Hypoxia-inducible factor 1 alpha (HIF-1 alpha) and the intracellular dioxin receptor mediate hypoxia and dioxin signalling, respectively. Both proteins are conditionally regulated basic helix-loop-helix (bHLH) transcription factors that, in addition to the bHLH motif, share a Per-Arnt-Sim (PAS) region of homology and form heterodimeric complexes with the common bHLH/PAS partner factor Arnt. Here we demonstrate that HIF-1 alpha required Arnt for DNA binding in vitro and functional activity in vivo. Both the bHLH and PAS motifs of Arnt were critical for dimerization with HIF-1 alpha. Strikingly, HIF-1 alpha exhibited very high affinity for Arnt in coimmunoprecipitation assays in vitro, resulting in competition with the ligand-activated dioxin receptor for recruitment of Arnt. Consistent with these observations, activation of HIF-1 alpha function in vivo or overexpression of HIF-1 alpha inhibited ligand-dependent induction of DNA binding activity by the dioxin receptor and dioxin receptor function on minimal reporter gene constructs. However, HIF-1 alpha- and dioxin receptor-mediated signalling pathways were not mutually exclusive, since activation of dioxin receptor function did not impair HIF-1 alpha-dependent induction of target gene expression. Both HIF-1 alpha and Arnt mRNAs were expressed constitutively in a large number of human tissues and cell lines, and these steady-state expression levels were not affected by exposure to hypoxia. Thus, HIF-1 alpha may be conditionally regulated by a mechanism that is distinct from induced expression levels, the prevalent model of activation of HIF-1 alpha function. Interestingly, we observed that HIF-1 alpha was associated with the molecular chaperone hsp90. Given the critical role of hsp90 for ligand binding activity and activation of the dioxin receptor, it is therefore possible that HIF-1 alpha is regulated by a similar mechanism, possibly by binding an as yet unknown class of ligands.

In response to hypoxia the hypoxia-inducible factor-1 (HIF-1) mediates transcriptional activation of a network of genes encoding erythropoietin, vascular endothelial growth factor, and several glycolytic enzymes. HIF-1 consists of a heterodimer of two basic helix-loop-helix PAS (Per/Arnt/Sim) proteins, HIF-1alpha and Arnt. HIF-1alpha and Arnt mRNAs are constitutively expressed and were not altered upon exposure of HeLa or HepG2 cells to hypoxia, suggesting that the activity of the HIF-1alpha-Arnt complex may be regulated by some as yet unknown posttranscriptional mechanism. In support of this model, we demonstrate here that Arnt protein levels were not increased under conditions that induce an hypoxic response in HeLa and HepG2 cells. However, under identical conditions, HIF-1alpha protein levels were rapidly and dramatically up-regulated, as assessed by immunoblot analysis. In addition, HIF-1alpha acquired a new conformational state upon dimerization with Arnt, rendering HIF-1alpha more resistant to proteolytic digestion in vitro. Dimerization as such was not sufficient to elicit the conformational change in HIF-1alpha, since truncated forms of Arnt that are capable of dimerizing with HIF-1alpha did not induce this effect. Moreover, the high affinity DNA binding form of the HIF-1alpha-Arnt complex was only generated by forms of Arnt capable of eliciting the allosteric change in conformation. In conclusion, the combination of enhanced protein levels and allosteric change by dimerization defines a novel mechanism for modulation of transcription factor activity.

Background Understanding the longitudinal trajectory of SARS-CoV-2 antibodies is crucial for diagnosis of prior infection and predicting future immunity. Methods We conducted a longitudinal analysis of COVID19 convalescents, using neutralizing antibody assays and SARS-CoV-2 serologic assay platforms employing SARS-CoV-2 spike (S) or nucleocapsid (N) antigens. Results Sensitivities of serologic assays to diagnose prior SARS-CoV-2 infection changed with time. One widely used commercial platform that had an initial sensitivity of >95% declined to 71% at 81-100 days post diagnosis. The trajectories of median binding antibody titers measured over ~3 to 4 months were not dependent on the use of SARS-CoV-2 N or S proteins as antigen. The median neutralization titer decreased by ~45% per month. Each serological assay gave quantitative antibody titers that correlated with SARS-CoV-2 neutralization titers, but S-based serological assay measurements better predicted neutralization potency. Correlation between S-binding and neutralization titers deteriorated with time and decreases in neutralization titers were not predicted by changes in S-binding antibody titers. Conclusions Different SARS-CoV-2 serologic assays are more or less well suited for surveillance versus prediction of serum neutralization potency. Extended follow up should facilitate the establishment of appropriate serologic correlates of protection against SARS-CoV-2 reinfection.

The dioxin/aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor regulating transcription of a battery of genes encoding xenobiotic metabolizing enzymes. Known receptor ligands are environmental pollutants including polycyclic aromatic hydrocarbons and polychlorinated dioxins. Loss-of-function (gene-disruption) studies in mice have demonstrated that the AhR is involved in toxic effects of dioxins but have not yielded unequivocal results concerning the physiological function of the receptor. Gain-of-function studies therefore were performed to unravel the biological functions of the AhR. A constitutively active AhR expressed in transgenic mice reduced the life span of the mice and induced tumors in the glandular part of the stomach, demonstrating the oncogenic potential of the AhR and implicating the receptor in regulation of cell proliferation.

Objectives:To investigate longitudinal trajectory of SARS-CoV-2 neutralising antibodies and the performance of serological assays in diagnosing prior infection and predicting serum neutralisation titres with time Design Retrospective longitudinal analysis of a COVID19 case cohort . Setting NHS outpatient clinics Participants Individuals with RT-PCR diagnosed SARS-CoV-2 infection that did not require hospitalization Main outcome measures The sensitivity with which prior infection was detected and quantitative antibody titres were assessed using four SARS-CoV-2 serologic assay platforms. Two platforms employed SARS-CoV-2 spike (S) based antigens and two employed nucleocapsid (N) based antigens. Serum neutralising antibody titres were measured using a validated pseudotyped virus SARS-CoV-2 neutralisation assay. The ability of the serological assays to predict neutralisation titres at various times after PCR diagnosis was assessed. Results The three of the four serological assays had sensitivities of 95 to100% at 21-40 days post PCR-diagnosis, while a fourth assay had a lower sensitivity of 85%. The relative sensitivities of the assays changed with time and the sensitivity of one assay that had an initial sensitivity of >95% declined to 85% at 61-80 post PCR diagnosis, and to 71% at 81-100 days post diagnosis. Median antibody titres decreased in one serologic assay but were maintained over the observation period in other assays. The trajectories of median antibody titres measured in serologic assays over this time period were not dependent on whether the SARS-CoV-2 N or S proteins were used as antigen source. A broad range of SARS-CoV-2 neutralising titres were evident in individual sera, that decreased over time in the majority of participants; the median neutralisation titre in the cohort decreased by 45% over 4 weeks. Each of the serological assays gave quantitative measurements of antibody titres that correlated with SARS-CoV-2 neutralisation titres, but, the S-based serological assay measurements better predicted serum neutralisation potency. The strength of correlation between serologic assay results and neutralisation titres deteriorated with time and decreases in neutralisation titres in individual participants were not well predicted by changes in antibody titres measured using serologic assays. Conclusions: SARS-CoV-2 serologic assays differed in their comparative diagnostic performance over time. Different assays are more or less well suited for surveillance of populations for prior infection versus prediction of serum neutralisation potency. Continued monitoring of declining neutralisation titres during extended follow up should facilitate the establishment of appropriate serologic correlates of protection against SARS-CoV-2 reinfection.

The intracellular dioxin receptor mediates signal transduction by dioxin and functions as a ligand-activated transcription factor. It contains a basic helix-loop-helix (bHLH) motif contiguous with a Per-Arnt-Sim (PAS) homology region. In extracts from nonstimulated cells the receptor is recovered in an inducible cytoplasmic form associated with the 90-kDa heat shock protein (hsp90), a molecular chaperone. We have reconstituted ligand-dependent activation of the receptor to a DNA-binding form by using the dioxin receptor and its bHLH-PAS partner factor Arnt expressed by in vitro translation in reticulocyte lysate. Deletion of the PAS domain of the receptor resulted in constitutive dimerization with Arnt. In contrast, this receptor mutant showed low levels of xenobiotic response element-binding activity, indicating that the PAS domain may be important for DNA-binding affinity and/or specificity of the receptor. It was not possible to reconstitute dioxin receptor function with proteins expressed in wheat germ lysate. In line with these observations, reticulocyte lysate but not wheat germ lysate promoted the association of de novo synthesized dioxin receptor with hsp90. At least two distinct domains of the receptor mediated interaction with hsp90: the ligand-binding domain located within the PAS region and, surprisingly, the bHLH domain. Whereas ligand-binding activity correlated with association with hsp90, bHLH-hsp90 interaction appeared to be important for DNA-binding activity but not for dimerization of the receptor. Several distinct roles for hsp90 in modulating dioxin receptor function are therefore likely: correct folding of the ligand-binding domain, interference with Arnt heterodimerization, and folding of a DNA-binding conformation of the bHLH domain. Thus, the dioxin receptor system provides a complex and interesting model of the regulation of transcription factors by hsp90.The intracellular dioxin receptor (also termed the aryl hydrocarbon receptor) is a ubiquitous basic helix-loop-helix (bHLH) factor (4, 10) that mediates signal transduction by the toxic environmental contaminant dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) (for recent reviews, see references 33, 42, and 47). In the absence of ligand the receptor exists in an inducible cytoplasmic form. Dioxin induces nuclear translocation of the receptor (reference 34 and references therein) and regulates dimerization with the bHLH partner factor Arnt, enabling both proteins to specifically recognize cognate response elements (xenobiotic response elements [XREs]) within regulated genes (9,24,38,46). Individually, neither the receptor nor Arnt shows any detectable affinity for this target sequence (23, 46). Moreover, they do not appear to bind the twofold symmetric CACGTG or CAGCTG E box motifs (23, 46) that are recognized by the great majority of bHLH and bHLH-leucine zipper (bHLH-Zip) proteins, including lymphoid transcription factors, the oncoproteins Myc and Max, and factors involved in vertebrate myogenesis and Drosophila neurogenesis (for recent r...

In response to dioxin, the nuclear basic helix-loop-helix (bHLH) dioxin receptor forms a complex with the bHLH partner factor Arnt that regulates target gene transcription by binding to dioxin-responsive sequence motifs. Previously, we have demonstrated that the latent form of dioxin receptor present in extracts from untreated cells is stably associated with molecular chaperone protein hsp9O, and Arnt is not a component of this complex. Here, we used a coimmunoprecipitation assay to demonstrate that the in vitro-translated dioxin receptor, but not Arnt, is stably associated with hsp9O. Although it showed ligand-binding activity, the in vitro-translated dioxin receptor failed to dissociate from hsp9O upon exposure to ligand. Addition of a specific fraction from wild-type hepatoma cells, however, to the in vitro-expressed receptor promoted dioxin-dependent release of hsp9O. This stimulatory elfect was mediated via the bHLH dimerization and DNA-binding motif of the receptor. Moreover, ligand-dependent release of hsp9O from the receptor was not promoted by fractionated cytosolic extracts from mutant hepatoma cells which are deficient in the function of bHLH dioxin receptor partner factor Arnt. Thus, our results provide a novel model for regulation of bHLH factor activity and suggest that derepression of the dioxin receptor by ligand-induced release of hsp9O may require bHLH-mediated concomitant recruitment of an additional cellular factor, possibly the structurally related bHLH dimerization partner factor Arnt. In support of this model, addition of in vitro-expressed wild-type Arnt, but not a mutated form of Arnt lacking the bHLH motif, promoted release of hsp9O from the dioxin receptor in the presence of dioxin.The nuclear dioxin receptor (also termed the aryl hydrocarbon receptor) mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin). Like other nuclear receptors, the dioxin receptor is a ligand-inducible transcriptional regulator that directly binds to cognate response elements within regulated genes (for a recent review, see reference 37). In contrast to members of the steroid receptor superfamily, however, the dioxin receptor harbors a basic helix-loop-helix (bHLH) motif (5, 17) that represents a dimerization and DNA-binding surface (18) of a broad class of gene regulatory proteins including Myc, its positive and negative regulators Max and Mad (1 and references therein), lymphoid transcription factors, and muscle-differentiating factors such as MyoD and myogenin (for a recent review, see reference 27).Dioxin and other receptor ligands (i.e., structurally related environmental pollutants) strictly regulate dioxin receptor function by modulating its DNA-binding activity in vivo (14,19,24,32) and in vitro (6). A physiological ligand, if any, of the dioxin receptor has not been identified (for a review, see reference 37). Although the detailed mechanism of ligandinduced activation of the dioxin receptor into a functional form remains unclear, this process involves several distinct steps. Mos...

The dioxin (aryl hydrocarbon) receptor is a ligand-dependent basic helix-loop-helix (bHLH)