Zhang Tianmin, Wang Xiaodong, Shinn Annie, Jin Jingjun, Chan William K
Department of Pharmaceutics and Medicinal Chemistry, University of the Pacific, Stockton, CA 95211, USA.
Biochem Pharmacol. 2010 Apr 15;79(8):1125-33. doi: 10.1016/j.bcp.2009.12.010. Epub 2009 Dec 16.
We have been studying the requirement for the aryl hydrocarbon receptor nuclear translocator (Arnt)-dependent DNA complex formation, which precedes the activation of gene transcription. Using DEAE chromatography, we have obtained a Sf9 insect fraction F5 that is highly enriched with beta-tubulin. F5 inhibits the formation of the AhR gel shift complex and this inhibition is sensitive to protease, suggesting that proteins that are present in this F5 fraction are responsible for the inhibition. Additional experiments have revealed that this inhibition is less pronounced in the presence of anti-beta-tubulin IgG and beta-tubulin enriched fraction from pig brain also inhibits the AhR gel shift formation. Sf9 beta-tubulin interacts with Arnt and suppresses the binding of the AhR/Arnt heterodimer to its corresponding enhancer. Human beta4-tubulin, which shares high sequence identity with Sf9 beta-tubulin, suppresses the AhR-dependent luciferase expression by reducing the nuclear Arnt content and retaining Arnt in the cytoplasm. Fluorescence studies using the GFP fusion of human beta4-tubulin have revealed that beta4-tubulin prevents the localization of Arnt in Sf9 cells. Here we have provided evidence suggesting that beta-tubulin may regulate the physiological content of Arnt.
我们一直在研究芳烃受体核转运蛋白（Arnt）依赖性DNA复合物形成的需求，这种复合物形成先于基因转录的激活。通过DEAE色谱法，我们获得了一个富含β-微管蛋白的Sf9昆虫组分F5。F5抑制芳烃受体凝胶迁移复合物的形成，并且这种抑制对蛋白酶敏感，这表明该F5组分中存在的蛋白质是造成这种抑制的原因。进一步的实验表明，在抗β-微管蛋白IgG存在的情况下，这种抑制作用不那么明显，并且来自猪脑的富含β-微管蛋白的组分也抑制芳烃受体凝胶迁移复合物的形成。Sf9β-微管蛋白与Arnt相互作用，并抑制芳烃受体/Arnt异二聚体与其相应增强子的结合。与人β4-微管蛋白具有高度序列同一性的人β4-微管蛋白，通过降低细胞核中Arnt的含量并将Arnt保留在细胞质中，抑制芳烃受体依赖性荧光素酶的表达。使用人β4-微管蛋白的绿色荧光蛋白融合体进行的荧光研究表明，β4-微管蛋白阻止了Arnt在Sf9细胞中的定位。在此我们提供了证据表明β-微管蛋白可能调节Arnt的生理含量。
