Molecular mechanism and prevention of drug dependence

给NG108-15细胞或小鼠长期给予吗啡可降低核因子(ssCRE-BP)与cAMP反应元件单链寡核苷酸(SsCRE)的结合活性。我们从小鼠小脑中提纯了单链Cre-BP，并分离了其cDNA克隆。我们发现，小鼠脑中的ssCRE-BP与Pura相同，Pura是一种结合富含嘌呤的单链DNA序列的蛋白，最初是作为复制因子分离出来的。SSCRE-BP在大脑皮层和小脑中含量丰富，而在其他组织中检测到很低的水平。通过检测慢性吗啡处理对小鼠脑内sCRE-BP含量的影响，免疫印迹分析表明，慢性吗啡处理对sCRE-BP无明显影响。虽然纯化的ssCRE-BP或其GST-融合蛋白的DNA结合活性很低，但加入约100 kDa、对胰酶敏感和热稳定的激活剂后，它们的DNA结合活性显著增强。酪蛋白可替代激活剂，使单链Cre-BP与DNA的亲和力提高一个数量级。为了确定DNA结合和激活剂相互作用的结构域，制备了一系列缺失突变体，发现它们都位于ssCRE-BP的AA50-215区域，虽然脑中含有丰富的ssCRE-BP，但激活剂普遍存在。该激活剂的特性有助于深入了解单链Cre-BP在吗啡依赖和耐受形成过程中的生理作用。

Chronic administration of morphine to NG108-15 cells or mice decreased the binding activity of a nuclear factor (ssCRE-BP) tosingle-stranded oligo-DNA of cAMP-response element (ssCRE). we have purified ssCRE-BP from mouse cerebella and isolated the cDNA clone. We found that ssCRE-BP from mouse brain was identical to Pura, which is a binding protein for single-stranded purine-rich DNA sequences that was originally isolated as a replication factor. ssCRE-BP was abundant in the cerebral cortex and cerebellum, but very low levels were detected in other tissues. Examination of the effect of chronic morphine treatment on the amount of ssCRE-BP in mouse brain showed that ssCRE-BP was not influenced by chronic morphine treatment significantly in western blot analysis. Although the DNA binding activities of purified ssCRE-BP or its GST-fusion protein were very low, they were markedly enhanced by the addition of an activator that was about 100kDa, trypsin sensitive and heat stable. The activator can be substituted with casein, which increased the affinity of ssCRE-BP for DNA by one order. A series of deletion mutants were prepared for determining the DNA binding-and activator-interacting domains and both of them were found to reside in AA 50-215 of ssCRE-BP.Although ssCRE-BP was abundant in the brain, the activator was ubiquitous. Characterization of the activator may provide insight into the physiological roles of ssCRE-BP which is involved in the development of morphine dependence and tolerance.