scholarly journals MOLECULAR CLONING, EXPRESSION AND FUNCTIONAL INTERACTION OF p48 SUBUNIT OF CHICKEN CHROMATIN ASSEMBLY FACTOR 1 WITH HISTONE DEACETYLASE 2 AND HISTONE ACETYLTRANSFERASE 1

2012 ◽  
Vol 12 (1) ◽  
pp. 77-83
Author(s):  
Ahyar Ahmad
Keyword(s):  
Amino Acids ◽  
Histone Deacetylase ◽  
Leucine Zipper ◽  
Histone Acetyltransferase ◽  
Leucine Zipper Motif ◽  
Histone Deacetylase 2 ◽  
Pulldown Assay ◽  
Assembly Factor ◽  
In Vitro Interaction

We cloned and sequenced cDNA encoding p48 subunit of the chicken CAF-1, chCAF-1p48, and histone acetyltransferase-1, chHAT-1 from chicken DT40 cell lines. We showed that the p48 subunit of CAF-1 tightly binds to two regions of chicken histone deacetylase 2, chHDAC-2, located between amino acid residues 82-180 and 245-314, respectively. We also established that two N-terminal, two C-terminal, or one N-terminal and one C-terminal WD repeat motif of chCAF-1p48 are required for this interaction. The GST pulldown assay, involving truncated and missense mutants of chCAF-1p48, revealed not only that a region containing the seventh WD dipeptide motif of chCAF-1p48, comprising amino acids 376-405, binds to chHAT-1 in vitro, but also that mutation of the motif has no influence on the in vitro interaction. We also established that the region, which is located between amino acids 380-408 of chHAT-1 and contains a leucine zipper motif, is required for its in vitro interaction with chCAF-1p48. Mutation on each of four Leu residues in the leucine zipper motif of chHAT-1 causes the disappearance of the interaction with chCAF-1p48. These results should be useful information for understanding the participation of chCAF-1p48 protein as histones chaperone in DNA-utilizing processes, such as replication, recombination, repair and gene expression in DT40 chicken B cell.

Inhibition of heat shock protein 70 blocks the development of cardiac hypertrophy by modulating the phosphorylation of histone deacetylase 2

2018 ◽  
Vol 115 (13) ◽  
pp. 1850-1860 ◽  
Author(s):  
Somy Yoon ◽  
Mira Kim ◽  
Hyun-Ki Min ◽  
Yeong-Un Lee ◽  
Duk-Hwa Kwon ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Cardiac Hypertrophy ◽  
Histone Deacetylase ◽  
Heat Shock Protein 70 ◽  
Specific Binding ◽  
Primary Cultures ◽  
Histone Deacetylase 2 ◽  
Ventricular Cardiomyocytes

Abstract Aims Previously, we reported that phosphorylation of histone deacetylase 2 (HDAC2) and the resulting activation causes cardiac hypertrophy. Through further study of the specific binding partners of phosphorylated HDAC2 and their mechanism of regulation, we can better understand how cardiac hypertrophy develops. Thus, in the present study, we aimed to elucidate the function of one such binding partner, heat shock protein 70 (HSP70). Methods and results Primary cultures of rat neonatal ventricular cardiomyocytes and H9c2 cardiomyoblasts were used for in vitro cellular experiments. HSP70 knockout (KO) mice and transgenic (Tg) mice that overexpress HSP70 in the heart were used for in vivo analysis. Peptide-precipitation and immunoprecipitation assay revealed that HSP70 preferentially binds to phosphorylated HDAC2 S394. Forced expression of HSP70 increased phosphorylation of HDAC2 S394 and its activation, but not that of S422/424, whereas knocking down of HSP70 reduced it. However, HSP70 failed to phosphorylate HDAC2 in the cell-free condition. Phosphorylation of HDAC2 S394 by casein kinase 2α1 enhanced the binding of HSP70 to HDAC2, whereas dephosphorylation induced by the catalytic subunit of protein phosphatase 2A (PP2CA) had the opposite effect. HSP70 prevented HDAC2 dephosphorylation by reducing the binding of HDAC2 to PP2CA. HSP70 KO mouse hearts failed to phosphorylate S394 HDAC2 in response to isoproterenol infusion, whereas Tg overexpression of HSP70 increased the phosphorylation and activation of HDAC2. 2-Phenylethynesulfonamide (PES), an HSP70 inhibitor, attenuated cardiac hypertrophy induced either by phenylephrine in neonatal ventricular cardiomyocytes or by aortic banding in mice. PES reduced HDAC2 S394 phosphorylation and its activation by interfering with the binding of HSP70 to HDAC2. Conclusion These results demonstrate that HSP70 specifically binds to S394-phosphorylated HDAC2 and maintains its phosphorylation status, which results in HDAC2 activation and the development of cardiac hypertrophy. Inhibition of HSP70 has possible application as a therapeutic.

scholarly journals Improved Cell Selectivity of Pseudin-2 via Substitution in the Leucine-Zipper Motif: In Vitro and In Vivo Antifungal Activity

Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 921
Author(s):  
Seong-Cheol Park ◽  
Heabin Kim ◽  
Jin-Young Kim ◽  
Hyeonseok Kim ◽  
Gang-Won Cheong ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Leucine Zipper ◽  
Pathogenic Fungi ◽  
Repeated Sequence ◽  
Antimicrobial Activities ◽  
Leucine Zipper Motif ◽  
Antifungal Activities ◽  
Cell Selectivity

Several antimicrobial peptides (AMPs) have been discovered, developed, and purified from natural sources and peptide engineering; however, the clinical applications of these AMPs are limited owing to their lack of abundance and side effects related to cytotoxicity, immunogenicity, and hemolytic activity. Accordingly, to improve cell selectivity for pseudin-2, an AMP from Pseudis paradoxa skin, in mammalian cells and pathogenic fungi, the sequence of pseudin-2 was modified by alanine or lysine at each position of two amino acids within the leucine-zipper motif. Alanine-substituted variants were highly selective toward fungi over HaCaT and erythrocytes and maintained their antifungal activities and mode of action (membranolysis). However, the antifungal activities of lysine-substituted peptides were reduced, and the compound could penetrate into fungal cells, followed by induction of mitochondrial reactive oxygen species and cell death. In vivo antifungal assays of analogous peptide showed excellent antifungal efficiency in a Candida tropicalis skin infection mouse model. Our results demonstrated the usefulness of selective amino acid substitution in the repeated sequence of the leucine-zipper motif for the design of AMPs with potent antimicrobial activities and low toxicity.

scholarly journals Loss of CFTR results in reduction of histone deacetylase 2 in airway epithelial cells

2009 ◽  
Vol 297 (1) ◽  
pp. L35-L43 ◽  
Author(s):  
Toni R. Bartling ◽  
Mitchell L. Drumm
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylase ◽  
Airway Epithelial Cells ◽  
Histone Deacetylation ◽  
Gene Promoters ◽  
Airway Epithelial ◽  
Histone Deacetylase 2 ◽  
Proinflammatory Gene ◽  
In Cells

Inflammatory cytokines, particularly the neutrophil chemoattractant IL-8, are elevated in the cystic fibrosis (CF) airway, even in the absence of detectable infection. The transcriptional regulation of many inflammatory genes, including IL8 ( CXCL8), involves chromatin remodeling through histone acetylation. NF-κB is known to facilitate histone acetylation of IL8 and other proinflammatory gene promoters, but we find that increased NF-κB activation cannot explain the elevated IL8 expression and promoter acetylation seen in CFTR-deficient cells. Recognized components of the NF-κB-coactivator complex, acetyltransferase CBP, p300, and the histone deacetylase HDAC1, are unchanged by CFTR activity. However, we find that the histone acetyltransferase (HAT)/HDAC balance is sensitive to CFTR function, as cells with reduced or absent CFTR function have decreased HDAC2 protein, resulting in hyperacetylation of the IL8 promoter and increased IL8 transcription. Reduced HDAC2 and HDAC2 activity, but not HDAC2 mRNA, is observed in cells deficient in CFTR. Suppressing HDAC2 expression with HDAC2 short hairpin RNA (shRNA) results in increased IL8 expression and promoter acetylation comparable with CFTR-deficient cells. Treating CFTR-deficient cells with N-acetyl-cysteine (NAC) increases HDAC2 expression to near control levels. Our data suggest that there is an intrinsic alteration in the HAT/HDAC balance in cells lacking CFTR function in vitro and in native CF tissue and that oxidative stress is likely contributing to this alteration. This mechanism, found in other inflammatory airway diseases, provides an explanation for the apparent dysregulation of inflammatory mediators seen in the CF airway, as reduced histone deacetylation would potentially influence many genes.

scholarly journals EXPRESSION AND FUNCTIONAL INTERACTION OF CHICKEN RbAp46 POLYPEPTIDE WITH HISTONES, HISTONE DEACETYLASE-1, AND HISTONE ACETYLTRANSFERASE-1

2013 ◽  
Vol 13 (2) ◽  
pp. 136-141
Author(s):  
Ahyar Ahmad ◽  
Harningsih Karim
Keyword(s):  
Amino Acids ◽  
Histone Acetyltransferase ◽  
Single Step ◽  
In Vitro Translation ◽  
Translation System ◽  
Functional Interaction ◽  
In Vitro Experiment ◽  
Histone Deacetylase 1 ◽  
Agarose Beads

In this study, we cloned and sequenced cDNA encoding the chicken p46 polypeptide, RbAp46. The cDNA encoding a protein consists of 424 amino acids is a member of the WD protein family, with seven WD repeat motifs, and exhibits 90.3% identity to RbAp48, and 94.3% identity to the human RbAp46. The RbAp46 fusion protein were synthesized by in vitro translation system and in Escherichia coli under induction by 50 µM IPTG and single step purified with glutathione-Agarose beads, showed that GST-tagged protein of approximately 72 kDa. The in vitro experiment established that RbAp46 interacts with chicken histones, chHDAC-1, and chHAT-1. The in vitro immunoprecipitation experiment, involving truncated mutants of RbAp46, revealed not only that two regions comprising amino acids 33-179 and 375-404 are necessary for its binding to H2B, but also that two regions comprising amino acids 1-32 and 405-424 are necessary for its binding to H4. Furthermore, the GST pulldown affinity assay, involving truncated mutants of RbAp46, revealed that a region comprising amino acids 359-404 binds to chHAT-1 in vitro. Taken together, these results indicate not only that RbAp46 should participate differentially in a number of DNA-utilizing processes through interactions of its distinct regions with histones and chHAT-1, but also that the proper propeller structure of RbAp46 is not necessary for its interaction with chHAT-1.

scholarly journals Positive Selection of Mutations Leading to Loss or Reduction of Transcriptional Activity of PrfA, the Central Regulator ofListeria monocytogenes Virulence

2001 ◽  
Vol 183 (19) ◽  
pp. 5562-5570 ◽  
Author(s):  
M. Herler ◽  
A. Bubert ◽  
M. Goetz ◽  
Y. Vega ◽  
J. A. Vazquez-Boland ◽  
...  
Keyword(s):  
Amino Acids ◽  
Positive Selection ◽  
Transcriptional Activity ◽  
Leucine Zipper ◽  
Stable Complex ◽  
Gene Encoding ◽  
Target Dna ◽  
Oflisteria Monocytogenes ◽  
Function Relationship

ABSTRACT Transcription factor PrfA controls the expression of virulence genes essential for Listeria monocytogenes pathogenesis. To gain insight into the structure-function relationship of PrfA, we devised a positive-selection system to isolate mutations reducing or abolishing transcriptional activity. The system is based on the observation that the listerial iap gene, encoding the p60 protein, is lethal if overexpressed in Bacillus subtilis. A plasmid in which the iap gene is placed under the control of the PrfA-dependent hlypromoter was constructed and introduced into B. subtilis. This strain was rapidly killed when expression ofiap was induced by introduction of a second plasmid carrying prfA. Two classes of B. subtilissurvivor mutants were identified: one carried mutations iniap, and the second carried mutations inprfA. Sequence analysis of the defectiveprfA genes identified mutations in three regions of the PrfA protein: region A, between amino acids 58 and 67 in the β-roll domain of PrfA; region B, between amino acids 169 and 193, which corresponds to the DNA-binding helix-turn-helix motif; and region C, comprising the 38 C-terminal amino acids of PrfA, which form a leucine zipper-like structure. PrfA proteins with mutations in regions B and C were unable to bind to the PrfA-binding site in the target DNA, while mutations in region A resulted in a protein still binding the target DNA but unable to form a stable complex with RNA polymerase and initiate transcription in vitro.

scholarly journals The leucine zipper of c-Myc is required for full inhibition of erythroleukemia differentiation.

1990 ◽  
Vol 10 (10) ◽  
pp. 5333-5339 ◽  
Author(s):  
M J Smith ◽  
D C Charron-Prochownik ◽  
E V Prochownik
Keyword(s):  
Leucine Zipper ◽  
Point Mutations ◽  
Leucine Zipper Motif ◽  
Wild Type ◽  
Protein Dimerization ◽  
Leucine Zipper Domain ◽  
Close Relationship ◽  
Type C ◽  
Murine Erythroleukemia

The leucine zipper motif has been observed in a number of proteins thought to function as eucaryotic transcription factors. Mutation of the leucine zipper interferes with protein dimerization and DNA binding. We examined the effect of point mutations in the leucine zipper of c-Myc on its ability to dimerize in vitro and to inhibit Friend murine erythroleukemia (F-MEL) differentiation. Glutaraldehyde cross-linking studies failed to provide evidence for homodimerization of in vitro-synthesized c-Myc protein, although it was readily demonstrated for c-Jun. Nevertheless, whereas transfected wild-type c-myc sequences strongly inhibited F-MEL differentiation, those with single or multiple mutations in the leucine zipper were only partially effective in this regard. Since the leucine zipper domain of c-Myc is essential for its cooperative effect in ras oncogene-mediated transformation, this study emphasizes the close relationship that exists between transformation and hematopoietic commitment and differentiation. c-Myc may produce its effects on F-MEL differentiation through leucine zipper-mediated heterodimeric associations rather than homodimeric ones.

scholarly journals The p55 subunit of Drosophila chromatin assembly factor 1 is homologous to a histone deacetylase-associated protein.

1996 ◽  
Vol 16 (11) ◽  
pp. 6149-6159 ◽  
Author(s):  
J K Tyler ◽  
M Bulger ◽  
R T Kamakaka ◽  
R Kobayashi ◽  
J T Kadonaga
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Replication ◽  
Histone Modification ◽  
Molecular Mass ◽  
Histone Deacetylase ◽  
Nuclear Extract ◽  
Chromatin Assembly ◽  
Chromatin Assembly Factor ◽  
Assembly Factor

To gain a better understanding of DNA replication-coupled chromatin assembly, we have isolated the cDNA encoding the smallest (apparent molecular mass, 55 kDa; termed p55) subunit of Drosophila melanogaster chromatin assembly factor 1 (dCAF-1), a multisubunit protein that is required for the assembly of nucleosomes onto newly replicated DNA in vitro. The p55 polypeptide comprises seven WD repeat motifs and is homologous to the mammalian RbAp48 protein, which is associated with the HD1 histone deacetylase. dCAF-1 was immunopurified by using affinity-purified antibodies against p55; the resulting dCAF-1 preparation possessed the four putative subunits of dCAF-1 (p180, p105, p75, and p55) and was active for DNA replication-coupled chromatin assembly. Moreover, dCAF-1 activity was specifically depleted with antibodies against p55. Thus, p55 is an integral component of dCAF-1. p55 is localized to the nucleus and is present throughout Drosophila development. Consistent with the homology between p55 and the HD1-associated RbAp48 protein, histone deacetylase activity was observed to coimmunoprecipitate specifically with p55 from a Drosophila nuclear extract. Furthermore, a fraction of the p55 protein becomes associated with the newly assembled chromatin following DNA replication. These findings collectively suggest that p55 may function as a link between DNA replication-coupled chromatin assembly and histone modification.

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