Characterization of PCSK9-mediated LDLR Degradation in Hepatic and Fibroblast Cells

Abstract

The discovery that proprotein convertase subtilisin/kexin type 9 (PCSK9) mediates degradation of low-density lipoprotein receptors (LDLR) indicates a critical role in LDL metabolism. PCSK9 is a secreted protein that binds to the epidermal growth factor-like (EGF)-A domain of LDLR and directs the receptor for degradation in lysosomes by an unknown mechanism. A gain-of-function mutation, D374Y, increases binding to LDLR EGF-A >10-fold and is associated with a severe form of hypercholesterolemia in humans. Similar to previous studies, data obtained in my project has established that PCSK9 was capable of promoting robust LDLR degradation in liver-derived cell lines; however, minimal effects on LDLR levels were detected in several lines of fibroblast cells despite normal LDLR-dependent cellular uptake of PCSK9. Importantly, a PCSK9 degradation assay showed that 125I-labeled wild-type PCSK9 was internalized and degraded equally in both hepatic and fibroblast cells, indicating dissociation of wild-type PCSK9 from recycling LDLRs in fibroblasts. Moreover, PCSK9 recycling assays confirmed that no recycling of wild-type PCSK9 to the cell surface could be detected in fibroblast cells. In contrast, more than 60% of internalized PCSK9-D374Y recycled to the cell surface in these cells, and thus had reduced ability to direct the LDLR for lysosomal degradation despite persistent binding. Co-localization studies indicated that PCSK9-D374Y trafficked to both lysosomes and recycling compartments in fibroblast cells, whereas wild-type PCSK9 exclusively trafficked to lysosomes. We conclude that two factors diminish PCSK9 activity in fibroblast cells: i) an increased dissociation from the LDLR in early endosomal compartments, and ii) a decreased ability of bound PCSK9 to direct the LDLR to lysosomes for degradation. Finally, an LDLR variant that binds to PCSK9 in a Ca2+-independent manner could partially restore wild-type PCSK9 activity, but not PCSK9-D374Y activity, in fibroblast cells.