William P. Sullivan

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.

Differential interactions of p23 and the TPR-containing proteins Hop, Cyp40, FKBP52 and FKBP51 with Hsp90 mutants

Hsp90 is required for the normal function of steroid receptors, but its binding to steroid receptors is mediated by Hsc70 and several hsp-associated accessory proteins. An assortment of Hsp90 mutants were tested for their abilities to interact with each of the following accessories: Hop, Cyp40, FKBP52, FKBP51, and p23. Of the 11 Hsp90 mutants tested, all were defective to some extent in associating with progestin (PR) complexes. In every case, however, reduced PR binding correlated with a defect in binding of one or more accessories. Co-precipitation of mutant Hsp90 forms with individual accessories was used to map Hsp90 sequences required for accessory protein interactions. Mutation of Hsp90s highly conserved C-terminal EEVD to AAVD resulted in diminished interactions with several accessory proteins, most particularly with Hop. Deletion of amino acids 661-677 resulted in loss of Hsp90 dimerization and also caused diminished interactions with all accessory proteins. Binding of p23 mapped most strongly to the N-terminal ATP-binding domain of Hsp90 while binding of TPR proteins mapped to the C-terminal half of Hsp90. These results and others further suggest that the N- and C-terminal regions of Hsp90 maintain important conformational links through intramolecular interactions and/or intermolecular influences in homodimers.

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.

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 β-strands in a compact antiparallel β-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.

Thiopurine S-methyltransferase pharmacogenetics: chaperone protein association and allozyme degradation.

Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs such as 6-mercaptopurine. A common genetic polymorphism for TPMT is associated with large individual variations in thiopurine drug toxicity and therapeutic efficacy. TPMT*3A, the most common variant allele in Caucasians, has two alterations in amino acid sequence, resulting in striking decreases in TPMT protein levels. This phenomenon results, in part, from rapid degradation through a ubiquitin-proteasome-mediated process. We set out to test the hypothesis that chaperone proteins might be involved in targeting TPMT for degradation. As a first step, hsp90, hsp70 and the cochaperone hop were immunoprecipitated from a rabbit reticulocyte lysate (RRL) that included radioactively labelled *3A and wild-type TPMT. TPMT*3A was much more highly associated with all three chaperones than was the wild-type enzyme. The RRL was also used to confirm the accelerated degradation of *3A compared to wild-type TPMT. Treatment of RRL with the hsp90 inhibitor geldanamycin resulted in enhanced association of hsp90 with wild-type TPMT, an observation that correlated with accelerated ubiquitin-dependent degradation of wild-type TPMT. Geldanamycin treatment of COS-1 cells transfected with FLAG-tagged wild-type also resulted in a time and geldanamycin concentration-dependent decrease in TPMT activity and protein, which was compatible with results obtained in the RRL. These observations indicate that TPMT is a client protein for hsp90 and suggest that chaperone proteins, especially hsp90, are involved in targeting both TPMT*3A and, in the presence of geldanamycin, the wild-type allozyme for degradation. Therefore, chaperone proteins play an important mechanistic role in this clinically significant example of pharmacogenetic variation in drug metabolism.

Identification of the 90 kDa substrate of rat liver type II casein kinase with the heat shock protein which binds steroid receptors.

It was recently reported that the type II casein kinase of rat liver cytosol co-purified with a major 90 kDa substrate when subjected to gel filtration at low ionic strength. The identity of the 90 kDa substrate was unknown. We have verified this report and have shown that the 90 kDa substrate is recognized by a monoclonal antibody prepared against the 90 kDa heat shock protein. This ubiquitous phosphoprotein is known to increase in abundance in cells subjected to heat stress and has been shown to complex steroid receptors and certain retrovirus tyrosine kinases.

Celastrol Inhibits Hsp90 Chaperoning of Steroid Receptors by Inducing Fibrillization of the Co-chaperone p23

Hsp90 is an ATP-dependent molecular chaperone. The best characterized inhibitors of Hsp90 target its ATP binding pocket, causing nonselective degradation of Hsp90 client proteins. Here, we show that the small molecule celastrol inhibits the Hsp90 chaperoning machinery by inactivating the co-chaperone p23, resulting in a more selective destabilization of steroid receptors compared with kinase clients. Our in vitro and in vivo results demonstrate that celastrol disrupts p23 function by altering its three-dimensional structure, leading to rapid formation of amyloid-like fibrils. This study reveals a unique inhibition mechanism of p23 by a small molecule that could be exploited in the dissection of protein fibrillization processes as well as in the therapeutics of steroid receptor-dependent diseases.

HORMONE-DEPENDENT PHOSPHORYLATION OF THE AVIAN PROGESTERONE RECEPTOR

Progesterone receptors are phosphoproteins, in which phosphorylation has been proposed as a control mechanism for some stages of hormone action. Progesterone administration was shown to increase phosphorylation of the receptor from both cytosol and nuclear extracts of whole cells. We have analyzed the receptor phosphopeptides generated by chemical and proteolytic cleavage to assess the number of phosphorylation sites and their approximate location in the receptor. Progesterone receptor was labeled in situ in the presence or absence of hormone in medium containing [32P] orthophosphate, isolated by immunoprecipitation, and then digested with several proteases. The resulting 32P-labeled peptides were resolved by either two-dimensional electrophoresis:chromatography or by reverse-phase high performance liquid chromatography. Multiple phosphopeptides (3-6) were detected after cleavage with trypsin, chymotrypsin, or V8 protease. Major increases in phosphorylation occurred at existing sites since after hormone treatment no new phosphopeptides were found. Individual phosphopeptides showed variable increases in phosphorylation of 1.5-5-fold. The A and B receptor forms showed identical phosphorylation patterns, indicating similar processing in vivo. The phosphopeptide pattern for receptor in nuclear extracts resembled that of cytosol receptor. Chemical cleavage was used to assess the distribution of phosphorylation sites. Cyanogen bromide produced a large 40-kDa polypeptide which contained all of the phosphorylation sites and comprised the residues 129-449. Hydroxylamine was used to cleave a unique bond, Asn-372-Gly-373, in the 40-kDa polypeptide. All of the phosphorylation sites were located on the amino-terminal side of the cleavage. Thus, all of the phosphorylation sites were localized to a specific region (Met-129 to Asn-372) of the progesterone receptor that does not include either the DNA or steroid binding domains.

Ataxia telangiectasia and rad3-related kinase contributes to cell cycle arrest and survival after cisplatin but not oxaliplatin

The DNA cross-linking agents cisplatin and oxaliplatin are widely used in the treatment of human cancer. Lesions produced by these agents are widely known to activate the G1 and G2 cell cycle checkpoints. Less is known about the role of the intra–S-phase checkpoint in the response to these agents. In the present study, two different cell lines expressing a dominant-negative kinase dead (kd) version of the ataxia telangiectasia and rad3-related (ATR) kinase in an inducible fashion were examined for their responses to these two platinating agents and a variety of other DNA cross-linking drugs. The expression of the kdATR allele markedly sensitized the cells to cisplatin, but not to oxaliplatin, as assessed by inhibition of colony formation, induction of apoptosis, and cell cycle analysis. Similar differences in survival were noted for melphalan (ATR dependent) and 4-hydroperoxycyclophosphamide (ATR independent). Further experiments showed that ATR function is not necessary for removal of Pt-DNA adducts. The predominant difference between the responses to the two platinum drugs was the presence of a drug-specific ATR-dependent S-phase arrest after cisplatin but not oxaliplatin. These results indicate that involvement of ATR in the response to DNA cross-linking agents is lesion specific. This observation might need to be taken into account in the development and use of ATR or Chk1 inhibitors. [Mol Cancer Ther 2009;8(4):855–63]

Characterization of plant p23-like proteins for their co-chaperone activities

The small acidic protein p23 is best described as a co-chaperone of Hsp90, an essential molecular chaperone in eukaryotes. p23 binds to the ATP-bound form of Hsp90 and stabilizes the Hsp90–client protein complex by slowing down ATP turnover. The stabilizing activity of p23 was first characterized in studies of steroid receptor–Hsp90 complexes. Earlier studies of the Hsp90 chaperone complex in plants suggested that a p23-like stabilizing activity was absent in plant cell lysates. Here, we show that p23-like proteins are present in plants and are capable of binding Hsp90, but unlike human p23 and yeast ortholog Sba1, the plant p23-like proteins do not stabilize the steroid receptor–Hsp90 complexes formed in wheat germ lysate. Furthermore, these proteins do not inhibit the ATPase activity of plant Hsp90. While transcripts of Arabidopsis thaliana p23-1 and Atp23-2 were detected under normal growing conditions, those of the closely related Brassica napus p23-1 were present only after moderate heat stress. These observations suggest that p23-like proteins in plants are conserved in their binding to Hsp90 but have evolved mechanisms of action different from their yeast and animal counterparts.