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D Toft

Publications and source records attributed to D Toft.

At least 19 recordsLinked to original sources
PubMed1998

The assembly of progesterone receptor-hsp90 complexes using purified proteins.

The progesterone receptor can be reconstituted into hsp90-containing complexes in vitro, and the resulting complexes are needed to maintain hormone binding activity. This process requires ATP/Mg2+, K+, and several axillary proteins. We have developed a defined system for the assembly of progesterone receptor complexes using purified proteins. Five proteins are needed to form complexes that are capable of maintaining hormone binding activity. These include hsp70 and its co-chaperone, hsp40, the hsp70/hsp90-binding protein, Hop, hsp90, and the hsp90-binding protein, p23. The proteins Hip and FKBP52 were not required for this in vitro process even though they have been observed in receptor complexes. Each of the five proteins showed a characteristic concentration dependence. Similar concentrations of hsp70, hsp90, and p23 were needed for optimal assembly, but hsp40 and Hop were effective at about 1/10 the concentration of the other proteins, suggesting that these two proteins act catalytically or are needed at levels similar to the receptor concentration. ATP was required for the functioning of both hsp70 and hsp90. The binding of hsp70 to the receptor requires hsp40 and about 10 microM ATP; however, hsp90 binding appears to occur subsequent to hsp70 binding and is optimal with 1 mM ATP. A three-step model is presented to describe the assembly process.

Animals↗
PubMed1998

Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with geldanamycin.

The molecular chaperone Hsp90 plays an essential role in the folding and function of important cellular proteins including steroid hormone receptors, protein kinases and proteins controlling the cell cycle and apoptosis. A 15 A deep pocket region in the N-terminal domain of Hsp90 serves as an ATP/ADP-binding site and has also been shown to bind geldanamycin, the only specific inhibitor of Hsp90 function described to date. We now show that radicicol, a macrocyclic antifungal structurally unrelated to geldanamycin, also specifically binds to Hsp90. Moreover, radicicol competes with geldanamycin for binding to the N-terminal domain of the chaperone, expressed either by in vitro translation or as a purified protein, suggesting that radicicol shares the geldanamycin binding site. Radicicol, as does geldanamycin, also inhibits the binding of the accessory protein p23 to Hsp90, and interferes with assembly of the mature progesterone receptor complex. Radicicol does not deplete cells of Hsp90, but rather increases synthesis as well as the steady-state level of this protein, similar to a stress response. Finally, radicicol depletes SKBR3 cells of p185erbB2, Raf-1 and mutant p53, similar to geldanamycin. Radicicol thus represents a structurally unique antibiotic, and the first non-benzoquinone ansamycin, capable of binding to Hsp90 and interfering with its function.

Animals↗
PubMed1997

Nucleotides and two functional states of hsp90.

Previous studies have demonstrated the ATP-dependent formation of a complex containing the heat shock protein hsp90, the unique hsp90 binding protein p23, and one of three high molecular weight immunophilins. In the present study, hsp90 and p23 are shown to form a complex that requires elevated temperature and ATP/Mg2+. Complex formation is strongly promoted by molybdate and by the nonionic detergent Nonidet P-40. ADP and the benzoquinone ansamycin, geldanamycin, are potent inhibitors of complex formation. The ATP-dependent process alters the state of hsp90, not p23, and influences the ability of hsp90 to bind to phenyl-Sepharose. Conversion of hsp90 to the ATP-bound state lowers its affinity for phenyl-Sepharose. These results show that hsp90 can exist in at least two functional states, one able to bind p23 and the other with a high affinity for hydrophobic resins. A model is presented where these states are dictated by the binding of either ATP or ADP.

Benzoquinones↗
PubMed1996

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.

Adenosine Triphosphate↗
PubMed1996

Mutational analysis of Hsp90 alpha dimerization and subcellular localization: dimer disruption does not impede "in vivo' interaction with estrogen receptor.

The molecular chaperone Hsp90 has been found ubiquitously as a predominantly cytoplasmic dimer. By interacting with cytoplasmic or nuclear proteins such as pp60v-src or steroid receptors, Hsp90 helps its targets to become competent for full biological activity. Mutational deletion analysis of some properties of chicken Hsp90 alpha was undertaken after transient transfection of the constructs in COS7 cells. First, Hsp90 mutants were analyzed for their ability to behave as cytosolic dimers. We confirmed that the C-terminal Hsp90 region (amino acids 446-728) was sufficient for dimerization, and found that deletion of three small subregions in the 200 C-terminal residues precluded Hsp90 dimer formation. Moreover, we demonstrated that the N-terminal region of the protein (1-442) was not involved in dimerization. Second, the subcellular localization of the wild-type (WT) protein and mutants was analyzed by specific immunodetection and confocal microscopy. Most of the mutants were cytoplasmic like Hsp90WT, a nuclear localization being barely detectable in the WT protein or in mutants with a C-terminal truncation equal to or shorter than 286 residues. Surprisingly a mutant encoding the N-terminal region (1-285) was nuclear localized. In addition, the in vivo interaction between the cytoplasmic Hsp90 and the nuclear ER was documented after coexpression of both proteins in the same cells: some Hsp90 was shifted into the nucleus via its interaction with ER. From an analysis of dimeric or monomeric cytoplasmic Hsp90 mutants, we found that disruption of Hsp90 dimer did not systematically impede its interaction with ER. Finally, Hsp90WT and cytoplasmic mutants were tested for their ability to rescue from lethality a yeast strain deleted of both Hsp90 genes. Interestingly, the delta 661-677 mutant that showed an impaired dimerization but interacted with ER was able to confer viability, while the mutant deleted of the 30 C-terminal residues (NC6) was monomeric, did not confer viability and did not interact with ER. We therefore suggest that Hsp90 properties analyzed here are not necessarily interdependent.

Animals↗
PubMed1995

Identification of a complex between centrin and heat shock proteins in CSF-arrested Xenopus oocytes and dissociation of the complex following oocyte activation.

Coimmunoprecipitation experiments using a monoclonal anti-centrin antibody (20H5) and cytostatic factor (CSF)-arrested Xenopus oocyte extracts specifically precipitates oocyte centrin (20-kDa) and two associated proteins of 70- and 90-kDa. Microsequence analysis of a tryptic peptide fragment of the 70-kDa protein reveals 100% identity with a 13-amino-acid peptide sequence from Xenopus heat shock protein hsp-70. Western blot analysis of immunoprecipitates using anti-hsp monoclonal antibodies (N27 and AC-88) confirms the identity of the 70-kDa protein as hsp-70 and identifies the 90-kDa protein as hsp-90. The centrin/hsp complex is also immunoprecipitated when anti-hsp-70 or anti-hsp-90 monoclonal antibodies (BB70 and 4F3, respectively) are used as primary antibodies during immunoprecipitation. The centrin/hsp complex is sensitive to pH and Ca2+ concentration. The complex shows differential dissociation of hsp-70 and hsp-90 under a variety of conditions, suggesting that each hsp can bind to centrin independently of the other. When oocytes are first activated by electric shock or ionophore treatment, followed by immunoprecipitation using anti-centrin monoclonal antibody 20H5, centrin precipitates with significantly reduced levels of hsp-70 in the complex, and these complexes contain no apparent hsp-90. We conclude that, in CSF-arrested oocytes, the centrosomal protein, centrin, is associated as a complex with the heat shock proteins, hsp-70 and hsp-90, and that this complex dissociates upon activation of the oocyte. The functional consequences of the formation of complexes between centrin and these hsps are unknown. However, based on the roles that have been defined for heat shock proteins in other systems, several possibilities are suggested.

Amino Acid Sequence↗
PubMed1989

Progesterone receptor concentration differences in the chick oviduct cells and apparent down-regulation by ligand. A semiquantitative immunohistochemical study.

A semiquantitative immunohistochemical technique was developed for identification of chick progesterone receptor (PR). The mouse monoclonal antibody PR6 was used. The nuclear PR concentration was analyzed with Leitz Orthoplan MPV-3 light microscope. The target tissue was chick oviduct, with epithelial, glandular, mesenchymal, smooth muscle and peritoneal cells analyzed separately. PR concentration varied between different cell types and also from cell to cell within a single cell type. A significant decrease of PR concentration, as noted by decrease in staining, was also observed in all studied cell types, 6 h after a single injection of progesterone. This technique allows for histological identification of biochemical events that should help lead to the understanding of the role of PR changes in a variety of experimental situations.

Animals↗
PubMed1979

Characterization of the avian progesterone receptor through the use of inhibitors.

Several chemical agents have been identified which block interaction of the avian progesterone receptor with isolated nuclei, ATP-Sepharose, DNA-cellulose or phosphocellulose. Four of these inhibitors, rifamycin AF/103, o-phenanthroline, aurintricarboxylic acid and pyridoxal 5-phosphate appear to block directly binding of the activated receptor complex to the above "acceptors." Another inhibitor, sodium molybdate, only blocks receptor interactions when added before receptor activation and therefore appears to interfere with the activation process. When nuclear receptor complexes were formed in vivo and labeled by nuclear exchange with [3H]progesterone in vitro, these complexes could not be disrupted by incubation of the nuclei with inhibitors. Therefore, the receptor complex bound in nuclei appears to be modified or masked in a way which resists the action of these chemical agents. These results indicate the value of inhibitors as chemical probes for the analysis of steroid receptors.

Animals↗