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Biomedical subjects

David Toft

Publications and source records attributed to David Toft.

9 recordsLinked to original sources
PubMed2007

An acetylation site in the middle domain of Hsp90 regulates chaperone function.

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 posttranslational modifications that affect its function, including acetylation. Histone deacetylase (HDAC) inhibitors and knockdown 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 WT 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.

Acetylation↗
PubMed2006

Defining the requirements for Hsp40 and Hsp70 in the Hsp90 chaperone pathway.

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.

Animals↗
PubMed2006

GCUNC-45 is a novel regulator for the progesterone receptor/hsp90 chaperoning pathway.

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.

Adaptor Proteins, Vesicular Transport↗
PubMed2005

Phase I trial of 17-allylamino-17-demethoxygeldanamycin in patients with advanced cancer.

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/m(2) 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 (t(1/2)) of 17-AAG were 11.6 L/h/m(2) and 4.15 hours, respectively; whereas the active metabolite 17-aminogeldanamycin had a t(1/2) 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/m(2). 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.

Adult↗
PubMed2005

Structures of the N-terminal and middle domains of E. coli Hsp90 and conformation changes upon ADP binding.

Hsp90 is an abundant molecular chaperone involved in many biological systems. We report here the crystal structures of the unliganded and ADP bound fragments containing the N-terminal and middle domains of HtpG, an E. coli Hsp90. These domains are not connected through a flexible linker, as often portrayed in models, but are intimately associated with one another. The individual HtpG domains have similar folding to those of DNA gyrase B but assemble differently, suggesting somewhat different mechanisms for the ATPase superfamily. ADP binds to a subpocket of a large site that is jointly formed by the N-terminal and middle domains and induces conformational changes of the N-terminal domain. We speculate that this large pocket serves as a putative site for binding of client proteins/cochaperones. Modeling shows that ATP is not exposed to the molecular surface, thus implying that ATP activation of hsp90 chaperone activities is accomplished via conformational changes.

Adenosine Diphosphate↗
PubMed2004

Requirement of heat shock protein 90 for human hepatitis B virus reverse transcriptase function.

The initiation of reverse transcription and nucleocapsid assembly in hepatitis B virus (HBV) depends on the specific recognition of an RNA signal (the packaging signal, epsilon) on the pregenomic RNA (pgRNA) by the viral reverse transcriptase (RT). RT-epsilon interaction in the duck hepatitis B virus (DHBV) was recently shown to require the molecular chaperone complex, the heat shock protein 90 (Hsp90). However, the requirement for RT-epsilon interaction in the human HBV has remained unknown due to the inability to obtain a purified RT protein active in specific epsilon binding. We now report that Hsp90 is also required for HBV RT-epsilon interaction. Inhibition of Hsp90 led to diminished HBV pgRNA packaging into nucleocapsids in cells, which depends on RT-epsilon interaction. Furthermore, using truncated HBV RT proteins purified from bacteria and five purified Hsp90 chaperone factors, we have developed an in vitro RT-epsilon binding assay. Our results demonstrate that Hsp90, in a dynamic process that was dependent on ATP hydrolysis, facilitated RT-epsilon interaction in HBV, as in DHBV. Specific epsilon binding required sequences from both the amino-terminal terminal protein and the carboxy-terminal RT domain. Only the cognate HBV epsilon, but not the DHBV epsilon, could bind the HBV RT proteins. Furthermore, the internal bulge, but not the apical loop, of epsilon was required for RT binding. The establishment of a defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT-epsilon interaction and chaperone activation.

DNA Replication↗
PubMed2003

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

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

Alleles↗
PubMed2002

In vitro reconstitution of functional hepadnavirus reverse transcriptase with cellular chaperone proteins.

Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, epsilon) 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-epsilon 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 epsilon 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.

Chaperonins↗