The acetyl-TESHHK-amide peptide, modeling a part of the C-terminal "tail" of histone H2A, was found previously by us to undergo at pH 7. 4 a Ni(II)-assisted hydrolysis of the E-S peptide bond with formation of a stronger Ni(II) complex with the SHHK-amide product [Bal, W., et al. (1998) Chem. Res. Toxicol. 11, 1014-1023]. To further characterize the hydrolysis and test the resulting Ni(II) complex for redox activity, bovine histone H2A and three peptides were investigated: acetyl-LLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK (H2A(34)), modeling the entire "C-tail" of H2A; SHHKAKGK (H2A(8)), modeling the cutoff product of hydrolysis; and acetyl-KTESHKAKGK (H2A(10)), modeling a putative Ni(II) binding site in a minor variant H2A.4 of human histone H2A. The Ni(II)-assisted hydrolysis of H2A and H2A(34) was found to proceed approximately 7-fold faster than that of the Ni(II)-acetyl-TESHHK-amide complex under comparable conditions. In both cases, the Ni(II) complex with H2A(8) was the smaller product of the hydrolysis, indicating a high site specificity of the reaction. Of three other metals tested with H2A(34), only Cu(II) cleaved the E-S bond, although much less efficiently than Ni(II); Co(II) and Zn(II) had no effect whatsoever. The H2A(10) peptide appeared to be fully resistant to hydrolytic cleavage and did not exhibit any redox activity versus H(2)O(2) in the presence of Ni(II) at pH 7.4. Likewise, redox-inactive was the Ni(II)-H2A(34) complex. In contrast, the Ni(II)-H2A(8) complex promoted oxidative damage of pUC19 DNA by H(2)O(2), evidenced by a significant increase in the number of single strand breaks and nucleobase modifications typical for a hydroxyl radical-like species attack on DNA. Interestingly, instead of 8-oxopurines, the corresponding formamidopyrimidines were the major products of the damage. The difference in redox activity between the Ni(II)-H2A(34) and Ni(II)-H2A(8) complexes is most likely associated with their different geometries: octahedral and square planar, respectively. Incubation of the Ni(II)-H2A(8) complex with H(2)O(2) also resulted in degradation of the peptide ligand, especially at its Ser and His residues. Thus, binding of Ni(II) to the ESHHK motif of the histone H2A C-tail is damaging to the histone C-terminal tail and to histone-associated DNA. The results support a dual mechanism of Ni(II)-induced carcinogenesis, including both genotoxic and epigenetic effects.
A combined pH-metric and spectroscopic (UV/vis, CD, NMR) study of the Ni(II) binding to CH3CO-Thr-Glu-Ser-His-His-Lys-NH2 (AcTESHHKam), a blocked hexapeptide modeling a part of the C-terminal sequence of the major variant of histone H2A (residues 120-125), revealed the formation of a pseudo-octahedral NiHL complex in weakly acidic and neutral solutions. Ni(II) is bound to the peptide through imidazole nitrogens on both of its histidine residues and the carboxylate of the side chain of glutamic acid. At higher pH, a series of square-planar complexes are formed. This process is accompanied by hydrolytic degradation of the peptide. At pH 7.4, the peptide hydrolyzes in a Ni(II)-assisted fashion, yielding the square-planar Ni(II) complex of SHHKam as the sole product detected by CD, MALDI-TOF MS, and HPLC. Quantitative analysis of complex stabilities indicates that the -TESHHK- motif is a very likely binding site for carcinogenic Ni(II) ions in the cell nucleus. The Ni(II)-assisted hydrolysis of the C-terminal chain of histone H2A may provide a novel mechanism of genotoxicity combining the damage to the nucleosome with the generation of further toxic Ni(II) species.
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