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

在过敏反应中起关键作用的分子的研究。

• 批准号: 12670732
• 负责人: MORIO Tomohiro
• 金额: $ 2.18万
• 依托单位: TOKYO MEDICAL AND DENTAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670732/
• 关键词: Ku70; 80; class switch; Fcε receptor; gene regulation; CD95; IL-4R; CD40; DNA-PK; IL-4; CD23; translocation; IgE

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.

当通过IL-4 R/CD 40刺激B细胞时，与CD 40缔合的Ku 70/80易位到细胞核中。相同的刺激也导致IgE的低亲和力受体（CD 23）的最大表达。这些数据表明Ku 70/80在CD 23表达的调节中起作用。我们构建了两种不同的Ku 80显性阴性（DN）构建体作为EGFP融合蛋白，将其转染到DND-39 B细胞系中，并检测了对IL-4 R/CD 40驱动的CD 23表达的影响。其中一个C端Ku 80缺失的DN构建体抑制IL-4 R/CD 40驱动的CD 23表达，我们采用报告基因分析来了解EBVRE是否作为CD 23表达的增强子。我们的研究表明，当B细胞被IL-4和抗-CD 40刺激时，围绕Ku结合位点的区域（EBVRE）显示增强效应。然而，我们不能证明Ku 70/80实际上占据EMSA测定的区域。本实验研究了Ku 70/80对CD 40驱动的CD 95（Fas）表达的影响，并确定了Ku 70/80在CD 95启动子区的结合位点。另一个兴趣是Ku 70/80的修饰及其对Ku 70/80功能的影响。我们的研究表明，应激反应导致Ku 70/80的降解是由丝氨酸蛋白酶或Ku 70/80的泛素化介导的。我们现在正在研究这种降解是否会导致细胞凋亡或过敏反应的停止。据报道，AID对免疫球蛋白类转换很重要。我们对16例非X连锁高IgM综合征患者的AID基因进行了测序，发现9例有突变。我们现在正在研究Ku 70/80和AID之间是否存在任何生理或功能相互作用。

项目成果

Ito S, et al.: "Mutations of the WASP gene in ten Japanese patients with WIskott-Aldrich syndrome and X-linked thrombocytopenia"International Hematology. 71. 79-83 (2000)

Ito S 等人：“10 名患有 WIskott-Aldrich 综合征和 X 连锁血小板减少症的日本患者中 WASP 基因的突变”国际血液学。

Kawai S, et al.: "Flow cytometric determination of intracytoplasmic Wiskott-Aldrich syndrome protein in peripheral blood lymphocyte subpopulations"Journal of Immunological Method. 260. 195-205 (2002)

Kawai S 等人：“外周血淋巴细胞亚群中胞浆内 Wiskott-Aldrich 综合征蛋白的流式细胞术测定”免疫学方法杂志。

Kumaki S,Ishii N,Ohashi Y, et al. (28名、8番目): "Characterization of the γc chain among 27 unrelated Japanese patients with X-linked severe combined immunodeficiency (X-SCID)"Human Genetics. 107. 406-408 (2000)

Kumaki S、Ishii N、Ohashi Y 等人（28 人，第 8 名）：“27 名不相关的日本 X 连锁严重联合免疫缺陷 (X-SCID) 患者的 γc 链特征”人类遗传学 107。 408（2000）

Nagasawa M, et al.: "ln vitro class switch of B cells after cord blood stem cell transplantation in severe combined immune deficient (SCID) patient"American Journal of Hematology. 65. 176-179 (2000)

Nagasawa M 等人：“严重联合免疫缺陷 (SCID) 患者脐带血干细胞移植后 B 细胞的体外类别转换”美国血液学杂志。

Kawai S: "Flow cytometric determination of intracytoplasmic Wiskott-Aldrich syndrome protein in peripheral blood lymphocyte subpopulations"Journal of Immunological Methods. 260. 195-205 (2002)

Kawai S：“外周血淋巴细胞亚群中胞浆内 Wiskott-Aldrich 综合征蛋白的流式细胞术测定”免疫学方法杂志。