Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nuclear receptor PPARgamma/RXRalpha heterodimer regulates glucose and lipid homeostasis and is the target for the antidiabetic drugs GI262570 and the thiazolidinediones (TZDs). We report the crystal structures of the PPARgamma and RXRalpha LBDs complexed to the RXR ligand 9-cis-retinoic acid (9cRA), the PPARgamma agonist rosiglitazone or GI262570, and coactivator peptides. The PPARgamma/RXRalpha heterodimer is asymmetric, with each LBD deviated approximately 10 degrees from the C2 symmetry, allowing the PPARgamma AF-2 helix to interact with helices 7 and 10 of RXRalpha. The heterodimer interface is composed of conserved motifs in PPARgamma and RXRalpha that form a coiled coil along helix 10 with additional charge interactions from helices 7 and 9. The structures provide a molecular understanding of the ability of RXR to heterodimerize with many nuclear receptors and of the permissive activation of the PPARgamma/RXRbeta heterodimer by 9cRA.
Mol Cell 2000 Mar
PMID:Asymmetry in the PPARgamma/RXRalpha crystal structure reveals the molecular basis of heterodimerization among nuclear receptors. 1088 39

The progressive myoclonus epilepsy of Lafora type is an autosomal recessive disorder caused by mutations in the EPM2A gene. EPM2A is predicted to encode a putative tyrosine phosphatase protein, named laforin, whose full sequence has not yet been reported. In order to understand the function of the EPM2A gene, we isolated a full-length cDNA, raised an antibody and characterized its protein product. The full-length clone predicts a 38 kDa laforin that was very close to the size detected in transfected cells. Recombinant laforin was able to hydrolyze phosphotyrosine as well as phosphoserine/threonine substrates, demonstrating that laforin is an active dual-specificity phosphatase. Biochemical, immunofluorescence and electron microscopic studies on the full-length laforin expressed in HeLa cells revealed that laforin is a cytoplasmic protein associated with polyribosomes, possibly through a conformation-dependent protein-protein interaction. We analyzed the intracellular targeting of two laforin mutants with missense mutations. Expression of both mutants resulted in ubiquitin-positive perinuclear aggregates suggesting that they were misfolded proteins targeted for degradation. Our results suggest that laforin is involved in translational regulation and that protein misfolding may be one of the molecular bases of the Lafora disease phenotype caused by missense mutations in the EPM2A gene.
Hum Mol Genet 2000 Sep 22
PMID:Laforin, defective in the progressive myoclonus epilepsy of Lafora type, is a dual-specificity phosphatase associated with polyribosomes. 1100 28

Here we review the results that have emerged from our structural studies on the oestrogen receptor ligand-binding domain (ER-LBD). The effects of agonists and antagonists on the structure of ERalpha- and ERbeta-LBDs are examined. In addition, the findings from structural studies of ER-LBD in complex with peptide fragments corresponding to the NR-box II and III modules of the p160 coactivator TIF2 are discussed in the context of the assembly of ER:coactivator complexes. Together these studies have broadened our understanding of ER function by providing a unique insight into ER's ligand specificity, it's ability to interact with coactivators and the structural changes that underlie receptor agonism and antagonism.
J Steroid Biochem Mol Biol 2000 Nov 30
PMID:A structural biologist's view of the oestrogen receptor. 1116 34

Mutations in the vitamin D receptor (VDR) cause hereditary vitamin D-resistant rickets (HVDRR), an autosomal recessive disease resulting in target organ resistance to 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. In this report, we describe the clinical case and molecular basis of HVDRR in an Asian boy exhibiting the typical clinical features of the disease including alopecia. Using cultured dermal fibroblasts from the patient, 1,25(OH)(2)D(3) resistance was demonstrated by a shift in the dose response required for 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) mRNA induction. Western blot showed that the cells express a normal size VDR but contained reduced levels of receptor compared to normal cells. At 24 degrees C, the affinity of the patient's VDR for [(3)H]1,25(OH)(2)D(3) was 50-fold lower than the VDR in normal fibroblasts. Sequence analysis identified a unique T to G missense mutation in exon 6 that changed phenylalanine to cysteine at amino acid 251 (F251C). The recreated F251C mutant VDR showed reduced transactivation activity using a 24-hydroxylase promoter-luciferase reporter. Maximal transactivation activity exhibited by the WT VDR was not achieved by the mutant VDR even when the cells were treated with up to 10(-6) M 1,25(OH)(2)D(3). However, the transactivation activity was partially rescued by addition of RXRalpha. In the yeast two-hybrid system and GST-pull-down assays, high concentrations of 1,25(OH)(2)D(3) were needed to promote F251C mutant VDR binding to RXRalpha, indicating defective heterodimerization. In conclusion, a novel mutation was identified in the VDR LBD that reduces VDR abundance and its affinity for 1,25(OH)(2)D(3) and interferes with RXRalpha heterodimerization resulting in the syndrome of HVDRR.
Mol Genet Metab 2001 Jun
PMID:A novel inborn error in the ligand-binding domain of the vitamin D receptor causes hereditary vitamin D-resistant rickets. 1138 49

SpSHR2 is a member of the nuclear receptor superfamily, expressed in embryos, larvae, and adult tissues of sea urchin. During embryonic development, two receptor isoforms are produced via alternative splicing. One exhibits the typical structure of nuclear receptors (SpSHR2-full length), whereas the other is missing the entire LBD (SpSHR2-splice variant). DNA-constructs encoding these isoforms and two additional in vitro generated deletion mutants were engineered in an expression vector carrying the myc-tag. Expression of the tagged isoforms in S. purpuratus embryos showed that the exogenous SpSHR2 full-length protein displays a similar subcellular localization as the endogenous receptor. In early cleavage stages (4-cells), the full-length isoform is predominantly localized in the nucleus, whereas two cell divisions later (16-cells) protein accumulations are detected in both the nucleus and cytoplasm. To the contrary, the SpSHR2-splice variant is confined in the embryonic nuclei both at 4- and 16-cell stage embryos. Analysis of the intracellular distribution of two receptor mutants, one having a deletion within the DBD (DeltaP) and the other a truncation of the C-terminal F-domain (DeltaF), revealed that DeltaP is localized similarly to full-length receptor, whereas DeltaF is maintained in the nucleus, similar to the SpSHR2 splice variant. Investigation of the DNA binding and dimerization properties of the two SpSHR2 isoforms demonstrated that they recognize and bind to a DR1-element as monomers, whereas DeltaP does not bind DNA and DeltaF binds to DR1 poorly. These results suggest that the receptor's putative LBD is responsible for the differential subcellular localization of the two natural SpSHR2-isoforms in early development.
Mol Reprod Dev 2001 Oct
PMID:Differential cellular compartmentalization of the nuclear receptor SpSHR2 splicing variants in early sea urchin embryos. 1155 12

The progressive myoclonus epilepsy of Lafora type (LD) is an autosomal recessive disorder caused by mutations in the EPM2A gene. We demonstrated recently that EPM2A encodes a dual-specificity phosphatase that is primarily associated with polyribosomes. In the present study, we screened for mutations in the EPM2A gene in 4 Japanese LD families and identified a novel mis-sense mutation, Ala46Pro (136G-->C), in heterozygous condition in one patient. In addition, sequence analyses in the patient and control DNA samples identified 4 single nucleotide polymorphisms (SNPs) (75G/A, 120G/T, 159C/G, 171C/T) in the coding region and a novel insertion/deletion polymorphic site (-483[T](11/10)[A](2/3)) and a SNP (-547A/G) in the putative regulatory region of the EPM2A gene. None of the sequence variants, however, co-segregated with the LD phenotype. Haplotype analysis for the 6q24 region in the affected families revealed lack of homozygosity at the EPM2A locus. Our studies suggest that EPM2A is not involved in the disease phenotype of the 4 families studied and that locus heterogeneity for LD may exist in Japanese population also. A simple test described for the detection of Ala46Pro mutation present heterozygously in Japanese population (allele frequency 0.026) can be used for screening this novel allele in a larger sample size.
Mol Cell Probes 2001 Oct
PMID:Mutation screening for Japanese Lafora's disease patients: identification of novel sequence variants in the coding and upstream regulatory regions of EPM2A gene. 1173

Mutations in the EPM2A gene encoding a dual-specificity phosphatase (laforin) cause Lafora disease (LD), a progressive and invariably fatal epilepsy with periodic acid-Schiff-positive (PAS+) cytoplasmic inclusions (Lafora bodies) in the central nervous system. To study the pathology of LD and the functions of laforin, we disrupted the Epm2a gene in mice. At two months of age, homozygous null mutants developed widespread degeneration of neurons, most of which occurred in the absence of Lafora bodies. Dying neurons characteristically exhibit swelling in the endoplasmic reticulum, Golgi networks and mitochondria in the absence of apoptotic bodies or fragmentation of DNA. As Lafora bodies become more prominent at 4-12 months, organelles and nuclei are disrupted. The Lafora bodies, present both in neuronal and non-neural tissues, are positive for ubiquitin and advanced glycation end-products only in neurons, suggesting different pathological consequence for Lafora inclusions in neuronal tissues. Neuronal degeneration and Lafora inclusion bodies predate the onset of impaired behavioral responses, ataxia, spontaneous myoclonic seizures and EEG epileptiform activity. Our results suggest that LD is a primary neurodegenerative disorder that may utilize a non-apoptotic mechanism of cell death.
Hum Mol Genet 2002 May 15
PMID:Targeted disruption of the Epm2a gene causes formation of Lafora inclusion bodies, neurodegeneration, ataxia, myoclonus epilepsy and impaired behavioral response in mice. 1201 6

Mutations in the EPM2A gene encoding a dual-specificity phosphatase (laforin) cause an autosomal recessive fatal disorder called Lafora's disease (LD) classically described as an adolescent-onset stimulus-sensitive myoclonus, epilepsy and neurologic deterioration. Here we related mutations in EPM2A with phenotypes of 22 patients (14 families) and identified two subsyndromes: (i) classical LD with adolescent-onset stimulus-sensitive grand mal, absence and myoclonic seizures followed by dementia and neurologic deterioration, and associated mainly with mutations in exon 4 (P = 0.0007); (ii) atypical LD with childhood-onset dyslexia and learning disorder followed by epilepsy and neurologic deterioration, and associated mainly with mutations in exon 1 (P = 0.0015). To understand the two subsyndromes better, we investigated the effect of five missense mutations in the carbohydrate-binding domain (CBD-4; coded by exon 1) and three missense mutations in the dual phosphatase domain (DSPD; coded by exons 3 and 4) on laforin's intracellular localization in HeLa cells. Expression of three mutant proteins (T194I, G279S and Y294N) in DSPD formed ubiquitin-positive cytoplasmic aggregates, suggesting that they were folding mutants set for degradation. In contrast, none of the three CBD-4 mutants showed cytoplasmic clumping. However, CBD-4 mutants W32G and R108C targeted both cytoplasm and nucleus, suggesting that laforin had diminished its usual affinity for polysomes. Our data, thus, represent the first report of a novel childhood syndrome for LD. Our results also provide clues for distinct roles for the CBD-4 and DSP domains of laforin in the etiology of two subsyndromes of LD.
Hum Mol Genet 2002 May 15
PMID:Genotype-phenotype correlations for EPM2A mutations in Lafora's progressive myoclonus epilepsy: exon 1 mutations associate with an early-onset cognitive deficit subphenotype. 1201 7

Nuclear receptors form strong dimers that are essential for their function as transcription factors, and it is thought that ligand binding can affect dimer stability. In this report, we describe convenient fluorescence resonance energy transfer (FRET)-based methods for measuring the thermodynamic and kinetic stability of dimers of the estrogen receptor-alpha ligand-binding domain (ERalpha-LBD). We have developed receptors that are chemically labeled with a single fluorophore in a site-specific manner. These fluorophore-labeled ERs are functional and can be used to measure directly the affinity and stability of ERalpha-LBD dimers. Our results indicate that unliganded ERalpha-LBDs exist as very stable dimers and that the dissociation rate of these dimers is slow (t(1/2)=39 +/- 3 min at 28 C) and is further slowed (< or =7-fold) by the addition of various ligands. Estrogen antagonists provide greater kinetic stabilization of the ER dimers than agonists. In addition, coactivator peptides containing the LXXLL motif selectively stabilize agonist-bound ERalpha-LBD dimers. These fluorescence-based assays for measuring the kinetic and thermodynamic stability of ER dimers provide a functional in vitro method for assessing the agonist or antagonist character of novel ligands.
Mol Endocrinol 2002 Dec
PMID:Estrogen receptor dimerization: ligand binding regulates dimer affinity and dimer dissociation rate. 1245 92

Besides the measurement of circulating conjugated metabolites of dihydrotestosterone (DHT), which reflects androgenic activity, only one assay to measure androgenic bioactivity in human serum has been proposed thus far. This recombinant bioassay is based on the androgen-dependent interaction between the LBD and NT domains of AR fused to the Gal 4 DNA-binding domain, but its construction is highly complex. We have developed a mammalian cell (CHO 515) bioassay that measures total androgen bioactivity in human serum. The AR-deficient Chinese hamster ovary (CHO) cells were stably transfected with pSG5-puro-hAR and pMMTV-neo-Luc. After selection with puromycin and neomycin, five highly inducible clones were isolated and one was selected. The expression of human androgen receptor (hAR) was confirmed by Western blot and steroid-binding assays on the whole cells. The transcriptional activity of the clone was measured after 24 h of incubation with increasing concentrations of various androgenic and non-androgenic steroid compounds in a 96-well plate. The EC50s for each tested androgenic steroid compound were 4 x 10(-11) M, 1.5 x 10(-11) M, 1 x 10(-9) M, 2 x 10(-10) M, 3 x 10(-10) M for testosterone, DHT, dehydroepiandrosterone (DHEA), delta5-androstenediol, and delta4-androstenedione, respectively. In the physiological concentrations of the non-androgenic steroids, estradiol, cortisol, aldosterone, and progesterone, no interference was noted with the AR transactivation level. Evaluation of androgenic bioactivity in human serum was performed by incubation of CHO 515 cells with 100 microl of patient serum, diluted at 1/100 = 1% in DMEM-F12 without phenol red. The sensitivity of the assay was < 0.3 ng/ml. The mean androgenic bioactivity expressed in testosterone equivalents was 0.6 +/- 0.2 ng/ml in normal prepubertal boys, and 12.4 +/- 2 and 1.7 +/- 0.1 ng/ml in normal pubertal boys and girls, respectively. In conclusion, this new recombinant cell bioassay is today the only assay that takes into account testosterone, DHT, DHEA, delta5-androstenediol, and delta4-androstenedione. It should be of particular use in male children with cryptorchidism, delayed puberty or hypogonadotrophic hypogonadism, i.e., in pediatric patients with low androgen levels.
Mol Cell Endocrinol 2002 Dec 30
PMID:Evaluation of androgenic bioactivity in human serum by recombinant cell line: preliminary results. 1257 22


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