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Biomedical subjects

Paul Webb

Publications and source records attributed to Paul Webb.

At least 19 recordsLinked to original sources
PubMed2006

Corepressor/coactivator paradox: potential constitutive coactivation by corepressor splice variants.

The functional consequences of the interaction of transcriptional coregulators with the human thyroid hormone receptor (TR) in mammalian cells are complex. We have used the yeast, Saccharomyces cerevisiae, which lack endogenous nuclear receptors (NRs) and NR coregulators, as a model to decipher mechanisms regulating transcriptional activation by TR. In effect, this system allows the reconstitution of TR mediated transcription complexes by the expression of specific combinations of mammalian proteins in yeast. In this yeast system, human adenovirus 5 early region 1A (E1A), a natural N-CoR splice variant (N-CoR(I)) or an artificial N-CoR truncation (N-CoR(C)) coactivate unliganded TRs and these effects are inhibited by thyroid hormone (TH). E1A contains a short peptide sequence that resembles known corepressor-NR interaction motifs (CoRNR box motif, CBM), and this motif is required for TR binding and coactivation. N-CoR(I) and N-CoR(C) contain three CBMs, but only the C-terminal CBM1 is critical for coactivation. These observations in a yeast model system suggest that E1A and N-CoR(I) are naturally occurring TR coactivators that bind in the typical corepressor mode. These findings also raise the possibility that alternative splicing events which form corepressor proteins containing only C-terminal CBM motifs could represent a novel mechanism in mammalian cells for regulating constitutive transcriptional activation by TRs.

Journal Article↗
PubMed2006

A three-compartment thermometry model for the improved estimation of changes in body heat content.

The aim of this study was to use whole body calorimetry to directly measure the change in body heat content (DeltaH(b)) during steady-state exercise and compare these values with those estimated using thermometry. The thermometry models tested were the traditional two-compartment model of "core" and "shell" temperatures, and a three-compartment model of "core," "muscle," and "shell" temperatures; with individual compartments within each model weighted for their relative influence upon DeltaH(b) by coefficients subject to a nonnegative and a sum-to-one constraint. Fifty-two participants performed 90 min of moderate-intensity exercise (40% of Vo(2 peak)) on a cycle ergometer in the Snellen air calorimeter, at regulated air temperatures of 24 degrees C or 30 degrees C and a relative humidity of either 30% or 60%. The "core" compartment was represented by temperatures measured in the esophagus (T(es)), rectum (T(re)), and aural canal (T(au)), while the "muscle" compartment was represented by regional muscle temperature measured in the vastus lateralis (T(vl)), triceps brachii (T(tb)), and upper trapezius (T(ut)). The "shell" compartment was represented by the weighted mean of 12 skin temperatures (T(sk)). The whole body calorimetry data were used to derive optimally fitting two- and three-compartment thermometry models. The traditional two-compartment model was found to be statistically biased, systematically underestimating DeltaH(b) by 15.5% (SD 31.3) at 24 degrees C and by 35.5% (SD 21.9) at 30 degrees C. The three-compartment model showed no such bias, yielding a more precise estimate of DeltaH(b) as evidenced by a mean estimation error of 1.1% (SD 29.5) at 24 degrees C and 5.4% (SD 30.0) at 30 degrees C with an adjusted R(2) of 0.48 and 0.51, respectively. It is concluded that a major source of error in the estimation of DeltaH(b) using the traditional two-compartment thermometry model is the lack of an expression independently representing the heat storage in muscle during exercise.

Absorptiometry, Photon↗
PubMed2006

The Snellen human calorimeter revisited, re-engineered and upgraded: design and performance characteristics.

The measurement of whole body heat loss in humans and the performance characteristics of a modified Snellen whole body air calorimeter are described. Modifications included the location of the calorimeter in a pressurized room, control of operating temperature over a range of - 15 to + 35 degrees C, control of ambient relative humidity over a range of 20-65%, incorporation of an air mass flow measuring system to provide real time measurement of air mass flow through the calorimeter, incorporation of a constant load 'eddy current' resistance ergometer and an open circuit, expired gas analysis calorimetry system. The performance of the calorimeter is a function of the sensitivity, precision, accuracy and response time characteristics of the fundamental measurement systems including: air mass flow; thermometry and hygrometry. Calibration experiments included a calibration of the air mass flow sensor, the response of the thermometric measurement system for dry heat loss and the response of the hygrometric measurement system for evaporative heat loss. The air mass flow system was evaluated using standard differential temperature procedures to demonstrate linearity and sensitivity of the device. A novel procedure based on differential hygrometry was developed to ascertain the absolute calibration of air mass flow by resolving the unique system coefficient K. The results of the hygrometric calibration demonstrate the air mass flow response of the system is linear over the range of air mass flows from 6 to 15 kg min(-1). R(2) was 0.995. The average half response time (tR50) was 14.5 +/- 2.1 s. Similarly the results of the thermometric calibration demonstrate that the response of the apparatus is linear over the range of power input measured (coefficient of linearity R(2)=0.9997) with a precision of 0.72 W and an accuracy to within 0.36 W. The average (tR50) over all conditions was 6.0 +/- 1.9 min. In summary, modifications brought to the Snellen calorimeter have significantly improved the precision, accuracy and response time characteristics of the previous system while extending its operating range.

Air Conditioning↗
PubMed2006

A high-throughput screening method to identify small molecule inhibitors of thyroid hormone receptor coactivator binding.

To provide alternative methods for regulation of gene transcription initiated by the binding of thyroid hormone (T3) to the thyroid receptor (TR), we have developed a high-throughput method for discovering inhibitors of the interaction of TR with its transcriptional coactivators. The screening method is based on fluorescence polarization (FP), one of the most sensitive and robust high-throughput methods for the study of protein-protein interactions. A fluorescently labeled coactivator is excited by polarized light. The emitted polarized light is a function of the molecular properties of the labeled coactivator, especially Brownian molecular rotation, which is very sensitive to changes in the molecular mass of the labeled complex. Dissociation of hormone receptor from fluorescently labeled coactivator peptide in the presence of small molecules can be detected by this competition method, and the assay can be performed in a high-throughput screening format. Hit compounds identified by this method are evaluated by several secondary assay methods, including a dose-response analysis, a semiquantitative glutathione-S-transferase assay, and a hormone displacement assay. Subsequent in vitro transcription assays can detect inhibition of thyroid signaling at low micromolar concentrations of small molecules in the presence of T3.

Acetyltransferases↗
PubMed2006

Negative regulation of superoxide dismutase-1 promoter by thyroid hormone.

The role of thyroid hormone [L-3,5,3'-triiodothyronine (T3)] and the thyroid hormone receptor (TR) in regulating growth, development, and metabolic homeostasis is well established. It is also emerging that T3 is associated with oxidative stress through the regulation of the activity of superoxide dismutase-1 (SOD-1), a key enzyme in the metabolism of oxygen free radicals. We found that T3 reverses the activation of the SOD-1 promoter caused by the free radical generators paraquat and phorbol 12-myristate 13-acetate through the direct repression of the SOD-1 promoter by liganded TR. Conversely, the SOD-1 promoter is significantly stimulated by unliganded TRs. This regulation requires the DNA-binding domain of the TR, which is recruited to an inhibitory element between -157 and +17 of the SOD-1 promoter. TR mutations, which abolish recruitment of coactivator proteins, block repression of the SOD-1 promoter. Conversely, a mutation that inhibits corepressor binding to the TR prevents activation. Together, our findings suggest a mechanism of negative regulation in which TR binds to the SOD-1 promoter but coactivator and corepressor binding surfaces have an inverted function. This effect may be important in T3 induction of oxidative stress in thyroid hormone excess.

Animals↗
PubMed2006

Insulation disks on the skin to estimate muscle temperature.

This study examined the use of insulation disks placed on the skin to estimate muscle temperature in resting subjects exposed to a thermoneutral (28 degrees C) ambient environment. The working hypothesis was that the skin temperature under each insulation disk would increase to a value corresponding to a specific muscle temperature measured by a control probe at 0.8+/-0.2, 1.3+/-0.2, 1.8+/-0.2, 2.3+/-0.2, and 2.8+/-0.2 cm below the skin surface. Eight subjects sat for 120 min while lateral thigh skin temperatures and vastus lateralis muscle temperature were directly measured. Vastus lateralis temperature was estimated non-invasively using two 5 cm diameter foam neoprene disks which were placed on top of the skin temperature probes (from time 60 to 120 min) located at 15.3 and 26.3 cm superior to the patella. The disks at the two locations were 3.2 and 4.8 mm thick, respectively. The placement of the 3.2- and 4.8-mm disks on the thigh for a minimum of 15 and 20 min, respectively, resulted in an increase in skin temperature under the disks which corresponded to the lateral thigh muscle temperature measured directly and invasively at 0.8+/-0.2 and 1.3+/-0.2 cm, respectively, below the skin.

Adult↗
PubMed2006

Structural rearrangements in the thyroid hormone receptor hinge domain and their putative role in the receptor function.

The thyroid hormone receptor (TR) D-domain links the ligand-binding domain (LBD, EF-domain) to the DNA-binding domain (DBD, C-domain), but its structure, and even its existence as a functional unit, are controversial. The D domain is poorly conserved throughout the nuclear receptor family and was originally proposed to comprise an unfolded hinge that facilitates rotation between the LBD and the DBD. Previous TR LBD structures, however, have indicated that the true unstructured region is three to six amino acid residues long and that the D-domain N terminus folds into a short amphipathic alpha-helix (H0) contiguous with the DBD and that the C terminus of the D-domain comprises H1 and H2 of the LBD. Here, we solve structures of TR-LBDs in different crystal forms and show that the N terminus of the TRalpha D-domain can adopt two structures; it can either fold into an amphipathic helix that resembles TRbeta H0 or form an unstructured loop. H0 formation requires contacts with the AF-2 coactivator-binding groove of the neighboring TR LBD, which binds H0 sequences that resemble coactivator LXXLL motifs. Structural analysis of a liganded TR LBD with small angle X-ray scattering (SAXS) suggests that AF-2/H0 interactions mediate dimerization of this protein in solution. We propose that the TR D-domain has the potential to form functionally important extensions of the DBD and LBD or unfold to permit TRs to adapt to different DNA response elements. We also show that mutations of the D domain LXXLL-like motif indeed selectively inhibit TR interactions with an inverted palindromic response element (F2) in vitro and TR activity at this response element in cell-based transfection experiments.

Amino Acid Motifs↗
PubMed2006

Molecular dynamics simulations of ligand dissociation from thyroid hormone receptors: evidence of the likeliest escape pathway and its implications for the design of novel ligands.

Steered molecular dynamics simulations of ligand dissociation from Thyroid hormone receptors indicate that dissociation is favored via rearrangements in a mobile part of the LBD comprising H3, the loop between H1 and H2, and nearby beta-sheets, contrary to current models in which the H12 is mostly involved. Dissociation is facilitated in this path by the interaction of the hydrophilic part of the ligand with external water molecules, suggesting strategies to enhance ligand binding affinity.

Computer Simulation↗
PubMed2006

Monitoring for bovine spongiform encephalopathy in sheep in Great Britain, 1998-2004.

Bovine spongiform encephalopathy (BSE) may have been transmitted to British sheep via contaminated feed in the 1980s. Strain-typing techniques based on immunohistochemical (IHC) detection of abnormal protein (PrP(d)) and the molecular analysis of proteinase-resistant protein (PrP(res)) by Western blotting (WB) can discriminate between natural or experimental scrapie and experimental BSE in sheep. Between 1 January 1998 and 31 October 2001, 1247 sheep, clinically suspected of scrapie, were found to be positive by statutory tests in Great Britain. Archived brain tissue from these cases was retested by using these discriminatory methods. Twelve brain samples showed PrP(res) WB patterns that were unlike those found in natural or experimental scrapie. Prospective screening of fresh tissue from a further 1121 scrapie cases was also carried out between 1 November 2001 and 31 May 2004. Two samples gave WB results with similarities to the results found for experimental BSE in sheep. When all 14 unusual cases were tested by IHC, no match to experimental BSE in sheep was found. There were uncertainties within the retrospective study, where some equivocal results were obtained due to poor tissue quality or the unavailability of the optimum brain region. However, for the samples where tissue condition was optimum, our results provide no evidence for the presence of BSE in sheep. Epidemiological interpretation of the 450 flocks sampled indicates that the maximum proportion of sheep transmissible spongiform encephalopathy cases that could be BSE is 0.66%. This estimate is lower than calculated previously (5%), when the analysis was based on the results of strain typing in mice.

Animals↗
PubMed2006

Human thyroid receptor forms tetramers in solution, which dissociate into dimers upon ligand binding.

Thyroid hormone nuclear receptors (TRs) bind to DNA and activate transcription as heterodimers with the retinoid X receptor (RXR) or as homodimers or monomers. RXR also binds to DNA and activates transcription as homodimers but can, in addition, self-associate into homotetramers in the absence of ligand and DNA templates. It is thought that homotetramer formation serves to sequester excess RXRs into an inactive pool within the cell. Here, we report systematic studies of the multimeric state of a recombinant human TRbeta1 truncation (hTRbeta1deltaAB) that encompasses the complete DNA binding domain and ligand binding domain in solution. Native gel electrophoresis, chemical crosslinking, gel filtration, and dynamic light scattering experiments reveal that hTRbeta1deltaAB forms a mixture of monomers, dimers, and tetramers. Like RXR, increasing protein concentration shifts the equilibrium between TR multimers toward tetramer formation, whereas binding of cognate thyroid hormone leads to dissociation of tetramers and increased formation of dimers. This work represents the first evidence that apo-hTRbeta1 forms homotetramers. The findings raise the possibility that tetramer formation provides an additional, and previously unsuspected, level of control of TR activity and that the capacity for homotetramer formation may be more widespread in the nuclear receptor family than previously thought.

Amino Acid Sequence↗
PubMed2005

Discovery of small molecule inhibitors of the interaction of the thyroid hormone receptor with transcriptional coregulators.

Thyroid hormone (3,5,3'-triiodo-L-thyronine, T3) is an endocrine hormone that exerts homeostatic regulation of basal metabolic rate, heart rate and contractility, fat deposition, and other phenomena (1, 2). T3 binds to the thyroid hormone receptors (TRs) and controls their regulation of transcription of target genes. The binding of TRs to thyroid hormone induces a conformational change in TRs that regulates the composition of the transcriptional regulatory complex. Recruitment of the correct coregulators (CoR) is important for successful gene regulation. In principle, inhibition of the TR-CoR interaction can have a direct influence on gene transcription in the presence of thyroid hormones. Herein we report a high throughput screen for small molecules capable of inhibiting TR coactivator interactions. One class of inhibitors identified in this screen was aromatic beta-aminoketones, which exhibited IC50 values of approximately 2 microm. These compounds can undergo a deamination, generating unsaturated ketones capable of reacting with nucleophilic amino acids. Several experiments confirm the hypothesis that these inhibitors are covalently bound to TR. Optimization of these compounds produced leads that inhibited the TR-CoR interaction in vitro with potency of approximately 0.6 microm and thyroid signaling in cellular systems. These are the first small molecules irreversibly inhibiting the coactivator binding of a nuclear receptor and suppressing its transcriptional activity.

Binding, Competitive↗
PubMed2005

Selective thyroid receptor modulation by GC-1 reduces serum lipids and stimulates steps of reverse cholesterol transport in euthyroid mice.

Thyroid hormones [predominantly 3,5,3'-triiodo-L-thyronine (T3)] regulate cholesterol and lipoprotein metabolism, but cardiac effects restrict their use as hypolipidemic drugs. T3 binds to thyroid hormone receptors (TRs) alpha and beta. TRbeta is the predominant isoform in liver, whereas T3 effects on heart rate are mediated mostly by TRalpha. Drugs that target TRbeta or exhibit tissue-selective uptake may improve plasma lipid levels while sparing the heart. Here, we asked how the TRbeta- and liver uptake-selective agonist GC-1 influences cholesterol and triglyceride metabolism in euthyroid mice. GC-1 treatment reduced serum cholesterol levels by 25% and serum triglycerides by 75% in chow-fed mice and also attenuated diet-induced hypercholesterolemia. GC-1 reduced plasma high-density lipoprotein cholesterol levels; increased expression of the hepatic high-density lipoprotein receptor, SR-BI; stimulated activity of cholesterol 7alpha-hydroxylase; and increased fecal excretion of bile acids. Collectively, these results suggest that GC-1 stimulates important steps in reverse cholesterol transport. Use of TRbeta and uptake selective agonists such as GC-1 should be further explored as a strategy to improve lipid metabolism in dyslipoproteinemia.

Acetates↗
PubMed2005

Novel, potent small-molecule inhibitors of the molecular chaperone Hsp90 discovered through structure-based design.

The crystal structure of a previously reported screening hit 1 (CCT018159) bound to the N terminal domain of molecular chaperone Hsp90 has been used to design 5-amide analogues. These exhibit enhanced potency against the target in binding and functional assays with accompanying appropriate cellular pharmacodynamic changes. Compound 11 (VER-49009) compares favorably with the clinically evaluated 17-AAG.

Adenosine Triphosphate↗
PubMed2005

Molecular dynamics simulations reveal multiple pathways of ligand dissociation from thyroid hormone receptors.

Nuclear receptor (NR) ligands occupy a pocket that lies within the core of the NR ligand-binding domain (LBD), and most NR LBDs lack obvious entry/exit routes upon the protein surface. Thus, significant NR conformational rearrangements must accompany ligand binding and release. The precise nature of these processes, however, remains poorly understood. Here, we utilize locally enhanced sampling (LES) molecular dynamics computer simulations to predict molecular motions of x-ray structures of thyroid hormone receptor (TR) LBDs and determine events that permit ligand escape. We find that the natural ligand 3,5,3'-triiodo-L-thyronine (T(3)) dissociates from the TRalpha1 LBD along three competing pathways generated through i), opening of helix (H) 12; ii), separation of H8 and H11 and the Omega-loop between H2 and H3; and iii), opening of H2 and H3, and the intervening beta-strand. Similar pathways are involved in dissociation of T(3) and the TRbeta-selective ligand GC24 from TRbeta; the TR agonist IH5 from the alpha- and beta-TR forms; and Triac from two natural human TRbeta mutants, A317T and A234T, but are detected with different frequencies in simulations performed with the different structures. Path I was previously suggested to represent a major pathway for NR ligand dissociation. We propose here that Paths II and III are also likely ligand escape routes for TRs and other NRs. We also propose that different escape paths are preferred in different situations, implying that it will be possible to design NR ligands that only associate stably with their cognate receptors in specific cellular contexts.

Algorithms↗
PubMed2005

Rearrangements in thyroid hormone receptor charge clusters that stabilize bound 3,5',5-triiodo-L-thyronine and inhibit homodimer formation.

In this study, we investigated how thyroid hormone (3,5',5-triiodo-l-thyronine, T3) inhibits binding of thyroid hormone receptor (TR) homodimers, but not TR-retinoid X receptor heterodimers, to thyroid hormone response elements. Specifically we asked why a small subset of TRbeta mutations that arise in resistance to thyroid hormone syndrome inhibit both T3 binding and formation of TRbeta homodimers on thyroid hormone response elements. We reasoned that these mutations may affect structural elements involved in the coupling of T3 binding to inhibition of TR DNA binding activity. Analysis of TR x-ray structures revealed that each of these resistance to thyroid hormone syndrome mutations affects a cluster of charged amino acids with potential for ionic bond formation between oppositely charged partners. Two clusters (1 and 2) are adjacent to the dimer surface at the junction of helices 10 and 11. Targeted mutagenesis of residues in Cluster 1 (Arg338, Lys342, Asp351, and Asp355) and Cluster 2 (Arg429, Arg383, and Glu311) confirmed that the clusters are required for stable T3 binding and for optimal TR homodimer formation on DNA but also revealed that different arrangements of charged residues are needed for these effects. We propose that the charge clusters are homodimer-specific extensions of the dimer surface and further that T3 binding promotes specific rearrangements of these surfaces that simultaneously block homodimer formation on DNA and stabilize the bound hormone. Our data yield insight into the way that T3 regulates TR DNA binding activity and also highlight hitherto unsuspected T3-dependent conformational changes in the receptor ligand binding domain.

Amino Acid Sequence↗
PubMed2005

E1A and a nuclear receptor corepressor splice variant (N-CoRI) are thyroid hormone receptor coactivators that bind in the corepressor mode.

Unliganded thyroid hormone (TH) receptors (TRs) and other nuclear receptors (NRs) repress transcription of hormone-activated genes by recruiting corepressors (CoRs), such as NR CoR (N-CoR) and SMRT. Unliganded TRs also activate transcription of TH-repressed genes. Some evidence suggests that these effects also involve TR/CoR contacts; however, the precise reasons that CoRs activate transcription in these contexts are obscure. Unraveling these mechanisms is complicated by the fact that it is difficult to decipher direct vs. indirect effects of TR-coregulator contacts in mammalian cells. In this study, we used yeast, Saccharomyces cerevisiae, which lack endogenous NRs and NR coregulators, to determine how unliganded TRs can activate transcription. We previously showed that adenovirus 5 early-region 1A coactivates unliganded TRs in yeast, and that these effects are blocked by TH. We show here that human adenovirus type 5 early region 1A (E1A) contains a short peptide (LDQLIEEVL amino acids 20-28) that resembles CoR-NR interaction motifs (CoRNR boxes), and that this motif is required for TR binding and coactivation. Although full-length N-CoR does not coactivate TR in yeast, a naturally occurring N-CoR variant (N-CoR(I)) and an artificial N-CoR truncation (N-CoR(C)) that retain CoRNR boxes but lack N-terminal repressor domains behave as potent and direct TH-repressed coactivators for unliganded TRs. We conclude that E1A and N-CoR(I) are naturally occurring TR coactivators that bind in the typical CoR mode and suggest that similar factors could mediate transcriptional activation by unliganded TRs in mammals.

Adenovirus E1A Proteins↗