Figure 1.
Lucanthone/Hycanthone promotes APE1 cleavage in presence of CHX and this cleavage is inhibited by 1% DMSO.
Western blot of total cell extract from APE1-5 overexpresser clone pretreated with 10 µg/ml of cycloheximide for 4 h followed by 25–100 µM lucanthone/hycanthone for 2 h (12.5 mg of total cell protein loaded per lane).
Figure 2.
Lucanthone promotes APE1 cleavage in presence of protease inhibitor.
Western blot of APE1 over expresser clone 5 pretreated with increasing (2.5–100 µM) concentration of lucanthone in presence of protease inhibitor cocktail for 2 h at 37°C (10 µg of total cell protein loaded per lane). An arrow indicates the corresponding increase in APE1 25 kDa fragment in last two lanes with a decrease in 35.5 kDa APE1 protein.
Figure 3.
Lucanthone/Hycanthone promote APE1 cleavage at lower concentration than CRT.
Western blot of APE1 over expresser clone 5 pretreated with increasing concentration of lucanthone (2.5–100 µM) and CRT0044876 (2.5–200 µM) in presence of protease inhibitor cocktail for 2 h at 37°C (10 µg of total cell protein loaded per lane). The corresponding decrease in 35.5 kDa APE1 protein band and increase in 25-kDa-degradation product is indicated by the arrows.
Figure 4.
Lucanthone promotes APE1 cleavage in vitro by possible non-competitive binding.
A. Recombinant APE1 protein (250 ng) treated with 10–50 µM lucanthone at 37°C for 2 h, the numbers in italic font represent fold change in APE1 and its 25 kDa fragment as measured by area analysis using Image J quantification program. B. Lineweaver-Burke plot for endonuclease assay determinations as detailed in Materials and Methods. Units of 1/v were min/fmoles of abasic sites incised and for 1/[S] was inverse of nM of depurinated plasmid DNA.
Figure 5.
Lucanthone promotes APE1 cleavage in vitro which is inhibited by TRIS.
Western blot of recombinant APE1 protein (250 ng) treated with 10–50 µM lucanthone at 37°C for 2 h in absence and presence of radical quencher, 10 mM Tris-HCl, pH 7.4.
Figure 6.
Thioxanthenones and CRT cleavage of APE1 is inhibited by Ascorbic acid but not by N-Acetyl cysteine.
Western blot of recombinant APE1 protein (250 ng) treated with 100 µM of radical quenchers, NAC and Ascorbic acid and 100 µM of lucanthone (L)/hycanthone (H) or 200 µM of CRT (C) at 37°C for 2 h.
Figure 7.
Lucanthone and hycanthone directly alter APE1 conformation.
CD spectra of APE1 in presence of lucanthone and hycanthone as described in materials and methods, which was analyzed by Dichroweb program CDSSTR.
Table 1.
Changes in APE1 conformation in presence of lucanthone and hycanthone.
Figure 8.
APE1 binds directly with lucanthone and hycanthone with different affinities.
APE1 protein (100 µg) (ligand) was immobilized on carboxymethyl-5 (CM-5) chip by amine coupling according to manufacturer's instructions. Hycanthone (top figure) (analyte) and lucanthone (analyte) (lower figure) (analyte) at different concentrations (as shown as numbers representing µM values) were tested for binding to APE1 on BAICORE 2000 SPR measurement system available at SUNYSB proteomics core facility. The observed maximum response (RU) was determined by direct curve fitting of the obtained data assuming a 1∶1 interaction model. The third sub figure shows plot of r (RU/RUmax) versus Cfree. Binding studies were carried out 3 times and data presented are representative of those 3 separate experiments.
Table 2.
Kinetics of lucanthone and hycanthone binding to APE1.
Figure 9.
Identification of an approximate lucanthone cleavage site in APE1.
LC/MS/MS identification of APE1 fragment after treatment with 100 µM of lucanthone for 2 h at 37°C. The data were analyzed with Inspect. A). Peptide aa64–73 analysis of 35.5 Kda (Sample 1) and 25 kDa (Sample 3). B). Elution profile of peptide aa64–73 in sample 1 and 3. C). Peptide aa282–299 analysis of 35.5 Kda (Sample 1) and 25 kDa (Sample 3). D). Peptide aa53–63 analysis of 35.5 Kda (Sample 1) and 25 kDa (Sample 3). B). Elution profile of peptide aa53–63 in sample 1 and 3.
Figure 10.
Lucanthone, Hycanthone and CRT cause APE1 degradation.
Mass spectroscopic (MALDI-TOF) analysis of APE1 in presence of lucanthone and CRT as described in materials and methods. APE1 (100 µM) was treated with 100 µM lucanthone and CRT for 2 h and 24 h and changes in its mass was determined as described in Materials & Methods.
Figure 11.
Lucanthone and hycanthone docks at hydrophobic site in APE1.
A) The lowest energy pose of docked APE1/lucanthone (gray) superposes with the structure of the complex post 30 ns of MD simulation with an r.m.s.d. of 1.8. Å. B) docked APE1/hycanhone (gray) superposes with the structure of this complex post 30 ns of molecular dynamics simulation with an r.m.s.d. of 1.7. Å.
Figure 12.
Molecular Dynamics simulation of APE1-bound Lucanthone and Hycanthone.
A: lucanthone (magenta sticks) binds in the hydrophobic pocket of Ape1, represented by its solvent accessible surface. Ape1 interacts with lucanthone mainly via apolar contacts with W280, pi-stacking with F266, and M270. Additionally, a hydrogen bond forms between the carbonyl oxygen and T268. B) Hycanthone (magenta sticks) binds in the DNA groove/hydrophobic pocket of APE1. In addition to apolar contacts and pi-stacking with F266, it forms a hydrogen bond with His309 and coordinates with the APE1-bound magnesium cation (represented as gray sphere).
Figure 13.
Lucanthone does not alter conformation of double hydrophobic site APE1 mutant, human Nth and bacterial Nfo proteins.
CD spectra of APE1 and its mutants in presence of lucanthone. APE1- hydrophobic site mutant proteins F266A/C, F266A/W280A, active site mutant D210N and non-related BSA protein or human Nth or E.coli Nfo(10 mg/ml), 50 µl (500 µg) (14 µM) in APE1 buffer (50 mM HEPES, 150 mM KCl, 5 mM MgCl2), was mixed with lucanthone (1 mg/ml), 50 µl (50 µg) (140 µM), incubated at 37°C for 60 min and far UV-CD spectra with specifications.
Figure 14.
Lucanthone causes cleavage of APE1 with intact hydrophobic site.
(A). Western blot of recombinant and mutant APE1 proteins (upper panel) treated with 100 µM lucanthone (lower panel) at 37°C for 2 h. (B). Endonuclease activity inhibition of wild type and F266A mutant of APE1 in presence of 100 µM of lucanthone for 2 h at 37°C.
Figure 15.
Lucanthone and Hycanthone do not significantly affect the DNA binding capacity of APE1.
Gel mobility shift assay for determining the effect of lucanthone and hycanthone on DNA binding capacity of APE1. APE1 at 10 nM and 25 nM was mixed with 100 µM lucanthone or 100 µM hycanthone in the buffer that contained 50 mM HEPES, 150 mM KCl, 0.1 mg/ml BSA, 0.5 mM EDTA and 1 mM DTT. The mixture was incubated at room temperature for 30 min. Subsequently, 10 nM radiolabeled substrate that contained an abasic site was added in the mixture and incubated with the enzyme for additional 30 min to allow binding of APE1 to the substrate. 8 µl binding mixture was subject to electrophoresis at 4°C, 100 V for 1.5 h in a 1% agarose–0.1% acrylamide gel. The gel was then dried on DE81 paper, and APE1-DNA complex was detected by phosphorimager as described previously [52].