Intracellular degradation of secretion defect‐type mutants of antithrombin is inhibited by proteasomal inhibitors
Intracellular degradation of secretion defect‐type mutants of antithrombin is inhibited by proteasomal inhibitors
复制标题
蛋白酶体抑制剂抑制抗凝血酶分泌缺陷型突变体的细胞内降解
DOI：
10.1016/s0014-5793(97)00745-x
复制
发表时间：
1997
期刊：
影响因子：
3.5
通讯作者：
T. Koide
中科院分区：
文献类型：
--
作者：
F. Tokunaga;H. Shirotani;K. Hara;Daisuke Kozuki;S. Ōmura;T. Koide
为了检查分泌缺陷型抗凝血酶缺陷的细胞基础，我们表达了两种突变体：P→stop（Pro429to 终止密码子）和ΔGlu（Glu313 的缺失）。使用稳定转染的 BHK 细胞进行的脉冲追踪实验表明，分泌很少（<5%）的 P→stop 突变体以及 ΔGlu 突变体，并且放射性总量显着降低，表明细胞内降解。布雷菲德菌素 A 不抑制降解，表明它发生在前高尔基体中。然而，降解受到蛋白酶体抑制剂的强烈抑制，例如苯甲酰基-l-亮氨酰-l-亮氨酰-l-亮氨酸（LLL）、苯甲酰基-l-亮氨酰-l-亮氨酰-l-正缬醛（LLnV）和乳胞素。通过内切糖苷酶 H 消化和免疫荧光染色，显示这些突变体定位于内质网 (ER)。这些结果表明，抗凝血酶的分泌缺陷型突变体是通过细胞内内质网相关的质量控制机制被蛋白酶体降解的。
To examine the cellular basis for secretion defect-type antithrombin deficiency, we expressed two mutants, P→stop (Pro429to stop codon) and ΔGlu (deletion of Glu313). Pulse-chase experiments using stably transfected BHK cells showed that little (<5%) of P→stop mutant as well as ΔGlu mutant was secreted and the total amount of radioactivity was significantly reduced, suggesting an intracellular degradation. The degradation was not inhibited by brefeldinA, indicating it occurring in a preGolgi apparatus. However, the degradation was strongly inhibited by proteasomal inhibitors, such as carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (LLL), carbobenzoxy-l-leucyl-l-leucyl-l-norvalinal (LLnV) and lactacystin. By endoglycosidaseH digestion and immunofluorescence staining, these mutants were shown to localize in the endoplasmic reticulum (ER). These results suggest that the secretion defect-type mutants of antithrombin are degraded by proteasome through the ER-associated quality control mechanism in the cells.