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. 2011 Jul 1;286(26):22855-63.
doi: 10.1074/jbc.M111.232926. Epub 2011 May 5.

Arsenite interacts selectively with zinc finger proteins containing C3H1 or C4 motifs

Xixi Zhou  1 , Xi Sun, Karen L Cooper, Feng Wang, Ke Jian Liu, Laurie G Hudson
Affiliations

Arsenite interacts selectively with zinc finger proteins containing C3H1 or C4 motifs

Xixi Zhou et al. J Biol Chem. .

Abstract

Arsenic inhibits DNA repair and enhances the genotoxicity of DNA-damaging agents such as benzo[a]pyrene and ultraviolet radiation. Arsenic interaction with DNA repair proteins containing functional zinc finger motifs is one proposed mechanism to account for these observations. Here, we report that arsenite binds to both CCHC DNA-binding zinc fingers of the DNA repair protein PARP-1 (poly(ADP-ribose) polymerase-1). Furthermore, trivalent arsenite coordinated with all three cysteine residues as demonstrated by MS/MS. MALDI-TOF-MS analysis of peptides harboring site-directed substitutions of cysteine with histidine residues within the PARP-1 zinc finger revealed that arsenite bound to peptides containing three or four cysteine residues, but not to peptides with two cysteines, demonstrating arsenite binding selectivity. This finding was not unique to PARP-1; arsenite did not bind to a peptide representing the CCHH zinc finger of the DNA repair protein aprataxin, but did bind to an aprataxin peptide mutated to a CCHC zinc finger. To investigate the impact of arsenite on PARP-1 zinc finger function, we measured the zinc content and DNA-binding capacity of PARP-1 immunoprecipitated from arsenite-exposed cells. PARP-1 zinc content and DNA binding were decreased by 76 and 80%, respectively, compared with protein isolated from untreated cells. We observed comparable decreases in zinc content for XPA (xeroderma pigmentosum group A) protein (CCCC zinc finger), but not SP-1 (specificity protein-1) or aprataxin (CCHH zinc finger). These findings demonstrate that PARP-1 is a direct molecular target of arsenite and that arsenite interacts selectively with zinc finger motifs containing three or more cysteine residues.

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Figures

FIGURE 1.
FIGURE 1.
Arsenite binds to both zinc finger peptides derived from the PARP-1 DNA-binding domain. Arsenite binding to PARP-1 zinc finger apopeptides was analyzed by MALDI-TOF as described under “Experimental Procedures.” A, arsenite (As) binding to the PARPzf1 peptide was detected by MALDI-TOF-MS at m/z = 3526, which reflects a +72 m/z shift against apoPARPzf1 (m/z = 3454). B, arsenite binding to the PARPzf2 peptide (m/z = 3502) also displays a +72 m/z shift relative to apoPARPzf2 (m/z = 3430).
FIGURE 2.
FIGURE 2.
Arsenite forms bonds with all three cysteine residues of the PARP-1 zinc finger. Shown are the results from MS/MS analysis of arsenite-bound PARPzf1. A, apoPARPzf1 was analyzed by MS/MS, and apo-b and apo-y ions are shown. B, arsenite-bound b and y ions were detected by MS/MS. Additional information regarding the bound ions is provided in supplemental Table S2.
FIGURE 3.
FIGURE 3.
Peptide substitution strategy to test arsenite binding selectivity. The schematic represents the native and mutant peptides tested in Figs. 4 and 5. Specific substitutions are indicated by underlined boldface characters. The full peptide sequences are presented in supplemental Table S1. P-like, PARP-1 like.
FIGURE 4.
FIGURE 4.
Arsenite binding selectivity for PARP-1-derived peptides. A–D, arsenite (As) binding to native and substituted PARPzf1 peptides was assessed by MALDI-TOF-MS analysis as described under “Experimental Procedures.” A, lack of arsenite binding to a PARPzf1-CHHC peptide mutant. B, lack of arsenite binding to a PARPzf1 peptide with a CCHH configuration that retains vicinal cysteine residues. C, arsenite binding to a PARPzf1-CCHC peptide with an insertion between the first two vicinal cysteine residues (CCHC-far) was detected at m/z = 3526. D, arsenite binding to a PARPzf1-CCCC peptide mutant was detected at m/z = 3491. E–G, cobalt spectrometry analysis of zinc binding to native and substituted PARPzf1 peptides. E, 100 μm native PARPzf1 peptide in 5 mm Tris (pH 7.4) was titrated with Co(II) over a range of 10–100 μm in increments of 10 μm. F, 100 μm native PARPzf1 peptide was saturated with 300 μm Co(II) and back-titrated with Zn(II) over a range of 10–50 μm in increments of 10 μm under argon. G, 100 μm mutant PARPzf1 peptide (CCHH) was saturated with 300 μm Co(II) and back-titrated with Zn(II) over a range of 10–40 μm in increments of 10 μm in each addition under argon. Similar results were obtained with each of the PARP-1 peptides shown in Fig. 3 (data not shown).
FIGURE 5.
FIGURE 5.
Arsenite binding to the aprataxin zinc finger peptide is conferred upon the addition of a cysteine residue. Arsenite (As) binding to native (CCHH) and mutant (CCHC) APTX zinc finger peptides was assessed by MALDI-TOF-MS as described under “Experimental Procedures.” A, arsenite did not bind to the APTX zinc finger peptide (CCHH configuration). B, arsenite binding to the CCHC mutant of the APTX zinc finger peptide was detected by MALDI-TOF-MS at m/z = 3356. C and D, 100 μm native (CCHH; C) or mutant (CCHC; D) APTX zinc finger peptide was saturated with 300 μm Co(II) and back-titrated with Zn(II) over a range of 10–50 μm in increments of 10 μm under argon.
FIGURE 6.
FIGURE 6.
Arsenite decreases the zinc content and DNA binding of PARP-1 isolated from cells. A–C, cells were treated with arsenite (As) or TPEN, and the indicated proteins were isolated by immunoprecipitation as described under “Experimental Procedures.” A, cells were treated with the indicated concentrations of arsenite for 40 h or with 5 μm TPEN for 24 h, PARP-1 was immunoprecipitated, and zinc content was measured as described under “Experimental Procedures.” Values are normalized to the untreated control and represent the mean ± S.D. of three independent experiments. *, p < 0.05. B, cells were treated as described for A, and PARP-1 DNA binding was measured by EMSA as described under “Experimental Procedures.” C, shown is the quantification of data in B based on densitometry. The concentration of zinc in the culture medium was 1.5 μm, so at the highest arsenite concentration, the molar ratio of arsenite to zinc in the tissue culture medium was 1.33:1.0. Values represent the mean ± S.D. of three independent experiments. *, p < 0.05.
FIGURE 7.
FIGURE 7.
Arsenite-induced zinc release is selective for zinc fingers containing three or more cysteine residues. A, HaCaT cells were cultured to ∼60% confluence in 15-cm plates and then treated with 2 μm arsenite (As) for 48 h or with 5 μm TPEN for 24 h. PARP-1, XPA, SP-1, and APTX were immunoprecipitated from cell lysates, and zinc content was measured as described under “Experimental Procedures.” Values represent the mean ± S.D. of three independent experiments. *, p < 0.05 (significantly different from similarly treated samples). B, HEKn cells were cultured to ∼50% confluence in 15-cm plates. Cells were then transfected with plasmids containing WT PARP-1, zinc finger mutant 1 (M1; CCHH), or zinc finger mutant 2 (M2; HCHC) as described under “Experimental Procedures” before exposure to arsenite (1 μm) for 24 h. Cell lysates were prepared, and expressed WT or mutant PARP-1 protein was isolated by immunoprecipitation using antibody directed against the DDK epitope tag. The zinc release assay was performed as described for A. The zinc content of PARP-1 mutants was not statistically different from that of the WT protein. The lower panels show the activity of the WT and zinc finger-mutated PARP-1 proteins based on DNA-dependent automodification. Modified PARP-1 (upper bands) was normalized to total PARP-1 for each construct to generate relative activity values. The results represent the findings from three independent experiments. Values are normalized to untreated (A) or untransfected (B) control samples (data not shown) and represent the mean ± S.D. of three independent experiments. *, p < 0.05 (significantly different from the no arsenite-matched sample).
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