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

当B细胞受到IL-4R/CD40刺激时，与CD40相关的Ku70/80易位进入细胞核。同样的刺激也会导致IgE低亲和力受体（CD23）的最大表达。这些数据提示Ku70/80在CD23表达调控中发挥作用。我们制备了两种不同的显性阴性（DN） Ku80作为egfp融合蛋白，将它们转染到ddn - 39b细胞系中，并检测了对IL-4R/ cd40驱动的CD23表达的影响。缺乏C端Ku80的DN构建体之一抑制IL-4R/ cd40驱动的CD23表达。我们采用报告基因试验来了解EBVRE是否具有CD23表达增强剂的功能。我们的研究表明，当B细胞受到IL-4和抗cd40刺激时，Ku结合位点周围的区域（EBVRE）表现出增强作用。然而，我们无法在EMSA实验中证明Ku70/80实际上占据了该区域。这些结果表明Ku70/80调控CD23的表达。我们已经开始观察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结合位点。另一个兴趣是Ku70/80的修改及其对Ku70/80函数的影响。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的泛素化介导的。我们现在正在研究降解是否导致细胞凋亡或停止过敏反应。据报道，AID对免疫球蛋白类转换很重要。我们对16例非x连锁超igm综合征的AID进行了测序，发现9例发生突变。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.