Internalized PCSK9 dissociates from recycling LDL receptors in PCSK9-resistant SV-589 fibroblasts

Abstract

Secreted PCSK9 binds to cell surface LDL receptor (LDLR) and directs the receptor for lysosomal degradation. PCSK9 is potent at inducing LDLR degradation in cultured liver-derived cells, but it is considerably less active in immortalized fibroblasts. We examined PCSK9 trafficking in SV-589 human skin fibroblasts incubated with purified recombinant wild-type PCSK9 or gain-of-function mutant PCSK9-D374Y with increased LDLR binding affinity. Despite LDLR-dependent PCSK9 uptake, cell surface LDLR levels in SV-589 fibroblasts were only modestly reduced by wild-type PCSK9, even at high nonphysiological concentrations (20 µg/ml). Internalized 125I-labeled wild-type PCSK9 underwent lysosomal degradation at high levels, indicating its dissociation from recycling LDLRs. PCSK9-D374Y (2 µg/ml) reduced cell surface LDLRs by approximately 50%, but this effect was still blunted compared with HepG2 hepatoma cells. Radioiodinated PCSK9-D374Y was degraded less efficiently in SV-589 fibroblasts, and Alexa488-labeled PCSK9-D374Y trafficked to both lysosomes and endocytic recycling compartments. Endocytic recycling assays showed that more than 50% of internalized PCSK9-D374Y recycled to the cell surface compared with less than 10% for wild-type PCSK9. These data support that wild-type PCSK9 readily dissociates from the LDLR within early endosomes of SV-589 fibroblasts, contributing to PCSK9-resistance. Although a large proportion of gain-of-function PCSK9-D374Y remains bound to LDLR in these cells, degradative activity is still diminished.

Proprotein Convertase Subtilisin Kexin Type 9 Promotes Intestinal Overproduction of Triglyceride-Rich Apolipoprotein B Lipoproteins Through Both Low-Density Lipoprotein Receptor–Dependent and –Independent Mechanisms

“…The observation that a portion of internalized PCSK9 is not degraded has been recently reported by us after finding human PCSK9 in liver of mice 4 hours after injection of exogenous human PCSK9 5 . Similarly, a recent study with human fibroblasts has documented that a portion of internalized PCSK9 is diverted from the lysosomal degradation pathway to endocytic recycling compartments 27 . It is unclear whether this mechanism involves dissociation of PCSK9 from LDLR or if it is a route for the coupled recycling for both protein partners.…”

Cell-associated heparin-like molecules modulate the ability of LDL to regulate PCSK9 uptake

“…On the basis of our data alone, however, it remains unclear whether the HLM-dependent mechanism also requires PCSK9:LDLR binding or the presence of the LDLR itself. It is tantalizing to conjecture that HLMs may offer a way to shuttle PCSK9 into an endocytic vesicle without the need for the PCSK9:LDLR interaction, whereby PCSK9 can then direct itself into acidified endosomes (50,59) and induce an interaction with the LDLR via its cysteine-histidine rich domain, which has known affinity for the LDLR at lower pH (60,61). This could effectively bypass the need for the interaction between the PCSK9 catalytic and the LDLR EGF-A domains at the cell surface (50).…”

On the function and homeostasis of PCSK9: Reciprocal interaction with LDLR and additional lipid effects

“…The underlying mechanism leading to these effects of PCSK9 are unknown. We, and others, have shown that a portion of PCSK9 entering the cell with LDLR remains intact and does not undergo lysosomal degradation for several hours (21, 75). A mechanism to explain how PCSK9 is able to alter intracellular processes is presented in Figure 2.…”