Explore the vast range of titles available.

Live cell imaging has revealed the rapid mobility of steroid hormone receptors within nuclei and their dynamic exchange at transcriptionally active target sites. Although a number of other proteins have been shown to be highly mobile within nuclei, the identity of soluble factors responsible for orchestrating nuclear trafficking remains unknown. We have developed a previously undescribed in situ subnuclear trafficking assay that generates transcriptionally active nuclei, which are depleted of soluble factors required for the nuclear mobility of glucocorticoid (GR) and progesterone receptors (PR). Using this system and a fluorescence recovery after photobleaching technique, we demonstrate that nuclear mobility of GR recovered on incubation with reticulocyte lysate was inhibited by geldanamycin, a drug that blocks the chaperone activity of heat-shock protein 90. Direct proof of molecular chaperone involvement in steroid receptor subnuclear trafficking was provided by the ATP-dependent recovery of nuclear mobility of GR and PR on incubation with various combinations of purified chaperone and/or cochaperone proteins. Additionally, for both receptors, the inclusion of hormone during the recovery period leads to a retardation of nuclear mobility. Thus, our results provide a description of soluble nuclear mobility factors and furthermore demonstrate a previously unrecognized role for molecular chaperones in the regulation of steroid receptor function within the nucleus.

Molecular chaperones are essential proteins that participate in the regulation of steroid receptors in eukaryotes. The steroid aporeceptor complex contains the molecular chaperones Hsp90 and Hsp70, p48, the cyclophilin Cyp-40, and the associated proteins p23 and p60. In vitro folding assays showed that Cyp-40 and p23 functioned as molecular chaperones in a manner similar to that of Hsp90 or Hsp70. Although neither Cyp-40 nor p23 could completely refold an unfolded substrate, both proteins interacted with the substrate to maintain a nonnative folding-competent intermediate. Thus, the steroid aporeceptor complexes have multiple chaperone components that maintain substrates in an intermediate folded state.

In the ligand-binding inactive state, the steroid receptor heterocomplex contains Hsp90, Hsp70, high–molecular weight immunophilins, and other proteins. Hsp90 acts in association with co-chaperones to maintain the native state of the receptor within the cells. It was reported earlier that Hsp90 might not be as important for the androgen receptor (AR) activity as for the glucocorticoid receptor (GR) and the progesterone receptor (PR) activities. We used the Hsp90 inhibitor geldanamycin (GA) to explore the role of Hsp90 in the function of the AR heterocomplex. GA selectively binds to Hsp90 and inhibits its activity, leading to the loss of steroid receptor activity, and frequently, its degradation. In our study, LNCaP prostate cancer cells were treated with GA for 30 minutes or 24 hours, in the presence of mibolerone, a synthetic androgen. GA reduced the androgen-induced AR protein levels to 15 % after 24 hours of treatment. Several androgen up-regulated genes, including immunophilin FKBP51 and prostate specific antigen (PSA), were reduced by GA treatment. In cells treated with GA after transfection with a PSA promoter or an androgen response element–driven reporter gene, AR-mediated transactivation of reporter gene expression was reversibly inhibited by GA. Loss of androgen-binding ability and AR levels was attributed to reduced transcription of AR-regulated gene expression. Degradation rate of 35S-labeled AR was significantly increased by GA in the presence or absence of mibolerone. GA induced the degradation of AR through the proteasomal pathway. AR in cells treated with proteasomal inhibitor lactacystin, was insoluble in Nonidet P-40 (NP40)-based buffer and could not restore the androgen-binding ability. We report here that GA treatment disrupted both hormone-binding activity and receptor protein stability, resulting in a dramatic loss of androgen-induced gene activation. These results show that Hsp90 activity is important for both the chaperone-mediated folding of the AR into a high-affinity ligand-binding conformation and the functional activity of the AR.

p23 is a small but important cochaperone for the Hsp90 chaperoning pathway. It appears to facilitate the adenosine triphosphate–driven cycle of Hsp90 binding to client proteins. It enters at a late stage of the cycle and enhances the maturation of client proteins. Although this role of p23 is fairly well established, recent studies suggest that it may have additional functions in the cell that merit further exploration.

Summary Purpose : To determine the maximum tolerated dose (MTD) and characterize the dose-limiting toxicities (DLT) of 17-AAG, gemcitabine and/or cisplatin. Levels of the proteins Hsp90, Hsp70 and ILK were measured in peripheral blood mononuclear cell (PMBC) lysates to assess the effects of 17-AAG. Experimental design : Phase I dose-escalating trial using a “3 + 3” design performed in patients with advanced solid tumors. Once the MTD of gemcitabine + 17-AAG + cisplatin was determined, dose escalation of 17-AAG with constant doses of gemcitabine and cisplatin was attempted. After significant hematologic toxicity occurred, the protocol was amended to evaluate three cohorts: gemcitabine and 17-AAG; 17-AAG and cisplatin; and gemcitabine, 17-AAG and cisplatin with modified dosing. Results : The 39 patients enrolled were evaluable for toxicity and response. The MTD for cohort A was 154 mg/m 2 of 17-AAG, 750 mg/m 2 of gemcitabine, and 40 mg/m 2 of cisplatin. In cohort A, DLTs were observed at the higher dose level and included neutropenia, hyperbilirubinemia, dehydration, GGT elevation, hyponatremia, nausea, vomiting, and thrombocytopenia. The MTD for cohort C was 154 mg/m 2 of 17-AAG and 750 mg/m 2 of gemcitabine, with one DLT observed (alkaline phosphatase elevation) observed. In cohort C, DLTs of thrombocytopenia, fever and dyspnea were seen at the higher dose level. The remaining cohorts were closed to accrual due to toxicity. Six patients experienced partial responses. Mean Hsp90 levels were decreased and levels of Hsp70 were increased compared to baseline. Conclusions : 17-AAG in combination with gemcitabine and cisplatin demonstrated antitumor activity, but significant hematologic toxicities were encountered. 17-AAG combined with gemcitabine is tolerable and has demonstrated evidence of activity at the MTD. The recommended phase II dose is defined as 154 mg/m 2 of 17-AAG and 750 mg/m 2 of gemcitabine, and is currently being investigated in phase II studies in ovarian and pancreatic cancers. There is no recommended phase II dose for the cisplatin-containing combinations.

Summary Purpose To determine the maximum tolerated dose (MTD) and characterize the dose-limiting toxicities (DLT) of tanespimycin when given in combination with bortezomib. Experimental design Phase I dose - escalating trial using a standard cohort “3+3” design performed in patients with advanced solid tumors. Patients were given tanespimycin and bortezomib twice weekly for 2 weeks in a 3 week cycle (days 1, 4, 8, 11 every 21 days). Results Seventeen patients were enrolled in this study, fifteen were evaluable for toxicity, and nine patients were evaluable for tumor response. The MTD was 250 mg/m 2 of tanespimycin and 1.0 mg/m 2 of bortezomib when used in combination. DLTs of abdominal pain (13 %), complete atrioventricular block (7 %), fatigue (7 %), encephalopathy (7 %), anorexia (7 %), hyponatremia (7 %), hypoxia (7 %), and acidosis (7 %) were observed. There were no objective responses. One patient had stable disease. Conclusions The recommended phase II dose for twice weekly 17-AAG and PS341 are 250 mg/m 2 and 1.0 mg/m 2 , respectively, on days 1, 4, 8 and 11 of a 21 day cycle.

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.

Hsp90 is an abundant and highly conserved chaperone that functions at later stages of protein folding to maintain and regulate the activity of client proteins. Using a recently described in vitro system to fold a functional model kinase Chk1, we performed a side-by-side comparison of the Hsp90-dependent chaperoning of Chk1 to that of the progesterone receptor (PR) and show that these distinct types of clients have different chaperoning requirements. The less stable PR required more total chaperone protein(s) and p23, whereas Chk1 folding was critically dependent on Cdc37. When the 2 clients were reconstituted under identical conditions, each client folding was dose dependent for Hsp90 protein levels and was inhibited by geldanamycin. Using this tractable system, we found that Chk1 kinase folding was more effective if we used a type II Hsp40 cochaperone, whereas PR is chaperoned equally well with a type I or type II Hsp40. Additional dissection of Chk1-chaperone complexes and the resulting kinase activity suggests that kinase folding, like that previously shown for PR, is a dynamic, multistep process. Importantly, the cochaperones Hop and Cdc37 cooperate as the kinase transitions from immature Hsp70- to mature Hsp90-predominant complexes.

To define the functional domains of the progesterone receptor required for gene regulation, complementary DNA (cDNA) clones encoding the chicken progesterone receptor have been isolated from a chicken oviduct $\\lambda $gt11 cDNA expression library. Positive clones expressed antigenic determinants that cross-reacted with six monospecific antibodies derived from two independent sources. A 36-amino acid peptide sequence obtained by microsequencing of purified progesterone receptor was encoded by nucleotide sequences in the longer cDNA clone. Analysis of the amino acid sequence of the progesterone receptor deduced from the cDNA clones revealed a cysteine-rich region that was homologous to a region found in the estrogen and glucocorticoid receptors and to the avian erythroblastosis virus gag-erb-A fusion protein. Northern blot analysis with chicken progesterone receptor cDNA's indicated the existence of at least three messenger RNA species. These messages were found only in oviduct and could be induced by estrogens.

Heat shock protein (hsp)90 functions in a complex chaperoning pathway where its activity is modulated by ATP and by interaction with several co-chaperones. One co-chaperone, p23, binds selectively to the ATP-bound state of hsp90. However, the isolated ATP-binding domain of hsp90 does not bind p23. In an effort to identify the p23-binding domain, we have constructed a series of hsp90 deletion mutants fused with glutathione-S-transferase (GST). Full-length GST-hsp90 is able to bind p23, and also, to chaperone assembly of progesterone receptor complexes. Truncations from the C terminus of GST-hsp90 reveal a C-terminal boundary for the p23-binding domain at approximately residue 490. This fragment contains, in order, the ATP-binding domain, a highly charged region, and 203 residues beyond the charged region. p23 binding is unaffected by deletion of the charged region, indicating that two noncontiguous regions of hsp90 are involved in p23 binding. These regions are only effective when hsp90 is in a dimeric state as shown by loss of p23 binding upon removal of GST or as shown by use of FK506-binding protein 12-hsp90 constructs that form dimers and bind p23 only in the presence of a bivalent drug. Thus, p23 binding requires an hsp90 dimer with close proximity between N-terminal regions of hsp90 and a conformation specified by ATP.