Questions
1,4-Naphthoquinone derivatives are naturally occurring compounds that are present in the Plumbago and Diospyros plant genera and have a variety of biological activities. One such derivative, plumbagin, is a potent inhibitor of a particular lysine acetyltransferase, KAT p300 lysine acetyltransferases 3B/3A (p300/CBP). Some of the inhibitors are toxic to cells as they react with free RSH groups in cells and generate reactive oxygen species (ROS). An ideal drug would be nontoxic and an effective inhibitor/activator of a specific enzyme. Investigators synthesized a derivative (PTK1) of plumbagin which inhibit p300 and with much lower toxicity that plumbagin. How does it work?
Fig 1A: To study the toxicity of plumbagin, investigators dissolved it in the solvent DMSO + N-acetylcysteine (NAC) which scavenges ROS. They added these reagents to three different human cells lines, SHSY-5Y, HEK293, and HeLa S3 as shown below.
a. What effect did plumbagin have on cell toxicity?
b. What effect did NAC have plumbagin cell effects?
c. What did the investigators conduct an experiment just DMSO alone?
Fig 1B. It has been shown that on DNA oxidative damage or breaks, one of the histones, H2AX is phosphorylated by a kinase, pI3K. Cells were treated as describe below (Plu = plumbagin_ and subjected to SDS-polyacrylamide gel electrophoresis. After the electrophoresis, a nitrocellluose membrane was placed on top and the proteins in the gel were electrophoresed into the membrane (a technique called Western blotting. Individual protein band were visualized by adding antibodies that recognized phosphorylated H2AXg, or histone H3 (without modification), followed by reagents that produce a dark band. Interpret the results. Why did the investigators do blots for unmodified H3?
Fig 1C. What affect did plumbagin have on histone acetylation? Immuoblots were performed after plumbagin treatment in the presence or absence of NAC in the indicated cell lines as above using antibodies that recognized acetylated Lys 9 on H3 (H3K9), or histone H3 (without modfication). Does Plu inhibit H3K9 acetylation? How does NAC affect Plumbagin effect on acetylation?
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Fig 1D: Another inhibitor of H3K9 acetylation, isogarcinol (IsoGar) was studied by western blotting. Did NAC have same effect on H3K9 acetylation of cells treated with IsoGAr as those treated with Plu, shown in Fig 1C?
NAC seems to behave differently in the presence of two acetylation inhibitors. These results offer two possibilities for its effects. • ROS could lead to decrease acetylation of histones, and NAC, by decreasing ROS, restore acetylation. • Alternatively, plumbagin reacts with the RSH of NAC, which decreases it effect on KAT and restores acetylation.
Draw cartoon models that would reflect these two different scenarios using a line with an arrow to indicate activation or promotion of an activity, and a line with a blunt end to show inhibition. Also use and these symbols:
Investigators found no increase in ROS with purified KAT in a cell free in vitro produced no free radicals. Which of your models is more likely?
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