Catalytic activity is not required for secreted PCSK9 to reduce low density lipoprotein receptors in HepG2 cells
Catalytic activity is not required for secreted PCSK9 to reduce low density lipoprotein receptors in HepG2 cells
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DOI：
10.1074/jbc.c700095200
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发表时间：
2007-07-20
影响因子：
4.8
通讯作者：
Horton, Jay D.
中科院分区：
文献类型：
--
作者：
McNutt, Markey C.;Lagace, Thomas A.;Horton, Jay D.
前蛋白转化酶枯草杆菌蛋白酶/kexin 9型（PCSK 9）是枯草杆菌蛋白酶K亚家族的成员，在结合肝细胞表面的受体后促进低密度脂蛋白受体（LDLR）的内化和降解。PCSK 9具有自催化活性，可释放蛋白质N末端的前结构域。当复合物通过分泌途径时，前结构域仍然与催化结构域紧密结合。尚不清楚PCSK 9的降LDLR作用是否需要酶活性。在这里，我们在细胞中表达了前结构域和催化失活的蛋白酶结构域，并从培养基中纯化了蛋白质。当以生理浓度添加到人肝癌HepG 2细胞的培养基中时，无催化活性的PCSK 9结合和降解LDLR的能力与使用野生型蛋白所观察到的相似。类似地，与催化活性对应物相比，功能获得性突变体PCSK 9（D374 Y）的催化死亡版本未显示活性损失;与野生型PCSK 9相比，两种蛋白质在细胞表面LDLR降解方面显示出相似的10倍增加的活性。我们得出结论，PCSK 9降解LDLR的能力是独立的催化活性，并建议PCSK 9作为伴侣蛋白，以防止LDLR回收和/或靶向LDLR的溶酶体降解。
Proprotein convertase subtilisin/kexin type 9 (PCSK9), a member of the proteinase K subfamily of subtilases, promotes internalization and degradation of low density lipoprotein receptors (LDLRs) after binding the receptor on the surface of hepatocytes. PCSK9 has autocatalytic activity that releases the prodomain at the N terminus of the protein. The prodomain remains tightly associated with the catalytic domain as the complex transits the secretory pathway. It is not known whether enzymatic activity is required for the LDLR-reducing effects of PCSK9. Here we expressed the prodomain together with a catalytically inactive protease domain in cells and purified the protein from the medium. The ability of the catalytically inactive PCSK9 to bind and degrade LDLRs when added to culture medium of human hepatoma HepG2 cells at physiological concentrations was similar to that seen using wild-type protein. Similarly, a catalytic-dead version of a gain-of-function mutant, PCSK9(D374Y), showed no loss of activity compared with a catalytically active counterpart; both proteins displayed similar to 10-fold increased activity in degradation of cell surface LDLRs compared with wild-type PCSK9. We conclude that the ability of PCSK9 to degrade LDLRs is independent of catalytic activity and suggest that PCSK9 functions as a chaperone to prevent LDLR recycling and/or to target LDLRs for lysosomal degradation.