Determination of binding constant of transcription factor myc-max/max-max and E-box DNA: the effect of inhibitors on the binding.
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 k(1)=(1.7+/-0.6)x10(5) M(-1) s(-1), k(-1)=(3.4+/-1.2)x10(-2) s(-1) for max-max and DNA or k(1)=(2.1+/-0.7)x10(5) M(-1) s(-1), k(-1)=(3.2+/-1.4)x10(-2) s(-1) for myc-max and DNA. The equilibrium binding constant (K(1)) was determined using these kinetic parameters [K(XXD)=(7.8+/-2.6)x10(6) M(-1) for max-max and DNA or K(XYD)=(6.9+/-2.2)x10(6) M(-1) for myc-max and DNA]. The binding constants of myc-max or max-max dimer formation were K(XX)=(2.6+/-0.9)x10(5) M(-1) or K(XY)=(1.3+/-0.4)x10(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 microM, 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.
Samsung Medical Center, 50 Ilwon-Dong, Kangnam-Ku, Seoul 135-710, South Korea.
Seyeon
S
Park
Sunah
S
Chung
Kyung-Mee
KM
Kim
Kyung-Chae
KC
Jung
Chihoon
C
Park
Eun-Ryeong
ER
Hahm
Chul-Hak
CH
Yang
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D045744:Cell Line, Tumor / Q000187:drug effects; D003474:Curcumin / Q000031:analogs & derivatives* / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D015536:Down-Regulation; D024721:E-Box Elements / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D016350:Leucine Zippers / Q000235:genetics; D009637:Masoprocol / Q000494:pharmacology; D008958:Models, Molecular; D015394:Molecular Structure; D009828:Oleanolic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C499330:MAX protein, human; C067850:Myc associated factor X; D016271:Proto-Oncogene Proteins c-myc; D014157:Transcription Factors; C408362:momordin I (Oleanolic Acid); D009828:Oleanolic Acid; D009637:Masoprocol; D003474:Curcumin