Protein modification by adenine propenal

Abstract

Base propenals are products of the reaction of DNA with oxidants such as peroxynitrite and bleomycin. The most reactive base propenal, adenine propenal, is mutagenic in Escherichia coli and reacts with DNA to form covalent adducts; however, the reaction of adenine propenal with protein has not yet been investigated. A survey of the reaction of adenine propenal with amino acids revealed that lysine and cysteine form adducts, whereas histidine and arginine do not. N(ε)-Oxopropenyllysine, a lysine-lysine cross-link, and S-oxopropenyl cysteine are the major products. Comprehensive profiling of the reaction of adenine propenal with human serum albumin and the DNA repair protein, XPA, revealed that the only stable adduct is N(ε)-oxopropenyllysine. The most reactive sites for modification in human albumin are K190 and K351. Three sites of modification of XPA are in the DNA-binding domain, and two sites are subject to regulatory acetylation. Modification by adenine propenal dramatically reduces XPA's ability to bind to a DNA substrate.

Figure 1

Schematic of base propenal and MDA production. Both species can react with dA or dG, resulting in oxypropenylation of the base. M₁dG is subsequently formed by ring closure of the oxypropenylated dG.

Figure 2

HPLC analysis of an N-α-acetyllysine and adenine propenal reaction mixture. N-α-Acetyllysine (50 mM) was incubated with adenine propenal (10 mM) in a 50 μL reaction mixture for 2 h at room temperature. Following incubation, 4 μL of the reaction mixture was analyzed by HPLC. The column eluate was monitored at 260 nm (A) or 300 nm (B). Analytes present in each peak were subjected to MS, UV, and NMR spectrometry. Numbers correspond to the identified products, represented in (C).

Figure 3

HPLC analysis of product formation in the reaction of adenine propenal and N-α-acetyllysine. The indicated amounts of N-α-acetyllysine (NAL) were incubated with 10 mM adenine propenal, and reactions were monitored hourly for 9–10 h. Peaks corresponding to 3 and 4 were monitored at 280 and 300 nm, respectively, by diode-array detection. Data show the mean ± SD of three determinations.

Figure 4

Proposed scheme for the reaction of adenine propenal with N-α-acetyllysine.

Figure 5

LC-MS analysis of the reduced reaction intermediate (6). (A) Representative LC-MS chromatogram of 6. In this example, 5 mM N-α-acetyllysine was reacted with 10 mM adenine propenal for 30 min followed by reduction with 50 mM NaCNBH₃. (B) Mass spectrum of the parent ion of the reduced intermediate displaying m/z 360 with detection in negative ion mode.

Figure 6

Human serum albumin modification by adenine propenal. (A) Modified lysine residues identified in Table 1 were mapped to the crystal structure of HSA (1AO6). Lysine residues with an additional mass of +54.0105 are indicated in red. (B) The most highly modified residues by individual electrophiles are highlighted with the modifying electrophile noted.

Figure 7

Adenine propenal modifies XPA and reduces its DNA-binding activity. (A) Lysine residues modified with an adduct mass of +54.0105 were mapped to the XPA NMR solution structure (1D4U). (B) Plots of the change in fluorescence anisotropy for a Y-shaped 8/12 ssDNA–dsDNA junction substrate (50 nM) versus added protein treated with DMSO, adenine propenal, or NHS-biotin. Each data point represents the mean ± SD of three titrations.