Functional analysis of a novel GTP-binding protein involved in apoptosis.

参与细胞凋亡的新型 GTP 结合蛋白的功能分析。

• 批准号: 12480213
• 负责人: INOUE Jun-ichiro
• 金额: $ 4.86万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 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/en/grant/KAKENHI-PROJECT-12480213/
• 关键词: GTP-binding protein; Cell cycle; Apoptosis; RNP K homology domain; RNA-binding; protein, DT40; 細胞増殖; アポートシス; 遺伝子破壊; Gタンパク質; 翻訳後修飾

We have cloned cDNAs encoding the entire coding region of a human homologue (H-ERA) and a mouse homologue (M-ERA) of ERA. The mammalian homologue of ERA consists of a typical GTPase/GTP-binding domain and a putative K homology (KH) domain, which is known as an RNA binding domain. We performed transfection experiments with wild-type H-ERA or various H-ERA mutants. H-ERA possessing the amino acid substitution mutation into the GTPase domain induced apoptosis of HeLa cells, which was blocked by Bcl-2 expression. Deletion of the C-terminus, which contains a part of the KH domain, alleviated apoptosis by the H-ERA mutant, suggesting the importance of this domain in the function of H-ERA. We have also shown the RNA binding activity of H-ERA by pull-down experiments using RNA homopolymer immobilized on beads or recombinant H-ERA proteins. Thus, H-ERA may play an important role in the regulation of apoptotic signalling with its GTPase/GTP binding domain. To elucidate the physiological function of eukaryotic ERA, we have performed a genetic analysis of chicken ERA (GdERA) in DT40 cells. Depletion of GdERA diminished the growth rate of the cells, accompanied by an accumulation of apoptotic cells. The analysis of cell cycle indicates that the elimination of GdERA caused arrest at G1 phase, but not at M phase, which highlights the distinct role of vertebrate ERA in the cell cycle progression compared to prokaryotic ERA. Furthermore, human ERA (HsERA) rescued the phenotype of GdERA-deficient cells, whereas a mutant of HsERA deprived of RNA binding activity did not. These data suggest that vertebrate ERA regulates the G1 phase progression via an as yet unknown molecular mechanism, which involves RNA recognition by ERA.

我们已经克隆了编码人类同源物（H-ERA）和ERA小鼠同源物（M-ERA）的整个编码区域的cDNA。 ERA的哺乳动物同源物由典型的GTPase/GTP结合域和推定的K同源域（KH）域组成，该结构域被称为RNA结合域。我们用野生型H-er时或各种H时代突变体进行了转染实验。 H-ERA具有氨基酸取代突变到GTPase结构域诱导的HeLa细胞凋亡，该凋亡被Bcl-2表达阻断。 c-末端的删除包含KH结构域的一部分，减轻了H时代突变体的凋亡，这表明该域在H时代的功能中的重要性。我们还通过使用固定在珠子或重组H-er蛋白上的RNA均聚物中的RNA均聚合物进行下拉实验显示了H-ERA的RNA结合活性。因此，H-era可能在其GTPase/GTP结合域的凋亡信号传导调节中起重要作用。为了阐明真核时代的生理功能，我们在DT40细胞中对鸡时代（GDERA）进行了遗传分析。 GDERA的耗竭降低了细胞的生长速率，伴随着凋亡细胞的积累。细胞周期的分析表明，消除GDERA在G1阶段引起了停滞，但并非在M期引起了停滞，这突出了脊椎动物时代与原核生物时代相比的细胞周期进程中的独特作用。此外，人类时代（HSERA）挽救了GDERA缺陷细胞的表型，而被RNA结合活性的Hsera突变体没有。这些数据表明，脊椎动物时代通过尚未知道的分子机制调节G1相进展，这涉及ERA识别RNA。

项目成果

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Matsumoto, N.：“源自环氧喹诺星 C 的 NF-κB 激活抑制剂的合成”Bioorg.Med.Chem.Lett.. 10. 865-869 (2000)

Kobayashi,N.: "Segregation of TRAF6-mediated signaling pathways clarifies its role in osteoclastogenesis"EMBO JOURNAL. (in press). (2001)

Kobayashi,N.：“TRAF6 介导的信号通路的分离阐明了其在破骨细胞生成中的作用”EMBO 杂志。

Inoue,J.: "Tumor necrosis factor receptor-associated factor (TRAF) family : adapter proteins that mediate cytokine signaling."Exp.Cell Res. 254. 14-24 (2000)

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Akiyama, T: "Mammalian homologue of E.Ras-like GTPase(ERA) is a possible apoptosis regulator with RNA binding activity"Cenes Cells. 6. 987-1001 (2001)

Akiyama, T：“E.Ras 样 GTP 酶 (ERA) 的哺乳动物同源物是一种可能具有 RNA 结合活性的细胞凋亡调节剂”Cenes Cells。

Jin Gohda: "Elimination of the vertebrate Escherichia coli Ras-like protein homologue leads to cell cycle arrest at G1 phase and apoptosis"Oncogene. (in press). (2003)

Jin Gohda：“消除脊椎动物大肠杆菌 Ras 样蛋白同源物导致细胞周期停滞在 G1 期并凋亡”癌基因。