phosphorylation may represent a fine mechanism for the regulation of cellular responses.     

Unique features of HIV-1 Rev protein phosphorylation by protein kinase CK2 ('casein kinase-2')

The HIV-1 Rev transactivator is phosphorylated in vitro by protein kinase CK2 at two residues, Ser-5 and Ser-8; these sites are also phosphorylated in vivo. Here we show that the mechanism by which CK2 phosphorylates Rev is unique in several respects, notably: (i) it is fully dependent on the regulatory, beta-subunit of CK2; (ii) it relies on the integrity of an acidic stretch of CK2 beta which down-regulates the phosphorylation of other substrates; (iii) it is inhibited in a dose-dependent manner by polyamines and other polycationic effectors that normally stimulate CK2 activity. In contrast, a peptide corresponding to the amino-terminal 26 amino acids of Rev, including the phosphoacceptor site, is readily phosphorylated by the catalytic subunit of CK2 even in the absence of the beta-subunit. These data, in conjunction with the observation that two functionally inactive derivatives of Rev with mutations in its helix-loop-helix motif are refractory to phosphorylation, indicate the phosphorylation of Rev by CK2 relies on conformational features of distinct regions that are also required for the transactivator's biological activity.     

Chaperone-like activity of the N-terminal region of a human small heat shock protein and chaperone-functionalized nanoparticles

Small heat shock proteins (sHsps) are molecular chaperones employed to interact with a diverse range of substrates as the first line of defense against cellular protein aggregation. The N-terminal region (NTR) is implicated in defining features of sHsps; notably in their ability to form dynamic and polydisperse oligomers, and chaperone activity. The physiological relevance of oligomerization and chemical-scale mode(s) of chaperone function remain undefined. We present novel chemical tools to investigate chaperone activity and substrate specificity of human HspB1 (B1NTR), through isolation of B1NTR and development of peptide-conjugated gold nanoparticles (AuNPs). We demonstrate that B1NTR exhibits chaperone capacity for some substrates, determined by anti-aggregation assays and size-exclusion chromatography. The importance of protein dynamics and multivalency on chaperone capacity was investigated using B1NTR-conjugated AuNPs, which exhibit concentration-dependent chaperone activity for some substrates. Our results implicate sHsp NTRs in chaperone activity, and demonstrate the therapeutic potential of sHsp-AuNPs in rescuing aberrant protein aggregation.     

StHsp14.0, a small heat shock protein of Sulfolobus tokodaii strain 7, protects denatured proteins from aggregation in the partially dissociated conformation

The small heat shock protein (sHsp), categorized into a class of molecular chaperones, binds and stabilizes denatured proteins for the purpose of preventing aggregation. The sHsps undergo transition between different oligomeric states to control their nature. We have been studying the function of sHsp of Sulfolobus tokodaii, StHsp14.0. StHsp14.0 exists as 24meric oligomer, and exhibits oligomer dissociation and molecular chaperone activity over 80°C. We constructed and characterized StHsp14.0 mutants with replacement of the C-terminal IKI to WKW, IKF, FKI and FKF. All mutant complexes dissociated into dimers at 50°C. Among them, StHsp14.0FKF is almost completely dissociated, probably to dimers. All mutants protected citrate synthase (CS) from thermal aggregation at 50°C. But, the activity of StHsp14.0FKF was the lowest. Then, we examined the complexes of StHsp14.0 mutants with denatured CS by SAXS. StHsp14.0WKW protects denatured CS by forming the globular complexes of 24 subunits and a substrate. StHsp14.0FKF also formed similar complex but the number of subunits in the complex is a little smaller. These results suggest that the dimer itself exhibits low chaperone activity, and a partially dissociated oligomer of StHsp14.0 protects a denatured protein from interacting with other molecules by surrounding it.     

The base of the proteasome regulatory particle exhibits chaperone-like activity

Protein substrates of the proteasome must apparently be unfolded and translocated through a narrow channel to gain access to the proteolytic active sites of the enzyme. Protein folding in vivo is mediated by molecular chaperones. Here, to test for chaperone activity of the proteasome, we assay the reactivation of denatured citrate synthase. Both human and yeast proteasomes stimulate the recovery of the native structure of citrate synthase. We map this chaperone-like activity to the base of the regulatory particle of the proteasome, that is, to the ATPase-containing assembly located at the substrate-entry ports of the channel. Denatured but not native citrate synthase is bound by the base complex. Ubiquitination of citrate synthase is not required for its binding or refolding by the base complex of the proteasome. These data suggest a model in which ubiquitin-protein conjugates are initially tethered to the proteasome by specific recognition of their ubiquitin chains; this step is followed by a nonspecific interaction between the base and the target protein, which promotes substrate unfolding and translocation.     

Ribosome-DnaK interactions in relation to protein folding

Bacterial ribosomes or their 50S subunit can refold many unfolded proteins. The folding activity resides in domain V of 23S RNA of the 50S subunit. Here we show that ribosomes can also refold a denatured chaperone, DnaK, in vitro, and the activity may apply in the folding of nascent DnaK polypeptides in vivo. The chaperone was unusual as the native protein associated with the 50S subunit stably with a 1:1 stoichiometry in vitro. The binding site of the native protein appears to be different from the domain V of 23S RNA, the region with which denatured proteins interact. The DnaK binding influenced the protein folding activity of domain V modestly. Conversely, denatured protein binding to domain V led to dissociation of the native chaperone from the 50S subunit. DnaK thus appears to depend on ribosomes for its own folding, and upon folding, can rebind to ribosome to modulate its general protein folding activity.     

ATP causes small heat shock proteins to release denatured protein.

Small heat-shock proteins (sHSPs) are a ubiquitous family of low molecular mass (15-30 kDa) stress proteins that have been found in all organisms. Under stress, sHSPs such as alpha-crystallin can act as chaperones binding partially denatured proteins and preventing further denaturation and aggregation. Recently, it has been proposed that the function of sHSPs is to stabilize stress-denatured protein and then act cooperatively with other HSPs to renature the partially denatured protein in an ATP-dependent manner. However, the process by which this occurs is obscure. As no significant phosphorylation of alpha-crystallin was observed during the renaturation, the role of ATP is not clear. It is now shown that ATP at normal physiological concentrations causes sHSPs to change their confirmation and release denatured protein, allowing other molecular chaperones such as HSP70 to renature the protein and renew its biological activity. In the absence of ATP, sHSPs such as alpha-crystallin are more efficient than HSP70 in preventing stress-induced protein aggregation. This work also indicates that in mammalian systems at normal cellular ATP concentrations, sHSPs are not effective chaperones.     

The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins

To maintain quality control in cells, mechanisms distinguish among improperly folded peptides, mature and functional proteins, and proteins to be targeted for degradation. The molecular chaperones, including heat-shock protein Hsp90, have the ability to recognize misfolded proteins and assist in their conversion to a functional conformation. Disruption of Hsp90 heterocomplexes by the Hsp90 inhibitor geldanamycin leads to substrate degradation through the ubiquitin-proteasome pathway, implicating this system in protein triage decisions. We previously identified CHIP (carboxyl terminus of Hsc70-interacting protein) to be an interaction partner of Hsc70 (ref. 4). CHIP also interacts directly with a tetratricopeptide repeat acceptor site of Hsp90, incorporating into Hsp90 heterocomplexes and eliciting release of the regulatory cofactor p23. Here we show that CHIP abolishes the steroid-binding activity and transactivation potential of the glucocorticoid receptor, a well-characterized Hsp90 substrate, even though it has little effect on its synthesis. Instead, CHIP induces ubiquitylation of the glucocorticoid receptor and degradation through the proteasome. By remodelling Hsp90 heterocomplexes to favour substrate degradation, CHIP modulates protein triage decisions that regulate the balance between protein folding and degradation for chaperone substrates.     

Polypeptide release by Hsp90 involves ATP hydrolysis and is enhanced by the co-chaperone p23

The molecular chaperone Hsp90 binds and hydrolyses ATP, but how this ATPase activity regulates the interaction of Hsp90 with a polypeptide substrate is not yet understood. Using the glucocorticoid receptor ligand binding domain as a substrate, we show that dissociation of Hsp90 from bound polypeptide depends on the Hsp90 ATPase and is blocked by geldanamycin, a specific ATPase inhibitor. The co-chaperone p23 greatly stimulates Hsp90 substrate release with ATP, but not with the non-hydrolysable nucleotides ATPgammaS or AMP-PNP. Point mutants of Hsp90 with progressively lower ATPase rates are progressively slower in ATP-dependent substrate release but are still regulated by p23. In contrast, ATPase-inactive Hsp90 mutants release substrate poorly and show no p23 effect. These results outline an ATP-driven cycle of substrate binding and release for Hsp90 which differs from that of other ATP-driven chaperones. Conversion of the ATP state of Hsp90 to the ADP state through hydrolysis is required for efficient release of substrate polypeptide. p23 couples the ATPase activity to polypeptide dissociation and thus can function as a substrate release factor for Hsp90.     

A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state.

The small heat shock proteins (sHSPs) recently have been reported to have molecular chaperone activity in vitro; however, the mechanism of this activity is poorly defined. We found that HSP18.1, a dodecameric sHSP from pea, prevented the aggregation of malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase heated to 45 degrees C. Under conditions in which HSP18.1 prevented aggregation of substrates, size-exclusion chromatography and electron microscopy revealed that denatured substrates coated the HSP18.1 dodecamers to form expanded complexes. SDS-PAGE of isolated complexes demonstrated that each HSP18.1 dodecamer can bind the equivalent of 12 MDH monomers, indicating that HSP18.1 has a large capacity for non-native substrates compared with other known molecular chaperones. Photoincorporation of the hydrophobic probe 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) into a conserved C-terminal region of HSP18.1 increased reversibly with increasing temperature, but was blocked by prior binding of MDH, suggesting that bis-ANS incorporates proximal to substrate binding regions and that substrate-HSP18.1 interactions are hydrophobic. We also show that heat-denatured firefly luciferase bound to HSP18.1, in contrast to heat-aggregated luciferase, can be reactivated in the presence of rabbit reticulocyte or wheat germ extracts in an ATP-dependent process. These data support a model in which sHSPs prevent protein aggregation and facilitate substrate refolding in conjunction with other molecular chaperones.     

Identification of small molecules that modify the protein folding activity of heat shock protein 70

Molecular chaperones, such as heat shock protein 70 (Hsp70) and its bacterial ortholog DnaK, play numerous important roles in protein folding. In vitro, this activity can be observed by incubating purified chaperones with denatured substrates and measuring the recovery of properly folded protein. In an effort to rapidly identify small molecules that modify this folding activity, we modified an existing method for use in 96-well plates. In this assay, denatured firefly luciferase was treated with a mixture of DnaK and prospective chemical modulators. The luminescence of refolded luciferase was used to follow the reaction progress, and counterscreens excluded compounds that target luciferase; thus, hits from these screens modify protein folding via their effects on the function of the chaperone machine. Using this platform, we screened a pilot chemical library and found five new inhibitors of DnaK and one compound that promoted folding. These chemical probes may be useful in studies aimed at understanding the many varied roles of chaperones in cellular protein folding. Moreover, this assay provides the opportunity to rapidly screen for additional compounds that might regulate the folding activity of Hsp70.     

Tuning of DnaK chaperone action by nonnative protein sensor DnaJ and thermosensor GrpE

DnaK, an Hsp70 molecular chaperone, processes its substrates in an ATP-driven cycle, which is controlled by the co-chaperones DnaJ and GrpE. The kinetic analysis of substrate binding and release has as yet been limited to fluorescence-labeled peptides. Here, we report a comprehensive kinetic analysis of the chaperone action with protein substrates. The kinetic partitioning of the (ATP x DnaK) x substrate complexes between dissociation and conversion into stable (ADP x DnaK) x substrate complexes is determined by DnaJ. In the case of substrates that allow the formation of ternary (ATP x DnaK) x substrate x DnaJ complexes, the cis-effect of DnaJ markedly accelerates ATP hydrolysis. This triage mechanism efficiently selects from the (ATP x DnaK) x substrate complexes those to be processed in the chaperone cycle; at 45 degrees C, the fraction of protein complexes fed into the cycle is 20 times higher than that of peptide complexes. The thermosensor effect of the ADP/ATP exchange factor GrpE retards the release of substrate from the cycle at higher temperatures; the fraction of total DnaK in stable (ADP x DnaK) x substrate complexes is 2 times higher at 45 degrees C than at 25 degrees C. Monitoring the cellular situation by DnaJ as nonnative protein sensor and GrpE as thermosensor thus directly adapts the operational mode of the DnaK system to heat shock conditions.     

Hsp90 chaperone complexes are required for the activity and stability of yeast protein kinases Mik1, Wee1 and Swe1.

The Wee1 protein kinase negatively regulates entry into mitosis by mediating the inhibitory tyrosine phosphorylation of Cdc2-cyclin B kinase. The stability and activity of Wee1 from the fission yeast Schizosaccharomyces pombe is critically dependent on functional Hsp90 chaperones. Here we identify two related tyrosine protein kinases, Mik1 from fission yeast and its Saccharomyces cerevisiae homolog Swe1, as Hsp90 substrates and show that the kinase domain is sufficient to mediate this interaction. Morphological and biochemical defects arising from overexpression of the kinases in fission yeast are suppressed in the conditional Hsp90 mutant swo1-26. A subset of all three kinases is associated with the Hsp90 cochaperones cyclophilin 40 and p23. Under conditions of impaired chaperone function or treatment with the Hsp90 inhibitory drug geldanamycin, intracellular levels of the kinases are reduced and the proteins become rapidly degraded by the proteasome machinery, indicating that Wee1, Mik1 and Swe1 require Hsp90 heterocomplexes for their stability and maintenance of function.     

HspB8 and Bag3: A new chaperone complex targeting misfolded proteins to macroautophagy

Protein quality control involves molecular chaperones that recognize misfolded proteins thereby preventing their aggregation, and associated co-chaperones that modulate substrate sorting between renaturation and proteasomal degradation. We recently described a new chaperone complex that stimulates degradation of protein substrates by macroautophagy. The complex is formed of HspB8, a member of the HspB family of molecular chaperones, which is found mutated in neuromuscular diseases, and Bag3, a member of the co-chaperone family of Bag domain-containing proteins. In this complex, Bag3 was shown to be responsible for macroautophagy stimulation. Here we analyzed the role of the three Bag3 canonical protein interaction domains. We show that the proline-rich region is essential for the Bag3-mediated stimulation of mutated huntingtin clearance. Surprisingly, deletion of the BAG domain that mediates Bag3 interaction with Hsp70 and Blc-2, did not affect its activity. We propose that in the HspB8-Bag3 complex, HspB8 is responsible for recognizing the misfolded proteins whereas Bag3, at least in part through its proline-rich domain, might recruit and activate the macroautophagy machinery in close proximity to the chaperone-loaded substrates.

Addendum to: Carra S, Seguin SJ, Lambert H, Landry J. HSPB8 chaperone activity towards poly-Q containing proteins depends on its association with BAG3, a stimulator of macroautophagy. J Biol Chem 2007; In press.     

HspB8 and Bag3: a new chaperone complex targeting misfolded proteins to macroautophagy

Protein quality control involves molecular chaperones that recognize misfolded proteins thereby preventing their aggregation, and associated co-chaperones that modulate substrate sorting between renaturation and proteasomal degradation. We recently described a new chaperone complex that stimulates degradation of protein substrates by macroautophagy. The complex is formed of HspB8, a member of the HspB family of molecular chaperones, which is found mutated in neuromuscular diseases, and Bag3, a member of the co-chaperone family of Bag domain-containing proteins. In this complex, Bag3 was shown to be responsible for macroautophagy stimulation. Here we analyzed the role of the three Bag3 canonical protein interaction domains. We show that the proline-rich region is essential for the Bag3-mediated stimulation of mutated huntingtin clearance. Surprisingly, deletion of the BAG domain that mediates Bag3 interaction with Hsp70 and Blc-2, did not affect its activity. We propose that in the HspB8- Bag3 complex, HspB8 is responsible for recognizing the misfolded proteins whereas Bag3, at least in part through its proline-rich domain, might recruit and activate the macroautophagy machinery in close proximity to the chaperone-loaded substrates.     

Thermoresponsive controlled association of protein with a dynamic nanogel of hydrophobized polysaccharide and cyclodextrin: heat shock protein-like activity of artificial molecular chaperone

Dynamic CHP-CD nanogels, which consisted of a self-assembly of cholesteryl-group-bearing pullulan (CHP) and beta-cyclodextrin (CD), were characterized by SEC and SEC-MALS methods. The nanogels prevented the thermal aggregation of carbonic anhydrase B (CAB) by selective trapping of the heat-denatured protein. After the complex between the CHP-CD nanogels and CAB was cooled, the enzyme activity of CAB spontaneously recovered upon release from the complex. The dynamic nanogels self-regulated an association of heat denatured protein and dissociation of native protein depending on the concentration of CD. The thermal stability of CAB was improved by thermoresponsive controlled association between the proteins and the artificial molecular chaperone.     

Function of nucleophosmin/B23, a nucleolar acidic protein, as a histone chaperone

We previously identified and purified a nucleolar phosphoprotein, nucleophosmin/B23, as a stimulatory factor for replication from the adenovirus chromatin. We show here that nucleophosmin/B23 functions as a histone chaperone protein such as nucleoplasmin, TAF-I, and NAP-I. Nucleophosmin/B23 was shown to bind to histones, preferentially to histone H3, to mediate formation of nucleosome, and to decondense sperm chromatin. These activities of B23 were dependent on its acidic regions as other histone chaperones, suggesting that B23/nucleophosmin is a member of histone chaperone proteins.