Study on the Molecule That Plays Critical Role in Allergic Response.

当B细胞受到IL-4R/CD40的刺激时，与CD40相关的Ku70/80转位进入细胞核。同样的刺激也导致低亲和力IgE受体(CD23)的最大表达。这些数据表明Ku70/80在CD23表达的调节中起作用。我们已经为Ku80构建了两种不同的显性负性(DN)结构，作为EGFP融合蛋白，将它们导入DND-39B细胞系，并检测了它们对IL-4R/CD40驱动的CD23表达的影响。其中一个缺失C端的DNA结构抑制了IL-4R/CD40诱导的CD23的表达，我们用报告基因分析来了解EBVRE是否作为CD23表达的增强子。我们的研究表明，当IL-4和抗CD40刺激B细胞时，围绕Ku结合位点的区域(EBVRE)显示出增强效应。然而，我们不能证明Ku70/80在EMSA检测中确实占据了该区域。这些结果表明，Ku70/80调节CD23的表达。我们已经开始研究Ku70/80对CD40驱动的CD95(Fas)表达的影响，并确定了CD95启动子区域的Ku结合部位。另外，有趣的是Ku70/80的修饰及其对Ku70/80功能的影响。我们的研究表明，应激反应导致Ku70/80的降解，这种降解是由丝氨酸蛋白酶或Ku70/80泛素化所介导的。我们现在正在检查降解是否会导致细胞凋亡或过敏反应的停止。据报道，艾滋病对免疫球蛋白类别的转换很重要。我们对16例非X-连锁高IgM综合征患者进行了AID测序，其中9例发现突变。我们现在正在研究Ku70/80和AID之间是否存在任何生理或功能上的相互作用。

Ku70/80 that associates with CD40 translocates into the nucleus when B cells are stimulated through IL-4R/CD40. The same stimulation also leads to maximal expression of low affinity receptor for IgE (CD23). These data suggest that Ku70/80 plays a role in the regulation of CD23 expression. We have generated two different dominant negative (DN) constructs for Ku80 as EGFP-fusion proteins, transfected them into DND-39 B cell line, and examined the effect on IL-4R/CD40-driven CD23 expression. One of the DN constructs that lacks C terminal Ku80 inhibited IL-4R/CD40-driven CD23 expression.We have employed reporter gene assay to know whether EBVRE functions as enhancer of CD23 expression. Our study has shown that the region encompassing the Ku binding site (EBVRE) shows enhancing effect when B cells were stimulated with IL-4 and anti-CD40. We could not, however, show that Ku70/80 actually occupies the region in EMSA assay. These results suggest that Ku70/80 regulates expression of CD23.We have started looking at the effect of Ku70/80 on CD40-driven CD95 (Fas) expression and has determined Ku-binding site in the promoter region of CD95.Another interest is the modification of Ku70/80 and its effect on the Ku70/80function. Our study indicated that stress response leads to degradation of Ku70/80 that is mediated by serine protease or ubiquitination of Ku70/80. We are now examining whether the degradation results in apoptosis of the cells or in cessation of allergic response.AID is reported to be important for Immunoglobulin class switch. We have sequenced AID from 16 cases of non-X-linked hyper-IgM syndrome and found mutation in 9 cases. We are now examining whether there is any physiological or functional interaction between Ku70/80 and AID.