Identification of Proteins Associated with the Intracellular Degradation of Misfolded Clotting Factors

我们已经证明，在口服抗凝剂华法林存在下合成的维生素K依赖性凝血因子通过内质网（ER）中的质量控制机制在细胞内降解。这是药物诱导的ER相关降解的唯一模型。1996年，我们发现在华法林存在下合成的细胞内蛋白C在细胞内降解过程中与ER分子伴侣如GRP 94、GRP 78（BiP）和钙网蛋白相关。蛋白酶体抑制剂如lactacystin和Z-leu-Leu-Leu-H可抑制华法林诱导的蛋白C降解，提示蛋白酶体通过逆行转运将羧基化不足的蛋白C降解。1997年，我们比较了维生素K依赖蛋白对华法林的敏感性。在华法林存在下，HepG 2细胞中合成的蛋白C被广泛降解，而约50%的凝血酶原被分泌到培养基中。因此，我们构建了两个嵌合体的蛋白C具有凝血酶原Gla域（GDII/PC）和具有凝血酶原Gladomain的前原序列（PPGDII/PC），并使用BHK细胞检测嵌合体的分泌。出乎意料的是，即使在维生素K存在下，两种嵌合蛋白也不分泌。嵌合体的γ-羧化正常发生。这些结果表明，不仅Gla结构域的γ-羧化，而且某些Gla结构域与其它结构域的相互作用也是维生素K依赖蛋白质质量控制的重要因素。

We have shown that the vitamin K-dependent coagulation factors synthesized in the presence of warfarin, an oral anticoagulant, is degraded intracellularly through the quality control mechanism in the endoplasmic reticulum (ER). this is a sole model for the drug-induced ER-associated degradation. On 1996, We identified that intracellular protein C synthesized in the presence of warfarin was associated with ER molecular chaperones such as GRP94, GRP78(BiP), and calreticulin on the course of intracellular degradation. Moreover, proteasomal inhibitors such as lactacystin and Z-leu-Leu-Leu-H inhibited the warfarin-induced degradation of protein C,suggesting that under-gamma-carboxylated protein C is Degraded by proteasome in cytosol through the retrograde transport. On 1997, We compared the warfarin-sensitivity among vitamin K-dependent proteins. Protein C synthesized in HepG2 cells was extensively degraded in the presence of warfarin, whereas-50% of prothrombin was secreted into medium. This results suggested that prothrombin Gla domain is moro resistant to warfarin than that of protein C.Then, we constructed two chimeras of protein C having prothrombin Gla domain (GDII/PC) and having prepro-sequence to Gladomain of prothrombin (PPGDII/PC), and examined the secretion of chimeras using BHK cells. Unexpectedly, both chimeric proteins were not secreted even in the presence of vitamin K.gamma-Carboxylation of chimeras was occurred normally. These results suggested that not only gamma-carboxylation in the Gla domain, but the interaction of certain Gla domain to other domains is also an important factor in quuality control of vitamin K-dependent proteins.