scholarly journals Dioxin Disrupts Thyroid Hormone and Glucocorticoid Induction of klf9, a Master Regulator of Frog Metamorphosis

2021 ◽  
Author(s):  
David T Han ◽  
Weichen Zhao ◽  
Wade H Powell
Keyword(s):  
Thyroid Hormone ◽  
Protein Interactions ◽  
Fold Increase ◽  
Early Gene ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Protein Protein Interactions ◽  
Endocrine Control ◽  
Aryl Hydrocarbon ◽  
Glucocorticoid Induction

Frog metamorphosis, the development of an air-breathing froglet from an aquatic tadpole, is under endocrine control by thyroid hormone (TH) and glucocorticoids (GC). Metamorphosis is susceptible to disruption by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AHR) agonist. Krüppel-Like Factor 9 (klf9), an immediate early gene in the endocrine-controlled cascade of expression changes that govern metamorphosis, can be synergistically induced by both hormones. This process is mediated by an upstream enhancer cluster, the klf9 synergy module (KSM). klf9 is also a target of the AHR. We measured klf9 mRNA expression following combined exposures to triiodothyronine (T3), corticosterone (CORT), and TCDD in the Xenopus laevis cell line XLK-WG. klf9 was induced 6-fold by 50 nM T3, 4-fold by 100 nM CORT, and 3-fold by 175 nM TCDD. Co-treatments of CORT and TCDD or T3 and TCDD induced klf9 mRNA 7- and 11-fold, respectively, while treatment with all 3 agents induced a 15-fold increase. Transactivation assays examined regulatory sequences from the Xenopus tropicalisklf9 upstream region. KSM-containing segments mediated a strong T3 response and a larger T3/CORT response, while induction by TCDD was mediated by a region ~1 kb farther upstream containing 5 AHR response elements. Unexpectedly, this region also supported a CORT response in the absence of readily- identifiable glucocorticoid responsive elements, suggesting mediation by protein-protein interactions. A similar AHRE cluster is positionally conserved in the human genome, and klf9 was induced by TCDD and TH in HepG2 cells. These results indicate that AHR binding to an upstream AHRE cluster represents an initiating event in TCDD disruption of klf9 expression and metamorphosis.

Dependence of KCC2 K-Cl cotransporter activity on a conserved carboxy terminus tyrosine residue

2000 ◽  
Vol 279 (3) ◽  
pp. C860-C867 ◽  
Author(s):  
Kevin Strange ◽  
Thomas D. Singer ◽  
Rebecca Morrison ◽  
Eric Delpire
Keyword(s):  
Protein Interactions ◽  
Regulatory Protein ◽  
Phosphorylation Site ◽  
Fold Increase ◽  
Tyrosine Residue ◽  
Carboxy Terminus ◽  
Protein Protein Interactions ◽  
Pharmacological Studies ◽  
Osmotic Homeostasis ◽  
Tyrosine Phosphorylation Site

K-Cl cotransporters (KCC) play fundamental roles in ionic and osmotic homeostasis. To date, four mammalian KCC genes have been identified. KCC2 is expressed exclusively in neurons. Injection of Xenopus oocytes with KCC2 cRNA induced a 20-fold increase in Cl−-dependent, furosemide-sensitive K+ uptake. Oocyte swelling increased KCC2 activity 2–3 fold. A canonical tyrosine phosphorylation site is located in the carboxy termini of KCC2 (R1081–Y1087) and KCC4, but not in other KCC isoforms. Pharmacological studies, however, revealed no regulatory role for phosphorylation of KCC2 tyrosine residues. Replacement of Y1087 with aspartate or arginine dramatically reduced K+ uptake under isotonic and hypotonic conditions. Normal or near-normal cotransporter activity was observed when Y1087 was mutated to phenylalanine, alanine, or isoleucine. A tyrosine residue equivalent to Y1087 is conserved in all identified KCCs from nematodes to humans. Mutation of the Y1087 congener in KCC1 to aspartate also dramatically inhibited cotransporter activity. Taken together, these results suggest that replacement of Y1087 and its congeners with charged residues disrupts the conformational state of the carboxy terminus. We postulate that the carboxy terminus plays an essential role in maintaining the functional conformation of KCC cotransporters and/or is involved in essential regulatory protein-protein interactions.

scholarly journals Retention Time Prediction Using Neural Networks Increases Identifications in Crosslinking Mass Spectrometry

2021 ◽  
Author(s):  
Sven H. Giese ◽  
Ludwig R. Sinn ◽  
Fritz Wegner ◽  
Juri Rappsilber
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Retention Time ◽  
Fold Increase ◽  
Reversed Phase ◽  
Fanconi Anaemia ◽  
Chromatographic Retention ◽  
Protein Protein Interactions ◽  
Noisy Information ◽  
Strong Anion Exchange

AbstractCrosslinking mass spectrometry (Crosslinking MS) has developed into a robust technique that is increasingly used to investigate the interactomes of organelles and cells. However, the incomplete and noisy information in the spectra limits the numbers of protein-protein interactions (PPIs) that can be confidently identified. Here, we successfully leveraged chromatographic retention time (RT) information to aid the identification of crosslinked peptides from spectra. Our Siamese machine learning model xiRT achieved highly accurate RT predictions of crosslinked peptides in a multi-dimensional separation of crosslinked E. coli lysate. We combined strong cation exchange (SCX), hydrophilic strong anion exchange (hSAX) and reversed-phase (RP) chromatography and reached R2 0.94 in RP and a margin of error of 1 fraction for hSAX in 94%, and SCX in 85% of the predictions. Importantly, supplementing the search engine score with retention time features led to a 1.4-fold increase in PPIs at a 1% false discovery rate. We also demonstrate the value of this approach for the more routine analysis of a crosslinked multiprotein complexes. An increase of 1.7-fold in heteromeric crosslinked residue-pairs was achieved at 1% residue-pair FDR for Fanconi anaemia monoubiquitin ligase complex, solely using reversed-phase RT. Retention times are a powerful complement to mass spectrometric information to increase the sensitivity of Crosslinking MS analyses.

Towards an Understanding of Plant Gene Regulation: The Action of Nuclear Factors

1991 ◽  
Vol 46 (1-2) ◽  
pp. 1-11 ◽  
Author(s):  
Kurt Weising ◽  
Günter Kahl
Keyword(s):  
Protein Interactions ◽  
Regulation Of Gene Expression ◽  
Regulatory Proteins ◽  
Regulatory Sequences ◽  
Protein Protein Interactions ◽  
Cis Acting ◽  
Plant Gene ◽  
Eukaryotic Genes ◽  
Genes Encoding ◽  
Plant Genes

Abstract Over the last decade an intensive research on the regulation of gene expression in viral and animal systems has led to the discovery of cis-acting regulatory sequences, the identification of sequence-specific DNA -binding proteins (trans-acting factors), the characterization of protein domains involved in DNA -protein recognition and binding as well as in protein -protein interactions, and the cloning and sequencing of genes encoding regulatory proteins. The tre­mendous progress in this field is now being complemented by advances in our understanding of how plant genes are regulated. A wealth of data has accumulated in the past few years witnessing basic similarities in the transcriptional regulation of various eukaryotic genes, but also specific features of plant genes. This article collects presently available data, focusses on DNA -protein interactions in plant genes, particularly in light-regulated and “constitutively expressed” genes, reports on the isolation of plant genes encoding regulatory proteins, an dismeant to induce further activities in plant gene research.

scholarly journals Nuclear receptor corepressors activate rather than suppress basal transcription of genes that are negatively regulated by thyroid hormone.

1997 ◽  
Vol 17 (5) ◽  
pp. 2642-2648 ◽  
Author(s):  
T Tagami ◽  
L D Madison ◽  
T Nagaya ◽  
J L Jameson
Keyword(s):  
Thyroid Hormone ◽  
Nuclear Receptor ◽  
Protein Interactions ◽  
Thyroid Hormone Receptor ◽  
Thyroid Stimulating Hormone ◽  
Dependent Manner ◽  
Protein Protein Interactions ◽  
Retinoid Receptors ◽  
Regulated Promoters ◽  
Stimulation Of

A group of transcriptional cofactors referred to as corepressors (CoRs) were recently shown to play a central role in basal silencing of genes that contain positive triiodothyronine (T3) response elements. In a reciprocal manner, negatively regulated genes are stimulated by unliganded thyroid hormone receptor (TR) and repressed upon the addition of T3. We used a TR beta mutant, called P214R, which fails to interact with CoRs, to examine whether CoRs also play a role in the control of genes that are negatively regulated in response to T3. In studies of three negatively regulated genes (the pituitary thyroid-stimulating hormone alpha-subunit [TSH alpha], TSH beta, and hypothalamic thyrotropin-releasing hormone [TRH] genes), stimulation of basal promoter activity by unliganded TR beta was impaired by introducing the P214R CoR mutation. Coexpression of each of the CoRs SMRT (silencing mediator for retinoid receptors and TRs) and NCoR (nuclear receptor CoR) enhanced basal stimulation of the negatively regulated promoters in a TR-dependent manner, but this effect was not seen with the P214R TR mutant. The mechanism of CoR effects on negatively regulated promoters was explored further with a series of GAL4-TR chimeric receptors and mutants that allowed TR effects to be assessed independently of receptor interactions with DNA. These experiments revealed that, like the negative regulation of genes by wild-type TR, basal activation occurred with GAL4-TR, but not with the GAL4-P214R mutant, and was reversed by the addition of T3. These results suggest that TR interactions with negatively regulated genes may be driven through protein-protein interactions. We conclude that a subset of negatively regulated genes are controlled by a novel mechanism that involves TR-mediated recruitment and basal activation by SMRT and NCoR. Addition of T3 reverses basal activation, perhaps by dissociation of CoRs.

scholarly journals Aryl Hydrocarbon Receptor Deficiency Alters Circadian and Metabolic Rhythmicity

2017 ◽  
Vol 32 (2) ◽  
pp. 109-120 ◽  
Author(s):  
Cassie Jaeger ◽  
Ali Q. Khazaal ◽  
Canxin Xu ◽  
Mingwei Sun ◽  
Stacey L. Krager ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Aryl Hydrocarbon Receptor ◽  
Protein Interactions ◽  
Clock Genes ◽  
Environmental Sensors ◽  
Protein Protein Interactions ◽  
Aryl Hydrocarbon ◽  
Metabolic Gene Expression ◽  
Circadian Clock Genes ◽  
Circadian Clock Proteins

PAS domain–containing proteins can act as environmental sensors that capture external stimuli to allow coordination of organismal physiology with the outside world. These proteins permit diverse ligand binding and heterodimeric partnership, allowing for varied combinations of PAS-dependent protein-protein interactions and promoting crosstalk among signaling pathways. Previous studies report crosstalk between circadian clock proteins and the aryl hydrocarbon receptor (AhR). Activated AhR forms a heterodimer with the circadian clock protein Bmal1 and thereby functionally inhibits CLOCK/Bmal1 activity. If physiological activation of AhR through naturally occurring, endogenous ligands inhibits clock function, it seems plausible to hypothesize that decreased AhR expression releases AhR-induced inhibition of circadian rhythms. Because both AhR and the clock are important regulators of glucose metabolism, it follows that decreased AhR will also alter metabolic function. To test this hypothesis, rhythms of behavior, metabolic outputs, and circadian and metabolic gene expression were measured in AhR-deficient mice. Genetic depletion of AhR enhanced behavioral responses to changes in the light-dark cycle, increased rhythmic amplitude of circadian clock genes in the liver, and altered rhythms of glucose and insulin. This study provides evidence of AhR-induced inhibition that influences circadian rhythm amplitude.

scholarly journals Why FRET over genomics?

1999 ◽  
Vol 1 (2) ◽  
pp. 93-99 ◽  
Author(s):  
DINO A. DE ANGELIS
Keyword(s):  
Protein Interactions ◽  
Complex Traits ◽  
High Capacity ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Regulatory Sequences ◽  
Gene Products ◽  
Nucleotide Polymorphisms ◽  
Protein Protein Interactions ◽  
Individual Gene

De Angelis, Dino A. Why FRET over genomics? Physiol. Genomics 1: 93–99, 1999.—Genetic information is being uncovered quickly and in vast amounts through the largely automated sequencing of genomes from all kinds of organisms. As this information becomes available, enormous challenges are emerging on three levels: first, functions will have to be assigned to individual gene products; second, factors that influence the expression level of these gene products will have to be identified; and third, allelic variants that act alone or in combination to give rise to complex traits will have to be characterized. Because of the sheer size of genomes, methods that can streamline or automate these processes are highly desirable. Fluorescence is an attractive readout for such high-throughput tasks because of the availability of equipment designed to detect light-emitting compounds with great speed and high capacity. The following is an overview of the achievements and potential of fluorescence resonance energy transfer (FRET) as applied in three areas of genomics: the identification of single-nucleotide polymorphisms, the detection of protein-protein interactions, and the genomewide analysis of regulatory sequences.

scholarly journals Discovery of Selective Small-Molecule CD80 Inhibitors

2007 ◽  
Vol 12 (4) ◽  
pp. 464-472 ◽  
Author(s):  
Kristina Uvebrant ◽  
Dorthe Da Graça Thrige ◽  
Anna Rosén ◽  
Mats Åkesson ◽  
Helena Berg ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Critical Role ◽  
Fold Increase ◽  
Initial Step ◽  
Protein Protein Interactions ◽  
Cellular Events ◽  
A Cell ◽  
Direct Binding ◽  
Further Development ◽  
Costimulatory Pathway

Protein-protein interactions are widely found in biological systems controlling diverse cellular events. Because these interactions are implicated in many diseases such as autoimmunity and cancer, regulation of protein-protein interactions provides ideal targets for drug intervention. The CD80-CD28 costimulatory pathway plays a critical role in regulation of the immune response and thus constitutes an attractive target for therapeutic manipulation of autoimmune diseases. The objective of this study is to identify small compounds disrupting these pivotal protein-protein interactions. Compounds that specifically blocked binding of CD80 to CD28 were identified using a strategy involving a cell-based scintillation proximity assay as the initial step. Secondary screening (e.g., by analyzing the direct binding of these compounds to the target immobilized on a biosensor surface) revealed that these compounds are highly selective CD80 binders. Screening of structurally related derivatives led to the identification of the chemical features required for inhibition of the CD80-CD28 interaction. In addition, the optimization process led to a 10-fold increase in binding affinity of the CD80 inhibitors. Using this approach, the authors identify low-molecular-weight compounds that specifically and with high potency inhibit the interaction between CD80 and CD28. These compounds serve as promising starting points for further development of CD80 inhibitors as potential immunomodulatory drugs. ( Journal of Biomolecular Screening 2007:464-472)

scholarly journals Extended Recognition of the Histone H3 Tail by Histone Demethylase KDM5A

2019 ◽  
Author(s):  
Nektaria Petronikolou ◽  
James E. Longbotham ◽  
Danica Galonić Fujimori
Keyword(s):  
Protein Interactions ◽  
Substrate Recognition ◽  
Methylation Status ◽  
Histone H3 ◽  
Fold Increase ◽  
Protein Protein Interactions ◽  
Alanine Scanning Mutagenesis ◽  
Post Translational Modifications ◽  
Lysine Demethylase ◽  
Demethylation Activity

ABSTRACTHuman lysine demethylase KDM5A is a chromatin modifying enzyme associated with transcriptional regulation due to its ability to catalyze removal of methyl groups from methylated lysine 4 of histone H3 (H3K4me3). Amplification of KDM5A is observed in a number of cancers, including breast cancer, prostate cancer, hepatocellular carcinoma, lung cancer and gastric cancer. In this study, we employed alanine scanning mutagenesis to investigate substrate recognition of KDM5A and identify the H3 tail residues necessary for KDM5A-catalyzed demethylation. Our data show that the H3Q5 residue is critical for substrate recognition by KDM5A. Our data also reveal that the protein-protein interactions between KDM5A and the histone H3 tail extend beyond the amino acids proximal to the substrate mark. Specifically, demethylation activity assays show that deletion or mutation of residues at positions 14-18 on the H3 tail results in an 8-fold increase in the KMapp compared to wild-type 18mer peptide, suggesting this distal epitope is important in histone engagement. Finally, we demonstrate that post-translational modifications on this distal epitope can modulate KDM5A-dependent demethylation. Our findings provide insights into H3K4-specific recognition by KDM5A as well as how chromatin context can regulate KDM5A activity and H3K4 methylation status.

scholarly journals Disulfide-compatible phage-assisted continuous evolution in the periplasmic space

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mary S. Morrison ◽  
Tina Wang ◽  
Aditya Raguram ◽  
Colin Hemez ◽  
David R. Liu
Keyword(s):  
Protein Interactions ◽  
Disulfide Bonds ◽  
Fold Increase ◽  
Soluble Expression ◽  
Important Research ◽  
Protein Protein Interactions ◽  
E Coli ◽  
Continuous Evolution ◽  
Defective Mutant ◽  
Covalent Interactions

AbstractThe directed evolution of antibodies has yielded important research tools and human therapeutics. The dependence of many antibodies on disulfide bonds for stability has limited the application of continuous evolution technologies to antibodies and other disulfide-containing proteins. Here we describe periplasmic phage-assisted continuous evolution (pPACE), a system for continuous evolution of protein-protein interactions in the disulfide-compatible environment of the E. coli periplasm. We first apply pPACE to rapidly evolve novel noncovalent and covalent interactions between subunits of homodimeric YibK protein and to correct a binding-defective mutant of the anti-GCN4 Ω-graft antibody. We develop an intein-mediated system to select for soluble periplasmic expression in pPACE, leading to an eight-fold increase in soluble expression of the Ω-graft antibody. Finally, we evolve disulfide-containing trastuzumab antibody variants with improved binding to a Her2-like peptide and improved soluble expression. Together, these results demonstrate that pPACE can rapidly optimize proteins containing disulfide bonds, broadening the applicability of continuous evolution.

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