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

• 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; 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

(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）ER中蛋白质的折叠涉及半胱氨酸的氧化。为了阐明氧化的机理，我们开发了一种新型的实验系统，该系统有选择地标记半胱氨酸蛋白形成新合成的转铁蛋白的二硫键。通过使用这种MECHBD，我们发现ER管腔的氧化似乎是不均匀和新合成的转铁蛋白的氧化。该过程的分析被两种特定化学物质抑制，发现了两种独特的机制。一种负责旋转氧化的原因，另一个用于翻译后。（2）我们证明酪氨酸酶的适当折叠取决于钙结合蛋白。众所周知，该酶的突变负责OCA1。接下来，我们报道说，它们在蛋白酶体从冠canlexin释放后立即降解。（3）已表明，蛋白酶体中ER中错误折叠蛋白的降解需要甘露糖的修剪，尤其是从man9结构到man8。通过搜索由应激诱导的新分子，我们报道了EDEM与A-甘露糖苷酶同源，但缺乏酶促活性。 EDEM与错误折叠的抗胰蛋白酶及其降解通过过表达EDEM加速，表明EDEM可能是ER中错误折叠蛋白的受体。（4）我们先前报道说，我们先前报道了Calmeegin KO-MOUSE中的精子无法与EGS结合。在这项研究中，我们确定了KO小鼠中细胞表面分子的杂聚糖化受损，这表明Calmegin是钙钙蛋白的睾丸同工型，是组装特定细胞表面受体所必需的。