Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70.

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.     

Molecular cloning of human FKBP51 and comparisons of immunophilin interactions with Hsp90 and progesterone receptor.

A cDNA for human FKBP51 has been cloned and sequenced, and protein products have been expressed in both in vitro and bacterial systems. The deduced amino acid sequence for human FKBP51 is 90% identical to sequences of recently described murine proteins and is 55% identical to the sequence of human FKBP52. Human FKBP51 mRNA is expressed in a wide range of tissues, and the protein has peptidylprolyl isomerase activity that is inhibited by FK506 but not cyclosporine. FKBP51 is the same as a previously described progesterone receptor-associated immunophilin that, similar to FKBP52 and cyclophilin 40, is an Hsp90-binding protein and appears in functionally mature steroid receptor complexes along with Hsp90 and p23. Each of the three receptor-associated immunophilins displays interactions with progesterone receptor that are more dynamic than Hsp90-receptor interactions. Whereas FKBP52 and FKBP51 compete about equally well for binding to Hsp90 in a purified system, FKBP51 accumulates preferentially in progesterone receptor complexes assembled in a cell-free system. This observation provides a precedent for differential interactions between Hsp90-associated immunophilins and target proteins such as steroid receptors.     

Crystal structure and activity of human p23, a heat shock protein 90 co-chaperone

p23 is a co-chaperone for the heat shock protein, hsp90. This protein binds hsp90 and participates in the folding of a number of cell regulatory proteins, but its activities are still unclear. We have solved a crystal structure of human p23 lacking 35 residues at the COOH terminus. The structure reveals a disulfide-linked dimer with each subunit containing eight beta-strands in a compact antiparallel beta-sandwich fold. In solution, however, p23 is primarily monomeric and the dimer appears to be a minor component. Conserved residues are clustered on one face of the monomer and define a putative surface region and binding pocket for interaction(s) with hsp90 or protein substrates. p23 contains a COOH-terminal tail that is apparently less structured and is unresolved in the crystal structure. This tail is not needed for the binding of p23 to hsp90 or to complexes with the progesterone receptor. However, the tail is necessary for optimum active chaperoning of the progesterone receptor, as well as the passive chaperoning activity of p23 in assays measuring inhibition of heat-induced protein aggregation.     

The hsp90-related protein TRAP1 is a mitochondrial protein with distinct functional properties

The hsp90 family of molecular chaperones was expanded recently due to the cloning of TRAP1 and hsp75 by yeast two-hybrid screens. Careful analysis of the human TRAP1 and hsp75 sequences revealed that they are identical, and we have cloned a similar protein from Drosophila. Immunofluorescence data show that human TRAP1 is localized to mitochondria. This mitochondrial localization is supported by the existence of mitochondrial localization sequences in the amino termini of both the human and Drosophila proteins. Due to the striking homology of TRAP1 to hsp90, we tested the ability of TRAP1 to function as an hsp90-like chaperone. TRAP1 did not form stable complexes with the classic hsp90 co-chaperones p23 and Hop (p60). Consistent with these observations, TRAP1 had no effect on the hsp90-dependent reconstitution of hormone binding to the progesterone receptor in vitro, nor could it substitute for hsp90 to promote maturation of the receptor to its hormone-binding state. However, TRAP1 is sufficiently conserved with hsp90 such that it bound ATP, and this binding was sensitive to the hsp90 inhibitor geldanamycin. In addition, TRAP1 exhibited ATPase activity that was inhibited by both geldanamycin and radicicol. Thus, TRAP1 has functions that are distinct from those of hsp90.     

The genome of Trypanosoma cruzi contains a constitutively expressed, tandemly arranged multicopy gene homologous to a major heat shock protein.

cDNA libraries have been constructed in the plasmid vector pUC18 with mRNA isolated from both epimastigotes and trypomastigotes of the Peru strain of Trypanosoma cruzi. Pools of randomly selected clones were analyzed by hybridization-selection-translation. Translation products were immunoprecipitated either with normal human sera or with sera from patients with Chagas' disease (chagasic sera), and the immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this approach, a cDNA clone (pEC5) was identified which encodes a portion of an 85,000-Mr polypeptide. A genomic clone was subsequently isolated (FG1) by using oligonucleotide probes derived from the DNA sequence of this cDNA clone. A portion of this clone was isolated and sequenced, and the coding region for the protein was identified. Computer analysis of the predicted protein sequence indicates that this protein is closely related to the 83,000-Mr heat shock protein (hsp83) of Drosophila melanogaster, the hsp90 of Saccharomyces cerevisiae, and the hsp90 of chicken. This gene is tandemly organized in the T. cruzi genome as a cluster of 6 to 10 copies.     

The common 90-kd protein component of non-transformed ''8S'' steroid receptors is a heat-shock protein.

Non-transformed steroid receptors have an approximately 8S sedimentation coefficient that corresponds to an oligomeric structure of 250-300 kd which includes a non-hormone binding 90-kd protein. A monoclonal antibody BF4 raised against the purified, molybdate-stabilized, 8S progesterone receptor (8S-PR) from chick oviduct, recognizes 8S forms of all steroid hormone receptors. BF4 was found specific for a 90-kd protein present in great abundance in all chicken tissues, including that present in 8S-forms of steroid receptors. Here, using immunological and biochemical techniques, we demonstrate that this ubiquitous BF4-positive 90-kd protein is in fact the chicken 90 kd heat-shock protein (hsp 90): it increased in heat-shocked chick embryo fibroblasts, and displayed identical migration in two-dimensional gel electrophoresis and the same V8 peptide map as the already described hsp 90. We discuss the possibility that the interaction between hsp 90 and steroid hormone-binding subunits may play a role in keeping the receptor in an inactive form.     

Binding of heat shock proteins to the avian progesterone receptor.

The protein composition of the avian progesterone receptor was analyzed by immune isolation of receptor complexes and gel electrophoresis of the isolated proteins. Nonactivated cytosol receptor was isolated in association with the 90-kilodalton (kDa) heat shock protein, hsp90, as has been described previously. A 70-kDa protein was also observed and was shown by Western immunoblotting to react with an antibody specific to the 70-kDa heat shock protein. Thus, two progesterone receptor-associated proteins are identical, or closely related, to heat shock proteins. When the two progesterone receptor species, A and B, were isolated separately in the absence of hormone, both were obtained in association with hsp90 and the 70-kDa protein. However, activated receptor isolated from oviduct nuclear extracts was associated with the 70-kDa protein, but not with hsp90. A hormone-dependent dissociation of hsp90 from the cytosolic form of the receptor complex was observed within the first hour of in vivo progesterone treatment, which could explain the lack of hsp90 in nuclear receptor complexes. In a cell-free system, hsp90 binding to receptor was stabilized by molybdate but disrupted by high salt. These treatments, however, did not alter the binding of the 70-kDa protein to receptor. Association of the 70-kDa protein with the receptor could be disrupted by the addition of ATP at elevated temperatures (23 degrees C). The receptor-associated 70-kDa protein is an ATP-binding protein, as demonstrated by its affinity labeling with azido[32P]ATP. These results indicate that the two receptor-associated proteins interact with the progesterone receptor by different mechanisms and that they are likely to affect the structure or function of the receptor in different ways.     

Constitutively expressed rat mRNA encoding a 70-kilodalton heat-shock-like protein.

A nearly full-length cDNA clone isolated from the rat pheochromocytoma cell line, PC12, revealed extensive nucleotide sequence similarity between the rat cDNA and the Drosophila melanogaster hsp70 gene. The rat recombinant clone encodes a 71,000-dalton protein that is 70% identical with the dipteran hsp70 protein. Remarkably, a truncated segment of this cDNA clone was originally isolated by immunoreactivity with antisera raised to catecholamine-synthesizing enzymes, suggesting that this heat shock protein and these catecholamine enzymes shared antigenic determinants. The rat hsp70-related mRNA is responsible for the production of a constitutive hsp70 protein, because it is present in abundant amounts in various tissues at normal growth temperatures and is only minimally induced by hyperthermia. The rat hsp70-related sequence is part of a multigene family that extends across species to mice and humans.     

Composition, assembly and activation of the avian progesterone receptor

When isolated from chick oviduct cytosol by antibody adsorption, the inactive progesterone receptor is associated with the two heat shock proteins, hsp90 and hsp70, plus three additional proteins termed p54, p50, and p23 according to their molecular weights. While their functions remain unknown, all of these receptor associated proteins are dissociated upon receptor activation in intact cells. To better understand the assembly and activation mechanisms of progesterone receptor complexes, we have developed a cell-free system for studying receptor interactions with hsp90 and hsp70 and have used this system to examine requirements for hsp90 binding to the receptor. Purified receptor, free of hsp90 and immobilized on an antibody affinity resin, will rebind hsp90 in rabbit reticulocyte lysate when several conditions are met. These include: (1) absence of progesterone, (2) elevated temperature (30°C), (3) presence of ATP, and (4) presence of Mg²⁺. We have obtained maximal hsp90 binding to receptor when lysate is supplemented with 3 mM MgCl₂ and an ATP regenerating system. ATP depletion of lysate by dialysis or ATPase addition blocks hsp90 binding to the receptor. When progesterone is added to pre-formed receptor complexes in reticulocyte lysate it promotes activation and the dissociation of hsp90. This process is also dependent upon ATP. Thus, both the assembly, and activation of the progesterone receptor can be accomplished in the reticulocyte lysate system.     

The involvement of p23, hsp90, and immunophilins in the assembly of progesterone receptor complexes

To better understand the assembly mechanism for the progesterone receptor (PR), we have developed cell-free systems for studying interactions of PR, hsp90, and other associated proteins. When PR is incubated in rabbit reticulocyte lysate, its association with hsp90, hsp70, the three immunophilins FKBP54, FKBP52 and CyP-40, and with p23 is observed. These interactions require ATP/Mg²⁺ and when ATP is limiting the PR complex is altered to one containing the proteins p60 and p48, but lacking immunophilins and p23. We have studied two pre-formed hsp90 complexes that may participate in the assembly of PR complexes. One contains hsp90 bound to hsp70 and p60 and this complex forms spontaneously in the absence of ATP. A second complex contains hsp90 bound to p23 plus the three immunophilins and some hsp70. The formation of this complex requires ATP. In further studies we have shown that purified hsp90 can bind to purified p23 and this interaction requires both ATP and molybdate. This explains, in part, the known effects of ATP and molybdate on assembly of PR complexes.     

The non-DNA-binding heterooligomeric form of mammalian steroid hormone receptors contains a hsp90-bound 59-kilodalton protein

Untransformed cytosol receptors for progesterone (PR), androgen (AR), estrogen (ER), and glucocorticosteroid (GR) in rabbit tissues contain a 59-kDa protein (p59) (Tai, P.K.K., Maeda, Y., Nakao, K., Wakim, N.G., Duhring, J.L., and Faber, L.E. (1986) Biochemistry 25, 5269-5275) and a 90-kDa heat shock protein (hsp90). In the present study, receptors from calf uterus (PR, AR, ER, and GR) and from human breast cancer MCF7 cells (PR and GR) were also shown to be comprised of hsp90 and p59. These heterooligomer receptor complexes were stabilized both by transition metal oxyanions (molybdate and tungstate) and chemical cross-linking with dimethylpimelimidate. In 0.4 M KCl, tungstate-stabilized (but not molybdate-stabilized) PR, AR, ER, and GR retained hsp90, but lost p59. Dimethylpimelimidate cross-linking prevented p59 dissociation from hsp90-receptor complexes. Stabilization with tungstate and/or cross-linking permitted immunoaffinity purification of untransformed rabbit as well as calf PR and ER on EC1-Affi-Gel 10 column (an anti-p59 immunoadsorbant). Combined immunoaffinity purification and cross-linking experiments indicated that p59 is bound to hsp90 in the cytosol. We propose that in the nontransformed steroid receptor, p59 interacts with hsp90 rather than with the hormone binding subunit.     

Progesterone receptor structure and function altered by geldanamycin, an hsp90-binding agent.

The assembly of progesterone receptor (PR) heterocomplexes in vitro involves at least eight components of the molecular chaperone machinery, and as earlier reports have shown, these proteins exhibit complex, dynamic, but ordered, interactions with one another and PR. Using the selective hsp90 binding agent geldanamycin (GA), we have found that PR assembly in vitro can be arrested at a previously observed intermediate assembly step. Like mature PR complexes, the intermediate complexes contain hsp90, but they differ from mature complexes by the presence of hsp70, p60, and p48 and the absence of immunophilins and p23. Arrest of PR assembly is likely due to GA's ability to directly block binding of p23 to hsp90. An important functional consequence of GA-mediated assembly arrest in vitro is the inability of the resulting PR complexes to bind progesterone, despite the presence of hsp90 in the receptor complexes. The biological significance of the in vitro observations is demonstrated by GA's ability to (i) rapidly block PR's hormone binding capacity in intact cells and (ii) alter the composition of COS cell PR complexes in a manner similar to that observed during in vitro reconstitutions. An updated model for the cyclic assembly pathway of PR complexes that incorporates the present findings with earlier results is presented.     

Cloning and characterization of chicken adipocyte fatty acid binding protein gene

Fatty acid binding proteins (FABPs) are members of a superfamily of lipid-binding proteins and occur intracellularly in vertebrates and invertebrates. This study was designed to clone and characterize the adipocyte fatty acid binding protein (A-FABP) gene in the chicken. PCR primers were designed according to mammalian A-FABP gene sequence to amplify partial cDNA of A-FABP gene from chicken adipose tissues, and the full length of the gene was cloned by 5'RACE and 3'RACE. Analysis of sequence showed that the cDNA of the chicken A-FABP gene was 74 and 73% homologous with porcine and human A-FABP gene, respectively. The similarity was 77, 28, and 23% at the predicted amino acid level with human A-FABP, human L-FABP, and human I-FABP, respectively. RT-PCR and Northern blot analysis indicated that the chicken A-FABP gene, similar to that of the mammal, is only expressed in fat tissues. This is the first report to identify and characterize A-FABP gene in the chicken.     

Structure and chromosomal location of the human gene encoding cartilage matrix protein

Cartilage matrix protein (CMP) is a major component of the extracellular matrix of nonarticular cartilage. The structure and chromosomal location of the human gene encoding CMP was determined by molecular cloning analysis. We used a partial chicken CMP cDNA probe to isolate three overlapping human genomic clones. From one of these clones, a probe containing 2 human CMP exons was isolated and used to map the gene to chromosome 1p35 and to screen a human retina cDNA library. Two overlapping cDNA clones were isolated. The predicted protein sequence of 496 amino acids includes a 22-residue signal peptide and a 474-residue mature protein of Mr 51,344. The human CMP gene and polypeptide are strikingly similar to the chicken CMP gene and polypeptide. Human CMP is 79% identical to chicken CMP and contains two homologous domains separated by an epidermal growth factor-like domain. One potential N-glycosylation site is conserved between the two species. The human CMP gene spans 12 kilobase pairs with 8 exons and 7 introns which are similar in size to those of the chicken CMP gene. Both RNA splice junctions of intron G in the human and chicken CMP genes are nonconforming to the consensus splice sequences. This suggests that the CMP gene utilizes a new RNA splicing mechanism.     

Sequencing of the chicken non-erythroid spectrin cDNA reveals an internal repetitive structure homologous to the human erythrocyte spectrin.

Immunological screening of a chicken gizzard cDNA expression library was used to isolate two clones encoding a part of the non-erythroid spectrin-like protein. Clones were identified by immunoblotting of the polypeptides synthesized in Escherichia coli cells transformed with cDNA cloned in the pUC8 plasmid vector using polyclonal rabbit antibodies raised against bovine non-erythroid spectrin. The sequence of an approximately 1.5-kb cDNA insert of one clone was determined. Analysis of the predicted amino acid sequence reveals that, despite differences in immunological cross-reactivity and peptide maps, the chicken non-erythroid and the human erythrocyte spectrins are highly homologous proteins. Like the human erythrocyte spectrin, the chicken smooth muscle spectrin appears also to be constructed from repeated, homologous structures of 106 amino acid residues. This is probably a universal structure motif of spectrins.     

P59, an hsp 90-binding protein. Cloning and sequencing of its cDNA and preparation of a peptide-directed polyclonal antibody.

The primary sequence of the rabbit liver cDNA coding for protein p59 has been determined. The protein binds to the 90-kDa heat shock protein (hsp 90) and is associated with it, including when hsp 90 participates in hetero-oligomeric complexes of untransformed mammalian steroid receptors that sediment at 8-10 S. The cloned cDNA codes for an open reading frame of 458 amino acids defining a yet unknown protein. However, 55% amino acid homology to peptidyl-prolyl isomerase is found between amino acids 41 and 137, suggesting rotamase activity for p59, which speculatively may apply to bound hsp 90 and thus be implied in the intracellular trafficking of hetero-oligomeric forms of steroid hormone receptors. A polyclonal antibody derived from the COOH-terminal peptide 441-458 demonstrates a good affinity for rabbit, rat, and human "p59" protein. It interacts with at least one epitope, available in 8-10 S untransformed steroid receptor complexes and different from that recognized by the monoclonal antibody KN382/EC-1.     

The importance of ATP binding and hydrolysis by hsp90 in formation and function of protein heterocomplexes.

The chaperone hsp90 is capable of binding and hydrolyzing ATP. Using information on a related ATPase, DNA gyrase B, we selected three conserved residues in hsp90's ATP-binding domain for mutation. Two of these mutations eliminate nucleotide binding, while the third retains nucleotide binding but is apparently deficient in ATP hydrolysis. We first analyzed how these mutations affect hsp90's binding to the co-chaperones p23 and Hop, and to the hydrophobic resin, phenyl-Sepharose. These experiments showed that ATP's effects, specifically, increased affinity for p23 and decreased affinity for Hop and phenyl-Sepharose, are brought on by ATP binding alone. We also tested the ability of hsp90 mutants to assist hsp70, hsp40, and Hop in the refolding of denatured firefly luciferase. While hsp90 is capable of participating in this process in a nucleotide-independent manner, the ability to hydrolyze ATP markedly potentiates hsp90's effect. Finally, we assembled progesterone receptor heterocomplexes with hsp70, hsp40, Hop, p23, and wild type or mutant hsp90. While neither ATP binding nor hydrolysis was necessary to bind hsp90 to the receptor, mature complexes containing p23 and capable of hormone binding were only obtained with wild type hsp90.     

A 50-kDa cytosolic protein complexed with the 90-kDa heat shock protein (hsp90) is the same protein complexed with pp60v-src hsp90 in cells transformed by the Rous sarcoma virus.

We have previously shown that a 50-kDa protein is one component of a heteromeric complex immunoprecipitated by the 90-kDa heat shock protein (hsp90) monoclonal antibodies 8D3 and 3G3 (Perdew, G. H., and Whitelaw, M. L. (1991) J. Biol. Chem. 266, 6708-6713). In this report, we compare the 50-kDa protein with that found in pp60v-src-hsp90-p50 complexes immunoprecipitated from Rous sarcoma virus-transformed cells with antibodies to pp60v-src. 35S- and 32P-labeled p50 proteins from each system were identical in their mobilities by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. The profile of N-chlorosuccinimide cleavage products derived from each 32P-labeled p50 protein were also identical when resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have developed a mouse monoclonal antibody, 3M/1B5p50, capable of detecting p50 on Western blots. This antibody detected the 50-kDa protein which co-purified with the pa104 pp60v-src mutant of the avian sarcoma virus oncoprotein in 44A rat fibroblasts. We did not detect p50 in association with native glucocorticoid receptor in L cells or with the overexpressed glucocorticoid receptor in Chinese hamster ovary cells. Two experiments utilizing immunochemical staining implied that essentially all cytosolic p50 is associated with hsp90. Firstly, immunoprecipitating hsp90 from Hepa 1 cytosol with monoclonal antibody 3G3 left the cytosol depleted of p50. Secondly, cytosol fractionated by sucrose gradient revealed that p50 cosedimented with hsp90, confirming the existence of p50 only in association with hsp90.