权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Collaborative Research on Quality Control Mechanism of Newly Synthesized Proteins

新合成蛋白质质量控制机制合作研究

基本信息

批准号：
11694094
负责人：
KOIDE Takehiko
金额：
$ 3.2万
依托单位：
Himeji Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

We investigated the quality control mechanism of newly synthesized proteins in the endoplasmic reticulum (ER) by using under-γ-carboxylated protein C (uc-PC), antithrombin (AT) ΔGlu313 and Pro429stop mutants and cogTg (thyroglobulin Leu2266Pro mutant) as models of misfolded proteins. [Results] 1. Analyses of the effects of high level co-expression of molecular chaperones (BiP, ERp72, ER94 or PDI) in CHO cells on the secretion and degradation of misfolded proteins. 1) In cells expressing a high level of BiP, PDI or ERp72, even wild-type PC was degraded without secretion. 2) Degradation of uc-PC was enhanced with co-expression of ERp72. In contrast, it was rather suppressed in cases of BiP and PDI.3) Wild-type AT was also partially degraded in CHO cells co-expressing BiP or ERp72. 4) Degradation of ΔGlu313 AT was enhanced with co-expression of BiP or ERp72, while that of Pro429stop AT was enhanced only with ERp72. 5) CogTg co-expressed with BiP or ER94 was mostly retained and accumulated in the ER.Thus, the effects of over-expression of molecular chaperones were quite variable among proteins.2. Mannose trimming of N-linked oligosaccharide(s) is proposed to be important for quality control of newly synthesized proteins and formation of Man8B form is critical for targeting misfolded glycoproteins to the degradation pathway. We examined the effects of a high level of co-expression of mannosidase I (whose action generates a Man8B form) or Edem (mannosidase I homologue) on the degradations of ΔGlu313 AT and Pro429stop AT.The result showed that degradations of both AT mutants were enhanced in CHO cells co-expressing a high level of Edem. This result strongly suggests that Edem is a key factor which introduces misfolded glycoproteins into the degradation pathway.

以γ-羧基化不足的蛋白C（uc-PC）、抗凝血酶（AT）Δ Glu 313和Pro429 stop突变体以及甲状腺球蛋白Leu 2266 Pro突变体（cogTg）为错误折叠蛋白质模型，探讨内质网（ER）中新合成蛋白质的质量控制机制。[结果] 1.分析CHO细胞中高水平共表达分子伴侣（BiP、ERp 72、ER 94或PDI）对错误折叠蛋白质的分泌和降解的影响。1)在表达高水平BiP、PDI或ERp 72的细胞中，甚至野生型PC也被降解而不分泌。2)ERp 72的共表达增强了uc-PC的降解。相比之下，在BiP和PDI的情况下，它受到相当大的抑制。3）野生型AT在共表达BiP或ERp 72的CHO细胞中也被部分降解。4)BiP或ERp 72共表达可增强Δ Glu 313 AT的降解，而只有ERp 72可增强Pro429 stop AT的降解。5)与BiP或ER 94共表达的CogTg大部分保留并积累在ER中，因此，分子伴侣过表达的影响在蛋白质之间存在很大差异.提出N-连接寡糖的甘露糖修剪对于新合成蛋白质的质量控制是重要的，并且Man 8B形式的形成对于将错误折叠的糖蛋白靶向至降解途径是关键的。我们检测了高水平共表达甘露糖苷酶I（其作用产生Man 8B形式）或Edem（甘露糖苷酶I同源物）对Δ Glu 313 AT和Pro429 stop AT的降解的影响。结果表明，在共表达高水平Edem的CHO细胞中，两种AT突变体的降解增强。这一结果强烈表明Edem是将错误折叠的糖蛋白引入降解途径的关键因素。