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1.
Mol Cell ; 62(2): 207-221, 2016 04 21.
Article in English | MEDLINE | ID: mdl-27105116

ABSTRACT

Nucleosome remodeling and deacetylation (NuRD) complexes are co-transcriptional regulators implicated in differentiation, development, and diseases. Methyl-CpG binding domain (MBD) proteins play an essential role in recruitment of NuRD complexes to their target sites in chromatin. The related SHREC complex in fission yeast drives transcriptional gene silencing in heterochromatin through cooperation with HP1 proteins. How remodeler and histone deacetylase (HDAC) cooperate within NuRD complexes remains unresolved. We determined that in SHREC the two modules occupy distant sites on the scaffold protein Clr1 and that repressive activity of SHREC can be modulated by the expression level of the HDAC-associated Clr1 domain alone. Moreover, the crystal structure of Clr2 reveals an MBD-like domain mediating recruitment of the HDAC module to heterochromatin. Thus, SHREC bi-functionality is organized in two separate modules with separate recruitment mechanisms, which work together to elicit transcriptional silencing at heterochromatic loci.


Subject(s)
Chromatin Assembly and Disassembly , Gene Silencing , Heterochromatin/metabolism , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Protein Processing, Post-Translational , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/enzymology , Acetylation , Binding Sites , CpG Islands , DNA, Fungal/metabolism , Gene Expression Regulation, Fungal , Heterochromatin/chemistry , Heterochromatin/genetics , Mi-2 Nucleosome Remodeling and Deacetylase Complex/chemistry , Mi-2 Nucleosome Remodeling and Deacetylase Complex/genetics , Models, Molecular , Nucleosomes/enzymology , Nucleosomes/genetics , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , RNA, Fungal/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/genetics , Structure-Activity Relationship , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic
2.
Hum Mutat ; 33(1): 86-90, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21922595

ABSTRACT

Bicaudal C homologue 1 (Bicc1) knockout in mice causes polycystic kidney disease and pancreas development defects, including a reduction in insulin-producing ß-cells and ensuing diabetes. We therefore screened 137 patients with renal abnormalities or association of early-onset diabetes and renal disease for genetic alterations in BICC1. We identified two heterozygous mutations, one nonsense in the first K Homology (KH) domain and one missense in the sterile alpha motif (SAM) domain. In mice, Bicc1 blocks canonical Wnt signaling, mostly via its SAM domain. We show that the human BICC1, similar to its mouse counterpart, blocks canonical Wnt signaling. The nonsense mutation identified results in a complete loss of Wnt inhibitory activity. The point mutation in the SAM domain has a similar effect to a complete SAM domain deletion, resulting in a 22% loss of activity.


Subject(s)
Carrier Proteins/genetics , Codon, Nonsense , Kidney/metabolism , Mutation, Missense , Polycystic Kidney Diseases/genetics , RNA-Binding Proteins/genetics , Wnt Signaling Pathway/genetics , Animals , Child, Preschool , Exons , Genetic Testing , Humans , Infant , Infant, Newborn , Introns , Kidney/pathology , Mice , Protein Structure, Tertiary
3.
J Biol Chem ; 281(17): 11787-91, 2006 Apr 28.
Article in English | MEDLINE | ID: mdl-16497675

ABSTRACT

Using a substituted cysteine accessibility scan, we have investigated the structures that form the internal pore of the acid-sensing ion channel 1a. We have identified the amino acid residues Ala-22, Ile-33, and Phe-34 in the amino terminus and Arg-43 in the first transmembrane helix, which when mutated into cysteine, were modified by intracellular application of MTSET, resulting in channel inhibition. The inhibition of the R43C mutant by internal MTSET requires opening of the channel. In addition, binding of Cd2+ ions to R43C slows the channel inactivation. This indicates that the first transmembrane helix undergoes conformational changes during channel inactivation. The effect of Cd2+ on R43C can be obtained with Cd2+ applied at either the extracellular or the intracellular side, indicating that R43C is located in the channel pore. The block of the A22C, I33C, and F34C mutants by MTSET suggests that these residues in the amino terminus of the channel also participate to the internal pore.


Subject(s)
Cysteine/metabolism , Ion Channel Gating/physiology , Membrane Proteins , Mutation/genetics , Nerve Tissue Proteins , Sodium Channels , Acid Sensing Ion Channels , Animals , Cadmium/metabolism , Cysteine/chemistry , Cysteine/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microinjections , Mutagenesis, Site-Directed , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Oocytes/metabolism , Patch-Clamp Techniques , Protein Conformation , Sodium Channels/genetics , Sodium Channels/metabolism , Xenopus laevis/metabolism
4.
J Biol Chem ; 280(9): 7739-47, 2005 Mar 04.
Article in English | MEDLINE | ID: mdl-15623528

ABSTRACT

The epithelial sodium channel ENaC is physiologically important in the kidney for the regulation of the extracellular fluid volume, and in the lungs for the maintenance of the appropriate airway surface liquid volume that lines the pulmonary epithelium. Besides the regulation of ENaC by hormones, intracellular factors such as Na(+) ions, pH, or Ca(2+) are responsible for fast adaptive responses of ENaC activity to changes in the intracellular milieu. In this study, we show that ENaC is rapidly and reversibly inhibited by internal sulfhydryl-reactive molecules such as methanethiosulfonate derivatives of different sizes, the metal cations Cd(2+) and Zn(2+), or copper(II) phenanthroline, a mild oxidizing agent that promotes the formation of disulfide bonds. At the single channel level, these agents applied intracellularly induce the appearance of long channel closures, suggesting an effect on ENaC gating. The intracellular reducing agent dithiothreitol fully reverses the rundown of ENaC activity in inside-out patches. Our observations suggest that changes in intracellular redox potential modulate ENaC activity and may regulate ENaC-mediated Na(+) transport in epithelia. Finally, substitution experiments reveal that multiple cysteine residues in the amino and carboxyl termini of ENaC subunits are responsible for this thiol-mediated inhibition of ENaC.


Subject(s)
Calcium/chemistry , Egtazic Acid/analogs & derivatives , Sodium Channels/metabolism , Sodium Channels/physiology , Sodium/chemistry , Sulfhydryl Compounds/metabolism , Animals , Cadmium/chemistry , Cell Membrane/metabolism , Chelating Agents/pharmacology , Copper/chemistry , Cysteine/chemistry , Dithiothreitol/pharmacology , Edetic Acid/pharmacology , Egtazic Acid/pharmacology , Electrophysiology , Epithelial Sodium Channels , Female , Hydrogen-Ion Concentration , Ions , Mesylates/chemistry , Models, Biological , Mutagenesis, Site-Directed , Oocytes/metabolism , Oxygen/chemistry , Protein Conformation , Protein Structure, Tertiary , RNA, Complementary/metabolism , Time Factors , Xenopus laevis , Zinc/chemistry
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