Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice
Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice
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DOI：
10.1172/jci29383
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发表时间：
2006-11-01
影响因子：
15.9
通讯作者：
Horton, Jay D.
中科院分区：
文献类型：
--
作者：
Lagace, Thomas A.;Curtis, David E.;Horton, Jay D.
蛋白转化酶枯草菌素/酮素9型（PCSK9）是枯草酶K亚家族的一员，通过一种未明确的转录后机制减少肝脏中LDL受体（ldlr）的数量。我们发现，纯化的PCSK9添加到HepG2细胞培养基中，以剂量和时间依赖的方式减少细胞表面ldlr的数量。对于导致高胆固醇血症的功能获得突变体PCSK9(D374Y)，这种活性大约高出10倍。PCSK9的结合和摄取在很大程度上依赖于LDLRs的存在。共免疫沉淀和配体印迹研究表明PCSK9与LDLR直接相关；这两种蛋白都定位于内吞细胞晚期。在缺乏常染色体隐性高胆固醇血症（ARH）的肝细胞中，纯化的PCSK9对细胞表面ldlr没有影响，ARH是受体内吞作用所需的一种衔接蛋白。在肝脏中过度表达人PCSK9的转基因小鼠将大量的蛋白质分泌到血浆中，使血浆LDL胆固醇浓度升高到与ldlr敲除小鼠相似的水平。为了确定PCSK9是否在血浆中有活性，将转基因PCSK9小鼠与野生型凋落物杂交。异种共生后，分泌的PCSK9被转移到野生型小鼠的循环中，使肝脏ldlr的数量减少到几乎无法检测到的水平。我们得出结论，分泌的PCSK9与LDLR相关，并降低肝脏LDLR蛋白水平。
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proteinase K subfamily of subtilases that reduces the number of LDL receptors (LDLRs) in liver through an undefined posttranscriptional mechanism. We show that purified PCSK9 added to the medium of HepG2 cells reduces the number of cell-surface LDLRs in a dose- and time-dependent manner. This activity was approximately 10-fold greater for a gain-of-function mutant, PCSK9(D374Y), that causes hypercholesterolemia. Binding and uptake of PCSK9 were largely dependent on the presence of LDLRs. Coimmunoprecipitation and ligand blotting studies indicated that PCSK9 and LDLR directly associate; both proteins colocalized to late endocytic compartments. Purified PCSK9 had no effect on cell-surface LDLRs in hepatocytes lacking autosomal recessive hypercholesterolemia (ARH), an adaptor protein required for endocytosis of the receptor. Transgenic mice overexpressing human PCSK9 in liver secreted large amounts of the protein into plasma, which increased plasma LDL cholesterol concentrations to levels similar to those of LDLR-knockout mice. To determine whether PCSK9 was active in plasma, transgenic PCSK9 mice were parabiosed with wild-type littermattes. After parabiosis, secreted PCSK9 was transferred to the circulation of wild-type mice and reduced the number of hepatic LDLRs to nearly undetectable levels. We conclude that secreted PCSK9 associates with the LDLR and reduces hepatic LDLR protein levels.