Studies on mechanisms for maturation and dynamics of nascent proteins in the early secretory pathway

早期分泌途径中新生蛋白的成熟和动力学机制研究

基本信息

批准号：
10480160
负责人：
WADA Ikuo
金额：
$ 8.13万
依托单位：
Sapporo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10480160/
关键词：
secretion folding disulfide bonds oxidation endoplasmic reticulum calnexin calmegin 分泌 folding ERGIC53 ERGIC time-lapse analysis ゴルジ体リサイクリング

项目摘要

(1) Folding of proteins in the ER involves oxidation of cystein. To elucidate the mechanism of oxidation, we have developed a novel experimental system which selectively labels cysteins forming disulfide bonds of newly synthesized transferrin. By using this methbd, we found that oxidation of the ER lumen appeared to proceeds unevenly and newly synthesized transferrin was rather selectively oxidized. Analysis of the process, which was inhibited by two specific chemicals, revealed two distinctive mechanisms ; one which is responsible for cotranslational oxidation and the other for posttranslational.(2) We demonstrated that proper folding of tyrosinase depends on calnexin. It is known that mutations of this enzyme is responsible for OCA1. We next reported that they were immediately degraded upon release from canlexin by proteasome.(3) It has been shown that degradation of misfolded proteins in the ER by proteasome requires mannose trimming, particularly from Man9 structures to Man8. By searching novel molecules which are induced by stress, we reported EDEM which has homologous to a-mannosidase but lacks the enzymatic activity. EDEM bound to misfolded antitrypsin and its degradation was accelerated by overexpressing EDEM, indicating that EDEM may be an acceptor for misfolded proteins in the ER.(4) We previously reported that sperm in calmegin KO-mouse were unable to bind to egs. In this study, we identified that heterooligomerazation of a cell surface molecule is impaired in the KO mouse, suggesting that calmegin, a testis isoform of calnexin, is required for assembly of a specific cell surface receptor.

(1) 内质网中蛋白质的折叠涉及半胱氨酸的氧化。为了阐明氧化机制，我们开发了一种新的实验系统，该系统选择性地标记新合成的转铁蛋白形成二硫键的半胱氨酸。通过使用这种方法，我们发现内质网腔的氧化似乎进行得不均匀，并且新合成的转铁蛋白被相当选择性地氧化。对受两种特定化学物质抑制的过程的分析揭示了两种独特的机制；一个负责共翻译氧化，另一个负责翻译后氧化。(2)我们证明酪氨酸酶的正确折叠取决于钙联蛋白。众所周知，这种酶的突变导致了 OCA1。接下来我们报道称，它们在从 Canlexin 中释放后立即被蛋白酶体降解。(3) 已经表明，蛋白酶体降解内质网中错误折叠的蛋白质需要甘露糖修剪，特别是从 Man9 结构到 Man8。通过寻找应激诱导的新分子，我们报道了与α-甘露糖苷酶同源但缺乏酶活性的EDEM。 EDEM 与错误折叠的抗胰蛋白酶结合，并且过表达 EDEM 会加速其降解，表明 EDEM 可能是 ER 中错误折叠蛋白的受体。(4) 我们之前报道过 calmegin KO 小鼠的精子无法与 egs 结合。在这项研究中，我们发现 KO 小鼠中细胞表面分子的异源寡聚化受到损害，这表明钙连接蛋白的睾丸亚型 calmegin 是特定细胞表面受体组装所必需的。