The following questions are based on data, graphs, and figures from the following article: Naphthoquinone-mediated Inhibition of Lysine Acetyltransferase KAT3B/p300, Basis for Non-toxic Inhibitor Synthesis. Mohankrishna Dalvoy Vasudevarao et al. The Journal of Biological Chemistry, 289, 7702-7717. March 14, 2014 . doi: 10.1074/jbc.M113.486522
Fig 5 (not shown): The authors used UV-Vis and fluorescence spectroscopy to study the effect of NAC on the spectra of 1,4-naphthoquinone analogs. What likely effects would you expect to observe on the spectrum of lawsone?
None since lawsone does not react with the thiol-containing NAC
Fig 6A: The ultimate goal of the investigators was to make a 1,4-naphthoquinone derivative that was not cytotoxic which presumably results from its interactions with free thiols. They synthesized PTK1 as shown below. Why would this reaction produce a potential inhibitor that did not react with free thols in cells?
It has a methyl at C3 which would inhibit its reaction with free thiols
FIGURE 6B. Investigators studied the effects of PTK1 on P300 acetylation of HeLa core histones with different concentrations of PTK1 in the absence and presence of NAC (Figure B) as well the presence of free thiol in the presence or absence of PTK1 (Figure C).
What can you conclude from these graphs?
As expected, PTIK1 inhibits P300 acetylation with no effects from the addition of NAC.
Figure 6D: The figure below shows the UV-visible absorption spectrum of PTK1 in the in presence or absence of NAC?
What conclusions can you draw?
PTK1 does not react with NAC, as expected.
Fig 6E: The figure below shows immunoblotting analysis (IB) of cell lysates upon 24 h of treatment by PTK1 using H3K9 acetylation antibodies. The concentrations of PTK1 used are indicated in the figure E. Error bars, S.D. What conclusions can you draw from the blots?
PTK1 acts as a potent inhibitor of acetylation of H3K9 in vivo.