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Characterization of ubiquitin-like polypeptide acceptor protein, a novel pro-apoptotic member of the Bcl-2 family

Bcl-2 家族新型促凋亡成员泛素样多肽受体蛋白的表征

基本信息

批准号：
15570118
负责人：
NAKAMURA Morihiko
金额：
$ 2.3万
依托单位：
Shimane University (2004)Shimane Medical University (2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15570118/
关键词：
Ubiquitin Apoptosis Bcl-G Suppressor MALDI Bcl

项目摘要

Monoclonal nonspecific suppressor factor(MNSF) is a cytokine with antigen nonspecific suppressive activity. MNSFβ (a subunit of MNSF) is a 14.5-kDa fusion protein consisting of a protein with 36% identity with ubiquitin and ribosomal protein S30. The ubiquitin-like segment (Ubi-L) may be cleaved from MNSFβ in the cytosol. Recently, we have observed that Ubi-L covalently binds to intracellular proteins in mitogen-activated murine T helper type 2 clone, D.10 cells. In this study, we purified a 33.5-kDa Ubi-L adduct from D.10 cell lysates by sequential chromatography on DEAE, anti-Ubi-L antibody conjugated Sepharose, and hydroxylapatite. MALDI-TOF-Mass fingerprinting revealed that this Ubi-L adduct consists of 8.5-kDa Ubi-L and Bcl-2-like protein, murine orthologue of previously cloned human BCL-G gene product with pro-apoptotic function. Murine Bcl-G mRNA was highly expressed in testis and significantly in spleen. In addition, the level of Bcl-G mRNA expression was increased in concanavalin A- and interferon γ-activated D.10 cells. The 33.5-kDa-Ubi-L adduct was expressed in spleen but not in testis, even though Bcl-G protein was highly expressed in this tissue. The antisense oligonucleotide to Bcl-G significantly decreased the level of the Ubi-L-adduct formation in concanavalin A-activated D.10 cells and the proliferative response of the D.10 cells. These results suggest that the posttranslational modification of Bcl-G by Ubi-L might be implicated in T cell activation.

单克隆非特异性抑制因子（MNSF）是一种具有抗原非特异性抑制活性的细胞因子。MNSFβ（MNSF的一个亚基）是一个14.5-kDa的融合蛋白，由与泛素和核糖体蛋白S30具有36%同一性的蛋白组成。泛素样片段（Ubi-L）可以在胞质溶胶中从MNSFβ裂解。最近，我们已经观察到，Ubi-L共价结合细胞内的蛋白质在有丝分裂原激活的小鼠辅助性T细胞2型克隆，D.10细胞。在这项研究中，我们纯化了33.5 kDa的Ubi-L加合物从D. 10细胞裂解物通过连续色谱上的DEAE，抗Ubi-L抗体共轭琼脂糖凝胶，和羟基磷灰石。MALDI-TOF-Mass指纹图谱显示，该Ubi-L加合物由8.5-kDa Ubi-L和Bcl-2样蛋白组成，Bcl-2样蛋白是先前克隆的具有促凋亡功能的人BCL-G基因产物的小鼠直向同源物。Bcl-G mRNA在小鼠睾丸中有高表达，在脾脏中也有显著表达。此外，伴刀豆球蛋白A和γ干扰素激活的D.10细胞中Bcl-G mRNA表达水平增加。33.5-kDa的Ubi-L加合物在脾脏中表达，而在睾丸中不表达，尽管Bcl-G蛋白在该组织中高度表达。Bcl-G反义寡核苷酸显著降低伴刀豆球蛋白A激活的D.10细胞中Ubi-L-加合物的形成水平和D.10细胞的增殖反应。这些结果表明Ubi-L对Bcl-G的翻译后修饰可能与T细胞活化有关。