Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain

<div><p>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 <i>PCSK9</i> 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 <i>trans</i>-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 <i>via</i> 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-ΔCTD 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.</p></div

Fusion of monomeric fluorescent proteins to PCSK9 and LDLR.

<p>(A) HEK293 cells were transiently transfected with plasmids encoding WT PCSK9-mCherry (mC) or its GOF D374Y mutant and expression was analyzed by fluorescence microscopy with a 20X objective. (B) HEK293 cells were transfected without (Empty) or with WT LDLR or its EGF-A D331E mutant, incubated for 24 h (left panels) or 4 h (right panels) in DMEM or with conditioned media from PCSK9-mC transfected HEK293 cells. LDLR was revealed under non-permeabilizing conditions and protein localization was analyzed by confocal immunofluorescence microscopy. Magnified images of area within dashed lines are shown (bottom panels). <i>Scale bars</i>; 20 μm. (C) Immunoblots (IB) of conditioned media of PCSK9 natural mutants and truncation variants obtained from transiently transfected HEK293 cells (<i>input media</i>). Corresponding media were incubated overnight on HepG2 cells and LDLR and β-actin (loading control) protein levels were analyzed by IB. (D) HEK293 cells were transfected with LDLR-EGFP without (Empty) or with PCSK9 D374Y-mC and both proteins were analyzed by confocal microscopy (<i>left panels</i>) and IB (<i>right panels</i>). Scale bar = 20 μM. Data are representative of at least three independent experiments.</p

PCSK9 CTD is required for efficient LDLR-mediated endocytosis of PCSK9.

<p>(A) HepG2 cells transfected with LDLR-EGFP were incubated 5h with conditioned media obtained from HEK293 cells transected without (Empty) or with PCSK9 WT-, D374Y-, ΔCTD- or CTD-mC. PCSK9-mC constructs and LDLR-EGFP were analyzed by live-cell confocal microscopy. Selected regions (dashed squares) were digitally zoomed 5X (MAG). Scale bars = 20 μM. (B) Quantification of the number of intracellular red fluorescent puncta per cell expressing LDLR-EGFP for each PCSK9 mCherry fusion construct described in (A) after incubation for 0, 60, 120, 180, 240 and 300 min. PCSK9 WT-mC (n = 37 cells), D374Y-mC (n = 62 cells), ΔCTD-mC (n = 31 cells) or CTD-mC (n = 52 cells). (C) HEK293 cells were transfected without (-) or with WT LDLR or its EGF-A D331E mutant and incubated for 6 h with DMEM alone (-) or with normalized conditioned media obtained from FL or ΔCTD PCSK9-mC transfected cells (input media). Cell-associated PCSK9-mC, LDLR and β-actin (loading control) protein levels were analyzed by IB. Data are representative of at least three independent experiments.</p

Deletion of PCSK9 hinge region does not affect CTD localization at the TGN.

<p>PCSK9 molecular structure was extracted from PDB 2P4E using MacPymol software. Prodomain (PRO), catalytic domain (CAT) and its LDLR-interacting residues (in pink), hinge region in blue and position R434 in red (site of LOF R434W) and C-terminal domain (CTD) are indicated. HepG2 cells were transfected with V5-tagged ΔHinge-CTD (Δaa 422-439-CTD), immunolabeled with the anti-V5 and anti-Golgin-97 antibodies and their localization was analyzed by confocal microscopy (<i>lower panels</i>). Colocalization was quantified using Pearson’s correlation coefficient (Rcoloc) (minimum of 10 cells were analyzed). Data are representative of at least three independent experiments. <i>Scale bar</i>, 10 μm.</p

PCSK9 localizes at the TGN <i>via</i> its CTD.

<p>(A) Ultrastructural localization of PCSK9 in normal human liver tissue sections shows the immunogold labeling over the rough endoplasmic reticulum (RER; left panel), Golgi apparatus (G), mulitvesicular bodies (MVB) and late endosomes (LE) (middle and left panels) of hepatocytes. Arrowheads denotes inward budding vesicles that are characteristic of MVBs (right panel). Note the presence of very few gold particles over mitochondria (M) and glycogen (Gly) indicating the specificity of the labeling. Omission of the primary antibody resulted in the absence of specific labeling (control experiment, Ctl; inset). Magnification X 25000. (B) Co-localization of endogenous PCSK9 with the TGN marker Golgin-97 as visualized by confocal microscopy (<i>left panels</i>, <i>white arrows</i>). Specificity of PCSK9 immunolabeling was tested following overnight pre-incubation of the anti-PCSK9 Ab with 1 μg/ml recombinant human PCSK9 (<i>right panels</i>, <i>immunoadsorption</i>). (C) HepG2 cells were transfected with V5-tagged FL PCSK9, CTD, ΔCTD or LOF R434W mutant and co-localization with Golgin-97 was analyzed by confocal microscopy. (B, C) Colocalization was quantified using Pearson’s correlation coefficient (Rcoloc) (minimum of 10 cells were analyzed). Data are representative of at least three independent experiments. <i>Scale bars</i>, 20 μm.</p

Lysosomal targeting of PCSK9-ΔCTD bypasses the need of CTD to induce LDLR degradation.

<p>(A-B) HepG2 cells were transfected without (Empty) or with V5-tagged PCSK9 full-length (FL), ΔCTD, CTD alone or PCSK9-TM-CT-Lamp1 chimeras (FL, F379A, ΔCTD, CTD) or Timp1-TM-CT-Lamp1 (herein used as control). Forty-eight hours post-transfection, LDLR, PCSK9 and β-actin (loading control) protein levels were analyzed by IB. Data are representative of at least three independent experiments</p

Effect of PCSK9 natural mutants on TGN localization and trafficking dynamics by FRAP and iFRAP.

<p>(A) HepG2 cells were transfected with PCSK9-mC WT, GOF S127R, D129G, D374Y or LOF R46L mutants (red) and TGN localization was determined by colocalization (arrows) with Golgin-97 (green) and analysis by confocal microscopy. Nuclei were labeled with TO-PRO-3 Iodide (cyan). (B) Half time (T_1/2; min) and mobile fraction (%) calculated from FRAP (ER ➜ Golgi) and iFRAP (Golgi ➜ plasma membrane) experiments in living HepG2 cells for corresponding PCSK9-mC constructs shown in (A). Dashed square represent typical TGN localization of PCSK9-mC before bleach for which fluorescence recovery after photobleaching (FRAP) values were obtained (FRAP). Inverse FRAP (iFRAP) studies in the presence of 200 μg/ml cycloheximide to block protein synthesis (as described in Materials and Methods) were performed for corresponding PCSK9-mC shown in (A). For each PCSK9-mC constructs, mobile fraction (%) at the TGN from FRAP and iFRAP were calculated as described in <i>Materials and</i> Methods. Data are representative of at least three independent experiments are shown as the mean ± S.E.M. Statistical significance: *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01, ***<i>p</i> ≤ 0.001. <i>Scale bars</i>, 10 μm.</p

Image1_Human induced pluripotent stem cells (hiPSCs) derived cells reflect tissue specificity found in patients with Leigh syndrome French Canadian variant (LSFC).pdf

Leigh syndrome French Canadian type (LSFC) is a recessive neurodegenerative disease characterized by tissue-specific deficiency in cytochrome c oxidase (COX), the fourth complex in the oxidative phosphorylation system. LSFC is caused by mutations in the leucine rich pentatricopeptide repeat containing gene (LRPPRC). Most LSFC patients in Quebec are homozygous for an A354V substitution that causes a decrease in the expression of the LRPPRC protein. While LRPPRC is ubiquitously expressed and is involved in multiple cellular functions, tissue-specific expression of LRPPRC and COX activity is correlated with clinical features. In this proof-of-principle study, we developed human induced pluripotent stem cell (hiPSC)-based models from fibroblasts taken from a patient with LSFC, homozygous for the LRPPRC*354V allele, and from a control, homozygous for the LRPPRC*A354 allele. Specifically, for both of these fibroblast lines we generated hiPSC, hiPSC-derived cardiomyocytes (hiPSC-CMs) and hepatocyte-like cell (hiPSC-HLCs) lines, as well as the three germ layers. We observed that LRPPRC protein expression is reduced in all cell lines/layers derived from LSFC patient compared to control cells, with a reduction ranging from ∼70% in hiPSC-CMs to undetectable levels in hiPSC-HLC, reflecting tissue heterogeneity observed in patient tissues. We next performed exploratory analyses of these cell lines and observed that COX protein expression was reduced in all cell lines derived from LSFC patient compared to control cells. We also observed that mutant LRPPRC was associated with altered expression of key markers of endoplasmic reticulum stress response in hiPSC-HLCs but not in other cell types that were tested. While this demonstrates feasibility of the approach to experimentally study genotype-based differences that have tissue-specific impacts, this study will need to be extended to a larger number of patients and controls to not only validate the current observations but also to delve more deeply in the pathogenic mechanisms of LSFC.</p