DNA strand breakage induced by CuII and NiII, in the presence of peptide models of histone H2B

In the present study we used the plasmid relaxation assay, a very sensitive method for detection of DNA strand breaks in vitro, in order to evaluate the role of peptide fragments of histone H2B in DNA strand breakage induced by copper and nickel. We have found that in the presence of peptides modeling the histone fold domain (H2B_32-62 and H2B_63-93) as well as the N-terminal tail (H2B_1-31) of histone H2B there is an increased DNA damage by Cu²⁺/H₂O₂ and Ni²⁺/H₂O₂ reaction mixtures. On the contrary, the C-terminal tail (H2B_94-125) seems to have a protective role on the attack of ROS species to DNA. We have rendered our findings to the interactions of the peptides with DNA, as well as with the metal.     

Histone H3 lysine 4 mono-methylation does not require ubiquitination of histone H2B

The yeast Set1-complex catalyzes histone H3 lysine 4 (H3K4) methylation. Using N-terminal Edman sequencing, we determined that 50% of H3K4 is methylated and consists of roughly equal amounts of mono, di and tri-methylated H3K4. We further show that loss of either Paf1 of the Paf1 elongation complex, or ubiquitination of histone H2B, has only a modest effect on bulk histone mono-methylation at H3K4. Despite the fact that Set1 recruitment decreases in paf1delta cells, loss of Paf1 results in an increase of H3K4 mono-methylation at the 5' coding region of active genes, suggesting a Paf1-independent targeting of Set1. In contrast to Paf1 inactivation, deleting RTF1 affects H3K4 mono-methylation at the 3' coding region of active genes and results in a decrease of global H3K4 mono-methylation. Our results indicate that the requirements for mono-methylation are distinct from those for H3K4 di and tri-methylation, and point to differences among members of the Paf1 complex in the regulation of H3K4 methylation.     

Histone H4 and H2B genes in rainbow trout (Salmo gairdnerii)

Summary The complete nucleotide sequence of the 3.0-kb BamH I-Sst I restriction fragment contained within the rainbow trout genomic clone TH2 has been determined. This region contains the rainbow trout H4 and H2B histone genes and 5 and 3 flanking and spacer sequences, and represents the 5 half of the histone-gene cluster; the remaining half has been characterized previously. The genes are uninterrupted, and are transcribed from the same strand. The protein sequence of H4, as determined from the nucleic acid sequence, is the same as that derived for other vertebrate H4 proteins, although comparison of nucleotide sequences shows a great deal of sequence divergence, especially in the third base position. The amino acid sequence of H2B, though largely homologous to those of other vertebrate H2B proteins, displays some characteristic differences in primary structure. Consensus sequences noted in many other eukaryotic genes, as well as histone-specific consensus sequences, have been identified. An unusual feature of the spacer region between the H4 and H2B genes is the presence of a duplicated sequence 87 bp in length. The 5 and 3 ends of each repeat are complementary, and each repeat contains smaller repeated sequences internally, as well as a possible cruciform structure.     

Copper effective binding with 32-62 and 94-125 peptide fragments of histone H2B

In an attempt to investigate the role of histone H2B in Cu(II) induced toxicity and carcinogenesis, we synthesized the terminally blocked peptides H2B_32-62 (SRKESYSVYVYKVLKQVH₄₈PDTGISSKAMGIM) and Η2Β_94-125 (IQTAVRLLLPGELAKH₁₁₀AVSEGTKAVTKYTSS), mimicking the N-terminal histone-fold domain and C-terminal tail of histone H2B, respectively and studied their interaction with Cu(II) ions by means of potentiometric titrations and spectroscopic techniques (UV-visible, CD and EPR). Both peptides, H2B_32-62 and H2B_94-125, interacted efficiently with Cu(II) ions, forming several species from pH 4 to 11, with His₄₈ and His₁₁₀ serving as anchors for metal binding. In H2B_32-62, the effective Cu(II) binding is emphasized by the formation of a soluble Cu(II)-H2B_32-62 complex, unlike the unbound peptide that precipitated over pH 7.9. At physiological pH, both peptides form tetragonal 3N species with a {N_Im, 2N⁻} coordination mode. At this pH, H2B_32-62 presented the formation of coordination isomers, differentiated by the presence in one of them, of an axial coordination of the carboxylate group of Asp₅₀. Copper binding with both H2B_32-62 and H2B_94-125 may induce a conformational change in the peptides' original structure. At physiological conditions, this effect may interfere with nucleosome's structure and dynamics, including the ubiquitination of Lys₁₂₀ which is linked to gene silencing.     

Transition metal-saccharide chemistry: d-glucose complexes of Mn(II), Co(II), Ni(II), Cu(II) and Zn(II)

Metal complexes of d-glucose (d-Glc) from large cation containing dibromo-dichloro salts of dipositive metals [NEt₄]₂[MBr₂Cl₂] (M = Mn, Co, Ni, Cu and Zn) and the disodium salt of glucose were synthesized from a MeOH:MeCN mixture. The complexes were characterized by UV-vis absorption, circular dichroism, IR and proton magnetic resonance spectroscopies, and by elemental analysis, and were found to be Na[M(d-Glc)(OMe)Cl]. Cyclic voltammetric studies of these complexes, in the acidic to neutral pH range, indicated no dissociation, even in highly acidic conditions.This paper is dedicated to Professor Richard H. Holm (Harvard University) on the occasion of his 60th birthday.     

Preparation, structural, magnetic and thermal properties of two heterobimetallic cyano-bridged nickel(II)-copper(II)/platinum(II) coordination polymers

Two unique bimetalic Pt(II) coordination polymers of composition [Ni(hydeten)₂Pt(CN)₄] (Ni-Pt) and [Cu(hydeten)₂Pt(CN)₄] (Cu-Pt) [hydeten = N-(2-hydroxyethyl-ethylenediamine) or 2-(2-aminoethylamino)ethanol] have been synthesized and structurally characterized by various methods in this study. The crystal structure of Cu-Pt was determined by single-crystal X-ray diffraction analysis. The structure of Cu-Pt forms of infinite 2,2-TT type [-Cu(hydeten)₂-NC-Pt(CN)₂-CN-] chains containing paramagnetic copper atoms bridged by tetracyanoplatinate species. In this complex, Cu(II) centers display an axially elongated octahedron with two chelating hydeten molecules in the equatorial positions and N-bonded bridging cyano groups in the axial positions, whereas Pt(II) centers are four coordinate with four cyanide-carbon atoms in a square-planar arrangement. The decrease of the moments of these complexes in temperature range of 50 305 K can assigned to the antiferromagnetic interactions in the structures. The thermal decomposition of Cu-Pt comprise of five distinguished stages, while the thermal decomposition of Ni-Pt take place four different stages.     

Synthesis and characterization of nickel(II) and copper(II) complexes with tetradentate Schiff base ligands

The 1:1 condensation of 1,2-diaminopropane and 1-phenylbutane-1,3-dione at high dilution gives a mixture of two positional isomers of terdentate mono-condensed Schiff bases 6-amino-3-methyl-1-phenyl-4-aza-2-hepten-1-one (HAMPAH) and 6-amino-3,5-dimethyl-1-phenyl-4-aza-2-hexen-1-one (HADPAH). The mixture of the terdentate ligands has been used for further condensation with pyridine-2-carboxaldehyde or 2-acetylpyridine to obtain the unsymmetrical tetradentate Schiff base ligands. The tetradentate Schiff bases are then allowed to react with the methanol solution of copper(II) and nickel(II) perchlorate separately. The X-ray diffraction confirms the structures of two of the complexes and shows that the condensation site of the diamine with 1-phenylbutane-1,3-dione is the same.     

Inhibition of protein-tyrosine phosphatase 1B (PTP1B) mediates ubiquitination and degradation of Bcr-Abl protein

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-Abl, a chimeric protein with deregulated tyrosine kinase activity. The protein-tyrosine phosphatase 1B (PTP1B) is up-regulated in Bcr-Abl-expressing cells, suggesting a regulatory link between the two proteins. To investigate the interplay between these two proteins, we inhibited the activity of PTP1B in Bcr-Abl-expressing TonB.210 cells by either pharmacological or siRNA means and examined the effects of such inhibition on Bcr-Abl expression and function. Herein we describe a novel mechanism by which the phosphatase activity of PTP1B is required for Bcr-Abl protein stability. Inhibition of PTP1B elicits tyrosine phosphorylation of Bcr-Abl that triggers the degradation of Bcr-Abl through ubiquitination via the lysosomal pathway. The degradation of Bcr-Abl consequently inhibits tyrosine phosphorylation of Bcr-Abl substrates and the downstream production of intracellular reactive oxygen species. Furthermore, PTP1B inhibition reduces cell viability and the IC(50) of the Bcr-Abl inhibitor imatinib mesylate. Degradation of Bcr-Abl via PTP1B inhibition is also observed in human CML cell lines K562 and LAMA-84. These results suggest that inhibition of PTP1B may be a useful strategy to explore in the development of novel therapeutic agents for the treatment of CML, particularly because host drugs currently used in CML such as imatinib focus on inhibiting the kinase activity of Bcr-Abl.     

BRHIS1 suppresses rice innate immunity through binding to monoubiquitinated H2A and H2B variants

In the absence of pathogen attack, organisms usually suppress immune responses to reduce the negative effects of disease resistance. Monoubiquitination of histone variants at specific gene loci is crucial for gene expression, but its involvement in the regulation of plant immunity remains unclear. Here, we show that a rice SWI/SNF2 ATPase gene BRHIS1 is downregulated in response to the rice blast fungal pathogen or to the defense‐priming‐inducing compound BIT (1,2‐benzisothiazol‐3(2h)‐one,1, 1‐dioxide). The BRHIS1‐containing complex represses the expression of some disease defense‐related genes, including the pathogenesis‐related gene OsPBZc and the leucine‐rich‐repeat (LRR) receptor‐like protein kinase gene OsSIRK1. This is achieved through BRHIS1 recruitment to the promoter regions of target genes through specific interaction with monoubiquitinated histone variants H2B.7 and H2A.Xa/H2A.Xb/H2A.3, in the absence of pathogen attack or BIT treatment. Our results show that rice disease defense genes are initially organized in an expression‐ready state by specific monoubiquitination of H2A and H2B variants deposited on their promoter regions, but are kept suppressed by the BRHIS1 complex, facilitating the prompt initiation of innate immune responses in response to infection through the stringent regulation of BRHIS1.     

Imidazole derivatives of binuclear copper (II) and nickel (II) complexes incorporating bis(1,4,7-triazacyclononan-1-yl) ligands

A series of new binuclear copper (II) and nickel (II) complexes of the macrocyclic ligands bis(1,4,7-triazacyclononan-1-yl)butane (L^but) and bis(1,4,7-triazacyclononan-1-yl)-m-xylene (L^mx) have been synthesized: [Cu₂L^butBr₄] (1), [Cu₂L^but(imidazole)₂Br₂](ClO₄)₂ (2), [Cu₂L^mx(μ-OH)(imidazole)₂](ClO₄)₃ (3), [Cu₂L^but(imidazole)₄](ClO₄)₄ · H₂O (4), [Cu₂L^mx(imidazole)₄](ClO₄)₄ (5), [Ni₂ L^but(H₂O)₆](ClO₄)₄ · 2H₂O (6), [Ni₂L^but(imidazole)₆](ClO₄)₄ · 2H₂O (7) and [Ni₂L^mx (imidazole)₄(H₂O)₂](ClO₄)₄ · 3H₂O (8). Complexes 1, 2, 7 and 8 have been characterized by single crystal X-ray studies. In each of the complexes, the two tridentate 1,4,7-triazacyclononane rings of the ligand facially coordinate to separate metal centres. The distorted square-pyramidal coordination sphere of the copper (II) centres is completed by bromide anions in the case of 1 and/or monodentate imidazole ligands in complexes 2, 4 and 5. Complex 3 has been formulated as a monohydroxo-bridged complex featuring two terminal imidazole ligands. Complexes 6-8 feature distorted octahedral nickel (II) centres with water and/or monodentate imidazole ligands occupying the remaining coordination sites. Within the crystal structures, the ligands adopt trans conformations, with the two metal binding compartments widely separated, perhaps as a consequence of electrostatic repulsion between the cationic metal centres. The imidazole-bearing complexes may be viewed as simple models for the coordinative interaction of the binuclear complexes of bis (tacn) ligands with protein molecules bearing multiple surface-exposed histidine residues.     

New template reactions of salicylaldehyde S-methyl-isothiosemicarbazone with 2-formylpyridine promoted by Ni(II) or Cu(II) metal ions

The template reaction between salicylaldehyde S-methyl-isothiosemicarbazone and 2-formylpyridine in presence of nickel(II) or copper(II) salts yields two new coordination compounds with general formula [NiL¹]₂(1) and [CuL²]₂(2) (L¹ = the dianionic (N¹-salicylidene)(N⁴-(hydroxy(pyridin-2-yl)methyl) S-methyl-isothiosemicarbazide) ligand and L² = the doubly deprotonated (N¹-salicylidene)(N⁴-(picolinoyl) S-methyl-isothiosemicarbazide) ligand). In the complex 1, the formed L¹ ligand appears as result of an addition reaction of the precursors, while for 2 a redox mechanism is implicated in the formation of L². Despite the fact that the initial organic precursors are the same, the resulting ligands obtained in the template reaction are different. In 1, the Ni(II) metal ion adopts a square-planar geometry and the [NiL¹] units are forming dimerized chains through weak Ni···Ni interactions (3.336 and 3.632 Å). In 2, the Cu(II) metal ions adopt a square-pyramidal geometry and form dinuclear species through weak Cu···O (phenoxo) interactions. The magnetic susceptibility measurements of the complexes reveal the diamagnetic nature of 1 as expected for a square planar Ni(II) complex and a paramagnetic behavior for 2 with weak intra-dimer antiferromagnetic interaction (J/k_B = −2.1(1) K).     

Polynuclear nickel (II) and copper (II) complexes of hexaazamacrocycles incorporating pairs of diethylenetriamine subunits separated by aromatic spacers

A series of Ni(II) and Cu(II) complexes of the hexaaza macrocycles, 3,6,9,17,20,23-hexaazatricyclo[23.3.1.1^11,15]triaconta-1(29),11(30),12,14,25,27-hexaene (L¹) and 3,6,9,16,19,22-hexaazatricyclo[22.2.2.2^11,14]triaconta-1(26),11(29),12,14(30),24(28),25-hexaene (L²), have been prepared and the crystal structures determined for [Ni₂L¹(O₂CCH₃)₂(H₂O)₂](ClO₄)₂ (1), [Ni₂L²(DMF)₆](ClO₄)₄ · 2H₂O (2), {[Cu₂L²Br(O₂CCH₃)](ClO₄)₂}_n (3), [Cu₂L²(μ-CO₃)(H₂O)₂]₂(ClO₄)₄ · 8H₂O (4), [Cu₂L²(O₂CCH₃)₂](BF₄)₂ (5), and [Cu₂L¹(μ-imidazolate)Br]₂Br₄ · 6H₂O (6). In these complexes, two metal centers are bound per ligand; in 1 and 3-6, the N₃ subunits of L¹ or L² coordinate meridionally to the metal centers, whilst in 2, each N₃ subunit in L² adopts a facial mode of coordination. The binuclear cations in 1 and 2 have chair-like conformations, with the distorted octahedral Ni(II) coordination spheres completed by terminal water and a bidentate acetate ligand in 1 and three DMF ligands in 2. The Cu(II) centers in 3-6 generally reside in square planar environments, although a weakly binding ligand enters the coordination sphere in some cases, generating a distorted square pyramidal geometry. The binuclear [Cu₂L²]⁴⁺ units in 3, 4 and 5 adopt similar bowl-shaped conformations, stabilized by H-bonding interactions between pairs of amine groups from L² and a perchlorate or tetrafluoroborate anion. In 3, the binuclear units are linked through acetate groups, bridging in a syn-anti fashion, to produce a zig-zag polymeric chain structure, whilst 4 incorporates a tetrameric cation consisting of two binuclear units linked via a pair of carbonate bridges. Compound 6 features an imidazolate bridge between the two Cu(II) centers bound by L¹. Pairs of [Cu₂L¹(μ-imidazolate)]³⁺ units are then weakly linked through a pair of bromide anions.     

Trypanosoma cruzi bromodomain factor 2 (BDF2) binds to acetylated histones and is accumulated after UV irradiation

Histone tail post-translational modifications (acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation) regulate many cellular processes. Among these modifications, phosphorylation, methylation and acetylation have already been described in trypanosomatid histones. Bromodomains, together with chromodomains and histone-binding SANT domains, were proposed to be responsible for “histone code” reading. The Trypanosoma cruzi genome encodes four coding sequences (CDSs) that contain a bromodomain, named TcBDF1-4. Here we show that one of those, TcBDF2, is expressed in discrete regions inside the nucleus of all the parasite life cycle stages and binds H4 and H2A purified histones from T. cruzi. Immunolocalization experiments using both anti-histone H4 acetylated peptides and anti-TcBDF2 antibodies determined that TcBDF2 co-localizes with histone H4 acetylated at lysines K10 and K14. TcDBF2 and K10 acetylated H4 interaction was confirmed by co-immunoprecipitation. It is also shown that TcBDF2 was accumulated after UV irradiation of T. cruzi epimastigotes. These results suggest that TcBDF2 could be taking part in a chromatin remodelling complex in T. cruzi.