151 results on '"David O. Toft"'
Search Results
2. Data from P-Glycoprotein–Mediated Resistance to Hsp90-Directed Therapy Is Eclipsed by the Heat Shock Response
- Author
-
Charles Erlichman, David O. Toft, Robert B. Jenkins, Karla V. Ballman, Bruce W. Morlan, Bridget Stensgard, Cynthia J. TenEyck, and Andrea K. McCollum
- Abstract
Despite studies that show the antitumor activity of Hsp90 inhibitors, such as geldanamycin (GA) and its derivative 17-allylamino-demethoxygeldanamycin (17-AAG), recent reports indicate that these inhibitors lack significant single-agent clinical activity. Resistance to Hsp90 inhibitors has been previously linked to expression of P-glycoprotein (P-gp) and the multidrug resistant (MDR) phenotype. However, the stress response induced by GA treatment can also cause resistance to Hsp90-targeted therapy. Therefore, we chose to further investigate the relative importance of P-gp and the stress response in 17-AAG resistance. Colony-forming assays revealed that high expression of P-gp could increase the 17-AAG IC50 6-fold in cells transfected with P-gp compared with parent cells. A549 cells selected for resistance to GA overexpressed P-gp, but verapamil did not reverse the resistance. These cells also overexpressed Hsp27, and Hsp70 was induced with 17-AAG treatment. When the GA and 17-AAG resistant cells were transfected with Hsp27 and/or Hsp70 small interfering RNA (siRNA), the 17-AAG IC50 decreased 10-fold compared with control transfected cells. Transfection with siRNA directed against Hsp27, Hsp70, or Hsp27 and Hsp70 also increased sensitivity to EC78, a purine scaffold-based Hsp90 inhibitor that is not a P-gp substrate. We conclude that P-gp may contribute, in part, to resistance to 17-AAG, but induction of stress response proteins, such as Hsp27 and Hsp70, by Hsp90-targeted therapy plays a larger role. Taken together, our results indicate that targeting of Hsp27 and Hsp70 should be exploited to increase the clinical efficacy of Hsp90-directed therapy. [Cancer Res 2008;68(18):7419–27]
- Published
- 2023
3. Supplementary Figure 2 from P-Glycoprotein–Mediated Resistance to Hsp90-Directed Therapy Is Eclipsed by the Heat Shock Response
- Author
-
Charles Erlichman, David O. Toft, Robert B. Jenkins, Karla V. Ballman, Bruce W. Morlan, Bridget Stensgard, Cynthia J. TenEyck, and Andrea K. McCollum
- Abstract
Supplementary Figure 2 from P-Glycoprotein–Mediated Resistance to Hsp90-Directed Therapy Is Eclipsed by the Heat Shock Response
- Published
- 2023
4. Supplementary Figure 1 from P-Glycoprotein–Mediated Resistance to Hsp90-Directed Therapy Is Eclipsed by the Heat Shock Response
- Author
-
Charles Erlichman, David O. Toft, Robert B. Jenkins, Karla V. Ballman, Bruce W. Morlan, Bridget Stensgard, Cynthia J. TenEyck, and Andrea K. McCollum
- Abstract
Supplementary Figure 1 from P-Glycoprotein–Mediated Resistance to Hsp90-Directed Therapy Is Eclipsed by the Heat Shock Response
- Published
- 2023
5. Phosphorylation of the Avian Progesterone Receptor
- Author
-
David O. Toft and William P. Sullivan
- Subjects
medicine.medical_specialty ,Endocrinology ,Chemistry ,Internal medicine ,Progesterone receptor ,medicine ,Phosphorylation - Published
- 2018
6. Phase I study of tanespimycin in combination with bortezomib in patients with advanced solid malignancies
- Author
-
Michael E. Menefee, Daniel Satele, R. Qin, David O. Toft, Matthew M. Ames, Erin L. Schenk, Donald W. Northfelt, Andrea E. Wahner Hendrickson, and Charles Erlichman
- Subjects
Male ,medicine.medical_specialty ,Abdominal pain ,Maximum Tolerated Dose ,Lactams, Macrocyclic ,Encephalopathy ,Antineoplastic Agents ,Anorexia ,Tanespimycin ,Gastroenterology ,Article ,Bortezomib ,chemistry.chemical_compound ,Neoplasms ,Internal medicine ,Antineoplastic Combined Chemotherapy Protocols ,Benzoquinones ,medicine ,Humans ,Pharmacology (medical) ,Protein Kinase Inhibitors ,Aged ,Aged, 80 and over ,Pharmacology ,Dose-Response Relationship, Drug ,business.industry ,Middle Aged ,medicine.disease ,Boronic Acids ,Surgery ,Treatment Outcome ,Oncology ,chemistry ,Pyrazines ,Toxicity ,Cohort ,Female ,medicine.symptom ,Hyponatremia ,business ,medicine.drug - Abstract
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/m2 of tanespimycin and 1.0 mg/m2 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/m2 and 1.0 mg/m2, respectively, on days 1, 4, 8 and 11 of a 21 day cycle.
- Published
- 2013
7. P-Glycoprotein–Mediated Resistance to Hsp90-Directed Therapy Is Eclipsed by the Heat Shock Response
- Author
-
Charles Erlichman, Karla V. Ballman, Cynthia J. TenEyck, Bruce W. Morlan, Bridget Stensgard, Andrea K. McCollum, Robert B. Jenkins, and David O. Toft
- Subjects
Cancer Research ,animal structures ,Lactams, Macrocyclic ,KB Cells ,Article ,Hsp90 inhibitor ,chemistry.chemical_compound ,Cell Line, Tumor ,Heat shock protein ,Benzoquinones ,polycyclic compounds ,Humans ,ATP Binding Cassette Transporter, Subfamily B, Member 1 ,HSP90 Heat-Shock Proteins ,Heat-Shock Proteins ,P-glycoprotein ,A549 cell ,biology ,Transfection ,Geldanamycin ,Hsp90 ,Up-Regulation ,Multiple drug resistance ,Oncology ,Biochemistry ,chemistry ,Drug Resistance, Neoplasm ,biology.protein ,Cancer research ,Heat-Shock Response - Abstract
Despite studies that show the antitumor activity of Hsp90 inhibitors, such as geldanamycin (GA) and its derivative 17-allylamino-demethoxygeldanamycin (17-AAG), recent reports indicate that these inhibitors lack significant single-agent clinical activity. Resistance to Hsp90 inhibitors has been previously linked to expression of P-glycoprotein (P-gp) and the multidrug resistant (MDR) phenotype. However, the stress response induced by GA treatment can also cause resistance to Hsp90-targeted therapy. Therefore, we chose to further investigate the relative importance of P-gp and the stress response in 17-AAG resistance. Colony-forming assays revealed that high expression of P-gp could increase the 17-AAG IC50 6-fold in cells transfected with P-gp compared with parent cells. A549 cells selected for resistance to GA overexpressed P-gp, but verapamil did not reverse the resistance. These cells also overexpressed Hsp27, and Hsp70 was induced with 17-AAG treatment. When the GA and 17-AAG resistant cells were transfected with Hsp27 and/or Hsp70 small interfering RNA (siRNA), the 17-AAG IC50 decreased 10-fold compared with control transfected cells. Transfection with siRNA directed against Hsp27, Hsp70, or Hsp27 and Hsp70 also increased sensitivity to EC78, a purine scaffold-based Hsp90 inhibitor that is not a P-gp substrate. We conclude that P-gp may contribute, in part, to resistance to 17-AAG, but induction of stress response proteins, such as Hsp27 and Hsp70, by Hsp90-targeted therapy plays a larger role. Taken together, our results indicate that targeting of Hsp27 and Hsp70 should be exploited to increase the clinical efficacy of Hsp90-directed therapy. [Cancer Res 2008;68(18):7419–27]
- Published
- 2008
8. An Acetylation Site in the Middle Domain of Hsp90 Regulates Chaperone Function
- Author
-
Dongxia Wang, Len Neckers, Bradley T. Scroggins, Shinji Tsutsumi, Robert J. Cotter, Sara J. Felts, David O. Toft, Kenneth Robzyk, Kristin Beebe, Larry M. Karnitz, Neal Rosen, and Monica G. Marcu
- Subjects
Lysine ,Molecular Sequence Data ,Plasma protein binding ,Saccharomyces cerevisiae ,SAP30 ,Article ,Mice ,Chlorocebus aethiops ,polycyclic compounds ,Animals ,Humans ,Amino Acid Sequence ,HSP90 Heat-Shock Proteins ,Molecular Biology ,biology ,Acetylation ,Cell Biology ,HDAC6 ,Hsp90 ,Protein Structure, Tertiary ,Biochemistry ,Chaperone (protein) ,COS Cells ,Checkpoint Kinase 1 ,Mutation ,biology.protein ,NIH 3T3 Cells ,Mutant Proteins ,Histone deacetylase ,Protein Kinases ,Protein Binding - Abstract
Heat shock protein 90 (Hsp90) chaperones a key subset of signaling proteins and is necessary for malignant transformation. Hsp90 is subject to an array of post-translational modifications which affect its function, including acetylation. Histone deacetylase (HDAC) inhibitors and knock-down of HDAC6 induce Hsp90 acetylation and inhibit its activity. However, direct determination of the functional consequences of Hsp90 acetylation has awaited mapping of specific sites. We now demonstrate that Hsp90 K294 is acetylated. Mutational analysis of K294 shows that its acetylation status is a strong determinant of client protein and cochaperone binding. In yeast, Hsp90 mutants that cannot be acetylated at K294 have reduced viability and chaperone function compared to wild type or to mutants that mimic constitutive acetylation. These data suggest that acetylation/deacetylation of K294 plays an important role in regulating the Hsp90 chaperone cycle.
- Published
- 2007
- Full Text
- View/download PDF
9. Up-regulation of Heat Shock Protein 27 Induces Resistance to 17-Allylamino-Demethoxygeldanamycin through a Glutathione-Mediated Mechanism
- Author
-
Charles Erlichman, David O. Toft, Brian M. Sauer, Cynthia J. TenEyck, and Andrea K. McCollum
- Subjects
endocrine system ,Cancer Research ,animal structures ,Lactams, Macrocyclic ,Biology ,chemistry.chemical_compound ,Hsp27 ,Heat shock protein ,Cellular stress response ,Benzoquinones ,polycyclic compounds ,Humans ,Buthionine sulfoximine ,RNA, Small Interfering ,Heat-Shock Proteins ,Glutathione ,Geldanamycin ,Molecular biology ,Hsp90 ,female genital diseases and pregnancy complications ,Up-Regulation ,Cell biology ,Oncology ,chemistry ,Drug Resistance, Neoplasm ,embryonic structures ,Cancer cell ,biology.protein ,Drug Screening Assays, Antitumor ,HeLa Cells - Abstract
17-Allylamino-demethoxygeldanamycin (17-AAG), currently in phase I and II clinical trials as an anticancer agent, binds to the ATP pocket of heat shock protein (Hsp90). This binding induces a cellular stress response that up-regulates many proteins including Hsp27, a member of the small heat shock protein family that has cytoprotective roles, including chaperoning of cellular proteins, regulation of apoptotic signaling, and modulation of oxidative stress. Therefore, we hypothesized that Hsp27 expression may affect cancer cell sensitivity to 17-AAG. In colony-forming assays, overexpression of Hsp27 increased cell resistance to 17-AAG whereas down-regulation of Hsp27 by siRNA increased sensitivity. Because Hsp27 is known to modulate levels of glutathione (GSH), we examined cellular levels of GSH and found that it was decreased in cells transfected with Hsp27 siRNA when compared with control siRNA. Treatment with buthionine sulfoximine, an inhibitor of GSH synthesis, also sensitized cells to 17-AAG. Conversely, treatment of Hsp27 siRNA–transfected cells with N-acetylcysteine, an antioxidant and GSH precursor, reversed their sensitivity to 17-AAG. A cell line selected for stable resistance to geldanamycin relative to parent cells showed increased Hsp27 expression. When these geldanamycin- and 17-AAG-resistant cells were transfected with Hsp27 siRNA, 17-AAG resistance was dramatically diminished. Our results suggest that Hsp27 up-regulation has a significant role in 17-AAG resistance, which may be mediated in part through GSH regulation. Clinical modulation of GSH may therefore enhance the efficacy of Hsp90-directed therapy. (Cancer Res 2006; 66(22): 10967-75)
- Published
- 2006
10. A Phase I Trial of Twice-Weekly 17-Allylamino-Demethoxy-Geldanamycin in Patients with Advanced Cancer
- Author
-
Alex A. Adjei, Sumithra J. Mandrekar, Grzegorz S. Nowakowski, Matthew M. Ames, Andrea K. McCollum, Stephanie L. Safgren, Charles Erlichman, David O. Toft, and Joel M. Reid
- Subjects
Adult ,Cancer Research ,Lactams, Macrocyclic ,Antineoplastic Agents ,Tanespimycin ,Pharmacology ,Peripheral blood mononuclear cell ,chemistry.chemical_compound ,Life Expectancy ,Pharmacokinetics ,Neoplasms ,Heat shock protein ,Benzoquinones ,medicine ,Humans ,HSP70 Heat-Shock Proteins ,Infusions, Intravenous ,Neoplasm Staging ,business.industry ,Patient Selection ,Cancer ,Geldanamycin ,medicine.disease ,Hsp70 ,Oncology ,chemistry ,Immunology ,Toxicity ,business - Abstract
Purpose: To determine the maximum tolerated dose (MTD), dose-limiting toxicity, and pharmacokinetics of 17-allylamino-demethoxy-geldanamycin (17-AAG) administered on days 1, 4, 8, and 11 every 21 days and to examine the effect of 17-AAG on the levels of chaperone and client proteins. Experimental Design: A phase I dose escalating trial in patients with advanced solid tumors was done. Toxicity and tumor responses were evaluated by standard criteria. Pharmacokinetics were done and level of target proteins was measured at various points during cycle one. Results: Thirteen patients were enrolled in the study. MTD was defined as 220 mg/m2. Dose-limiting toxicities were as follows: dehydration, diarrhea, hyperglycemia, and liver toxicity. At the MTD, the mean clearance of 17-AAG was 18.7 L/h/m2. There was a significant decrease in integrin-linked kinase at 6 hours after infusion on day 1 but not at 25 hours in peripheral blood mononuclear cells. Treatment with 17-AAG on day 1 significantly increased pretreatment levels of heat shock protein (HSP) 70 on day 4, which is consistent with the induction of a stress response. In vitro induction of a stress response and up-regulation of HSP70 resulted in an increased resistance to HSP90-targeted therapy in A549 cells. Conclusions: The MTD of 17-AAG on a twice-weekly schedule was 220 mg/m2. Treatment at this dose level resulted in significant changes of target proteins and also resulted in a prolonged increase in HSP70. This raises the possibility that HSP70 induction as part of the stress response may contribute to resistance to 17-AAG.
- Published
- 2006
11. Defining the Requirements for Hsp40 and Hsp70 in the Hsp90 Chaperone Pathway
- Author
-
David O. Toft and Nela S. Cintron
- Subjects
Plasma protein binding ,Biochemistry ,Mice ,ATP hydrolysis ,Animals ,Humans ,Point Mutation ,Protein Isoforms ,HSP70 Heat-Shock Proteins ,HSP90 Heat-Shock Proteins ,Binding site ,Receptor ,Molecular Biology ,Progesterone ,Binding Sites ,biology ,Cell Biology ,HSP40 Heat-Shock Proteins ,Hsp90 ,Chaperone (protein) ,biology.protein ,Chaperone complex ,Signal transduction ,Receptors, Progesterone ,HeLa Cells ,Molecular Chaperones ,Protein Binding ,Signal Transduction - Abstract
The Hsp90 chaperoning pathway and its model client substrate, the progesterone receptor (PR), have been used extensively to study chaperone complex formation and maturation of a client substrate in a near native state. This chaperoning pathway can be reconstituted in vitro with the addition of five proteins plus ATP: Hsp40, Hsp70, Hop, Hsp90, and p23. The addition of these proteins is necessary to reconstitute hormone-binding capacity to the immuno-isolated PR. It was recently shown that the first step for the recognition of PR by this system is binding by Hsp40. We compared type I and type II Hsp40 proteins and created point mutations in Hsp40 and Hsp70 to understand the requirements for this first step. The type I proteins, Ydj1 and DjA1 (HDJ2), and a type II, DjB1 (HDJ1), act similarly in promoting hormone binding and Hsp70 association to PR, while having different binding characteristics to PR. Ydj1 and DjA1 bind tightly to PR whereas the binding of DjB1 apparently has rapid on and off rates and its binding cannot be observed by antibody pull-down methods using either purified proteins or cell lysates. Mutation studies indicate that client binding, interactions between Hsp40 and Hsp70, plus ATP hydrolysis by Hsp70 are all required to promote conformational maturation of PR via the Hsp90 pathway.
- Published
- 2006
12. HDAC6 Regulates Hsp90 Acetylation and Chaperone-Dependent Activation of Glucocorticoid Receptor
- Author
-
Jeffrey J. Kovacs, Patrick J. Murphy, Xuan Zhao, Stephanie Gaillard, June-Tai Wu, Christopher V. Nicchitta, David O. Toft, William B. Pratt, Minoru Yoshida, and Tso-Pang Yao
- Subjects
Transcription, Genetic ,Biology ,Tubulin deacetylase activity ,Histone Deacetylase 6 ,Dexamethasone ,Histone Deacetylases ,Cell Line ,Mice ,Receptors, Glucocorticoid ,Glucocorticoid receptor ,Heat shock protein ,polycyclic compounds ,Animals ,Humans ,HSP90 Heat-Shock Proteins ,Glucocorticoids ,Molecular Biology ,Acetylation ,Cell Biology ,Hsp90 ,Cell biology ,Biochemistry ,Chaperone (protein) ,biology.protein ,Chaperone complex ,Signal transduction ,Signal Transduction - Abstract
The molecular chaperone heat shock protein 90 (Hsp90) and its accessory cochaperones function by facilitating the structural maturation and complex assembly of client proteins, including steroid hormone receptors and selected kinases. By promoting the activity and stability of these signaling proteins, Hsp90 has emerged as a critical modulator in cell signaling. Here, we present evidence that Hsp90 chaperone activity is regulated by reversible acetylation and controlled by the deacetylase HDAC6. We show that HDAC6 functions as an Hsp90 deacetylase. Inactivation of HDAC6 leads to Hsp90 hyperacetylation, its dissociation from an essential cochaperone, p23, and a loss of chaperone activity. In HDAC6-deficient cells, Hsp90-dependent maturation of the glucocorticoid receptor (GR) is compromised, resulting in GR defective in ligand binding, nuclear translocation, and transcriptional activation. Our results identify Hsp90 as a target of HDAC6 and suggest reversible acetylation as a unique mechanism that regulates Hsp90 chaperone complex activity.
- Published
- 2005
13. Hsp90: from structure to phenotype
- Author
-
Christine Queitsch, Sophie E. Jackson, and David O. Toft
- Subjects
EGF-like domain ,Biology ,Phenotype ,Hsp90 ,Cell biology ,Protein structure ,Biochemistry ,Structural Biology ,Cyclic nucleotide-binding domain ,EVH1 domain ,biology.protein ,Signal transduction ,Molecular Biology ,Binding domain - Abstract
A recent international conference focused on Hsp90, a molecular chaperone that plays a critical role in a diverse array of cellular processes including the assembly and maturation of some important 'client' proteins, many of which are involved in signal transduction.
- Published
- 2004
14. Hsp90 Inhibition Depletes Chk1 and Sensitizes Tumor Cells to Replication Stress
- Author
-
Alex K. Eapen, Sonnet J.H. Arlander, Robert J. McDonald, Benjamin T. Vroman, David O. Toft, and Larry M. Karnitz
- Subjects
DNA Replication ,Antimetabolites, Antineoplastic ,CDC25A ,Time Factors ,animal structures ,Cell Survival ,DNA damage ,Lactams, Macrocyclic ,Immunoblotting ,Protein Serine-Threonine Kinases ,Deoxycytidine ,environment and public health ,Biochemistry ,Cell Line ,S Phase ,Hsp90 inhibitor ,Cell Line, Tumor ,Benzoquinones ,polycyclic compounds ,Humans ,cdc25 Phosphatases ,HSP90 Heat-Shock Proteins ,CHEK1 ,Molecular Biology ,biology ,DNA replication ,Cell Biology ,Precipitin Tests ,Gemcitabine ,Hsp90 ,Cell biology ,enzymes and coenzymes (carbohydrates) ,Rifabutin ,Cell culture ,Checkpoint Kinase 1 ,biology.protein ,biological phenomena, cell phenomena, and immunity ,Signal transduction ,Protein Kinases ,DNA Damage ,HeLa Cells ,Protein Binding ,Signal Transduction - Abstract
DNA damage and replication stress activate the Chk1 signaling pathway, which blocks S phase progression, stabilizes stalled replication forks, and participates in G2 arrest. In this study, we show that Chk1 interacts with Hsp90, a molecular chaperone that participates in the folding, assembly, maturation, and stabilization of specific proteins known as clients. Consistent with Chk1 being an Hsp90 client, we also found that Chk1 but not Chk2 is destabilized in cells treated with the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). 17-AAG-mediated Chk1 loss blocked the ability of Chk1 to target Cdc25A for proteolytic destruction, demonstrating that the Chk1 signaling pathway was disrupted in the 17-AAG-treated cells. Finally, 17-AAG-mediated disruption of Chk1 activation dramatically sensitized various tumor cells to gemcitabine, an S phase-active chemotherapeutic agent. Collectively, our studies identify Chk1 as a novel Hsp90 client and suggest that pharmacologic inhibition of Hsp90 may sensitize tumor cells to chemotherapeutic agents by disrupting Chk1 function during replication stress.
- Published
- 2003
15. Novel Activation Step Required for Transcriptional Competence of Progesterone Receptor on Chromatin Templates
- Author
-
Steven K. Nordeen, Varykina G. Thackray, and David O. Toft
- Subjects
Embryo, Nonmammalian ,Reticulocytes ,Transcription, Genetic ,Biology ,law.invention ,chemistry.chemical_compound ,Endocrinology ,Transcription (biology) ,law ,Progesterone receptor ,Animals ,Humans ,Receptor ,Molecular Biology ,Progesterone ,Base Sequence ,DNA ,Templates, Genetic ,General Medicine ,Geldanamycin ,Molecular biology ,Chromatin ,Recombinant Proteins ,Cell biology ,chemistry ,Recombinant DNA ,Drosophila ,Protein folding ,Rabbits ,Receptors, Progesterone - Abstract
To elucidate the earliest molecular steps in the activation of transcription by the progesterone receptor (PR), we investigated its activity in a cell-free transcription system utilizing chromatin templates. PR prepared as a ligand-free, recombinant protein failed to induce transcription on chromatin templates. However, transcriptional competence could be restored by coincubation with rabbit reticulocyte lysate (RRL). The interaction of PR with chaperones results in a receptor conformation competent to bind ligand and RRL contains abundant chaperone-mediated protein folding activity. Blocking this activity with the specific inhibitor geldanamycin inhibited receptor-dependent transcriptional activity. However, recombinant chaperones could not replace RRL in the restoration of transcriptional activity on chromatin templates, suggesting the presence of an additional activity in the lysate. Under chromatin assembly conditions, PR could bind naked DNA and RRL did not increase that binding. In contrast, PR bound to a chromatin template only poorly. Interestingly, RRL stimulated sequence-specific binding by PR to target sites in chromatin and the concomitant recruitment of the steroid receptor coactivator 1 to the promoter. Thus, our results indicate that a novel protein-mediated activity in RRL is involved in an additional, heretofore unrecognized, activation step required for PR to become transcriptionally competent on chromatin templates.
- Published
- 2003
16. Thiopurine S-methyltransferase pharmacogenetics
- Author
-
David O. Toft, Richard M. Weinshilboum, Liewei Wang, and William J. Sullivan
- Subjects
Methyltransferase ,biology ,Thiopurine methyltransferase ,Geldanamycin ,Hsp90 ,Hsp90 inhibitor ,Thiopurine S-Methyltransferase ,chemistry.chemical_compound ,Biochemistry ,chemistry ,Heat shock protein ,Genetics ,biology.protein ,General Pharmacology, Toxicology and Pharmaceutics ,Pharmacogenetics - Abstract
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.
- Published
- 2003
17. Regulation of Signaling Protein Function and Trafficking by the hsp90/hsp70-Based Chaperone Machinery
- Author
-
William B. Pratt and David O. Toft
- Subjects
0301 basic medicine ,Conformational change ,Macromolecular Substances ,Receptors, Cell Surface ,Plasma protein binding ,Biology ,Models, Biological ,General Biochemistry, Genetics and Molecular Biology ,Structure-Activity Relationship ,03 medical and health sciences ,0302 clinical medicine ,polycyclic compounds ,Animals ,Drosophila Proteins ,Humans ,HSP70 Heat-Shock Proteins ,HSP90 Heat-Shock Proteins ,Immunophilins ,Janus Kinases ,Nucleotides ,Protein-Tyrosine Kinases ,Hsp90 ,Transport protein ,Cell biology ,Protein Transport ,030104 developmental biology ,CDC37 ,Multiprotein Complexes ,030220 oncology & carcinogenesis ,Chaperone (protein) ,Hsp33 ,biology.protein ,Signal transduction ,Molecular Chaperones ,Protein Binding ,Signal Transduction ,Transcription Factors - Abstract
Nearly 100 proteins are known to be regulated by hsp90. Most of these substrates or “client proteins” are involved in signal transduction, and they are brought into complex with hsp90 by a multlprotein hsp90/hsp70-based chaperone machinery. In addition to binding substrate proteins at the chaperone site(s), hsp90 binds cofactors at other sites that are part of the heterocomplex assembly machinery as well as immunophllins that connect assembled substrate·hsp90 complexes to protein-trafficking systems. In the 5 years since we last reviewed this subject, much has been learned about hsp90 structure, nucleotide-binding, and cochaperone interactions; the most important concept is that ATP hydrolysis by an intrinsic ATPase activity results in a conformational change in hsp90 that is required to induce conformational change in a substrate protein. The conformational change induced in steroid receptors is an opening of the steroid-binding cleft so that it can be accessed by steroid. We have now developed a minimal system of five purified proteins—hsp90, hsp70, Hop, hsp40, and p23—that assembles stable receptor·hsp90 heterocomplexes. An hsp90·Hop·hsp70·hsp40 complex opens the cleft in an ATP-dependent process to produce a receptor·hsp90 heterocomplex with hsp90 in its ATP-bound conformation, and p23 then interacts with the hsp90 to stabilize the complex. Stepwise assembly experiments have shown that hsp70 and hsp40 first interact with the receptor in an ATP-dependent reaction to produce a receptor·hsp70·hsp40 complex that is “primed” to be activated to the steroid-binding state in a second ATP-dependent step with hsp90, Hop, and p23. Successful use of the five-protein system with other substrates Indicates that it can assemble signal protein-hsp90 heterocomplexes whether the substrate is a receptor, a protein kinase, or a transcription factor. This purified system should facilitate understanding of how eukaryotlc hsp70 and hsp90 work together as essential components of a process that alters the conformations of substrate proteins to states that respond in signal transduction.
- Published
- 2003
18. The Assembly and Intermolecular Properties of the hsp70-Hop-hsp90 Molecular Chaperone Complex
- Author
-
M. Patricia Hernández, David O. Toft, and William P. Sullivan
- Subjects
Macromolecular Substances ,Dimer ,Biology ,Biochemistry ,Hop (networking) ,chemistry.chemical_compound ,Adenosine Triphosphate ,Animals ,Humans ,HSP70 Heat-Shock Proteins ,HSP90 Heat-Shock Proteins ,Binding site ,Molecular Biology ,Heat-Shock Proteins ,Cell-Free System ,Signal transducing adaptor protein ,Cell Biology ,HSP40 Heat-Shock Proteins ,Hsp90 ,Hsp70 ,Folding (chemistry) ,chemistry ,biology.protein ,Biophysics ,Signal transduction ,Molecular Chaperones ,Protein Binding - Abstract
The highly coordinated interactions of several molecular chaperones, including hsp70 and hsp90, are required for the folding and conformational regulation of a variety of proteins in eukaryotic cells, such as steroid hormone receptors and many other signal transduction regulators. The protein called Hop serves as an adaptor protein for hsp70 and hsp90 and is thought to optimize their functional cooperation. Here we characterize the assembly of the hsp70-Hop-hsp90 complex and reveal interactions that cause conformational changes between the proteins in the complex. We found that hsp40 plays an integral role in the assembly by enhancing the binding of hsp70 to the Hop complex. This is accomplished by stimulating the conversion of hsp70-ATP to hsp70-ADP, the hsp70 conformation favored for Hop binding. The hsp70-Hop-hsp90 complex is highly dynamic, as has been observed previously for hsp90 in its interaction with client proteins. Nonetheless, hsp90 binds with high affinity to Hop (K d = 90 nm), and this binding is not affected by hsp70. hsp70 binds with lower affinity to Hop (K d = 1.3 μm) on its own, but this affinity is increased (K d = 250 nm) in the presence of hsp90. hsp90 also reduces the number of hsp70 binding sites on the Hop dimer from two sites in the absence of hsp90 to one site in its presence. Hop can inhibit the ATP binding and p23 binding activity of hsp90, yet this can be reversed if hsp70 is present in the complex. Taken together, our results suggest that the assembly of hsp70-Hop-hsp90 complexes is selective and influences the conformational state of each protein.
- Published
- 2002
19. Regulation of Heat Shock Protein 90 ATPase Activity by Sequences in the Carboxyl Terminus
- Author
-
David O. Toft, Sara J. Felts, Barbara A.L. Owen, and William P. Sullivan
- Subjects
ATPase ,Mutant ,Biochemistry ,Gene Expression Regulation, Enzymologic ,chemistry.chemical_compound ,Adenosine Triphosphate ,ATP hydrolysis ,Heat shock protein ,polycyclic compounds ,Animals ,Humans ,HSP90 Heat-Shock Proteins ,Binding site ,Molecular Biology ,Adenosine Triphosphatases ,chemistry.chemical_classification ,Binding Sites ,Dose-Response Relationship, Drug ,biology ,Hydrolysis ,Thrombin ,Cell Biology ,Hsp90 ,Molecular biology ,Protein Structure, Tertiary ,Amino acid ,Kinetics ,chemistry ,Mutation ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Chickens ,Adenosine triphosphate ,Gene Deletion ,Protein Binding - Abstract
Hsp90, in addition to being an abundant and pivotal cytoplasmic chaperone protein, has been shown to be a weak ATPase. In an effort to characterize the ATPase activity of hsp90, we have observed marked differences in activities among various species of hsp90. Chicken or human hsp90 hydrolyzed ATP with a k(cat) of 0.02 min(-1) and a K(m) greater than 300 microm. In contrast, yeast hsp90 and TRAP1, an hsp90 homologue found in mitochondria, were 10-100-fold more active as ATPases. Sedimentation studies confirmed that all are dimeric proteins. Chicken hsp90 mutants were then analyzed to identify regions within the protein that influence ATPase activity. A truncation mutant of chicken hsp90, N1-573, was found to be monomeric, and yet the catalytic efficiency (k(cat)/K(m)) was greater than 100 times that of the full-length protein (k(cat) of 0.24 min(-1) and K(m) of 60 microm). In contrast, an internal deletion mutant, Delta661-677, was also monomeric but failed to hydrolyze ATP. Finally, deletion of the last 30 amino acids resulted in a dimeric protein with an ATPase activity very similar to full-length hsp90. These data indicate that sequences within the last one-fourth of hsp90 regulate ATP hydrolysis.
- Published
- 2002
20. In Vitro Reconstitution of Functional Hepadnavirus Reverse Transcriptase with Cellular Chaperone Proteins
- Author
-
Jianming Hu, David O. Toft, Dana Anselmo, and Xingtai Wang
- Subjects
Chaperonins ,Immunology ,Duck hepatitis B virus ,Priming (immunology) ,RNA ,RNA-Directed DNA Polymerase ,Biology ,Hepadnaviridae ,Virus Replication ,biology.organism_classification ,Microbiology ,Hsp90 ,Molecular biology ,Recombinant Proteins ,Reverse transcriptase ,Virus-Cell Interactions ,Cell biology ,Viral replication ,Virology ,Insect Science ,Chaperone (protein) ,Heat shock protein ,biology.protein ,Humans ,HSP90 Heat-Shock Proteins - Abstract
Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, ε) on the pregenomic RNA template by the viral reverse transcriptase (RT) and is primed by the RT itself (protein priming). We have previously shown that the RT-ε interaction and protein priming require the cellular heat shock protein, Hsp90. However, additional host factors required for these reactions remained to be identified. We now report that five cellular chaperone proteins, all known cofactors of Hsp90, were sufficient to reconstitute a duck hepatitis B virus RT active in ε binding and protein priming in vitro. Four proteins, Hsp90, Hsp70, Hsp40, and Hop, were required for reconstitution of RT activity, and the fifth protein, p23, further enhanced the kinetics of reconstitution. RT activation by the chaperone proteins is a dynamic process dependent on ATP hydrolysis and the Hsp90 ATPase activity. Thus, our results have defined a minimal complement of host factors necessary and sufficient for RT activation. Furthermore, this defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT activation and chaperone functions.
- Published
- 2002
21. Evidence for a Mechanism of Repression of Heat Shock Factor 1 Transcriptional Activity by a Multichaperone Complex
- Author
-
Mary Fenna, Yongle Guo, Frank Boellmann, David O. Toft, David F. Smith, William B. Pratt, Richard Voellmy, and Toumy Guettouche
- Subjects
Protein Denaturation ,Transcription, Genetic ,Repressor ,Plasma protein binding ,Biology ,Biochemistry ,DNA-binding protein ,Tacrolimus Binding Proteins ,Heat Shock Transcription Factors ,Humans ,HSP90 Heat-Shock Proteins ,Binding site ,HSF1 ,Molecular Biology ,Psychological repression ,Transcription factor ,DNA Primers ,Binding Sites ,Base Sequence ,Cell Biology ,Hsp90 ,Cell biology ,DNA-Binding Proteins ,Repressor Proteins ,biology.protein ,Protein Binding ,Transcription Factors - Abstract
In the absence of stress, human heat shock factor 1 (hHSF1) is in its unactivated form. hHSF1 polypeptide is in a dynamic heterocomplex with Hsp90 and is incapable of specifically binding DNA. When cells are stressed, heterocomplex assembly is disrupted. Unbound hHSF1 homotrimerizes, acquires DNA binding activity, and concentrates in the nucleus, but remains transcriptionally inactive. A subsequent reaction converts this inactive, trimeric form into the active, hyperphosphorylated transcription factor. Subsequent to the stressful event, hHSF1 is deactivated and eventually returned to its unactivated form. Evidence is presented herein that trimeric hHSF1 has the propensity to dynamically associate with an Hsp90-immunophilin-p23 complex through its regulatory domain. Formation of this heterocomplex results in repression of the transcriptional activity of trimeric hHSF1. Stress-denatured proteins effectively compete with trimeric hHSF1 for Hsp90-immunophilin-p23 complex, counteracting assembly of the heterocomplex and repression of hHSF1 transcriptional activity. This repression mechanism may be required for a proportional transcriptional response to stress. Formation of the heterocomplex may also represent the first step toward returning the hHSF1 to its unactivated form.
- Published
- 2001
22. hsp70 Interacting Protein Hip Does Not Affect Glucocorticoid Receptor Folding by the hsp90-Based Chaperone Machinery Except To Oppose the Effect of BAG-1
- Author
-
William B. Pratt, Kimon C. Kanelakis, Shinichi Takayama, Yoshihiro Morishima, John C. Reed, Patrick J. Murphy, Mario D. Galigniana, and David O. Toft
- Subjects
Protein Folding ,Reticulocytes ,Macromolecular Substances ,Lactams, Macrocyclic ,Plasma protein binding ,Spodoptera ,Biochemistry ,DNA-binding protein ,Cell Line ,Cell-free system ,Mice ,Receptors, Glucocorticoid ,Glucocorticoid receptor ,Reticulocyte ,Benzoquinones ,medicine ,Animals ,Humans ,HSP70 Heat-Shock Proteins ,HSP90 Heat-Shock Proteins ,COS cells ,Cell-Free System ,biology ,Tumor Suppressor Proteins ,Quinones ,Hsp90 ,DNA-Binding Proteins ,medicine.anatomical_structure ,Chaperone (protein) ,COS Cells ,biology.protein ,Rabbits ,Carrier Proteins ,Protein Binding ,Signal Transduction ,Transcription Factors - Abstract
Reticulocyte lysate contains a chaperone system that assembles glucocorticoid receptor (GR).hsp90 heterocomplexes. Using purified proteins, we have prepared a five-protein heterocomplex assembly system consisting of two proteins essential for heterocomplex assembly-hsp90 and hsp70-and three proteins that act as co-chaperones to enhance assembly-Hop, hsp40, p23 [Morishima, Y., Kanelakis, K. C., Silverstein, A. M., Dittmar, K. D., Estrada, L., and Pratt, W. B. (2000) J. Biol. Chem. 275, 6894-6900]. The hsp70 co-chaperone Hip has been recovered in receptor.hsp90 heterocomplexes at an intermediate stage of assembly in reticulocyte lysate, and Hip is also thought to be an intrinsic component of the assembly machinery. Here we show that immunodepletion of Hip from reticulocyte lysate or addition of high levels of Hip to the purified five-protein system does not affect GR.hsp90 heterocomplex assembly or the activation of steroid binding activity that occurs with assembly. Despite the fact that Hip does not affect assembly, it is recovered in GR.hsp90 heterocomplexes assembled by both systems. In the five-protein system, Hip prevents inhibition of assembly by the hsp70 co-chaperone BAG-1, and cotransfection of Hip with BAG-1 opposes BAG-1 reduction of steroid binding activity in COS cells. We conclude that Hip is not a component of the assembly machinery but that it could play a regulatory role in opposition to BAG-1.
- Published
- 2000
23. The Importance of ATP Binding and Hydrolysis by Hsp90 in Formation and Function of Protein Heterocomplexes
- Author
-
Brian D. Johnson, David O. Toft, and James P. Grenert
- Subjects
Protein Folding ,ATPase ,Mutant ,Plasma protein binding ,Biochemistry ,Adenosine Triphosphate ,ATP hydrolysis ,polycyclic compounds ,Animals ,HSP90 Heat-Shock Proteins ,Binding site ,Luciferases ,Molecular Biology ,Heat-Shock Proteins ,Binding Sites ,biology ,Sepharose ,Wild type ,Cell Biology ,Hsp90 ,Chaperone (protein) ,Mutation ,biology.protein ,Receptors, Progesterone ,Chickens ,Molecular Chaperones ,Protein Binding - Abstract
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.
- Published
- 1999
24. ATP sensitive tryptophans of hsp90
- Author
-
Bence B Bartha, Thomas P. Burghardt, Katalin Ajtai, and David O. Toft
- Subjects
Biophysics ,Photochemistry ,Biochemistry ,Fluorescence ,chemistry.chemical_compound ,Adenosine Triphosphate ,Tryptophan fluorescence ,Humans ,Nucleotide ,HSP90 Heat-Shock Proteins ,chemistry.chemical_classification ,Acrylamides ,Quenching (fluorescence) ,biology ,Organic Chemistry ,Tryptophan ,Hsp90 ,Dissociation constant ,Spectrometry, Fluorescence ,chemistry ,Acrylamide ,biology.protein ,Thermodynamics ,Algorithms - Abstract
The nature of the interaction between the nucleotide ATP and hsp90 was investigated by observing fluorescence quenching of the four tryptophan residues in hsp90 as a function of quencher type and temperature. ATP and acrylamide quench the fluorescence from tryptophan free in solution principally by static and collisional mechanisms, respectively. Acrylamide quenching of tryptophan fluorescence in hsp90 is also principally collisional and identifies two classes of residues, one readily accessible to quenching the other less accessible. ATP quenching of tryptophan fluorescence in hsp90 is more complex exhibiting no overall preferred mechanism. However, ATP competitively inhibits acrylamide quenching of the readily accessible class of tryptophan residues by static quenching with the quenching constant providing an upper limit for the ATP dissociation constant. The ATP-free tryptophan dissociation constant is more than a factor of three larger than that for ATP–hsp90 suggesting that the ATP–hsp90 interaction is specific. The static quenching of tryptophan fluorescence in hsp90 by ATP implies that the nucleotide binds in close proximity to one or more of the tryptophan residues.
- Published
- 1998
25. The Amino-terminal Domain of Heat Shock Protein 90 (hsp90) That Binds Geldanamycin Is an ATP/ADP Switch Domain That Regulates hsp90 Conformation
- Author
-
Leonard M. Neckers, Jenny Clark, Theodor W. Schulte, David O. Toft, William P. Sullivan, Hans Joachim Ochel, Timothy A.J. Haystead, Patrick Fadden, James P. Grenert, Edward A. Sausville, Henry C. Krutzsch, and Edward G. Mimnaugh
- Subjects
Protein Conformation ,Lactams, Macrocyclic ,Molecular Sequence Data ,Plasma protein binding ,Biochemistry ,chemistry.chemical_compound ,Adenosine Triphosphate ,Protein structure ,Heat shock protein ,Benzoquinones ,polycyclic compounds ,Humans ,Amino Acid Sequence ,HSP90 Heat-Shock Proteins ,Molecular Biology ,Sequence Deletion ,biology ,Chemistry ,Quinones ,Cell Biology ,Geldanamycin ,Hsp90 ,Adenosine Diphosphate ,Mutagenesis ,Chaperone (protein) ,biology.protein ,Biophysics ,ATP–ADP translocase ,Protein Binding ,Binding domain - Abstract
Many functions of the chaperone, heat shock protein 90 (hsp90), are inhibited by the drug geldanamycin that specifically binds hsp90. We have studied an amino-terminal domain of hsp90 whose crystal structure has recently been solved and determined to contain a geldanamycin-binding site. We demonstrate that, in solution, drug binding is exclusive to this domain. This domain also binds ATP linked to Sepharose through the gamma-phosphate. Binding is specific for ATP and ADP and is inhibited by geldanamycin. Mutation of four glycine residues within two proposed ATP binding motifs diminishes both geldanamycin binding and the ATP-dependent conversion of hsp90 to a conformation capable of binding the co-chaperone p23. Since p23 binding requires regions outside the 1-221 domain of hsp90, these results indicate a common site for nucleotides and geldanamycin that regulates the conformation of other hsp90 domains.
- Published
- 1997
26. Steroid Receptor Interactions with Heat Shock Protein and Immunophilin Chaperones*
- Author
-
William B. Pratt and David O. Toft
- Subjects
Molybdenum ,Receptors, Steroid ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Immune receptor ,Biology ,FKBP52 ,Steroid hormone ,Endocrinology ,Nuclear receptor ,Biochemistry ,medicine ,Enzyme-linked receptor ,Animals ,Humans ,Estrogen-related receptor gamma ,HSP90 Heat-Shock Proteins ,Carrier Proteins ,Receptor ,Heat-Shock Proteins ,Immunosuppressive Agents ,Molecular Chaperones ,G protein-coupled receptor - Abstract
We have provided a historical perspective on a body of steroid receptor research dealing with the structure and physiological significance of the untransformed 9S receptor that has often confused both novice and expert investigators. The frequent controversies and equivocations of earlier studies were due to the fact that the native, hormone-free state of these receptors is a large multiprotein complex that resisted description for many years because of its unstable and dynamic nature. The untransformed 9S state of the steroid and dioxin receptors has provided a unique system for studying the function of the ubiquitous, abundant, and conserved heat shock protein, hsp90. The hormonal control of receptor association with hsp90 provided a method of manipulating the receptor heterocomplex in a manner that was physiologically meaningful. For several steroid receptors, binding to hsp90 was required for the receptor to be in a native hormone-binding state, and for all of the receptors, hormone binding promoted dissociation of the receptor from hsp90 and conversion of the receptor to the DNA-binding state. Although the complexes between tyrosine kinases and hsp90 were discovered earlier, the hormonal regulation or steroid receptor association with hsp90 permitted much more rapid and facile study of hsp90 function. The observations that hsp90 binds to the receptors through their HBDs and that these domains can be fused to structurally different proteins bringing their function under hormonal control provided a powerful linkage between the hormonal regulation of receptor binding to hsp90 and the initial step in steroid hormone action. Because the 9S receptor hsp90 heterocomplexes could be physically stabilized by molybdate, their protein composition could be readily studied, and it became clear that these complexes are multiprotein structures containing a number of unique proteins, such as FKBP51, FKBP52, CyP-40, and p23, that were discovered because of their presence in these structures. Further analysis showed that hsp90 itself exists in a variety of native multiprotein heterocomplexes independent of steroid receptors and other 'substrate' proteins. Cell-free systems can now be used to study the formation of receptor heterocomplexes. As we outlined in the scheme of Fig. 1, the multicomponent receptor-hsp90 heterocomplex assembly system is being reconstituted, and the importance of individual proteins, such as hsp70, p60, and p23, in the assembly process is becoming recognized. It should be noted that our understanding of the mechanism and purpose of steroid receptor heterocomplex assembly is still at an early stage. We can now speculate on the roles of receptor-associated proteins in receptor action, both as individuals and as a group, but their actual functions are still vague or unknown. We can make realistic models about the chaperoning and trafficking of steroid receptors, but we don't yet know how these processes occur, we don't know where chaperoning occurs in the cell (e.g. Is it limited to the cytoplasm? Is it a diffuse process or does chaperoning occur in association with structural elements?), and, with the exception of the requirement for hormone binding, we don't know the extent to which the hsp90-based chaperone system impacts on steroid hormone action. It is not yet clear how far the discovery of this hsp90 heterocomplex assembly system will be extended to the development of a general understanding of protein processing in the cell. Because this assembly system is apparently present in all eukaryotic cells, it probably performs an essential function for many proteins. The bacterial homolog of hsp90 is not an essential protein, but hsp90 is essential in eukaryotes, and recent studies indicate that the development of the cell nucleus from prokaryotic progenitors was accompanied by the duplication of genes for hsp90 and hsp70 (698). (ABSTRACT TRUNCATED)
- Published
- 1997
27. The involvement of p23, hsp90, and immunophilins in the assembly of progesterone receptor complexes
- Author
-
Bridget Stensgard, Ronald Corbisier, David O. Toft, and Jill L. Johnson
- Subjects
Reticulocytes ,Macromolecular Substances ,Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,Biology ,Biochemistry ,DNA-binding protein ,Cell-free system ,Tacrolimus Binding Proteins ,Cyclophilins ,Mice ,chemistry.chemical_compound ,Adenosine Triphosphate ,Endocrinology ,Immunophilins ,Reticulocyte ,Progesterone receptor ,medicine ,Animals ,Humans ,HSP70 Heat-Shock Proteins ,HSP90 Heat-Shock Proteins ,Molecular Biology ,Heat-Shock Proteins ,Amino Acid Isomerases ,Prostaglandin-E Synthases ,Molybdenum ,Peptidylprolyl isomerase ,Cell-Free System ,Antibodies, Monoclonal ,Cell Biology ,Peptidylprolyl Isomerase ,Phosphoproteins ,Hsp90 ,DNA-Binding Proteins ,Intramolecular Oxidoreductases ,medicine.anatomical_structure ,chemistry ,biology.protein ,Molecular Medicine ,Rabbits ,Carrier Proteins ,Receptors, Progesterone ,Chickens ,Adenosine triphosphate ,Cyclophilin D ,Molecular Chaperones - Abstract
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 Mg 2+ 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.
- Published
- 1996
28. Interaction of the progesterone receptor with binding proteins for FK506 and cyclosporin A
- Author
-
William J. Sullivan, David O. Toft, E Diehl, Dean P. Edwards, M Altmann, S Nordeen, and Magdy P. Milad
- Subjects
Immunoprecipitation ,Breast Neoplasms ,DNA-binding protein ,Tacrolimus ,Tacrolimus Binding Proteins ,Mice ,Endocrinology ,Cyclosporin a ,Heat shock protein ,Progesterone receptor ,Tumor Cells, Cultured ,Animals ,Humans ,Molecular Biology ,Heat-Shock Proteins ,biology ,Carcinoma ,Receptor Aggregation ,Gene Transfer Techniques ,General Medicine ,Hsp90 ,Molecular biology ,Hsp70 ,DNA-Binding Proteins ,Blot ,Cyclosporine ,biology.protein ,Carrier Proteins ,Receptors, Progesterone ,Chickens ,Immunosuppressive Agents - Abstract
T47D human breast carcinoma cells and the chicken oviduct were used to study the structure of the nonactivated progesterone receptor (PR) complex. Immunoprecipitation of PR (B form) from cytosol extracts was performed using monoclonal antibody PR6, a cross-reactive antibody prepared to chicken PR. Analysis of the PR complex by sodium dodecyl sulfate gels and Western immuno-blotting revealed the presence of several specific copurifying proteins. Consistent with previous reports, the two heat shock proteins, hsp90 and hsp70, were shown to be present. A third 59-kilodalton (kDa) protein observed previously was confirmed to be p59 (also called hsp56 or FKBP52), which has been shown to bind the immunosuppressant drug FK506. Two additional PR-associated proteins were observed that had not been previously recognized with human PR. These have molecular masses of 54-kDa and 23-kDa and have been shown by Western blotting to be related to the proteins p54 and p23 that are associated with chicken PR. P23 is a novel protein of unknown function and p54 or FKBP54 has been recently shown to be another FK506-binding protein related to p59. Finally, the cyclosporin A-binding protein, CyP-40, could be detected in isolated chicken PR complexes and in PR complexes that were reconstituted in vitro, but this protein was not detected in human PR complexes, which are less stable than chicken PR complexes in cytosol extracts. The functional significance of FK506 and cyclosporin A-binding proteins to hormone action was tested using a T47D cell line that contained a progestin reporter gene, MMTV-CAT. Treatment with cyclosporin A had no effect on the basal level of CAT expression, but it caused a dramatic increase in the sensitivity and magnitude of the response to the synthetic progestin, R5020. The enhanced response elicited by drug treatment was blocked by the antiprogestin RU486 indicating that this effect was receptor-mediated. While cyclosporin A enhanced progestin action in T47D cells, it inhibited a PR/reporter gene system in L cells. The drugs FK506 and rapamycin had no effect on progestin action in T47D cells, but they stimulated glucocorticoid action in T47D cells. Thus, the effects of these immunosuppressant drugs vary with the cell type and hormonal system that is tested. Whether these drug effects relate directly to the immunophilins bound in receptor complexes remains unknown.
- Published
- 1995
29. Accelerated recovery of postischemic stunned myocardium after induced expression of myocardial heat-shock protein (HSP70)
- Author
-
James J. Morris, Barbara L. Robinson, David O. Toft, and Terumasa Morita
- Subjects
Pulmonary and Respiratory Medicine ,Ischemia ,Myocardial Reperfusion ,Dogs ,Heart rate ,medicine ,Animals ,HSP70 Heat-Shock Proteins ,cardiovascular diseases ,Artery occlusion ,Circumflex ,Myocardial Stunning ,Cardioprotection ,business.industry ,Myocardium ,Stroke Volume ,medicine.disease ,Myocardial Contraction ,Preload ,medicine.anatomical_structure ,Anesthesia ,Ventricular pressure ,Surgery ,Cardiology and Cardiovascular Medicine ,business ,Artery - Abstract
In vitro studies suggest that interventions targeted at myocardial gene regulation of endogenous cytoprotective elements, such as heat-shock protein, may attenuate myocardial ischemic injury. We tested the hypothesis that heat shock-induced expression of myocardial heat-shock protein before ischemia accelerates functional recovery of postischemic stunned myocardium in the intact circulation. Sixteen dogs underwent partial femoral arteriovenous bypass and core temperature was raised to 42° C for 15 minutes in eight dogs (heat-shocked) and maintained at 37° C in eight dogs (nonheat-shocked). After 24 hours dogs were studied to measure myocardial segment length in the circumflex artery region with ultrasonic dimension transducers, left ventricular pressure with a micromanometer, and circumflex coronary flow with an ultrasonic probe. Regional contractile function was quantified by the area beneath the linear preload recruitable stroke work relationship at baseline and at intervals during reperfusion after a 15-minute circumflex artery occlusion followed by 3 hours of reperfusion. Baseline and peak reperfusion hyperemic circumflex flows were 37 ± 9 ml/min and 154 ± 33 ml/min, respectively, in heat-shocked dogs (p < 0.001) and 46 ± 24 ml/min and 171 ± 57 ml/min, respectively, in nonheat-shocked dogs (p < 0.001), with no differences between groups (p = not significant) at any time during reperfusion. Heart rate and left ventricular peak pressure, end-diastolic pressure, and first derivative of left ventricular pressure were similar (all p = not significant) in heat-shocked and nonheat-shocked dogs during ischemia and reperfusion. Before ischemia, preload recruitable stroke work relationship did not differ (p = not significant) in heat-shocked and nonheat-shocked dogs. Ischemia reduced preload recruitable stroke work relationship to 32% ± 8% control (p < 0.001) in heat-shocked dogs and to 19% ± 15% control in nonheat-shocked dogs (p < 0.001) at 15 minutes of reperfusion, indicating a similar (p = not significant) initial degree of injury. During 3 hours of reperfusion, preload recruitable stroke work relationship returned to 80% ± 38% control in heat-shocked dogs but to only 33% ± 13% control in nonheat-shocked dogs (p < 0.0001). Myocardial expression of heat-shock protein, quantified by optical densitometry of Western blots using an antibody specific for HSP70, was greater in heat-shocked than in nonheat-shocked dogs (108 ± 27 versus 71 ± 14 densitometry units, p < 0.005). Exact causal mechanisms remain to be defined, but these data indicate (1) hyperthermic bypass triggers induction of myocardial heat-shock protein and (2) elevated myocardial heat-shock protein is associated with accelerated recovery of stunned myocardium. Promotion of endogenous molecular cytoprotective systems represents a novel and potentially useful strategy for myocardial protection. (J THORAC CARDIOVASC SURG 1995;109:753-64)
- Published
- 1995
30. ATP-dependent chaperoning activity of reticulocyte lysate
- Author
-
Robert J. Schumacher, Nancy J. McMahon, David O. Toft, William P. Sullivan, Robert L. Matts, and Robin Hurst
- Subjects
Lysis ,biology ,Cell Biology ,Biochemistry ,Molecular biology ,Hsp70 ,Cell-free system ,medicine.anatomical_structure ,Reticulocyte ,Chaperone (protein) ,Heat shock protein ,biology.protein ,medicine ,Luciferase ,Molecular Biology ,Protein Renaturation - Abstract
We have developed an assay for chaperone-mediated protein renaturation using thermally denatured Firefly luciferase. Dilution of denatured luciferase (> 99% loss of activity) into reticulocyte lysate typically results in recovery of 5-15% activity. Addition of an ATP-regenerating system increases yields to > 60%, while heat shock or the addition of denatured proteins inhibits the chaperoning activity. Reticulocyte lysate contains abundant quantities of the heat shock proteins, hsp90 and hsp70, and a 60-kDa protein homologous to the yeast stress protein, STI1. Immune isolated samples of these three proteins support recovery of up to 35% of luciferase activity in an ATP-dependent manner, suggesting that these or associated proteins are involved in the renaturation of luciferase. Furthermore, we observed a correlation between luciferase renaturation activity and the levels of hsp70 and hsp90 in reticulocyte lysate preparations. Purified hsp90 and hsp70, along with an ATP-regenerating system, are able to renature luciferase to greater than 20% of its original activity. This renaturation is most efficient when hsp90 and hsp70 are at about a 2:1 ratio and at concentrations similar to those found in reticulocyte lysate. This study provides evidence for an ATP-dependent chaperoning activity in reticulocyte lysate that involves a cooperative action of hsp70 and hsp90.
- Published
- 1994
31. Mutational analysis of hsp90 binding to the progesterone receptor
- Author
-
William P. Sullivan and David O. Toft
- Subjects
chemistry.chemical_classification ,medicine.medical_treatment ,Mutant ,Cell Biology ,Biology ,Biochemistry ,Hsp90 ,Amino acid ,Steroid hormone ,medicine.anatomical_structure ,Reticulocyte ,chemistry ,Heat shock protein ,Progesterone receptor ,polycyclic compounds ,medicine ,biology.protein ,Receptor ,Molecular Biology - Abstract
The 90-kDa heat shock protein, hsp90, is known to associate with steroid receptors that are in the inactive state. While the biochemical function of hsp90 is unclear, this association is believed to be significant because dissociation of hsp90 occurs when receptors are activated by hormone. Complexes between hsp90 and the progesterone receptor can be formed in vitro in rabbit reticulocyte lysate. This has been shown to be an ATP-dependent process, and dissociation of the complex occurs when progesterone is added to the system. We now show that hsp90 synthesized by in vitro translation in reticulocyte lysate can form complexes with progesterone receptor that are sensitive to hormone. This system was used to analyze several mutant forms of hsp90. A series of NH2-terminal deletions showed that amino acids 1-380 can be removed from hsp90 without substantial loss of receptor binding activity. However, several deletions in the COOH-terminal half of hsp90 resulted in a partial or complete loss of this activity. Two regions, amino acids 381-441 and 601-677, appear to be particularly important for receptor binding. These studies describe a convenient and reliable method for the initial screening of hsp90 mutants, and they provide important clues to the identification of domains on hsp90 that interact with other proteins.
- Published
- 1993
32. P59 (FK506 binding protein 59) interaction with heat shock proteins is highly conserved and may involve proteins other than steroid receptors
- Author
-
Mark W. Albers, Hong Chang, David O. Toft, Stuart L. Schreiber, Ping Kaung K. Tai, and Lee E. Faber
- Subjects
Receptors, Steroid ,Oviducts ,Saccharomyces cerevisiae ,Biochemistry ,Tacrolimus ,Tacrolimus Binding Proteins ,HSPA4 ,Heat shock protein ,Animals ,Cells, Cultured ,Heat-Shock Proteins ,biology ,Binding protein ,Uterus ,Hsp90 ,Actins ,Hsp70 ,FKBP ,Chaperone (protein) ,biology.protein ,Chaperone complex ,Female ,Rabbits ,Carrier Proteins ,Chickens ,Immunosuppressive Agents ,Protein Binding - Abstract
P59 (also known as FK506 binding protein 59 (FKBP59) or heat shock protein 56 (hsp56)l and heat shock proteins 90 and 70 (hsp90 and hsp70) associate with steroid receptors and are believed to maintain the receptors in an inactive state. Recently, we showed that p59 purified from human lymphocytes is an immunophilin (FKBP59) which binds both FK506 and rapamycin. It was also demonstrated that immunosuppressant-FKBP59 complexes associate with hsp90, hsp70, and the glucocorticoid receptor (Tai, P.-K. K., Albers, M. W., Chang, H., Faber, L. E., & Schreiber, S. L. (1992) Science 256, 1315-13181. Here we provide evidence that rabbit uterine p59 also binds FK506 and rapamycin and that p59 or its homologue is associated with nontransformed progesterone receptors of rabbit uterus and chicken oviduct, This suggests that the immunophilin-heat shock protein-steroid receptor interaction is ubiquitous and not limited to immune systems. A FKBP59 homologue complexed with hsp90-hsp70 was also detected in yeast, which suggests that the immunophilin-heat shock protein association has been evolutionarily conserved. In addition, we found that the FKBP59-hsp complexes are more complicated than previously thought, involving other proteins such as actin and a 63-kDa protein, p63. The association of p63 to the p59 complex was inhibited by FK506 and rapamycin, suggesting that p63 could be a potential target for the immunosuppressive actions of these two drugs. Since both immunophilin and heat shock proteins have been suggested to be responsible for protein folding and assembly of new synthesized polypeptides, it is reasonable to propose that p59, in association with hsp70 and hsp90, forms the core structure of a universal molecular chaperone. This chaperone complex may be responsible for recognition, folding, assembly, and disassembly of steroid receptors or other regulatory proteins at or near the end of translation.
- Published
- 1993
33. Biological response of the female strain Achlya ambisexualis 734 to dehydro-oogoniol and analogues
- Author
-
Trevor C. McMorris, Moon Surksik, Wang Wen, and David O. Toft
- Subjects
Oogonium ,Strain (chemistry) ,Plant Science ,General Medicine ,Achlya ,Horticulture ,Biology ,biology.organism_classification ,Biochemistry ,medicine.anatomical_structure ,Botany ,medicine ,Molecular Biology ,Achlya ambisexualis - Abstract
Oogonium development in female strains of the water mould Achlya is initiated in nature by a steroid mixture of oogoniol, dehydro-oogoniol and thei
- Published
- 1993
34. FKBP51 promotes assembly of the Hsp90 chaperone complex and regulates androgen receptor signaling in prostate cancer cells
- Author
-
Bryce M. Paschal, Chun-Song Yang, David O. Toft, Daniel Gioeli, Li Ni, and Henry F. Frierson
- Subjects
Male ,Neoplasms, Hormone-Dependent ,medicine.drug_class ,Transplantation, Heterologous ,Mice, SCID ,Biology ,Models, Biological ,Small hairpin RNA ,Tacrolimus Binding Proteins ,Prostate cancer ,Mice ,Cell Line, Tumor ,medicine ,Animals ,Humans ,HSP90 Heat-Shock Proteins ,Molecular Biology ,DNA Primers ,Prostaglandin-E Synthases ,Base Sequence ,Androgen binding ,Prostatic Neoplasms ,Cell Biology ,Articles ,Androgen ,medicine.disease ,Hsp90 ,Molecular biology ,Recombinant Proteins ,Cell biology ,Up-Regulation ,Androgen receptor ,Intramolecular Oxidoreductases ,Receptors, Androgen ,Multiprotein Complexes ,biology.protein ,Chaperone complex ,Signal transduction ,Neoplasm Transplantation ,Signal Transduction - Abstract
Prostate cancer progression to the androgen-independent (AI) state involves acquisition of pathways that allow tumor growth under low-androgen conditions. We hypothesized that expression of molecular chaperones that modulate androgen binding to AR might be altered in prostate cancer and contribute to progression to the AI state. Here, we report that the Hsp90 cochaperone FKBP51 is upregulated in LAPC-4 AI tumors grown in castrated mice and describe a molecular mechanism by which FKBP51 regulates AR activity. Using recombinant proteins, we show that FKBP51 stimulates recruitment of the cochaperone p23 to the ATP-bound form of Hsp90, forming an FKBP51-Hsp90-p23 superchaperone complex. In cells, FKBP51 expression promotes superchaperone complex association with AR and increases the number of AR molecules that undergo androgen binding. FKBP51 stimulates androgen-dependent transcription and cell growth, and FKBP51 is part of a positive feedback loop that is regulated by AR and androgen. Finally, depleting FKBP51 levels by short hairpin RNA reduces the transcript levels of genes regulated by AR and androgen. Because the superchaperone complex plays a critical role in determining the ligand-binding competence and transcription function of AR, it provides an attractive target for inhibiting AR activity in prostate cancer cells.
- Published
- 2010
35. Composition, assembly and activation of the avian progesterone receptor
- Author
-
David F. Smith and David O. Toft
- Subjects
Reticulocytes ,Macromolecular Substances ,Endocrinology, Diabetes and Metabolism ,ATPase ,Clinical Biochemistry ,Oviducts ,Biology ,Biochemistry ,Cytosol ,Endocrinology ,Reticulocyte ,Progesterone receptor ,medicine ,Animals ,Receptor ,Molecular Biology ,Heat-Shock Proteins ,Estradiol ,Cell Biology ,Interleukin-13 receptor ,Hsp90 ,Hsp70 ,Molecular Weight ,medicine.anatomical_structure ,Protein Biosynthesis ,biology.protein ,Molecular Medicine ,Electrophoresis, Polyacrylamide Gel ,Female ,Rabbits ,Receptors, Progesterone ,Chickens - Abstract
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 Mg 2+ . We have obtained maximal hsp90 binding to receptor when lysate is supplemented with 3 mM MgCl 2 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.
- Published
- 1992
36. Assembly of progesterone receptor with heat shock proteins and receptor activation are ATP mediated events
- Author
-
David F. Smith, Bridget Stensgard, David O. Toft, and W J Welch
- Subjects
chemistry.chemical_classification ,Cell Biology ,Biology ,Biochemistry ,Hsp90 ,Hsp70 ,Cell-free system ,Enzyme ,medicine.anatomical_structure ,chemistry ,Reticulocyte ,Heat shock protein ,Progesterone receptor ,polycyclic compounds ,medicine ,biology.protein ,Receptor ,Molecular Biology - Abstract
To better understand assembly mechanisms of progesterone receptor (PR) complexes, we have developed a cell-free system for studying PR interactions with the 90- and 70-kDa heat shock proteins (hsp90 and hsp70), and we have used this system to examine requirements for hsp90 binding to PR. Purified chick PR, 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 degrees 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 by enzymatic means blocks hsp90 binding to PR; likewise, addition of EDTA to lysate blocks hsp90 binding, but binding is restored by the addition of excess Mg2+. Addition to lysate of monoclonal antibody against hsp70 inhibits hsp90 binding to PR and destabilizes preformed complexes. Stabilization of hsp90-receptor complexes also requires ATP, indicating that ATP and hsp70 are needed to form and to maintain hsp90 complexes. Hormone-dependent activation of reconstituted receptor complexes was also examined. The addition of progesterone to the reticulocyte lysate promotes dissociation of hsp90 and hsp70 from the receptor. This also appears to require ATP and dissociation is most efficient in the presence of an ATP-regenerating system. In conclusion, these studies indicate that PR-hsp90 complexes do not self-assemble; instead, assembly is probably a multistep process requiring ATP and other cellular factors.
- Published
- 1992
37. Phase I study of 17-allylamino-17 demethoxygeldanamycin, gemcitabine and/or cisplatin in patients with refractory solid tumors
- Author
-
Charles Erlichman, Bridget Stensgard, Gretchen N. Batzel, Joleen M. Hubbard, Sara J. Felten, Rui Qin, David O. Toft, Cynthia Ten Eyck, Paul Haluska, and S. Percy Ivy
- Subjects
Adult ,Male ,medicine.medical_specialty ,Nausea ,Lactams, Macrocyclic ,Antineoplastic Agents ,Pharmacology ,Neutropenia ,Protein Serine-Threonine Kinases ,Gastroenterology ,Deoxycytidine ,Article ,Cohort Studies ,chemistry.chemical_compound ,Refractory ,Internal medicine ,Neoplasms ,Antineoplastic Combined Chemotherapy Protocols ,polycyclic compounds ,medicine ,Benzoquinones ,Biomarkers, Tumor ,Humans ,Pharmacology (medical) ,HSP70 Heat-Shock Proteins ,HSP90 Heat-Shock Proteins ,Aged ,Cisplatin ,Aged, 80 and over ,Dose-Response Relationship, Drug ,business.industry ,Middle Aged ,medicine.disease ,Gemcitabine ,female genital diseases and pregnancy complications ,Treatment Outcome ,Oncology ,chemistry ,Toxicity ,Female ,medicine.symptom ,business ,Hyponatremia ,medicine.drug - Abstract
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/m2 of 17-AAG, 750 mg/m2 of gemcitabine, and 40 mg/m2 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/m2 of 17-AAG and 750 mg/m2 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/m2 of 17-AAG and 750 mg/m2 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.
- Published
- 2009
38. Characterization of progesterone receptor binding to the 90- and 70-kDa heat shock proteins
- Author
-
Alan D. W. Dobson, O. M. Conneely, Nita J. Maihle, William P. Sullivan, David O. Toft, D. B. Schowalter, and B. W. O'malley
- Subjects
animal structures ,biology ,Progesterone receptor binding ,Cell Biology ,Biochemistry ,Hsp90 ,Cell biology ,Mutant protein ,Heat shock protein ,Progesterone receptor ,biology.protein ,5-HT5A receptor ,Receptor ,Molecular Biology ,Binding domain - Abstract
In this study a model system for expression of the chicken progesterone receptor in cultured cells was developed using a quail fibroblast cell line, QT6. The chicken progesterone receptor form A expressed in QT6 cells was evaluated and determined to have a number of similarities to receptor isolated from chicken oviduct. These include hormone binding, sedimentation profile, phosphorylation pattern, heat shock protein (hsp) 70 and hsp90 associations and the ability to stimulate a reporter gene construct. Therefore, the receptor expressed in this system functioned adequately for further evaluation of the particular region (or regions) involved in hsp70 and hsp90 binding. Several receptor deletion mutants were tested for hsp70/hsp90 binding; only the d369-659 mutant, which has the entire steroid-binding domain deleted, was unable to bind hsp90 and hsp70. Three separate regions of the steroid-binding domain were found to partially restore hsp90 and hsp70 binding to the d369-659 mutant protein. However, hsp binding was not abolished when these or other regions of the steroid binding domain were deleted individually. These findings indicate that hsp90 and hsp70 both bind to the steroid-binding domain of the receptor through interactions at multiple locations or through some structural quality that is distributed throughout this region of the protein.
- Published
- 1991
39. Cisplatin abrogates the geldanamycin-induced heat shock response
- Author
-
Charles Erlichman, David O. Toft, Cynthia J. TenEyck, Wilma L. Lingle, Andrea K. McCollum, and Kara B. Lukasiewicz
- Subjects
Cancer Research ,endocrine system ,Lactams, Macrocyclic ,Antineoplastic Agents ,Biology ,Article ,chemistry.chemical_compound ,Hsp27 ,Heat Shock Transcription Factors ,Heat shock protein ,Cell Line, Tumor ,medicine ,Benzoquinones ,Humans ,HSP70 Heat-Shock Proteins ,HSP90 Heat-Shock Proteins ,Heat shock ,Melphalan ,Cisplatin ,Drug Synergism ,Geldanamycin ,Molecular biology ,female genital diseases and pregnancy complications ,Chromatin ,Hsp70 ,Up-Regulation ,Heat shock factor ,DNA-Binding Proteins ,Oncology ,chemistry ,Drug Resistance, Neoplasm ,biology.protein ,Camptothecin ,Heat-Shock Response ,medicine.drug ,Protein Binding ,Transcription Factors - Abstract
Benzoquinone ansamycin antibiotics such as geldanamycin (GA) bind to the NH2-terminal ATP-binding domain of heat shock protein (Hsp) 90 and inhibit its chaperone functions. Despite in vitro and in vivo studies indicating promising antitumor activity, derivatives of GA, including 17-allylaminogeldanamycin (17-AAG), have shown little clinical efficacy as single agents. Thus, combination studies of 17-AAG and several cancer chemotherapeutics, including cisplatin (CDDP), have begun. In colony-forming assays, the combination of CDDP and GA or 17-AAG was synergistic and caused increased apoptosis compared with each agent alone. One measurable response that results from treatment with Hsp90-targeted agents is the induction of a heat shock factor-1 (HSF-1) heat shock response. Treatment with GA + CDDP revealed that CDDP suppresses up-regulation of HSF-1 transcription, causing decreased levels of stress-inducible proteins such as Hsp27 and Hsp70. However, CDDP treatment did not prevent trimerization and nuclear localization of HSF-1 but inhibited DNA binding of HSF-1 as shown by chromatin immunoprecipitation. Melphalan, but not camptothecin, caused similar inhibition of GA-induced HSF-1–mediated Hsp70 up-regulation. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt cell survival assays revealed that deletion of Hsp70 caused increased sensitivity to GA (Hsp70+/+ IC50 = 63.7 ± 14.9 nmol/L and Hsp70−/− IC50 = 4.3 ± 2.9 nmol/L), which confirmed that a stress response plays a critical role in decreasing GA sensitivity. Our results suggest that the synergy of GA + CDDP is due, in part, to CDDP-mediated abrogation of the heat shock response through inhibition of HSF-1 activity. Clinical modulation of the HSF-1–mediated heat shock response may enhance the efficacy of Hsp90-directed therapy. [Mol Cancer Ther 2008;7(10):3256–64]
- Published
- 2008
40. Minireview: the intersection of steroid receptors with molecular chaperones: observations and questions
- Author
-
David O. Toft and David F. Smith
- Subjects
Protein Folding ,Receptors, Steroid ,biology ,Protein Conformation ,General Medicine ,Plasma protein binding ,Bioinformatics ,Co-chaperone ,Endocrinology ,Protein structure ,Chaperone (protein) ,Heat shock protein ,Multiprotein Complexes ,biology.protein ,Animals ,Humans ,Protein folding ,Minireview ,Receptor ,Molecular Biology ,Neuroscience ,Hormone ,Molecular Chaperones ,Protein Binding - Abstract
An involvement of molecular chaperones in the action and well-being of steroid receptors was recognized early in the molecular era of hormone research. However, this has continued to be a topic of much enquiry and some confusion. All steroid receptors associate with heat shock protein 90, the main character of a series of multiprotein chaperone complexes generally referred to as the "heat shock protein 90 chaperoning machine." Receptor association with chaperones occurs in an ordered, step-wise fashion and is necessary for the maintenance of unliganded receptor in a state ready to bind and respond to hormone. Chaperones additionally modulate how receptors respond to hormone and activate target genes. Although much is known about the participants in this chaperoning process and the consequences of chaperoning, many key questions remain unanswered, particularly those concerning molecular mechanisms, cellular dynamics, and the functions of an array of cochaperone proteins. Here, we point out several areas in need of investigation to encourage new ideas and participants in this burgeoning field.
- Published
- 2008
41. GCUNC45 is the first Hsp90 co-chaperone to show alpha/beta isoform specificity
- Author
-
Sara J. Felts, David O. Toft, and Ahmed Chadli
- Subjects
Gene isoform ,Accelerated Publication ,Plasma protein binding ,Biology ,Spodoptera ,Biochemistry ,Substrate Specificity ,Progesterone receptor ,Animals ,Humans ,Protein Isoforms ,Gene Silencing ,HSP90 Heat-Shock Proteins ,Receptor ,Molecular Biology ,Gene knockdown ,Intracellular Signaling Peptides and Proteins ,Cell Biology ,Hsp90 ,In vitro ,Cell biology ,Co-chaperone ,biology.protein ,Receptors, Progesterone ,HeLa Cells ,Protein Binding - Abstract
Hsp90 is an essential molecular chaperone required for the normal functioning of many key regulatory proteins in eukaryotic cells. Vertebrates have two closely related isoforms of cytosolic Hsp90 (Hsp90alpha and Hsp90beta). However, specific functions for each isoform are largely unknown, and no Hsp90 co-chaperone has been reported to distinguish between the two isoforms. In this study, we show that the Hsp90 co-chaperone GCUNC45 bound preferentially to the beta isoform of Hsp90 in vitro. GCUNC45 efficiently blocked the progression of progesterone receptor chaperoning in an in vitro functional system when Hsp90beta was used, but did so with much less efficacy when Hsp90alpha was used. Knockdown experiments in HeLa cells showed that GCUNC45 is required for the normal cellular distribution of Hsp90beta, but not Hsp90alpha. This is the first example of a co-chaperone with isoform selectivity, and this approach may open novel avenues to understanding the functional differences between Hsp90 isoforms.
- Published
- 2008
42. Partners in surveillance and quality control: G. L. Blatch (ed): Networking of chaperones by co-chaperones. 2007, Landes Bioscience/Eurekah.com and Springer Science+Business Media, LLC, ISBN: 978-0-387-49308-4
- Author
-
David O. Toft
- Subjects
Genetics ,biology ,Cell Biology ,Computational biology ,DNAJ Protein ,Biochemistry ,Hsp90 ,GroEL ,Book Review ,FKBP ,CDC37 ,Chaperone (protein) ,biology.protein ,Protein folding ,Cellular localization - Abstract
Cellular proteins need varying degrees of instruction and assistance during their appearance in the cell and in conducting their activities. They also have to bow out of the job at the appropriate time. Key figures in the control of protein expression and homeostasis are the molecular chaperones. Many of these proteins are essential to all cells and they serve a number of different purposes. They usually work together as chaperone machines to assist in the processes of protein folding, the repair of improperly folded proteins, the degradation of unwanted proteins, and in some cases, the stabilization and control of key cell regulators. Within these chaperoning processes, important decisions must be made. Upon handling of a difficult client, some component of the chaperone machine must determine when to pass off a client to another system, direct the client toward degradation, or simply try it again. Thus, client proteins must be selected and sorted in various ways. To fulfill these tasks, most chaperone systems are aided by a number of cochaperones—proteins that add additional information or a specialized function to the chaperoning system. Cochaperones add much diversity to chaperone machines, and a growing number of proteins in this category are being recognized. “Networking of chaperones by co-chaperones” is the first published volume dedicated solely to cochaperones. This is a timely addition and perhaps overdue since this field has grown dramatically over the past 10–15 years. This volume contains 11 chapters on separate topics contributed by authorities in the field. The chapters are surprisingly similar in quality and design, which is a credit to its editor, Gregory Blatch. Each contains a brief, but very informative introduction to the topic; an excellent aid for newcomers to the field. This is followed by an update on our current knowledge on the subject. This is also quite brief, but very informative with excellent reference to the literature. This reviewer found all of the chapters to be very readable and thought-provoking. Although knowledge on all cochaperone systems is still incomplete, the systems selected for this volume are somewhat advanced and biased toward the Hsp70 and Hsp90 chaperone pathways. Seven of the chapters concern cochaperones for the Hsp70 family. Abundant chaperones of the Hsp70 family exist in all cell compartments and are needed for many routine activities such as the folding of nascent proteins, the transport of proteins through membranes, the uncoating of clathrin-coated vesicles, the assembly of multisubunit proteins, and the transport of proteins to the proteasome for degradation This system is complex with a confusing number of J protein cochaperones (Hsp40s) and nucleotide exchange factors (NEFs). These are needed to support the ATP-dependent Hsp70 chaperoning cycle. This chaperone network is clearly predominant and essential for cell viability, and there is little overlap among the chapters. Little was known about eukaryotic NEFs until recently, but some have emerged (Bag1, HspBP1) with quite curious properties (J. Brodsky and A. Bacher). Rather than simply accelerating the Hsp70 cycle, their roles may be more regulatory and they may promote or inhibit a particular chaperoning event. Numerous Hsp40 proteins have been identified (M.F.N. Rosser and D.M. Cyr). While they stimulate the Hsp70 cycle though their J domains, their roles in client recognition and their possible chaperoning functions on their own are actively being investigated. A chapter on the ER compartment concerns mainly the Hsp70 family and their Hsp40 and NEF cochaperones (J. Dudek et al.). Much remains unknown in this complex system as to the way these proteins interact with one another and their functional roles, other than in protein import. The Hsp70 systems are also the focus in the mitochondria (D. Bursac and T. Lithgow). While the useful analogies to bacterial chaperoning are evident, mitochondria require additional chaperoning machinery, particularly for protein import. Of the many cochaperones for Hsp90, four very distinct groups were included in this volume. The first concerns the large FKBP immunophilins (M.B. Cox and D.F. Smith). These were identified early and much is known on their structures and biological importance. However, their mechanisms of action still remain vague. Similar statements can be made for Cdc37, the first Hsp90 cochaperone to be recognized (A.J. Caplan). While this protein is essential for the chaperoning of protein kinases, its functions in relation to the other components of the chaperone machine remain unclear. The cochaperone UNC-45 (O.O. Odunuga and H.F. Epstein) is in a novel group of proteins having “UCS” domains which are involved in the chaperoning of myosins, and UNC-45 impacts many cellular processes such as muscle function, development, and cytokinesis. A more thoroughly studied protein, Hop, is a cochaperone for both Hsp70 and Hsp90 and it appears to coordinate their functions (S. Daniel et al.). However, recent observations on the interaction of Hop with additional proteins and the changes in its phosphorylation and cellular localization suggest more expanded roles for this protein. Of the remaining chapters, there is one chapter on GroES, the cochaperone for GroEL (H. Liu and P.A. Lund). GroES is the best understood cochaperone in terms of structure/function relationships, but there are still many unknowns, most notably with homologous systems in cell organelles. The last two chapters are particularly thought-provoking since they are on subjects that are very incomplete but are growing rapidly. Chapter 10 is on the link between chaperones and the ubiquitin/proteasome degradation system (J. Hohfeld et al.). While chaperone functions clearly support this system, much remains unknown on the cochaperone participants and the principles that dictate the fate of client proteins. The final chapter addresses our knowledge on chaperones and protein folding diseases (J. van der Spuy et al.). This topic is at a more descriptive phase that has focused mainly on Hsp70. However, some interesting examples are noted which correlate mutations in chaperones or cochaperones to disease states. There are no weak chapters in this volume and it is highly recommended for both newcomers and senior investigators in this field. Perhaps, its only significant shortcoming is that it could easily have been expanded to two or three times its size and scope. We now know of many chaperone systems that are important to various cellular processes and each has a battery of supporting cochaperones. For examples, there are about two dozen known cochaperones that just serve the Hsp90 machine, and full coverage would be too much to ask and probably would not be desirable. Perhaps a sequel on the subject will be considered. While this volume is available in hard copy, the chapters may also be obtained from the Eurekah Bioscience Database at Eurekah.com.
- Published
- 2008
43. Functioning of the Hsp90 machine in chaperoning checkpoint kinase I (Chk1) and the progesterone receptor (PR)
- Author
-
Sara J, Felts, Larry M, Karnitz, and David O, Toft
- Subjects
Homeodomain Proteins ,Protein Folding ,Chaperonins ,Lactams, Macrocyclic ,Tumor Suppressor Proteins ,Cell Cycle Proteins ,Original Articles ,HSP40 Heat-Shock Proteins ,Models, Biological ,Protein Transport ,Checkpoint Kinase 1 ,Benzoquinones ,Animals ,Humans ,HSP70 Heat-Shock Proteins ,HSP90 Heat-Shock Proteins ,Receptors, Progesterone ,Chickens ,Protein Kinases ,Protein Binding - Abstract
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.
- Published
- 2008
44. Analysis of Hsp90 cochaperone interactions reveals a novel mechanism for TPR protein recognition
- Author
-
Bridget Stensgard, Elizabeth S. Bruinsma, David O. Toft, and Ahmed Chadli
- Subjects
Models, Molecular ,Plasma protein binding ,Biochemistry ,Models, Biological ,Protein Structure, Secondary ,Tacrolimus Binding Proteins ,Protein structure ,polycyclic compounds ,HSP90 Heat-Shock Proteins ,Binding site ,chemistry.chemical_classification ,Binding Sites ,biology ,Intracellular Signaling Peptides and Proteins ,FKBP52 ,Hsp90 ,Amino acid ,Cell biology ,Protein Structure, Tertiary ,Tetratricopeptide ,chemistry ,Chaperone (protein) ,biology.protein ,Mutagenesis, Site-Directed ,Receptors, Progesterone ,Molecular Chaperones ,Protein Binding - Abstract
The chaperone Hsp90 is required for the appropriate regulation of numerous key signaling molecules, including the progesterone receptor (PR). Many important cochaperones bind Hsp90 through their tetratricopeptide repeat (TPR) domains. Two such proteins, GCUNC45 and FKBP52, assist PR chaperoning and are thought to interact sequentially with PR-Hsp90 complexes. TPR proteins bind to the C-terminal MEEVD sequence of Hsp90, but GCUNC45 has been shown also to bind to a novel site near the N-terminus. We now show that FKBP52 is also able to bind to this site, and that these two cochaperones act competitively, through Hsp90, to modulate PR activity. The N-terminal site involves noncontiguous amino acids within or near the ATP binding pocket of Hsp90. TPR interactions at this site are thus strongly regulated by nucleotide binding and Hsp90 conformation. We propose an expanded model for client chaperoning in which the coordinated use of TPR recognition sites at both N- and C-terminal ends of Hsp90 enhances its ability to coordinate interactions with multiple TPR partners.
- Published
- 2008
45. Purification of unactivated progesterone receptor and identification of novel receptor-associated proteins
- Author
-
David O. Toft, Lee E. Faber, and David F. Smith
- Subjects
Gel electrophoresis ,Chemistry ,Binding protein ,Cell Biology ,Biochemistry ,Molecular biology ,In vitro ,Cytosol ,Heat shock protein ,Progesterone receptor ,Receptor ,Molecular Biology ,Polyacrylamide gel electrophoresis - Abstract
Progesterone receptor complexes were purified from crude cytosol in a rapid, gentle, one-step procedure using anti-receptor monoclonal antibodies covalently attached to an agarose resin. Five nonreceptor proteins specifically co-purified with unactivated avian progesterone receptor; these proteins had molecular masses of approximately 90, 70, 54, 50, and 23 kDa. The 90- and 70-kDa proteins have been previously identified as the 90-kDa heat shock protein and a member of the 70-kDa heat shock protein family, respectively. The 54-, 50-, and 23-kDa proteins have not been previously described as associated with avian progesterone receptor. Two-dimensional gel electrophoresis revealed charge heterogeneities for all five proteins. Except for p70, each could be dissociated from receptor by salt, a process inhibited by sodium molybdate. However, molybdate was not required for protein association with receptor in low ionic strength. Following progesterone treatment in vivo p70 still co-purified with cytosolic receptor although the other affiliated proteins were reduced, suggesting hormone-dependent dissociation in conjunction with receptor activation. One of the proteins, p54, displayed in vitro hormone-dependent dissociation which was not prevented by molybdate.
- Published
- 1990
46. Progesterone-Induced Avidin as a Marker of Cytodifferentiation in the Oviduct: Comparison to Ovalbumin*
- Author
-
Pentti Tuohimaa, Timo Ylikomi, R. A. Keinanen, David O. Toft, Markku S. Kulomaa, and Timo Joensuu
- Subjects
medicine.medical_specialty ,Ovalbumin ,medicine.drug_class ,Diethylstilbestrol ,Mitosis ,Oviducts ,Immunoenzyme Techniques ,Endocrinology ,Internal medicine ,Progesterone receptor ,medicine ,Animals ,Sexual Maturation ,Progesterone ,biology ,Histocytochemistry ,Cell Differentiation ,Epithelial Cells ,respiratory system ,Avidin ,Epithelium ,medicine.anatomical_structure ,Estrogen ,biology.protein ,Immunohistochemistry ,Oviduct ,Female ,Receptors, Progesterone ,Chickens ,medicine.drug - Abstract
Immunohistochemical analysis of avidin and ovalbumin expression in the normally developing chick oviduct was compared to those changes induced by exogenous estrogen. Oviduct maturation was found to occur in two consecutive phases: slow proliferation and rapid differentiation. Mitosis was induced in the epithelium by estrogen, whereas it was inhibited by progesterone. Endogenous progesterone may retard the proliferation and prevent the differentiation, an effect that is overridden by increased estrogen concentration at the beginning of differentiation. Short secondary stimulation was shown to closely mimic normal maturation. When chicks treated with diethylstilbestrol (DES) for 1 month were allowed to mature, there were marked alterations in oviduct histology and laying behavior. The tubular glands were found to form from the surface epithelium as budlike invaginations, and these cells also contained avidin and ovalbumin. Ovalbumin production was stable in tubular glands. In contrast, the intensity of avidin staining was variable between gland cells even in the same sections. It was conspicuous that the number of avidin-expressing gland cells diminished markedly when estrogen treatment was prolonged over 1 week. After 2-week stimulation with DES, avidin was expressed predominantly by cells of the basal layer of pseudostratified surface epithelium, and ovalbumin mainly by tubular glands and cells of the luminal layer of surface epithelium. Neither of these proteins was expressed by goblet cells. Expression of progesterone receptor, characterized by two antibodies (polyclonal IgG-RB and monoclonal PR6), did not explain the heterogeneity of expression of avidin and ovalbumin, but probably reflects various differentiation stages of epithelial cells.
- Published
- 1990
47. GCUNC-45 Is a Novel Regulator for the Progesterone Receptor/hsp90 Chaperoning Pathway
- Author
-
M. Greg Abel, Ahmed Chadli, Twila A. Jackson, J. Dinny Graham, David O. Toft, David F. Gordon, Kathryn B. Horwitz, William M. Wood, and Sara J. Felts
- Subjects
Cytoplasm ,Molecular Sequence Data ,Regulator ,Tacrolimus Binding Proteins ,Progesterone receptor ,Animals ,Humans ,Amino Acid Sequence ,HSP90 Heat-Shock Proteins ,Binding site ,Cloning, Molecular ,Receptor ,Molecular Biology ,Cells, Cultured ,Binding Sites ,biology ,Intracellular Signaling Peptides and Proteins ,Signal transducing adaptor protein ,Cell Biology ,Articles ,FKBP52 ,Hsp90 ,Heterotrimeric GTP-Binding Proteins ,Protein Structure, Tertiary ,Adaptor Proteins, Vesicular Transport ,Biochemistry ,biology.protein ,Signal transduction ,Receptors, Progesterone ,Molecular Chaperones ,Signal Transduction - Abstract
The hsp90 chaperoning pathway is a multiprotein system that is required for the production or activation of many cell regulatory proteins, including the progesterone receptor (PR). We report here the identity of GCUNC-45 as a novel modulator of PR chaperoning by hsp90. GCUNC-45, previously implicated in the activities of myosins, can interact in vivo and in vitro with both PR-A and PR-B and with hsp90. Overexpression and knockdown experiments show GCUNC-45 to be a positive factor in promoting PR function in the cell. GCUNC-45 binds to the ATP-binding domain of hsp90 to prevent the activation of its ATPase activity by the cochaperone Aha1. This effect limits PR chaperoning by hsp90, but this can be reversed by FKBP52, a cochaperone that is thought to act later in the pathway. These findings reveal a new cochaperone binding site near the N terminus of hsp90, add insight on the role of FKBP52, and identify GCUNC-45 as a novel regulator of the PR signaling pathway.
- Published
- 2006
48. Chaperoning checkpoint kinase 1 (Chk1), an Hsp90 client, with purified chaperones
- Author
-
Bridget Stensgard, Sara J. Felts, Jill M. Wagner, Sonnet J.H. Arlander, David O. Toft, and Larry M. Karnitz
- Subjects
animal structures ,Biology ,environment and public health ,Biochemistry ,Cell-free system ,Serine ,Heat shock protein ,Humans ,CHEK1 ,HSP90 Heat-Shock Proteins ,Casein Kinase II ,Molecular Biology ,Heat-Shock Proteins ,Cell-Free System ,Kinase ,Cell Biology ,Hsp90 ,enzymes and coenzymes (carbohydrates) ,CDC37 ,embryonic structures ,Checkpoint Kinase 1 ,biology.protein ,biological phenomena, cell phenomena, and immunity ,Casein kinase 2 ,Protein Kinases ,HeLa Cells ,Molecular Chaperones - Abstract
Checkpoint kinase 1 (Chk1), a serine/threonine kinase that regulates DNA damage checkpoints, is destabilized when heat shock protein 90 (Hsp90) is inhibited, suggesting that Chk1 is an Hsp90 client. In the present work we examined the interplay between Chk1 and Hsp90 in intact cells, identified a source of unchaperoned Chk1, and report the in vitro chaperoning of Chk1 in reticulocyte lysates and with purified chaperones and co-chaperones. We find that bacterially expressed Chk1 is post-translationally chaperoned to an active kinase. This reaction minimally requires Hsp90, Hsp70, Hsp40, Cdc37, and the protein kinase CK2. The co-chaperone Hop, although not essential for the activation of Chk1 in vitro, enhanced the chaperoning process, whereas the co-chaperone p23 did not stimulate the chaperoning reaction. Additionally, we found that the C-terminal regulatory domain of Chk1 affects the association of Chk1 with Hsp90. Collectively these results provide new insights into Hsp90-dependent chaperoning of a client kinase and identify a novel, biochemically tractable model system that will be useful to further dissect the Hsp90-dependent chaperoning of this important and ubiquitous class of Hsp90 clients.
- Published
- 2005
49. Phase I trial of 17-allylamino-17-demethoxygeldanamycin in patients with advanced cancer
- Author
-
Bridget Stensgard, Matthew M. Ames, Araba A. Adjei, Vlad C. Vasile, Matthew P. Goetz, David O. Toft, Jeff A. Sloan, Sandra Salazaar, Joel M. Reid, Pamela J. Atherton, Stephanie L. Safgren, Gary A. Croghan, Alex A. Adjei, and Charles Erlichman
- Subjects
Adult ,Male ,Cancer Research ,Maximum Tolerated Dose ,Anemia ,Bilirubin ,Nausea ,Lactams, Macrocyclic ,Antineoplastic Agents ,Pharmacology ,Tanespimycin ,Peripheral blood mononuclear cell ,Drug Administration Schedule ,chemistry.chemical_compound ,Pharmacokinetics ,Cytochrome P-450 Enzyme System ,Neoplasms ,polycyclic compounds ,medicine ,Benzoquinones ,NAD(P)H Dehydrogenase (Quinone) ,Cytochrome P-450 CYP3A ,Humans ,HSP90 Heat-Shock Proteins ,Aged ,Polymorphism, Genetic ,business.industry ,Middle Aged ,medicine.disease ,Oncology ,chemistry ,Rifabutin ,Immunology ,Toxicity ,Vomiting ,Female ,medicine.symptom ,business ,Biomarkers - Abstract
Purpose We determined the maximum-tolerated dose (MTD) and the dose-limiting toxicities (DLT) of 17-allylamino-17-demethoxygeldanamycin (17-AAG) when infused on days 1, 8, and 15 of a 28-day cycle in advanced solid tumor patients. We also characterized the pharmacokinetics of 17-AAG, its effect on chaperone and client proteins, and whether cytochrome P450 (CYP) 3A5 and NAD(P)H:quinone oxidoreductase 1 (NQO1) polymorphisms affected 17-AAG disposition or toxicity. Patients and Methods An accelerated titration design was used. Biomarkers were measured in peripheral-blood mononuclear cells (PBMCs) at baseline and on days 1 and 15, and pharmacokinetic analysis was performed on day 1 of cycle 1. CYP3A5*3 and NQO1*2 genotypes were determined and correlated with pharmacokinetics and toxicity. Results Twenty-one patients received 52 courses at 11 dose levels. DLTs at 431 mg/m2 were grade 3 bilirubin (n = 1), AST (n = 1), anemia (n = 1), nausea (n = 1), vomiting (n = 1), and myalgias (n = 1). No tumor responses were seen. 17-AAG consistently increased heat shock protein (Hsp) 70 levels in PBMCs. At the MTD, the clearance and half-life (t1/2) of 17-AAG were 11.6 L/h/m2 and 4.15 hours, respectively; whereas the active metabolite 17-aminogeldanamycin had a t1/2 of 7.63 hours. The CYP3A5*3 and NQO1*2 polymorphisms were not associated with 17-AAG toxicity. The CYP3A5*3 polymorphism was associated with higher 17-AAG clearance. Conclusion The MTD of weekly 17-AAG is 308 mg/m2. 17-AAG induced Hsp70 in PBMCs, indicating that Hsp90 has been affected. Further evaluation of 17-AAG is ongoing using a twice-weekly regimen, and this schedule of 17-AAG is being tested in combination with chemotherapy.
- Published
- 2005
50. Molecular Chaperone Machines: Chaperone Activities of the Cyclophilin Cyp-40 and the Steroid Aporeceptor-Associated Protein p23
- Author
-
Brian C. Freeman, David O. Toft, and Richard I. Morimoto
- Subjects
Multidisciplinary ,biology ,medicine.medical_treatment ,Hsp90 ,Co-chaperone ,Steroid hormone ,Biochemistry ,Nuclear receptor ,Chaperone (protein) ,Heat shock protein ,Hsp33 ,biology.protein ,medicine ,Cyclophilin - Abstract
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.
- Published
- 1996
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.