Steve Poirier

The proprotein convertase PCSK9 induces the degradation of low density lipoprotein receptor (LDLR) and its closest family members VLDLR and ApoER2.

The proprotein convertase PCSK9 gene is the third locus implicated in familial hypercholesterolemia, emphasizing its role in cardiovascular diseases. Loss of function mutations and gene disruption of PCSK9 resulted in a higher clearance of plasma low density lipoprotein cholesterol, likely due to a reduced degradation of the liver low density lipoprotein receptor (LDLR). In this study, we show that two of the closest family members to LDLR are also PCSK9 targets. These include the very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) implicated in neuronal development and lipid metabolism. Our results show that wild type PCSK9 and more so its natural gain of function mutant D374Y can efficiently degrade the LDLR, VLDLR, and ApoER2 either following cellular co-expression or re-internalization of secreted human PCSK9. Such PCSK9-induced degradation does not require its catalytic activity. Membrane-bound PCSK9 chimeras enhanced the intracellular targeting of PCSK9 to late endosomes/lysosomes and resulted in a much more efficient degradation of the three receptors. We also demonstrate that the activity of PCSK9 and its binding affinity on VLDLR and ApoER2 does not depend on the presence of LDLR. Finally, in situ hybridization show close localization of PCSK9 mRNA expression to that of VLDLR in mouse postnatal day 1 cerebellum. Thus, this study demonstrates a more general effect of PCSK9 on the degradation of the LDLR family that emphasizes its major role in cholesterol and lipid homeostasis as well as brain development.

The activation and physiological functions of the proprotein convertases

The mammalian secretory proprotein convertases are part of a family of nine serine proteinases of the subtilisin-type. Seven of them cleave after basic amino acids and are called PC1/3, PC2, furin, PC4, PC5/6, PACE4 and PC7. The two other convertases SKI-1/S1P and PCSK9 are implicated in cholesterol and/or fatty acid metabolism. The convertases PC5/6 and PACE4 are activated at the cell surface where they are tethered to heparan sulfate proteoglycans. This activation pathway is unique and differs from that of furin and PC7, which are activated in the trans-Golgi network and from PC1/3 and PC2 that are activated in dense core secretory granules. While some of the basic amino acid-specific convertases may display redundant cleavages of substrates, they uniquely process certain substrates in vivo. Indeed, the conditional knockout of the PC5/6 gene in the embryo proper in mice led to severe malformations, bone morphogenic defects and death at birth. This is likely due to the absence of processing of the growth differentiating factor 11 (Gdf11). Both complete and liver-specific knockout of Pcsk9 revealed that it is a major convertase that regulates the level of circulating low-density lipoproteins (LDL) via the degradation of the hepatic LDL-receptor. This apparently non-enzymatic mechanism implicates the enhanced degradation of the LDLR in endosomes/lysosomes. These data provide evidence that an inhibitor of PCSK9-LDLR interaction is a viable target for the development of a novel cholesterol lowering drug in conjunction with the classical statins.

Dissection of the Endogenous Cellular Pathways of PCSK9-induced Low Density Lipoprotein Receptor Degradation EVIDENCE FOR AN INTRACELLULAR ROUTE

Elevated levels of plasma low density lipoprotein (LDL)-cholesterol, leading to familial hypercholesterolemia, are enhanced by mutations in at least three major genes, the LDL receptor (LDLR), its ligand apolipoprotein B, and the proprotein convertase PCSK9. Single point mutations in PCSK9 are associated with either hyper- or hypocholesterolemia. Accordingly, PCSK9 is an attractive target for treatment of dyslipidemia. PCSK9 binds the epidermal growth factor domain A (EGF-A) of the LDLR and directs it to endosomes/lysosomes for destruction. Although the mechanism by which PCSK9 regulates LDLR degradation is not fully resolved, it seems to involve both intracellular and extracellular pathways. Here, we show that clathrin light chain small interfering RNAs that block intracellular trafficking from the trans-Golgi network to lysosomes rapidly increased LDLR levels within HepG2 cells in a PCSK9-dependent fashion without affecting the ability of exogenous PCSK9 to enhance LDLR degradation. In contrast, blocking the extracellular LDLR endocytosis/degradation pathway by a 4-, 6-, or 24-h incubation of cells with Dynasore or an EGF-AB peptide or by knockdown of endogenous autosomal recessive hypercholesterolemia did not significantly affect LDLR levels. The present data from HepG2 cells and mouse primary hepatocytes favor a model whereby depending on the dose and/or incubation period, endogenous PCSK9 enhances the degradation of the LDLR both extra- and intracellularly. Therefore, targeting either pathway, or both, would be an effective method to reduce PCSK9 activity in the treatment of hypercholesterolemia and coronary heart disease.

Gene Inactivation of Proprotein Convertase Subtilisin/Kexin Type 9 Reduces Atherosclerosis in Mice

Background— The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the low-density lipoprotein (LDL) receptor. PCSK9 gain of function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in ≈7-fold lower levels of LDL cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results— We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E–deficient, and LDL receptor–deficient mouse models. Circulating cholesterol levels, fast protein liquid chromatography profiles, aortic cholesteryl esters (CE), and plaque sizes were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (knockout [KO]), normal (WT), or high (transgenic [Tg]) levels of PCSK9 were fed a 12-month Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next we generated apolipoprotein E–deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e), or high (Tg/e) levels of PCSK9. After a 6-month regular diet, KO/e mice showed a 39% reduction compared with WT/e mice in aortic CE accumulation, whereas Tg/e mice showed a 137% increase. Finally, LDL receptor–deficient mice expressing no (KO/L), normal (WT/L), or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited levels of plasma cholesterol and CE accumulation similar to those of WT/L mice, suggesting that PCSK9 modulates atherosclerosis mainly via the LDL receptor. Conclusions— Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpression is proatherogenic, whereas its absence is protective.Background —The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the LDL receptor (LDLR). PCSK9 gain-of-function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in ~7-fold lower levels of LDL-cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results —We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E (apoE)-deficient and LDLR-deficient mouse models. Circulating cholesterol levels, FPLC profiles, aortic cholesteryl esters (CE) and plaque size were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (KO), normal (WT) or high (Tg) levels of PCSK9 were fed a 12 month-Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next, we generated apoE-deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e) or high (Tg/e) levels of PCSK9. Following a 6 month-regular diet, KO/e mice showed a 39% reduction compared to WT/e mice in aortic CE accumulation, while Tg/e mice showed a 137% increase. Finally, LDLR-deficient mice expressing no (KO/L), normal (WT/L) or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited similar levels of plasma cholesterol and CE accumulation to WT/L, suggesting that PCSK9 modulates atherosclerosis mainly via the LDLR. Conclusions —Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpression is pro-atherogenic, while its absence is protective.

Gene Inactivation of PCSK9 Reduces Atherosclerosis in Mice

Background— The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the low-density lipoprotein (LDL) receptor. PCSK9 gain of function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in ≈7-fold lower levels of LDL cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results— We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E–deficient, and LDL receptor–deficient mouse models. Circulating cholesterol levels, fast protein liquid chromatography profiles, aortic cholesteryl esters (CE), and plaque sizes were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (knockout [KO]), normal (WT), or high (transgenic [Tg]) levels of PCSK9 were fed a 12-month Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next we generated apolipoprotein E–deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e), or high (Tg/e) levels of PCSK9. After a 6-month regular diet, KO/e mice showed a 39% reduction compared with WT/e mice in aortic CE accumulation, whereas Tg/e mice showed a 137% increase. Finally, LDL receptor–deficient mice expressing no (KO/L), normal (WT/L), or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited levels of plasma cholesterol and CE accumulation similar to those of WT/L mice, suggesting that PCSK9 modulates atherosclerosis mainly via the LDL receptor. Conclusions— Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpression is proatherogenic, whereas its absence is protective.Background —The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the LDL receptor (LDLR). PCSK9 gain-of-function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in ~7-fold lower levels of LDL-cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results —We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E (apoE)-deficient and LDLR-deficient mouse models. Circulating cholesterol levels, FPLC profiles, aortic cholesteryl esters (CE) and plaque size were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (KO), normal (WT) or high (Tg) levels of PCSK9 were fed a 12 month-Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next, we generated apoE-deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e) or high (Tg/e) levels of PCSK9. Following a 6 month-regular diet, KO/e mice showed a 39% reduction compared to WT/e mice in aortic CE accumulation, while Tg/e mice showed a 137% increase. Finally, LDLR-deficient mice expressing no (KO/L), normal (WT/L) or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited similar levels of plasma cholesterol and CE accumulation to WT/L, suggesting that PCSK9 modulates atherosclerosis mainly via the LDLR. Conclusions —Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpression is pro-atherogenic, while its absence is protective.

Annexin A2 Is a C-terminal PCSK9-binding Protein That Regulates Endogenous Low Density Lipoprotein Receptor Levels

The proprotein convertase subtilisin/kexin-type 9 (PCSK9), which promotes degradation of the hepatic low density lipoprotein receptor (LDLR), is now recognized as a major player in plasma cholesterol metabolism. Several gain-of-function mutations in PCSK9 cause hypercholesterolemia and premature atherosclerosis, and thus, inhibition of PCSK9-induced degradation of the LDLR may be used to treat this deadly disease. Herein, we discovered an endogenous PCSK9 binding partner by Far Western blotting, co-immunoprecipitation, and pull-down assays. Following two-dimensional gel electrophoresis and mass spectrometry analysis, we demonstrated that PCSK9 binds to a ∼33-kDa protein identified as annexin A2 (AnxA2) but not to the closely related annexin A1. Furthermore, our functional LDLR assays and small hairpin RNA studies show that AnxA2 and the AnxA2·p11 complex could prevent PCSK9-directed LDLR degradation in HuH7, HepG2, and Chinese hamster ovary cells. Immunocytochemistry revealed that PCSK9 and AnxA2 co-localize at the cell surface, indicating a possible competition with the LDLR. Structure-function analyses demonstrated that the C-terminal cysteine-histidine-rich domain of PCSK9 interacts specifically with the N-terminal repeat R1 of AnxA2. Mutational analysis of this 70-amino acid-long repeat indicated that the RRTKK81 sequence of AnxA2 is implicated in this binding because its mutation to AATAA81 prevents its interaction with PCSK9. To our knowledge, this work constitutes the first to show that PCSK9 activity on LDLR can be regulated by an endogenous inhibitor. The identification of the minimal inhibitory sequence of AnxA2 should pave the way toward the development of PCSK9 inhibitory lead molecules for the treatment of hypercholesterolemia.

Implication of the proprotein convertase NARC-1/PCSK9 in the development of the nervous system

Neural apoptosis‐regulated convertase‐1/proprotein convertase subtilisin‐kexin like‐9 (NARC‐1/PCSK9) is a proprotein convertase recently described to play a major role in cholesterol homeostasis through enhanced degradation of the low‐density lipoprotein receptor (LDLR) and possibly in neural development. Herein, we investigated the potential involvement of this proteinase in the development of the CNS using mouse embryonal pluripotent P19 cells and the zebrafish as models. Time course quantitative RT–PCR analyses were performed following retinoic acid (RA)‐induced neuroectodermal differentiation of P19 cells. Accordingly, the mRNA levels of NARC‐1/PCSK9 peaked at day 2 of differentiation and fell off thereafter. In contrast, the expression of the proprotein convertases subtilisin kexin isozyme 1/site 1 protease and Furin was unaffected by RA, whereas that of PC5/6 and PC2 increased within and/or after the first 4 days of the differentiation period respectively. This pattern was not affected by the cholesterogenic transcription factor sterol regulatory element‐binding protein‐2, which normally up‐regulates NARC‐1/PCSK9 mRNA levels in liver. Furthermore, in P19 cells, RA treatment did not affect the protein level of the endogenous LDLR. This agrees with the unique expression pattern of NARC‐1/PCSK9 in the rodent CNS, including the cerebellum, where the LDLR is not significantly expressed. Whole‐mount in situ hybridization revealed that the pattern of expression of zebrafish NARC‐1/PCSK9 is similar to that of mouse both in the CNS and periphery. Specific knockdown of zebrafish NARC‐1/PCSK9 mRNA resulted in a general disorganization of cerebellar neurons and loss of hindbrain–midbrain boundaries, leading to embryonic death at ∼ 96 h after fertilization. These data support a novel role for NARC‐1/PCSK9 in CNS development, distinct from that in cholesterogenic organs such as liver.

The biology of PCSK9 from the endoplasmic reticulum to lysosomes: new and emerging therapeutics to control low-density lipoprotein cholesterol

Proprotein convertase subtilisin/kexin type 9 (PCSK9) directly binds to the epidermal growth factor-like repeat A domain of low-density lipoprotein receptor and induces its degradation, thereby controlling circulating low-density lipoprotein cholesterol (LDL-C) concentration. Heterozygous loss-of-function mutations in PCSK9 can decrease the incidence of coronary heart disease by up to 88%, owing to lifelong reduction of LDL-C. Moreover, two subjects with PCSK9 loss-of-function mutations on both alleles, resulting in a total absence of functional PCSK9, were found to have extremely low circulating LDL-C levels without other apparent abnormalities. Accordingly, PCSK9 could represent a safe and effective pharmacological target to increase clearance of LDL-C and to reduce the risk of coronary heart disease. Recent clinical trials using anti-PCSK9 monoclonal antibodies that block the PCSK9:low-density lipoprotein receptor interaction were shown to considerably reduce LDL-C levels by up to 65% when given alone and by up to 72% in patients already receiving statin therapy. In this review, we will discuss how major scientific breakthroughs in PCSK9 cell biology have led to the development of new and forthcoming LDL-C-lowering pharmacological agents.

Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

Background: It was reported that amyloid precursor-like protein 2 (APLP2) increases PCSK9-mediated low-density lipoprotein receptor (LDLR) degradation, and sortilin facilitates PCSK9 secretion. Results: APLP2 or sortilin deficiency/overexpression in cells/mice did not affect LDLR degradation by PCSK9. However, APLP2 binds sortilin, and PCSK9 enhances their degradation. Conclusion: APLP2/sortilin are not required for PCSK9 activity on LDLR, but their interaction may modulate APLP2 functions. Significance: APLP2 and sortilin do not affect LDLR levels. Amyloid precursor-like protein 2 (APLP2) and sortilin were reported to individually bind the proprotein convertase subtilisin/kexin type 9 (PCSK9) and regulate its activity on the low-density lipoprotein receptor (LDLR). The data presented herein demonstrate that mRNA knockdowns of APLP2, sortilin, or both in the human hepatocyte cell lines HepG2 and Huh7 do not affect the ability of extracellular PCSK9 to enhance the degradation of the LDLR. Furthermore, mice deficient in APLP2 or sortilin do not exhibit significant changes in liver LDLR or plasma total cholesterol levels. Moreover, cellular overexpression of one or both proteins does not alter PCSK9 secretion, or its activity on the LDLR. We conclude that PCSK9 enhances the degradation of the LDLR independently of either APLP2 or sortilin both ex vivo and in mice. Interestingly, when co-expressed with PCSK9, both APLP2 and sortilin were targeted for lysosomal degradation. Using chemiluminescence proximity and co-immunoprecipitation assays, as well as biosynthetic analysis, we discovered that sortilin binds and stabilizes APLP2, and hence could regulate its intracellular functions on other targets.

The epigenetic drug 5-Azacytidine interferes with cholesterol and lipid metabolism

Background: The anticancer drug 5-azacytidine acts through an incompletely understood mechanism. Results: 5-Azacytidine reprograms the glycerolipid biosynthesis pathway and prevents activation of master transcription factors that regulate lipid homeostasis. Conclusion: 5-Azacytidine deeply modifies how cells manage cholesterol and lipid synthesis. Significance: The findings unravel important insights into the mechanism of 5-azacytidine and highlight new potential cancer therapeutics. DNA methylation and histone acetylation inhibitors are widely used to study the role of epigenetic marks in the regulation of gene expression. In addition, several of these molecules are being tested in clinical trials or already in use in the clinic. Antimetabolites, such as the DNA-hypomethylating agent 5-azacytidine (5-AzaC), have been shown to lower malignant progression to acute myeloid leukemia and to prolong survival in patients with myelodysplastic syndromes. Here we examined the effects of DNA methylation inhibitors on the expression of lipid biosynthetic and uptake genes. Our data demonstrate that, independently of DNA methylation, 5-AzaC selectively and very potently reduces expression of key genes involved in cholesterol and lipid metabolism (e.g. PCSK9, HMGCR, and FASN) in all tested cell lines and in vivo in mouse liver. Treatment with 5-AzaC disturbed subcellular cholesterol homeostasis, thereby impeding activation of sterol regulatory element-binding proteins (key regulators of lipid metabolism). Through inhibition of UMP synthase, 5-AzaC also strongly induced expression of 1-acylglycerol-3-phosphate O-acyltransferase 9 (AGPAT9) and promoted triacylglycerol synthesis and cytosolic lipid droplet formation. Remarkably, complete reversal was obtained by the co-addition of either UMP or cytidine. Therefore, this study provides the first evidence that inhibition of the de novo pyrimidine synthesis by 5-AzaC disturbs cholesterol and lipid homeostasis, probably through the glycerolipid biosynthesis pathway, which may contribute mechanistically to its beneficial cytostatic properties.