Characterization of ubiquitin-like polypeptide acceptor protein, a novel pro-apoptotic member of the Bcl2 family
Characterization of ubiquitin-like polypeptide acceptor protein, a novel pro-apoptotic member of the Bcl2 family
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DOI：
10.1046/j.1432-1033.2003.03790.x
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发表时间：
2003-10-01
期刊：
影响因子：
--
通讯作者：
Tanigawa, Y
中科院分区：
文献类型：
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作者：
Nakamura, M;Tanigawa, Y
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。MNSF β（MNSF的亚基）是由与泛素和核糖体蛋白S30具有36%同一性的蛋白质组成的14.5kDa融合蛋白。泛素样片段（Ubi-L）可以在胞质溶胶中从MNSF β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促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）Ig-共轭琼脂糖凝胶，和羟基磷灰石。MALDI-TOF-MS指纹图谱显示，该Ubi-L加合物由8.5 kDa的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，Bcl-G mRNA的表达水平在刀豆球蛋白A和干扰素γ激活的D.10细胞中增加。33.5kDa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。
Monoclonal nonspecific suppressor factor (MNSF) is a cytokine with antigen nonspecific suppressive activity. MNSFbeta (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 MNSFbeta 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) Ig-conjugated Sepharose, and hydroxylapatite. MALDI-TOF-MS fingerprinting revealed that this Ubi-L adduct consists of an 8.5 kDa Ubi-L and a Bcl2-like protein, murine orthologue of a 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 gamma-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 post-translational modification of Bcl-G by Ubi-L might be implicated in T-cell activation.