Determination of binding constant of transcription factor myc–max/max–max and E-box DNA: the effect of inhibitors on the binding

摘要

The truncated myc and max proteins, only containing basic regions and helix–loop–helix/zipper (b/HLH/Zip) regions were over-expressed in E. coli and used for the determination of the binding constant and of the inhibitory mechanism on myc–max (or max–max)–DNA complex formation. The association kinetic constants (k_1 and k_(-1)) of truncated max–max or myc–max dimer and DNA were determined as k1=(1.7±0.6) * 10~5 M~(-1) s~(-1), k_(-1)=(3.4±1.2) * 10~(-2) s~(-1) for max–max and DNA or k1=(2.1±0.7) * 10~5 M~(-1) s~(-1), k_(-1)=(3.2±1.4) * 10~(-2) s~(-1) for myc–max and DNA. The equilibrium binding constant (K1) was determined using these kinetic parameters [KXXD=(7.8±2.6) * 10~6 M~(-1) for max–max and DNA or KXYD=(6.9±2.2) * 10~6 M~(-1) for myc–max and DNA]. The binding constants of myc–max or max–max dimer formation were KXX=(2.6±0.9) * 10~5 M~(-1) or KXY=(1.3±0.4) * 10~4 M~(-1), respectively. When truncated proteins were used, the max–max dimer formation was easier than the myc–max dimer formation, contrary to the physiologically determined case. This leads us to deduce that domains other than b/HLH/Zip are very important for the transcriptional regulatory activity in physiological conditions. The truncated myc and max proteins, which were expressed in E. coli and contained only b/HLH/Zip regions were also used for the screening of inhibitors of myc–max-DNA complex formation. A synthesized curcuminoid, 1,7-bis(4-methyl-3-nitrophenyl)-1,6-heptadiene-3,5-dione (curcuminoid 004), showed the most potent inhibition out of the synthesized curcuminoids, in competition with DNA. The dissociation constant of max–max dimer and the inhibitor was 9 M, when investigated using in vitro expressed b/HLH/Zip dimer proteins. The curcuminoid 004 showed an inhibitory effect on the binding of myc–max protein to the E-box element in SNU16 cells, and suppressed the expression of myc target genes including ornithine decarboxylase (ODC), cdc25a and c-myc in myc over-expressed human stomach cancer cell line SNU16.