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

Authors
 Seyeon Park  ;  Sunah Chung  ;  Chul-Hak Yang  ;  Eun-Ryeong Hahm  ;  Chihoon Park  ;  Kyung-Chae Jung  ;  Kyung-Mee Kim 
Citation
 BIOCHIMICA ET BIOPHYSICA ACTA , Vol.1670(3) : 217-228, 2004 
Journal Title
 BIOCHIMICA ET BIOPHYSICA ACTA 
ISSN
 0006-3002 
Issue Date
2004
MeSH
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ; Basic-Leucine Zipper Transcription Factors ; Cell Line, Tumor/drug effects ; Curcumin/analogs & derivatives* ; Curcumin/pharmacology ; DNA-Binding Proteins/biosynthesis ; DNA-Binding Proteins/genetics ; DNA-Binding Proteins/metabolism* ; Dimerization ; Down-Regulation ; E-Box Elements*/drug effects ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Gene Expression Regulation/drug effects ; Humans ; Kinetics ; Leucine Zippers/genetics ; Masoprocol/pharmacology ; Models, Molecular ; Molecular Structure ; Oleanolic Acid/analogs & derivatives* ; Oleanolic Acid/pharmacology ; Protein Binding/drug effects ; Protein Structure, Tertiary ; Proto-Oncogene Proteins c-myc/biosynthesis ; Proto-Oncogene Proteins c-myc/genetics ; Proto-Oncogene Proteins c-myc/metabolism* ; Transcription Factors/chemistry ; Transcription Factors/metabolism* ; Transcription, Genetic/drug effects*
Keywords
myc–max ; Binding constant ; Inhibitor ; SNU16 cell ; Target gene
Abstract
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.
Full Text
http://www.sciencedirect.com/science/article/pii/S030441650300271X
DOI
10.1016/j.bbagen.2003.12.007
Appears in Collections:
5. Research Institutes (연구소) > Anesthesia and Pain Research Institute (마취통증의학연구소) > 1. Journal Papers
Yonsei Authors
Park, Se Yeon(박세연)
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/111649
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