Regulation of cell cycle progression by the ubiquitin-proteasome system

泛素-蛋白酶体系统对细胞周期进程的调节

• 批准号: 08458225
• 负责人: YOKOSAWA Hideyoshi
• 金额: $ 5.38万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08458225/
• 关键词: cell cycle; ubiquitin; proteasome; budding yeast

1. Multiubiquitinated proteins were isolated from tempetature-sensitive nin1 mutant of budding yeast, which had been cultured at non-permissive temperature, by a procedure including Superose 6 gel filtration, FK2 antibody-agarose chromatography, and Mono Q FPLC.The isolated proteins were digested with lysylendopeptidase, and the digests were separated by reverse-phase HPLC,followed by amino acid sequence determination. By homology search, we propose that several proteins including porin are ubiquitinated.2. In cell-free extract of Xenopus eggs, proteasome inhibitors caused a delay of cell cycle progression and accumulation of multiubiquitinated proteins. The accumulated proteins were also isolated by the procedure described above.3. In heat-shock treated HeLa cells, multiubiquitinated proteins were accumulated in cytosol, while ubiquitinated histone H2A in nucleus was decreased.4. By enzymeimmunoassay for multiubiquitin chains, it was found that serum concentration of multiubiquitin chains was higher in some patients than in healthy subjects.5. Multiubiquitinated proteins were accumulated in temperature-sensitive sen3, nasl, and sun2 mutants, indicating that these components of the 26S proteasome play essential roles in degradation of multiubiquitinated proteins by the 26S proteasome.

1.从非允许温度下培养的芽殖酵母温度敏感突变株nin 1中，通过Superose 6凝胶过滤、FK 2抗体-琼脂糖层析和Mono Q FPLC等方法，分离得到多泛素化蛋白，用赖氨酰内肽酶酶切，反相高效液相色谱分离，并进行氨基酸序列测定。通过同源性搜索，我们推测包括孔蛋白在内的几种蛋白质是泛素化的.在非洲爪蟾卵的无细胞提取物中，蛋白酶体抑制剂引起细胞周期进程的延迟和多泛素化蛋白的积累。积累的蛋白质也通过上述方法分离。热休克处理后，HeLa细胞胞浆中多泛素化蛋白的积累，而细胞核中泛素化组蛋白H2 A的表达减少.通过对多泛素链的酶免疫分析，发现部分患者血清中多泛素链浓度高于健康人. Multiubiquitinated蛋白质积累在温度敏感的sen 3，nasl，sun 2突变体，表明这些组件的26 S蛋白酶体降解multiubiquitinated蛋白质的26 S蛋白酶体发挥重要作用。

项目成果

H.Aizawa, H.Kawahara, K.Tanaka, and H.Yokosawa.: "Activation of the proteasome suring Xenopus egg activation implies a link between proteasome activation and intracellular calcium release." Biochem. Biophys. Res. Commun.218. 224-228 (1996)

H.Aizawa、H.Kawahara、K.Tanaka 和 H.Yokosawa.：“确保非洲爪蟾卵激活的蛋白酶体激活意味着蛋白酶体激活与细胞内钙释放之间存在联系。”

Masahiro, Fujimuro: "Dynamics of ubiquitin conjugation during heat-shock response revealed by using a monoclonal antibody specific to multi-ubiquitin chains." European Journal of Biochemistry. 249. 427-433 (1997)

Masahiro, Fujimuro：“通过使用针对多泛素链的单克隆抗体揭示了热休克反应期间泛素结合的动力学。”

K.Kominami, N.Okura, M.Kawamura, G.N.DeMartino, C.A.Slaughter, N.Shimbara, C.H.Chung, M.Fujimuro, H.Yokosawa, Y.Shimizu, N.Tanahashi, K.Tanaka, and A.Toh-e.: "Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two m

K.Kominami、N.Okura、M.Kawamura、G.N.DeMartino、C.A.Slaughter、N.Shimbara、C.H.Chung、M.Fujimuro、H.Yokosawa、Y.Shimizu、N.Tanahashi、K.Tanaka 和 A.Toh-

Kin-ichiro, Kominami: "Yeast counterparts of subunits S5a and p58(S3)of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1." Molecular Biology of the Cell. 8. 171-187 (1997)

Kin-ichiro, Kominami：“人类 26S 蛋白酶体的 S5a 和 p58(S3) 亚基的酵母对应物由 nin1-1 的两个多拷贝抑制子编码。”

Koji, Takada: "Serum levels of free ubiquitin and multiubiquitin chains." Clinical Chemistry. 43. 1188-1195 (1997)

Koji, Takada：“游离泛素和多泛素链的血清水平。”