Park Seyeon, Chung Sunah, Kim Kyung-Mee, Jung Kyung-Chae, Park Chihoon, Hahm Eun-Ryeong, Yang Chul-Hak
Samsung Medical Center, 50 Ilwon-Dong, Kangnam-Ku, Seoul 135-710, South Korea.
Biochim Biophys Acta. 2004 Feb 24;1670(3):217-28. doi: 10.1016/j.bbagen.2003.12.007.
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.
仅包含碱性区域和螺旋-环-螺旋/拉链（b/HLH/Zip）区域的截短型myc和max蛋白在大肠杆菌中过表达，并用于测定结合常数以及对myc-max（或max-max）-DNA复合物形成的抑制机制。截短型max-max或myc-max二聚体与DNA的缔合动力学常数（k(1)和k(-1)）测定结果为：max-max与DNA的k(1)=(1.7±0.6)x10(5) M(-1) s(-1)，k(-1)=(3.4±1.2)x10(-2) s(-1)；或myc-max与DNA的k(1)=(2.1±0.7)x10(5) M(-1) s(-1)，k(-1)=(3.2±1.4)x10(-2) s(-1)。利用这些动力学参数确定平衡结合常数（K(1)）[max-max与DNA的K(XXD)=(7.8±2.6)x10(6) M(-1)；或myc-max与DNA的K(XYD)=(6.9±2.2)x10(6) M(-1)]。myc-max或max-max二聚体形成的结合常数分别为K(XX)=(2.6±0.9)x10(5) M(-1)或K(XY)=(1.3±0.4)x10(4) M(-1)。当使用截短蛋白时，与生理测定情况相反，max-max二聚体的形成比myc-max二聚体的形成更容易。这使我们推断，除b/HLH/Zip之外的结构域对于生理条件下的转录调节活性非常重要。在大肠杆菌中表达且仅包含b/HLH/Zip区域的截短型myc和max蛋白也用于筛选myc-max-DNA复合物形成的抑制剂。一种合成的姜黄素类化合物，1,7-双（4-甲基-3-硝基苯基）-1,6-庚二烯-3,5-二酮（姜黄素类化合物004），在与DNA竞争时，是合成姜黄素类化合物中抑制作用最强的。当使用体外表达的b/HLH/Zip二聚体蛋白进行研究时，max-max二聚体与抑制剂的解离常数为9 microM。姜黄素类化合物004对SNU16细胞中myc-max蛋白与E-box元件的结合具有抑制作用，并在myc过表达的人胃癌细胞系SNU16中抑制包括鸟氨酸脱羧酶（ODC）、细胞周期蛋白依赖性激酶25A（cdc25a）和c-myc在内的myc靶基因的表达。
