Lower DNA Binding Affinity Research Articles

Ku proteins promote DNA binding and condensation of cyclic GMP-AMP synthase.

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that plays a critical role in regulating antiviral signaling. cGAS binds to DNA and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which is essential for downstream signal transduction. The antiviral response is a rapid biological process; however, cGAS itself has relatively low DNA binding affinity, implying that formation of the cGAS-DNA complex requires an additional factor(s) that promotes cGAS-DNA binding, allowing efficient antiviral signal transduction. Here, we report that the Ku proteins (Ku80 and Ku70) directly interact with cGAS and positively regulate cGAS-mediated antiviral signaling. Mechanistically, we find that the interaction of the Ku proteins with cGAS significantly increases the DNA-binding affinity of cGAS and promotes cGAS condensation in the cytosol, thereby enhancing cGAS catalytic activity. Our results show that the Ku proteins are critical partners of cGAS in sensing DNA virus infection and ensuring efficient innate immune signal transduction.

Low DNA and high BSA binding affinity of cationic ruthenium(II) organometallic featuring pyridine and 2’-hydroxychalcone ligands

The chiral-at-metal, piano-stool ruthenium(II) racemic organometallic [Ru(cymene)(chalconato)(pyridine)]PF6 was prepared by a multistep solution synthesis and its molecular and crystal structure was determined by single crystal X-ray diffraction. The compound crystallizes in a centrosymmetric triclinic P-1 space group with two molecules of opposite chirality within the asymmetric unit. Ruthenium is embedded in an octahedral half-sandwich coordination environment with four different donors, which generates the chirality of the metal centre. The formation of the organometallic was monitored ex-situ by infrared spectroscopy. The pyridine coordination to Ru(II) was particularly analysed to find its characteristic marker bands. The complex was also characterized by elemental analysis and NMR spectroscopy in solution. Its interaction with CT DNA and bovine serum albumin (BSA) was investigated by electron spectroscopy and spectrofluorimetry. The organometallic binds to DNA predominantly by electrostatic forces with low Kb value. On the contrary, its high affinity for BSA was confirmed by strong fluorescence quenching. The synchronous emission spectra revealed that the microenvironment of tryptophan is more affected compared to the environment of tyrosine.

Molecular Alterations in Spermatozoa of a Family Case Living in the Land of Fires—A First Look at Possible Transgenerational Effects of Pollutants

In our previous work, we reported alterations in protamines/histones ratio, in DNA binding of these proteins and their involvement in DNA oxidative damage in 84% of the young men living in the Land of Fires. In the present work, we extended our findings, evaluating any alterations in spermatozoa of a family case, a father and son, living in this area, to also give a first look at the possibility of transgenerational inherited effects of environmental contaminants on the molecular alterations of sperm nuclear basic proteins (SNBP), DNA and semen parameters. In the father and son, we found a diverse excess of copper and chromium in the semen, different alterations in SNBP content and low DNA binding affinity of these proteins. In addition, DNA damage, in the presence of CuCl2 and H2O2, increased by adding both the father and son SNBP. Interestingly, son SNBP, unlike his father, showed an unstable DNA binding and were able to produce DNA damage even without external addition of CuCl2, in line with a lower seminal antioxidant activity than the father. The peculiarity of some characteristics of son semen could be a basis for possible future studies on transgenerational effects of pollutants on fertility.

Structural insights into repression of the Pneumococcal fatty acid synthesis pathway by repressor FabT and co-repressor acyl-ACP.

The Streptococcuspneumoniae fatty acid synthesis (FAS) pathway is globally controlled at the transcriptional level by the repressor FabT and its co-repressor acyl carrier protein (acyl-ACP), the intermediate of phospholipid synthesis. Here, we report the crystal structure of FabT complexed with a 23-bp dsDNA, which indicates that FabT is a weak repressor with low DNA-binding affinity in the absence of acyl-ACP. Modification of ACP with a long-chain fatty acid is necessary for the formation of a stable complex with FabT, mimicked invitro by cross-linking, which significantly elevates the DNA-binding affinity of FabT. Altogether, we propose a putative working model of gene repression under the double control of FabT and acyl-ACP, elucidating a distinct repression network for Pneumococcus to precisely coordinate FAS.

Seasonal dependence of cadmium molecular effects on Mytilus galloprovincialis (Lamarck, 1819) protamine-like protein properties.

Mussels have a seasonal reproduction and cadmium is a common stressor in estuarine and coastal environments. In previous studies, we have shown that exposure to subtoxic doses of cadmium produced alterations in the properties of winter Mytilus galloprovincialis sperm protamine-like (PL) proteins. In this study, it was analyzed the possibility that these cadmium effects may be seasonal. Winter and summer mussels were exposed to CdCl2 , and it was tested the PL-proteins for cadmium bioaccumulation, electrophoretic pattern, DNA binding, and potentiality to induce DNA oxidative damage. It was found that cadmium exposure did not produce the same effects on PL-proteins of summer mussels that were produced on PL-proteins of winter mussels, that is: cadmium bioaccumulation, alterations in the acetic acid-urea polyacrylamide gels (AU-PAGE) and sodium dodecyl sulfate-PAGE pattern, a reduced DNA binding affinity and the ability to induce DNA oxidative damage. PL-proteins from summer mussels, apart from not being affected by all the abovementioned effects of cadmium, also showed a very low DNA binding affinity, independent of cadmium exposure. This study reveals clock-associated seasonal responses to cadmium in M. galloprovincialis. Understanding the mechanisms through which environmental signals guide biological rhythms is fundamental to understanding the seasonal sensitivity of this bioindicator, to use M. galloprovincialis in appropriate seasonal periods.

G-quadruplex DNA interactions, docking and cell photocytotoxicity research of porphyrin dyes

5,10,15,20-tetra-(N-methyl-2-pyridyl)porphyrin (TMPyP2) was always paid little attention because it has relatively low DNA-binding affinity and photocytotoxicity compared with its positional isomer 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphyrin (TMPyP4). Inspired by the previous successful results on higher bioactivities of ligands with larger aromatic surfaces, herein, 5,10,15,20-tetra (phenyl-4-N-methyl-2-pyridyl) porphyrin (TPMPyP2), a TMPyP2-like porphyrin with enlarged planar substituents, was designed and successfully synthesized. Results from spectral experiments and molecular docking calculation suggest that TPMPyP2 could partially overcome the steric hindrance of TMPyP2, with more favorable DNA-binding mode and lower molecular binding energy (M.B.E). Meanwhile, cell photocytotoxicity research indicates that TPMPyP2 shows higher antitumor potency than TMPyP2, which allowed the previous conclusion that the higher G-Quadruplexes DNA binding affinity would be related to better photocytotoxicity.

Mechanisms of Translesion DNA Bypass Across (5′S)‐8,5′‐Cyclo‐2′‐Deoxyguanosine by Sulfolobus Solfataricus DNA Polymerases

The (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine (S‐cdG) DNA adduct is produced from reactions of DNA with hydroxyl radicals generated from ionizing radiation or endogenous oxidative metabolisms. An elevated level of S‐cdG has been detected in Xeroderma pigmentosum, Cockayne syndrome and breast cancer patients, and in aged mice. S‐dG blocks DNA replications in vitro and in human cells, and is repaired by synergetic actions of nucleotide excision repair and translesion DNA synthesis. S‐cdG induces G to A transition in E coli and in human cells, and causes S‐cdG:T mispair in vitro with several translesion DNA polymerases. However, the molecular mechanisms of translesion bypass of S‐cdG remain elusive. We examined kinetic and structural mechanisms of S‐cdG bypass using model DNA polymerases, Sulfolobus Solfataricus DNA Polymerase I (Dpo1) and IV (Dpo4). Both Dpo1 and Dpo4 can moderately bypass S‐cdG with much lower catalytic efficiencies compared to efficiencies with an undamaged template. Dpo1 and Dpo4 bypassed the lesion in a largely error‐free fashion. Pre‐steady‐state kinetics analyses revealed that the lowered catalytic efficiency of Dpo4 in the presence of template S‐cdG derived from a lower DNA binding affinity and an attenuated formation of the productive enzyme‐DNA‐nucleotide complex. To provide structural insights, we solved two X‐ray crystal structures containing Dpo4, S‐cdG‐containing DNA duplex, and the correct dCTP or incorrect dTTP. Together, our study provided mechanistic insights for the translesion bypass across a highly mutagenic cyclopurine lesion.

Oxidized DJ-1 Inhibits p53 by Sequestering p53 from Promoters in a DNA-Binding Affinity-Dependent Manner

DJ-1 is an oncogene and the causative gene for familial Parkinson's disease. Although the oxidative status of DJ-1 at cysteine 106 (C106) is thought to affect all of the activities of DJ-1 and excess oxidation leads to the onset of various diseases, the precise molecular mechanisms underlying the effects of oxidation of DJ-1 on protein-protein interactions of DJ-1 remain unclear. In this study, we found that DJ-1 bound to the DNA-binding region of p53 in a manner dependent on the oxidation of C106. Of the p53 target genes, the expression level and promoter activity of the DUSP1 gene, but not those of the p21 gene, were increased in H(2)O(2)-treated DJ-1(-/-) cells and were decreased in wild-type DJ-1- but not C106S DJ-1-transfected H1299 cells through sequestration of p53 from the DUSP1 promoter by DJ-1. DUSP1 downregulated by oxidized DJ-1 activated extracellular signal-regulated kinase (ERK) and decreased apoptosis. The DUSP1 and p21 promoters harbor nonconsensus and consensus p53 recognition sequences, respectively, which have low affinity and high affinity for p53. However, DJ-1 inhibited p21 promoter activity exhibited by p53 mutants harboring low DNA-binding affinity but not by wild-type p53. These results indicate that DJ-1 inhibits the expression of p53 target genes and depend on p53 DNA-binding affinity and oxidation of DJ-1 C106.

DNA binding and antiproliferative activity toward human carcinoma cells of copper(ii) and zinc(ii) complexes of a 2,5-diphenyl[1,3,4]oxadiazole derivative

The interaction of calf thymus DNA with [CuL(ClO(4))]ClO(4)·H(2)O (1) and [ZnLBr]Br·H(2)O (2) (L = 9,12,15,18,27,28-hexaaza-29-oxatetracyclo[24.2.1.0(2,7).0(20,25)]enneicosa-2,4,6,20,22,24,26,28(1)-octaene) dicationic complexes in aqueous solution at neutral pH, was investigated by variable-temperature UV-vis absorption, circular dichroism and fluorescence spectroscopy. The values of the DNA-binding constants of these complexes, determined by competitive binding spectrofluorimetric titrations of ethidium bromide (EB)-DNA solutions, are (6.7 ± 0.5) × 10(6) M(-1) for CuL(2+) and (4.7 ± 0.5) × 10(5) M(-1) for ZnL(2+). These data together with a through analysis of the spectroscopic behaviour consistently suggest that both compounds are effective DNA binders. Interestingly, the DNA-binding strength of these complexes has been found to be correlated to their in vitro cytotoxic activity toward human breast carcinoma cells, although the complex with lower DNA-binding affinity is more active. In fact, biological studies showed that when the compounds are delivered through the cell membrane by a lipidic carrier, the cell survival is sensibly reduced, up to 58% with 1 and to 31% with 2.

In vitro photobiochemical characterization of sulfobutylether-β-cyclodextrin formulation of bufexamac

The present study aimed to modulate the photoreactivity of bufexamac, with a focus on photostability and phototoxicity, by forming an inclusion complex with sulfobutylether-β-cyclodextrin (SBECD). The photobiochemical properties of bufexamac were evaluated by reactive oxygen species (ROS) assay and using in vitro photogenotoxic assessment tools. To assess the inclusion properties of SBECD complex with bufexamac, a UV absorption spectroscopic study was also carried out. The influence of SBECD on the photoreactivity of bufexamac was analyzed by ROS assay and photostability test. From the photobiochemical data, superoxide generation from irradiated bufexamac indicated its photoreactivity; however, the photogenotoxic risk of bufexamac was negligible owing to low DNA-binding affinity and DNA-photocleaving activity. SBECD complex of bufexamac was formed, and the association constant of the complex was calculated to be 620 M −1. On the basis of the photochemical data on bufexamac co-existing with SBECD, ROS generation from irradiated bufexamac (200 μM) was inhibited by SBECD at concentrations of over 20 μM. The degradation constant of bufexamac in SBECD was decreased ca. 30% compared with that of bufexamac, suggesting improvement of its photostability. The phototoxic risk of bufexamac might be attenuated by SBECD complexation, and cyclodextrin inclusion complexes might be a useful approach for modulating the phototoxicity of drugs.

Nucleotide and DNA-Induced Structural Transitions and the Coupling Between ATP and DNA Binding Sites in RecA

RecA is the prototype of ATPase proteins that mediates homologous DNA recombination. RecA requires ATP to promote the binding of DNA, as both ADP- and nucleotide-free states show low DNA binding affinity. We investigated how ATP binding affects the dynamics of DNA binding Loops, and activates RecA by the interactions between β-/γ-phosphate and Walker A and Walker B motifs, as well as through the contacts made between the C-terminal and the central domains within the protein. DNA binding results in the formation of the extended, active conformation of the RecA filament, which catalyzes strand exchange. We have performed a set of molecular dynamics simulations on the active RecA, with ATP/ADP/nucleotide-free bound, to investigate the conformational transitions between the active and inactive states. Our simulations have revealed that the structural changes upon ATP binding are confined to small motifs, while the conformational changes upon DNA binding involve larger scale rearrangement of the protein, namely the rotation of monomers with respect to each other. The results suggest that ATP binding stabilizes the L1 and L2 DNA binding loops, mediated through specific residues located between the ATP and DNA binding sites that sense the presence of γ-phosphate. Furthermore, DNA binding leads to monomer rotations to form the extended conformation by affecting the interfaces of the adjacent monomers with the help of the bound nucleotide. Based on these simulations, we propose that the ATP and DNA binding enhance the binding of each other by an allosteric mechanism mediated by a series of residues between the ATP and DNA binding sites.

Distinct p53 acetylation cassettes differentially influence gene-expression patterns and cell fate

The activity of the p53 gene product is regulated by a plethora of posttranslational modifications. An open question is whether such posttranslational changes act redundantly or dependently upon one another. We show that a functional interference between specific acetylated and phosphorylated residues of p53 influences cell fate. Acetylation of lysine 320 (K320) prevents phosphorylation of crucial serines in the NH2-terminal region of p53; only allows activation of genes containing high-affinity p53 binding sites, such as p21/WAF; and promotes cell survival after DNA damage. In contrast, acetylation of K373 leads to hyperphosphorylation of p53 NH2-terminal residues and enhances the interaction with promoters for which p53 possesses low DNA binding affinity, such as those contained in proapoptotic genes, leading to cell death. Further, acetylation of each of these two lysine clusters differentially regulates the interaction of p53 with coactivators and corepressors and produces distinct gene-expression profiles. By analogy with the “histone code” hypothesis, we propose that the multiple biological activities of p53 are orchestrated and deciphered by different “p53 cassettes,” each containing combination patterns of posttranslational modifications and protein–protein interactions.

A Bisanthracycline (WP631) Represses uPAR Gene Expression and Cell Migration of RKO Colon Cancer Cells by Interfering With Transcription Factor Binding to a Chromatin-Accessible −148/−124 Promoter Region

The urokinase receptor (uPAR), transcriptionally activated in several cancers, contributes to tumor progression by promoting cell migration and proteolysis, and repressing expression of this gene could be of therapeutic utility. Indeed, targeting regulatory element(s) in the promoter may represent an efficient means for reducing expression because only two alleles have to be neutralized. We previously identified the -148/-124 promoter region, bound with Sp1 and Sp3, as regulatory for uPAR expression in vitro. The purpose of this study was twofold: to determine (a) the accessibility of this region in its natural chromatin setting and (b) the efficacy of WP631, a bisintercalator favoring GC-rich DNA sequences, in repressing endogenous uPAR expression in RKO colon cancer cells. In these cells, DNaseI hypersensitivity, genomic footprinting, and chromatin immunoprecipitation experiments revealed that the -148/-124 uPAR promoter region was accessible in chromatin and bound with Sp1, thus validating it as a therapeutic target. WP631 treatment competed for transcription factor binding to this regulatory region and reduced uPAR mRNA/protein. However, a chemically related compound (WP629), with low DNA binding affinity, failed to diminish uPAR protein amount. GAPDH mRNA level was only modestly affected by WP631, arguing against the possibility that this bisanthracycline universally represses expression of GC-rich promoter-driven genes. Further, uPAR function, as assessed by migration of cells across a vitronectin-coated filter, was attenuated with WP631. Thus, we have shown that the chromatinized -148/-124 regulatory region of the uPAR promoter is accessible to small molecules and that WP631, which disrupts the interaction of DNA binding proteins with this region, diminishes uPAR expression and function.

The mutation G145S in PrfA, a key virulence regulator of Listeria monocytogenes, increases DNA‐binding affinity by stabilizing the HTH motif

Listeria monocytogenes, a Gram-positive, facultative intracellular human pathogen, causes systemic infections with high mortality rate. The majority of the known pathogenicity factors of L. monocytogenes is regulated by a single transcription factor, PrfA. Hyperhaemolytic laboratory strains of L. monocytogenes express the constitutively active mutant PrfA(G145S) inducing virulence gene overexpression independent of environmental conditions. PrfA belongs to the Crp/Fnr family of transcription factors generally activated by a small effector, such as cAMP or O(2). We present the crystal structures of wild-type PrfA, the first Gram-positive member of the Crp/Fnr family, and of the constitutively active mutant PrfA(G145S). Cap (Crp) has previously been described exclusively in the cAMP-induced (DNA-free and -bound) conformation. By contrast, the PrfA structures present views both of the non-induced state and of the mutationally activated form. The low DNA-binding affinity of wild-type PrfA is supported both structurally (partly disordered helix-turn-helix motif, overall geometry of the HTH alpha-helices deviates from Cap) and by surface plasmon resonance analyses (K(D) = 0.9 microM). In PrfA(G145S) the HTH motifs dramatically rearrange to adopt a conformation comparable to cAMP-induced Cap and hence favourable for DNA binding, supported by a DNA-binding affinity of 50 nM. Finally, the hypothesis that wild-type PrfA, like other Crp/Fnr family members, may require an as yet unidentified cofactor for activation is supported by the presence of a distinct tunnel in PrfA, located at the interface of the beta-barrel and the DNA-binding domain.

Interaction interface of human flap endonuclease-1 with its DNA substrates.

Flap endonuclease-1 or FEN-1 is a structure-specific and multifunctional nuclease critical for DNA replication, repair, and recombination; however, its interaction with DNA substrates has not been fully understood. In the current study, we have defined the borders of the interaction between the FEN-1 protein and its DNA substrates and identified six clusters of conserved positively charged amino acid residues, which are in direct contact with DNA substrate. To map further the corresponding interactions between FEN-1 residues and DNA substrates, we performed biochemical assays employing a series of flap DNA substrates lacking some structural components and a series of binding-deficient point mutants of FEN-1. It was revealed that Arg(47), Arg(70), and Lys(326)-Arg(327) of FEN-1 interact with the upstream duplex of DNA substrates, whereas Lys(244)-Arg(245) interact with the downstream duplex. This result indicates the orientation of the FEN-1-DNA interaction. Moreover, Arg(70) and Arg(47) were determined to interact with the sites around the 2nd nucleotide (Arg(70)) or the 5th/6th nucleotide (Arg(47)) of the template strand in the upstream duplex portion counting from the nick point of the flap substrate. Together with previously published data and the crystallographic ainformation from the FEN-1.DNA complex that we published recently (Chapados, B. R., Hosfield, D. J., Han, S., Qiu, J., Yelent, B., Shen, B., Tainer, J. A. (2004) Cell 116, 39-50) we are able to propose a reasonable model for how the human FEN-1 protein interacts with its DNA substrates.

Biochemical Characterization of an Invariant Histidine Involved in Escherichia coli DNA Topoisomerase I Catalysis

An invariant histidine residue, His-365 in Escherichia coli DNA topoisomerase I, is located at the active site of type IA DNA topoisomerases and near the active site tyrosine. Its ability to participate in the multistep catalytic process of DNA relaxation was investigated. His-365 was mutated to alanine, arginine, asparagine, aspartate, glutamate, and glutamine to study its ability to participate in general acid/base catalysis and bind DNA. The mutants were examined for pH-dependent DNA relaxation and cleavage, salt-dependent DNA relaxation, and salt-dependent DNA binding affinity. The mutants relax DNA in a pH-dependent manner and at low salt concentrations. The pH dependence of all mutants is different from the wild type, suggesting that His-365 is responsible for the pH dependence of the enzyme. Additionally, whereas the wild type enzyme shows pH-dependent oligonucleotide cleavage, cleavage by both H365Q and H365A is pH-independent. H365Q cleaves DNA with rates similar to the wild type enzyme, whereas H365A has a slower rate of DNA cleavage than the wild type but can cleave more substrate overall. H365A also has a lower DNA binding affinity than the wild type enzyme. The binding affinity was determined at different salt concentrations, showing that the alanine mutant displaces half a charge less upon binding DNA than an inactive form of topoisomerase I. These observations indicate that His-365 participates in DNA binding and is responsible for optimal catalysis at physiological pH.

Two Classes of Androgen Receptor Elements Mediate Cooperativity through Allosteric Interactions

Genes uniquely regulated by the androgen receptor (AR) typically contain multiple androgen response elements (AREs) that in isolation are of low DNA binding affinity and transcriptional activity. However, specific combinations of AREs in their native promoter context result in highly cooperative DNA binding by AR and high levels of transcriptional activation. We demonstrate that the natural androgen-regulated promoters of prostate specific antigen and probasin contain two classes of AREs dictated by their primary nucleotide sequence that function to mediate cooperativity. Class I AR-binding sites display conventional guanine contacts. Class II AR-binding sites have distinctive atypical sequence features and, upon binding to AR, the DNA structure is dramatically altered through allosteric interactions with the receptor. Class II sites stabilize AR binding to adjacent class I sites and result in synergistic transcriptional activity and increased hormone sensitivity. We have determined that the specific nucleotide variation within the AR binding sites dictate differential functions to the receptor. We have identified the role of individual nucleotides within class II sites and predicted consensus sequences for class I and II sites. Our data suggest that this may be a universal mechanism by which AR achieved unique regulation of target genes through complex allosteric interactions dictated by primary binding sequences.

Retinoic Acid Stimulation of the Human Surfactant Protein B Promoter Is Thyroid Transcription Factor 1 Site-dependent

Surfactant B (SP-B) is a 79-amino acid peptide critical to postnatal respiratory adaptation. Expression of SP-B by respiratory epithelial cells is regulated by developmental and hormonal influences at the level of gene transcription. Previous studies supported the role of retinoic acids (RA) and their receptors (RARs) in SP-B gene transcription. In the present study, RARalpha was detected in mouse alveolar type II epithelial cells where SP-B is synthesized and processed. Deletion and site-specific mutagenesis analysis identified clustered retinoic acid-responsive element sites in the 5'-flanking enhancer region of the hSP-B gene that bound RARalpha proteins. RAR coactivators ACTR, SRC-1, and transcriptional intermediary factor 2 (TIF2) stimulated human (h) SP-B promoter activity in a dose-dependent fashion in pulmonary adenocarcinoma H441 cells. In addition, an RAR-associated protein, CREB-binding protein (CBP), potentiated the effects of RAR on hSP-B promoter activity in H441 cells. Importantly, RA stimulation of the hSP-B promoter depends on tissue-specific thyroid transcription factor (TTF-1) DNA-binding sites. TTF-1 protein synergistically stimulated the hSP-B promoter with RARalpha, CBP, and nuclear receptor coactivators in H441 cells. In addition, TTF-1 interacted directly with RARalpha and TIF2 in the mammalian two-hybrid system. These findings support a model in which RAR/retinoid X receptor, TTF-1, coactivators, and CBP form a transcription activation complex in the upstream enhancer region of the hSP-B gene.

DNA binding affinity of hTRβ1 mutants as heterodimers with traps from different tissues

Patients with generalized resistance to thyroid hormone (GRTH) show various organ-specific features, for example mental retardation, growth abnormalities, liver damage, delayed bone age or cardiac disorders. Could this reflect aberrant mutant thyroid hormone receptor β1 (TRβ1) heterodimerization with specific TR auxiliary proteins (TRAPs) from different tissues, altering the mutant's ability to transactivate tissue-specific genes? To answer this question, we examined the heterodimerization of TRβ1 mutants and TRAPs of several rat tissues (cerebrum, cerebellum, liver, heart, lung, spleen, and kidney), and in vitro translated RXRα, β and γ by electrophoretic gel mobility shift assay (EMSA). Mutant TRβ1 proteins, synthesized in reticulocyte lysate, were incubated with 32P rat malic enzyme (rME) thyroid hormone response elements (TRE) and nuclear extracts of rat tissues. The TRβ1 mutants used were Mf (G345R), and GH (R316H). Both have non-detectable T3 binding affinity. GH has weak dominant negative effect and Mf has strong dominant negative effect. Two major bands were observed in EMSA. Cerebrum, cerebellum, lung and liver extracts formed a slower migrating band than a TR homodimer, while kidney extracts formed a faster migrating band, and heart and spleen extracts had both bands. There were no qualitative differences in heterodimerization between TRβ1wt, and TRβ1 mutants, when using tissue extracts and DNA in excess ratio to TR. We found that RXRα, β, and γ were differentially expressed in each rat tissue and formed heterodimer complexes with wild type (WT) TRβ1. Scatchard analysis of affinity and capacity of the binding of TR-TRAP heterodimers to response elements was performed by competing with 2.5-, 5-, 10-, 25-, and 250-fold excess non-radiolabeled rME-TRE. When using kidney extract, the DNA binding affinity of heterodimers was significantly decreased both in wild type and mutant TRs, suggesting that the DNA binding affinity of the faster migrating band was lower than that of the slower migrating band. Mutant GH, which causes `pituitary RTH' and shows weak dominant negative effect, tended to form heterodimers with lower DNA binding affinity than TRβ1wt with all extracts. Mutant Mf, which has strong dominant negative effect, tended to show higher DNA binding affinity than TRβ1WT. When the data were pooled for all tissues, GH and Mf were found to form heterodimers with significantly lower, or higher, affinity for TREs than TRβ1wt. These results indicate that: 1) differences of DNA binding affinity of mutant TR-TRAP heterodimers to response elements in DNA play a part in its reduced or strong dominant negative effect; and 2) differences in formation of heterodimers with TRAPs present in tissues do not appear to explain the apparent tissue-specific and mutant-specific variations seen in RTH.

Serine/Threonine Phosphorylation of Orphan Receptor Hepatocyte Nuclear Factor 4

We showed previously that hepatocyte nuclear factor 4 (HNF-4) defines a new subclass, Group IV, of nuclear receptors. In order to determine whether members of this subclass are phosphorylated, HNF-4 was overexpressed to high levels in insect cells using a baculovirus expression system. The baculovirus-expressed HNF-4 (HNF4.BV) was characterized and compared to HNF-4 overexpressed in transiently transfected mammalian (COS-7) cells (HNF4.COS). The results indicate that both HNF4.BV and HNF4.COS are phosphorylated although HNF4.BV was hypophosphorylated relative to HNF4.COS. Phosphoamino acid analysis showed that HNF-4 is phosphorylated mainly on serine and to a lesser extent on threonine residues. Phosphopeptide mapping revealed 13 phosphopeptides for HNF4.COS, only 9 of which were present in the HNF4.BV sample. DNA-binding studies also showed that HNF4.BV binds DNA with a lower specificity and affinity, as measured by the equilibrium dissociation constant (Kd), than does HNF4.COS. Partial proteolytic digestion experiments also revealed that HNF4.BV and HNF4.COS adopt somewhat different three-dimensional conformations. Since glycosylation of HNF4.BV was ruled out by a number of methods and since HNF-4 expressed in bacteria exhibited an even lower DNA-binding affinity than HNF4.BV, we propose that serine/theronine phosphorylation may play a role in the DNA-binding activity of HNF-4 and, therefore, possibly of other Group IV receptors as well.