Biomedical subjects

E Langley

Publications and source records attributed to E Langley.

12 recordsLinked to original sources

PubMed2001

Rapid purification and biochemical characterization of glucose kinase from Streptomyces peucetius var. caesius.

Glucose kinase catalyzes the ATP-dependent phosphorylation of glucose. Streptomyces peucetius var. caesius glucose kinase was purified 292-fold to homogeneity. The enzyme has cytosolic localization and is composed of four identical subunits, each of 31 kDa. The purified enzyme easily dissociates into dimers. However, in the presence of 100 mM glucose the enzyme maintains its tetrameric form. Maximum activity was found at 42 degrees C and pH 7.5. Isoelectric focusing of the enzyme showed a pl of 8.4. The N- and C-terminal amino acid sequences were MGLTIGVD and VYFAREPDPIM, respectively. The kinetic mechanism of S. peucetius var. caesius glucose kinase appears to be a rapid equilibrium ordered type, i.e., ordered addition of substrates to the enzyme, where the first substrate is d-glucose. The K(m) values for d-glucose and MgATP(2-) were 1.6 +/- 0.2 and 0.8 +/- 0.1 mM, respectively. Mg(2+) in excess of 10 mM inhibits enzyme activity.

Adenosine Triphosphate↗

PubMed2001

Differential localization of HDAC4 orchestrates muscle differentiation.

The class II histone deacetylases HDAC4 and HDAC5 interact specifically with the myogenic MEF2 transcription factor and repress its activity. Here we show that HDAC4 is cytoplasmic during myoblast differentiation, but relocates to the nucleus once fusion has occurred. Inappropriate nuclear entry of HDAC4 following overexpression suppresses the myogenic programme as well as MEF2-dependent transcription. Activation of the Ca(2+)/calmodulin signalling pathway via constitutively active CaMKIV prevents nuclear entry of HDAC4 and HDAC4-mediated inhibition of differentiation. Consistent with a role of phosphorylation in HDAC4 cytoplasmic localisation, HDAC4 binds to 14-3-3 proteins in a phosphorylation-dependent manner. Together these data establish a role for HDAC4 in muscle differentiation. Recently, HDAC5 has also been implicated in muscle differentiation. However, despite the functional similarities of HDAC4 and HDAC5, their intracellular localisations are opposed, suggesting a distinct role for these enzymes during muscle differentiation.

14-3-3 Proteins↗

PubMed2001

Purification and characterization of an extracellular lipase from Penicillium candidum.

Penicillium candidum produces and secretes a single extracellular lipase with a monomer molecular weight of 29 kDa. However, this enzyme forms dimers and higher molecular weight aggregates under nondenaturing conditions. The lipase from P. candidum was purified 37-fold using Octyl-Sepharose CL-4B and DEAE-Sephadex columns. The optimal assay conditions for lipase activity were 35 degrees C and pH 9. The lipase was stable in the pH range of 5-6 with a pl of 5.5, but rapid loss of the enzyme activity was observed above 25 degrees C. Tributyrin was found to be the best substrate for the P. candidum lipase, among those tested. Metal ions such as Fe2+ and Cu2+ inhibited enzymatic activity and only Ca2+ was able to slightly enhance lipase activity. Ionic detergents inhibited the activity of the enzyme, whereas nonionic detergents stimulated lipase activity.

Amino Acid Sequence↗

PubMed1999

Constitutive activation of toll-mediated antifungal defense in serpin-deficient Drosophila.

The antifungal defense of Drosophila is controlled by the spaetzle/Toll/cactus gene cassette. Here, a loss-of-function mutation in the gene encoding a blood serine protease inhibitor, Spn43Ac, was shown to lead to constitutive expression of the antifungal peptide drosomycin, and this effect was mediated by the spaetzle and Toll gene products. Spaetzle was cleaved by proteolytic enzymes to its active ligand form shortly after immune challenge, and cleaved Spaetzle was constitutively present in Spn43Ac-deficient flies. Hence, Spn43Ac negatively regulates the Toll signaling pathway, and Toll does not function as a pattern recognition receptor in the Drosophila host defense.

Animals↗

PubMed1999

MEF-2 function is modified by a novel co-repressor, MITR.

The MEF-2 proteins are a family of transcriptional activators that have been detected in a wide variety of cell types. In skeletal muscle cells, MEF-2 proteins interact with members of the MyoD family of transcriptional activators to synergistically activate gene expression. Similar interactions with tissue or lineage-specific cofactors may also underlie MEF-2 function in other cell types. In order to screen for such cofactors, we have used a transcriptionally inactive mutant of Xenopus MEF2D in a yeast two-hybrid screen. This approach has identified a novel protein expressed in the early embryo that binds to XMEF2D and XMEF2A. The MEF-2 interacting transcription repressor (MITR) protein binds to the N-terminal MADS/MEF-2 region of the MEF-2 proteins but does not bind to the related Xenopus MADS protein serum response factor. In the early embryo, MITR expression commences at the neurula stage within the mature somites and is subsequently restricted to the myotomal muscle. In functional assays, MITR negatively regulates MEF-2-dependent transcription and we show that this repression is mediated by direct binding of MITR to the histone deacetylase HDAC1. Thus, we propose that MITR acts as a co-repressor, recruiting a specific deacetylase to downregulate MEF-2 activity.

Amino Acid Sequence↗

PubMed1999

HDAC4 deacetylase associates with and represses the MEF2 transcription factor.

The acetylation state of histones can influence transcription. Acetylation, carried out by acetyltransferases such as CBP/p300 and P/CAF, is commonly associated with transcriptional stimulation, whereas deacetylation, mediated by the three known human deacetylases HDAC1, 2 and 3, causes transcriptional repression. The known human deacetylases represent a single family and are homologues of the yeast RPD3 deacetylase. Here we identify and characterize HDAC4, a representative of a new human histone deacetylase family, which is homologous to the yeast HDA1 deacetylase. We show that HDAC4, unlike other deacetylases, shuttles between the nucleus and the cytoplasm in a process involving active nuclear export. In the nucleus, HDAC4 associates with the myocyte enhancer factor MEF2A. Binding of HDAC4 to MEF2A results in the repression of MEF2A transcriptional activation, a function that requires the deacetylase domain of HDAC4. These results identify MEF2A as a nuclear target for HDAC4-mediated repression and suggests that compartmentalization may be a novel mechanism for controlling the nuclear activity of this new family of deacetylases.

Acetylation↗

PubMed1999

Distinguishing androgen receptor agonists and antagonists: distinct mechanisms of activation by medroxyprogesterone acetate and dihydrotestosterone.

Natural and pharmacological androgen receptor (AR) ligands were tested for their ability to induce the AR NH2-terminal and carboxyl-terminal (N/C) interaction in a two-hybrid protein assay to determine whether N/C complex formation distinguishes in vivo AR agonists from antagonists. High-affinity agonists such as dihydrotestosterone, mibolerone, testosterone, and methyltrienolone at concentrations between 0.1 and 1 nM induce the N/C interaction more than 40-fold. The lower affinity anabolic steroids, oxandrolone and fluoxymesterone, require concentrations of 10-100 nM for up to 23-fold induction of the N/C interaction. However no N/C interaction was detected in the presence of the antagonists, hydroxyflutamide, cyproterone acetate, or RU56187, at concentrations up to 1 microM, or with 1 microM estradiol, progesterone, or medroxyprogesterone acetate; each of these steroids at 1-500 nM inhibited the dihydrotestosterone-induced N/C interaction, with medroxyprogesterone acetate being the most effective. In transient and stable cotransfection assays using the mouse mammary tumor virus reporter vector, all ligands displayed concentration-dependent AR agonist activity that paralleled induction of the N/C interaction, with antagonists and weaker agonists failing to induce the N/C interaction. AR dimerization and DNA binding in mobility shift assays and AR stabilization reflected, but were not dependent on, the N/C interaction. The results indicate that the N/C interaction facilitates agonist potency at low physiological ligand concentrations as detected in transcription, dimerization/DNA binding, and stabilization assays. However the N/C interaction is not required for agonist activity at sufficiently high ligand concentrations, nor does its inhibition imply antagonist activity.

Androgen Antagonists↗

PubMed1998

Intermolecular NH2-/carboxyl-terminal interactions in androgen receptor dimerization revealed by mutations that cause androgen insensitivity.

Structural alignment of the human androgen receptor dimer was investigated by introducing steroid binding domain mutations that cause partial or complete androgen insensitivity into fusion proteins containing the full-length androgen receptor or the steroid binding domain. Most of the mutants had unchanged apparent equilibrium androgen binding affinity and increased dissociation rates of [3H]methyltrienolone and required increased dihydrotestosterone concentrations for transcriptional activation. In a 2-hybrid protein interaction assay in mammalian cells, the steroid binding domain interacts with an NH2-terminal-DNA binding domain fragment and with the full-length androgen receptor at physiological androgen concentrations in a dose-dependent manner. However, mutations at Val-889 and Arg-752 disrupt the NH2-/carboxyl-terminal interaction when introduced into the steroid binding domain fragment but not when present in the full-length androgen receptor. The N-C bimolecular interaction reduces the dissociation rate of bound androgen and slows the degradation rate of the carboxyl-terminal steroid binding domain fragment. The results suggest that steroid binding domain residues Val-889 and Arg-752 are critical to the NH2-/carboxyl-terminal interaction and that an intermolecular N-C interaction occurs during receptor dimerization that results in an antiparallel arrangement of androgen receptor monomers.

Androgens↗

PubMed1995

Evidence for an anti-parallel orientation of the ligand-activated human androgen receptor dimer.

Domain interactions of the human androgen receptor (AR) dimer were investigated using a protein-protein interaction assay in which the NH2- and carboxyl-terminal regions of human AR were fused to the Saccharomyces cerevisiae GAL4 DNA-binding domain and herpes simplex virus VP16 transactivation domain to produce chimeric proteins. Transcriptional activation of a GAL4 luciferase reporter vector up to 100-fold was greater than Fos/Jun leucine zipper binding, indicating stable AR interaction between AR NH2-terminal residues 1-503 and steroid-binding domain residues 624-919 that was specific for and dependent on androgen binding to the steroid-binding domain and was inhibited by anti-androgen binding. Deletion mutagenesis within the NH2-terminal region indicated transactivation domain residues 142-337 were not required for dimerization, whereas deletions near the NH2 terminus (delta 14-150) or NH2-terminal to the DNA-binding domain (delta 339-499) reduced or eliminated the AR interaction, respectively. An NH2-/NH2-terminal interaction was also observed, but no interaction was detected between ligand-free or bound steroid-binding domains. The results indicate that high affinity androgen binding promotes interactions between the NH2-terminal and steroid-binding domains of human AR, raising the possibility of an androgen-induced anti-parallel AR dimer.

Animals↗

PubMed1995

Drosophila immunity. A sequence homologous to mammalian interferon consensus response element enhances the activity of the diptericin promoter.

Bacterial challenge of larvae or adults of Drosophila induces the rapid transcription of several genes encoding antibacterial peptides with a large spectrum of activity. One of these peptides, the 82-residue anti-gram negative diptericin, is encoded by a single intronless gene and we are investigating the control of expression of this gene. Previous studies using both transgenic experiments and footprint analysis have highlighted the role in the induction of this gene of a 30 nucleotide region which contains three partially overlapping motifs with sequence homology to mammalian NF-kappa B and NF-IL6 response elements and to the GAAANN sequence present in the interferon consensus response elements of some mammalian interferon-induced genes. We now show that the latter sequence binds in immune responsive tissues (fat body, blood cells) of Drosophila a approximately 45 kDa polypeptide which cross-reacts with a polyserum directed against mammalian interferon Regulatory Factor-I. Using a transfection assay of Drosophila tumorous blood cells, we show that the GAAANN sequence positively regulates the activity of the diptericin promoter. We propose that this motif cooperatively interacts with the other response elements in the regulation of the diptericin gene expression.

Animals↗

PubMed1995

Hormone-dependent transactivation by the human androgen receptor is regulated by a dnaJ protein.

Genetic studies were performed to examine the role of eukaryotic dnaJ protein, Ydj1p, in the regulated activation of human androgen receptor (hAR) after heterologous expression in Saccharomyces cerevisiae. Hormone-dependent activation of hAR was measured as a function of lacZ reporter gene expression, which was defective in ydj1-151 and ydj1-2 delta null mutant strains compared to the wild type. This defect was not due to receptor misfolding, since hAR in both wild type and mutant strains had a similar capacity to bind hormone. The target for Ydj1p action was determined to be the hAR hormone binding domain since an N-terminal fragment lacking this region was constitutively active in both wild type and ydj1-151 mutant strains. These data correlate hormone dependence of hAR activation with a requirement for Ydj1p function and are consistent with a role for dnaJ proteins in signal transduction by steroid hormone receptors.

Bacterial Proteins↗

PubMed1993

Endometrial expression of progesterone receptor and uteroglobin genes during early pregnancy in the rabbit.

The progesterone receptor (PR) plays a pivotal role in the maturation process of the secretory endometrium, implantation and maintenance of pregnancy in rabbits. To determine the dynamics of PR gene expression and its physiological significance, the endometrial expression of PR and PR mRNA were evaluated and compared with the expression of the progesterone-regulated uteroglobin (UG) gene during 0-5 days post-coitus in rabbits. The results of immunoblot experiments indicated the presence of PR in endometrial cell extracts from days 1-4 of pregnancy with maximum PR immunostaining on day 2, followed by a marked diminution until its complete disappearance on day 5. When endometrial PR mRNA content was assessed by Northern blots, the results were similar to those of PR immunostaining, with maximal concentrations on the second day after mating. However, PR mRNA levels were still high on day 3, despite the concomitant decrease in immunostainable PR. Endometrial UG gene expression, on the other hand, exhibited a different time sequence. Thus, the UG content in uterine flushings progressively increased from day 3 after mating, reaching maximal levels on the fifth day. The endometrial UG mRNA content presented a similar profile, as its maximum concentration occurred on days 4-5. The overall results indicate that endometrial PR is down-regulated at both the mRNA and protein levels, possibly by endogenous progesterone during early pregnancy. The striking observation that maximal expression of endometrial UG gene products occurred when PR and its mRNA are no longer detectable suggests an important role for this progesterone-binding uterine protein during the preimplantation period.

Animals↗