Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain
Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain
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DOI：
10.1371/journal.pone.0157230
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发表时间：
2016-06-09
期刊：
影响因子：
3.7
通讯作者：
Mayer, Gaetan
中科院分区：
文献类型：
--
作者：
Poirier, Steve;Hamouda, Hocine Ait;Mayer, Gaetan
PCSK9是肝细胞中低密度脂蛋白受体（LDLR）的分泌配体和翻译后负调节因子。PCSK9的功能获得（GOF）或功能丧失（LOF）突变分别与血浆ldl -胆固醇水平高或低直接相关。因此，PCSK9是预防冠心病和中风的普遍降脂靶点。在此，我们将单体荧光蛋白与PCSK9和LDLR融合，通过活共聚焦显微镜观察它们在细胞内和细胞外的运输动态。光漂白后荧光恢复（FRAP）显示，PCSK9 LOF R46L突变体和GOF突变体S127R和D129G显著加速了PCSK9从内质网（ER）的退出，而LDLR高亲和突变体D374Y则没有。反向FRAP的定量分析显示，只有R46L从反式高尔基网络（TGN）转运到质膜的速度要慢得多，而且移动率较低，这可能表明在TGN积聚或延迟退出是潜在的机制。虽然PCSK9 c -末端结构域（CTD）并不主要参与LDLR的结合，但研究发现PCSK9 c -末端结构域（CTD）在细胞内过表达或通过细胞外途径诱导LDLR降解是必不可少的。我们的数据显示PCSK9 CTD是PCSK9在TGN定位和增加其ldlr介导的内吞作用所必需的。有趣的是，细胞内溶酶体靶向PCSK9-Delta CTD能够恢复其诱导LDLR降解的能力，强调了CTD在PCSK9-LDLR复合物向晚期内噬室分选中的作用。最后，我们通过使用PCSK9- ldlr阻断抗体来阻止PCSK9内化，从而验证了我们的双荧光系统是一种基于细胞的检测方法，该抗体可用于鉴定PCSK9的蛋白质、肽或小分子抑制剂。
PCSK9 is a secreted ligand and negative post-translational regulator of low-density lipoprotein receptor (LDLR) in hepatocytes. Gain-of-function (GOF) or loss-of-function (LOF) mutations in PCSK9 are directly correlated with high or low plasma LDL-cholesterol levels, respectively. Therefore, PCSK9 is a prevailing lipid-lowering target to prevent coronary heart diseases and stroke. Herein, we fused monomeric fluorescent proteins to PCSK9 and LDLR to visualize their intra-and extracellular trafficking dynamics by live confocal microscopy. Fluorescence recovery after photobleaching (FRAP) showed that PCSK9 LOF R46L mutant and GOF mutations S127R and D129G, but not the LDLR high-affinity mutant D374Y, significantly accelerate PCSK9 exit from the endoplasmic reticulum (ER). Quantitative analysis of inverse FRAP revealed that only R46L presented a much slower trafficking from the trans-Golgi network (TGN) to the plasma membrane and a lower mobile fraction likely suggesting accumulation or delayed exit at the TGN as an underlying mechanism. While not primarily involved in LDLR binding, PCSK9 C-terminal domain (CTD) was found to be essential to induce LDLR degradation both upon its overexpression in cells or via the extracellular pathway. Our data revealed that PCSK9 CTD is required for the localization of PCSK9 at the TGN and increases its LDLR-mediated endocytosis. Interestingly, intracellular lysosomal targeting of PCSK9-Delta CTD was able to rescue its capacity to induce LDLR degradation emphasizing a role of the CTD in the sorting of PCSK9-LDLR complex towards late endocytic compartments. Finally, we validated our dual fluorescence system as a cell based-assay by preventing PCSK9 internalization using a PCSK9-LDLR blocking antibody, which may be expended to identify protein, peptide or small molecule inhibitors of PCSK9.