1521-0081/69/1/33–52$25.00 http://dx.doi.org/10.1124/pr.116.012989 PHARMACOLOGICAL REVIEWS Pharmacol Rev 69:33–52, January 2017 Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics

ASSOCIATE EDITOR: RHIAN M. TOUYZ The Proprotein Convertases in Hypercholesterolemia and Cardiovascular Diseases: Emphasis on Proprotein Convertase Subtilisin/Kexin 9

Nabil G. Seidah, Marianne Abifadel, Stefan Prost, Catherine Boileau, and Annik Prat Laboratory of Biochemical Neuroendocrinology, Institut de Recherches Cliniques de Montréal, affiliated to Université de Montréal, QC, Canada (N.G.S., A.P.); LVTS, INSERM U1148, Hôpital Xavier-Bichat, Paris, France (M.A., C.B.); Laboratory of Biochemistry and Molecular Therapeutics, Faculty of Pharmacy, Pôle Technologie-Santé, Saint-Joseph University, Beirut, Lebanon (M.A.); Department of Integrative Biology, Center for Theoretical Evolutionary Genomics, University of California Berkeley, Berkeley, California (S.P.); Department of Biology, Stanford University, Stanford, California (S.P.); and Département de Génétique, AP-HP, CHU Xavier Bichat, and Université Paris Diderot, Paris, France (C.B.)

Abstract ...... 33 I. The Proprotein Convertases and Their Functions in Health and Disease ...... 34 II. Phylogenetic and Haplotype Analyses of PCSK9 in Human Species ...... 36 Downloaded from III. The Enzymatically Inactive Mature PCSK9 Regulates Circulating LDL Cholesterol Levels . . . 37 Role of PCSK9 in Lipoprotein (a) Regulation...... 38 IV. The Absence of PCSK9 Leads to a Sex- and Tissue-Specific Subcellular Distribution of the LDLR ...... 38 V. Role of PCSK9 in Inflammation, Sepsis, and Viral Infections...... 39 VI. Other Physiologic Roles of PCSK9...... 40 by guest on September 24, 2021 VII. Cellular Studies: Mechanism of Action of PCSK9 ...... 41 VIII. What Have We Learned from Key Natural Mutations on the Functions of PCSK9 and Its Partners? ...... 42 IX. Ongoing Outcome Clinical Trials ...... 44 X. Other Genes and Loci Implicated in LDL-c Regulation...... 45 XI. Implication of the Enzymatically Active PCs in CVD ...... 46 A. PC1/3 and PC2 in Diabetes and Obesity ...... 46 B. Furin and SKI-1/S1P in Cardiovascular Diseases ...... 47 C. PACE4 in Blood Pressure Regulation ...... 47 D. PC5/6 in Heart Development and Extracellular Matrix Remodeling ...... 47 E. PC7 in HDL, small dense LDL, and TG ...... 48 XII. Conclusions and Future Perspectives ...... 48 Acknowledgments...... 48 References ...... 48

Abstract——The secretory proprotein convertase (PC) factors at nonbasic residues. In contrast, PCSK9 cleaves family comprises nine members, as follows: PC1/3, PC2, itself once, and the secreted inactive protease drags the furin, PC4, PC5/6, paired basic amino acid cleaving low-density lipoprotein receptors (LDLR) and very enzyme 4, PC7, subtilisin kexin isozyme 1/site 1 protease LDLR (VLDLR) to endosomal/lysosomal degradation. (SKI-1/S1P), and PC subtilisin/kexin type 9 (PCSK9). The Inhibitory PCSK9 monoclonal antibodies are now first seven PCs cleave their substrates at single/paired prescribed to treat hypercholesterolemia. This review basic residues and exhibit specific and often essential focuses on the implication of PCs in cardiovascular functions during development and/or in adulthood. The functions and diseases, with a major emphasis on essential SKI-1/S1P cleaves membrane-bound transcription PCSK9. We present a phylogeny of the PCs and the

This work was supported by Canadian Institutes of Health Research [Grants MOP-102741 and CTP-82946]; Canada Research Chair 216684; and Leducq Foundation [Grant 13CVD03]. Address correspondence to: Dr. Nabil G. Seidah, Institut de Recherches Cliniques de Montréal, 110 Pine Avenue West, Montreal, QC H2W1R7, Canada. E-mail: [email protected] dx.doi.org/10.1124/pr.116.012989.

33 34 Seidah et al. analysis of PCSK9 haplotypes in modern and archaic mechanism(s) that enables PCSK9 to induce the human species. The absence of PCSK9 in mice led to degradation of receptors is reviewed, as well as the the discovery of a sex- and tissue-specific subcellu- consequences of its key natural mutations. The PCSK9 lar distribution of the LDLR and VLDLR. PCSK9 ongoing clinical trials are reviewed. Finally, how the inhibition may have other applications because it other PCs may impact cardiovascular disease and the reduces inflammation and sepsis in a LDLR-dependent metabolic syndrome, and become relevant targets, is manner. Our present understanding of the cellular discussed.

I. The Proprotein Convertases and Their Potentially the most advanced/promising clinical trials Functions in Health and Disease are for inhibitors of furin in cancer and viral infections, PACE4 in arthritis pain and prostate cancer, and PCSK9 Post-translational modifications of secretory proteins in hypercholesterolemia and septic shock (Fig. 1). fashion the final bioactive forms of primary protein The first seven basic amino acid–specific PCs were products in various tissues and cells during development discovered between 1990 and 1996 (Seidah and Prat, and in adulthood (Seidah and Guillemot, 2016). Of all 2012; Seidah, 2015). However, other precursors, such as post-translational modifications of proteins known, pep- brain-derived neurotrophic factor, cell surface glycopro- tide bond cleavage represents one of the most drastic and teins of hemorrhagic fever viruses, and membrane-bound irreversible reactions leading to two or more fragments transcription factors [sterol regulatory element-binding of distinct, but sometimes complementary, biologic proteins (SREBPs); endoplasmic reticulum (ER)–stress functions. From the time it was realized that secretory polypeptide hormones such as insulin (Steiner, 2011) protein activating transcription factor 6; or cAMP- and adrenocorticotropic hormone/b-endorphin (Chrétien, response element-binding proteins], are cleaved after 2011) were synthesized from inactive precursors, pro- nonbasic residues, predicting the implication of a distinct insulin and pro-opiomelanocortin, respectively, it took protease(s). This led to the simultaneous discovery and 23 years to identify the cognate proteases that generate cDNA cloning of site 1 protease (S1P) (Cheng et al., 1999; various peptide hormones and protein products (Seidah Espenshade et al., 1999), also known as subtilisin-kexin and Chrétien, 1999; Seidah and Prat, 2012). isozyme 1 (SKI-1) (Seidah et al., 1999). The mammalian genome encodes nine proprotein The transcription factors SREBP-1 and SREBP-2 are convertases (PCs). These serine proteases share sequence major regulators of cholesterol/low-density lipoprotein identity with bacterial subtilisin and yeast kexin (Julius (LDL) receptor (LDLR) and fatty acid synthesis, re- et al., 1984; Mizuno et al., 1988), and their genes are spectively. They are anchored in the secretory pathway – referred as proprotein convertases subtilisin kexin types via a luminal loop separating two transmembrane 1–9(PCSK1 to PCSK9), with two exceptions for members cytosolic domains. The initial cleavage by SKI-1/S1P in 3and8,respectively,namedFURIN and MBTPS1.The the luminal loop within the cis/medial Golgi is followed nine corresponding proteins are known as follows: PC1/3, by an intramembrane cleavage in the first transmem- PC2, furin, PC4, PC5/6, paired basic amino acid cleaving brane domain by the metalloprotease site-2 protease enzyme 4 (PACE4), PC7, subtilisin kexin isozyme 1/site in the medial Golgi (Rawson et al., 1997). The released 1 protease (SKI-1/S1P), and PCSK9. Except for PCSK9, cytosolic N-terminal basic helix-loop-helix leucine they all cleave secretory proteins, such as polypeptide zipper-containing domains of both factors are then hormones, receptors, growth factors, adhesion molecules, translocated to the nucleus, where they activate their membrane-bound transcription factors, and neuronal respective target genes. SKI-1/S1P was the first PC regulators, during embryonic development and in adult- implicated in the regulation of lipid metabolism, as its hood (Seidah and Prat, 2012). Such processing regulates inactivation in the liver decreased by 75% lipid synthe- essential cellular functions, and their dysregulation can sis (Yang et al., 2001). However, the complete Mbtps1 lead to the metabolic syndrome, obesity, diabetes, hyper- gene inactivation in mouse resulted in an early embry- tension, dyslipidemia, inflammation, cancer/metastasis, onic death with no epiblast formation (Yang et al., pain, memory deficits, anxiety/depression, neurodegen- 2001), thus hampering clinical applications of SKI- eration, iron dysregulation, and even viral and parasitic 1/S1P inhibitors. Only short treatments with the SKI- infections. Accordingly, a number of ongoing clinical trials 1/S1P small-molecule inhibitor PF-429242 (Hawkins are investigating the plausible roles of PCs in diseases. et al., 2008) were suggested for persistent arenavirus

ABBREVIATIONS: aa, amino acid; ADH, autosomal dominant hypercholesterolemia; apoB, apolipoprotein B; ApoER2, apolipoprotein E receptor 2; ARH, autosomal recessive hypercholesterolemia; CHRD, Cys-His–rich domain; CLP, cecal ligation and puncture; CT, cytosolic tail; CVD, cardiovascular disease; ecKO, endothelial cell–specific KO; ER, endoplasmic reticulum; FH, familial hypercholesterolemia; Gdf11, growth differentiation factor 11; GOF, gain-of-function; GWAS, genome-wide association studies; HDL, high-density lipoprotein; HepKO, hepatocyte-specific KO; KO, knockout; LDL, low-density lipoprotein; LDL-c, LDL cholesterol; LDLR, LDL receptor; LOF, loss-of-function; Lp(a), lipoprotein (a); LPS, lipopolysaccharide; LRP1, LDLR-related protein-1; mAb, monoclonal antibody; PACE4, paired basic amino acid cleaving enzyme 4; PC, proprotein convertase; PCSK, PC subtilisin/kexin; S1P, site 1 protease; SKI-1, subtilisin kexin isozyme 1; SNP, single- nucleotide polymorphism; SREBP, sterol regulatory element-binding protein; TG, triglyceride; VLDLR, very LDLR. Roles of PCSKs in Cholesterol and Triglyceride Metabolism 35

Fig. 1. Proprotein convertases in health and diseases. The clinical fields relevant to each PC are indicated. To date, only PCSK9 is clinically targeted using mAbs. These mAbs are used for the treatment of hypercholesterolemia and are very promising for the prevention of septic shock. In this schematic, overlaps between PC clinical fields are only qualitative. Diabetes is related to both PC1/3 and PC2 through insulin processing. Cancers are mainly regulated by furin, PC5/6, and PACE4 (prostate) via processing of growth factors and their receptors. Most viral surface glycoproteins are processed by furin and, in some cases, by SKI-1/S1P (arenaviruses). Cholesterol and TG synthesis are regulated by SKI-1/S1P and LDL-c uptake by PCSK9. The convertase PC4 has a unique role in fertility, and PC7 in iron metabolism and anxiety/depression. Finally, in humans, PC1/3, PC5/6, and PACE4 were genetically linked to obesity, developmental defects, and osteoarthritis pain, respectively.

(Pasquato et al., 2012; Pasquato and Kunz, 2016), the presence of any basic amino acid at the vicinity of the hepatitis C (Blanchet et al., 2015), and Dengue viral cleavage site. infections (Uchida et al., 2016). Mbtps1 inactivation in The most amazing and key feature that distinguishes osteoblasts further showed that SKI-1/S1P is critical for PCSK9 from the other PCs is its inability to get rid of its extracellular matrix signaling and axial elongation prodomain, leading to a secreted inactive protease. In- during somitogenesis and vertebral development, and deed, after autocatalytic cleavage of the prodomain, possibly linked to the caudal regression syndrome which acts as an intramolecular chaperone and potent (Achilleos et al., 2015). In addition, Mbtps1 inactivation but transient inhibitor in the other PCs (Seidah and in osteocytes stimulates soleus muscle regeneration and Prat, 2012), in mature PCSK9 it remains noncovalently increased muscle size and contractile force with age attached to its catalytic pocket (Seidah et al., 2003), (Gorski et al., 2016). Understanding bone–muscle cross- thereby preventing any in trans catalytic activity of the talk may provide a novel approach to prevention and enzyme (Cunningham et al., 2007). Thus, PCSK9 acts as treatment of age-related muscle loss. a protease only once in its lifetime, whereas the other PCs Although the first seven PCs cleave their substrates cleave and activate/inactivate secretory precursor pro- after basic residues often organized in pairs within the teins either in the cis/medial (SKI-1/S1P) or trans Golgi motif K/R-2Xn-K/R↓ [where Xn = 0, 1, 2, or 3 amino acids network, cell surface or endosomes (furin, PC4, PC5/6, (aa)], SKI-1/S1P recognizes the motif R-X-(aliphatic PACE4 and PC7), or immature secretory granules (PC1/3 residue)-X↓. In contrast, the last convertase, PCSK9 and PC2) (Seidah and Prat, 2007, 2012; Creemers and (Seidah et al., 2003), which is abundantly synthesized Khatib, 2008; Seidah et al., 2008, 2013; Turpeinen et al., by the liver and released into the circulation, is only 2013). In contrast, PCSK9 binds various receptors and found in some vertebrates. Interestingly, because the escorts them to endosomes/lysosomes for degradation bovine PCSK9 genome exhibits a premature stop (Seidah and Prat, 2012; Seidah et al., 2014). Interest- codon in exon 10 (Ding et al., 2007; Cameron et al., ingly, from 1990 to the present, a literature statistical 2008), this revealed that fetal bovine serum, widely analysis of the interest in PCs, and especially in PCSK9, used in cell biology, does not contain a PCSK9 protein. revealed that, since its discovery in 2003, it has stimu- Like all other PCs, PCSK9 undergoes in the ER an lated wide interest, as exemplified by the .1850 manu- autocatalytic cleavage between its pro and catalytic scripts published on PCSK9, compared with a total domains at the site VFAQ152↓ (Naureckiene et al., of .4100 manuscripts reported on all PCs (Fig. 2). 2003; Benjannet et al., 2004), where Gln152 is critical In this review, we will focus on the newly described (Benjannet et al., 2012). Note that, different from the aspects of the biology of PCSK9, its mechanism of eight other members of the family, it does not require action, as well as its validated and possibly new targets. 36 Seidah et al.

Fig. 2. Research interest of the nine PCs. Literature search revealed .1850 manuscripts published on PCSK9, compared with a total of .4100 manuscripts on all PCs. The discovery of PCSK9 in 2003, and especially the benefit of its inhibition in hypercholesterolemia, boosted the number of PC publications. The relative research interest was calculated as follows: weighted publications per year/total weighted publications per year (in PubMed), where weighted publications per year = number of publications per year multiplied by relevance factors (as defined by PubMed): https://www. researchgate.net/figure/255716646_fig1_Figure-2-Number-of-abstracts-and-relative-research-interest-from-the-Republic-of.

We will also discuss the consequences of PCSK9 natural with only ;13% carrying a Gly670 in the Chinese Han mutations on the metabolism of LDL cholesterol (LDL-c), population (He et al., 2016). We have consistently found a as well as the clinical applications of its inhibitors in Glu670 in all of our DNA sequences of PCSK9 to date. The pathologies. Finally, we will summarize the implication common His449 in modern humans and neandertal is of other PCs in cardiovascular diseases (CVD) and how replaced by Leu449 in the denisova individual (H449L). some of them can regulate PCSK9. The functional consequence of this replacement in the hinge domain (aa 422–452) (Cunningham et al., 2007; Saavedra et al., 2012) is the loss of a positive charge at the II. Phylogenetic and Haplotype Analyses of acidic pHs of endosomes/lysosomes and begs for more PCSK9 in Human Species detailed cellular analyses. In that context, a similar loss A phylogenetic tree based on the amino acid sequences of positive charge, R434W, in the same domain resulted in of the PCs revealed that SKI-1/S1P is the most ancient a loss-of-function (LOF) associated with lower levels of member of the family (Seidah et al., 1999) and is found circulating PCSK9 and LDL-c (Dubuc et al., 2010). in parasites and the plant kingdom (Barale et al., 1999). Given the possible LOF phenotype of the denisovan Although the closest members form couples, that is, PCSK9 protein (H449L variation), we investigated PC1/PC2; furin/PC4, PC5/PACE4; and SKI-1/PCSK9, whether its genomic haplotype could have been adaptively the highly conserved PC7 (Seidah et al., 1996) is difficult introgressed into modern humans. Recent studies have to place on the phylogenetic tree (Fig. 3A). demonstrated that several important genes show signs of Alignment of PCSK9 protein sequences of modern and adaptive introgression from neandertal and/or denisovans archaic human species revealed only three variations to modern humans, such as the EPAS1 gene important for between the modern human reference sequence and either high altitude adaptation in Tibetans (introgressed from the denisovan individual, H449L,V474I, and G670E, or the denisovans to modern humans) (Huerta-Sánchez et al., Altai neandertal individual, A53V, V474I, and G670E. 2014) or human Toll-like receptors important for our innate Interestingly, the benign polymorphic variants A53V and immunity (introgressed from both neandertals and deni- V474I were also detected in modern humans before. Also, sovans into modern humans) (Dannemann et al., 2016). G670E is a major common variant in modern humans, However, PCSK9 does not show a pattern consistent with Roles of PCSKs in Cholesterol and Triglyceride Metabolism 37

Fig. 3. Phylogenetic reconstruction of the nine PCs and haplotype structure of PCSK9 in modern and extinct human species. (A) Phylogenetic tree displaying the relationship among the nine PCs. The phylogeny was inferred using maximum likelihood (indicated numbers) with 100 bootstrap replicates. (B) Output from haplostrips software (Racimo et al., 2016) showing haplotype clustering of PCSK9 in modern and archaic humans. Haplotypes were first clustered and then ordered by decreasing similarity to the denisova individual (at the top). The x-axis shows the haplotype sequence (sites). Black rectangles represent sites with derived mutations, and white rectangles indicate sites with the ancestral nucleotide. The colors on the y-axis show which population the respective haplotype originates from among the 683 individuals in total. adaptive introgression for the denisovan haplotype (H449L as baits led us to report in February 2003 (Seidah et al., variation, Fig. 3B). In case of adaptive introgression, we 2003) the cDNA cloning and protein analysis of the ninth would have expected this haplotype to be present in high and last member of the PC-family, a putative convertase, (or higher) frequency within certain populations in our originally called neural apoptosis-regulated convertase- data, as well as genetic distance to the archaic haplotype to 1. Millennium Pharmaceuticals had released part of its increase with a sharp incline. cDNA in a patent database as belonging to a group of In conclusion, only minor changes in PCSK9 primary genes upregulated upon induction of apoptosis in pri- protein sequence occurred within the years that sepa- mary cerebellar neurons by serum withdrawal. Neural rate modern humans from either denisovans (estimated apoptosis-regulated convertase-1, now known as PCSK9, separation ;380,000–473,000 years ago) (Prüfer et al., belongs to the proteinase K family of subtilases, and its 2014) or neandertals (estimated separation ;550,000– catalytic domain exhibits ;25% of sequence identity with 765,000 years ago) (Prüfer et al., 2014). However, we that of its closest family member SKI-1/S1P (Seidah et al., found one interesting amino acid change (H449L) in 2003) (Fig. 3A). Human PCSK9 mRNA (NM_174936.3) denisovans that may result in a LOF of PCSK9. In spans 3710 bp over 12 exons encoding a 692 amino-acid contrast to the conserved protein sequence of PCSK9 protein (NP_777596.2). between hominids, their genomic sequences are quite Chromosomal mapping of the PCSK9 gene revealed it variable in individuals outside of East Asia. Further- to be located on chromosome 1p32 (Seidah et al., 2003), more, it does not show any pattern consistent with close to a predicted third major gene locus [familial adaptive introgression. More in-depth analyses (includ- hypercholesterolemia (FH)3] for autosomal dominant ing all nine PCs and more extensive sampling) will be familial hypercholesterolemia (ADH) located at 1p34.1- needed to get a more detailed picture of the evolutionary p32 in a large French family in whom the two genes history of PCSK9 and the other eight PCs. knowntobeimplicatedinthisdisease,theLDLR and APOB genes, had been excluded (Varret et al., 1999). This locus was previously associated with an increase in the III. The Enzymatically Inactive Mature PCSK9 hepatic secretion of LDL, but not high-density lipoprotein Regulates Circulating LDL Cholesterol Levels (HDL) cholesterol, or triglycerides (TG) (Varret et al., Screening of nucleotide databases with conserved 1999; Hunt et al., 2000). Armed with this information and sequences encoding the active site residues of S1P/SKI-1 the major expression of PCSK9 in liver (Seidah et al., 38 Seidah et al.

2003), we contacted a French team that was actively levels (Kotowski et al., 2006). Finally, four individuals studying this region of chromosome 1. Intensive genetic with complete LOF of PCSK9 have undetectable circu- analyses in 23 French non-LDLR/non-APOB families had lating PCSK9 and LDL-c values of ;0.4 mM (;15 mg/dL) allowed the identification of a new multiplex family that (Zhao et al., 2006; Hooper et al., 2007; Cariou et al., 2009; was crucial in refining the boundaries of the genetic Mayne et al., 2011). region on chromosome 1. This positional cloning approach The fact that complete LOF of human PCSK9 resulted using genetic mapping, physical mapping, and sequenc- in an unprecedented large decrease in LDL-c levels ing of several genes led to the identification of a common without seemingly adverse effects encouraged pharma- mutation, S127R, in the 22-kb PCSK9 gene in two large ceutical companies to develop the first inhibitor approach families and another mutation, F216L, in a third family. using PCSK9 monoclonal antibodies (mAbs) to reduce The very fruitful collaboration between the Canadian the levels of circulating PCSK9. As will be discussed team working on PCs and the French one working on the later, these mAbs are now s.c. administered to FH patients genetics of hypercholesterolemia culminated in the first that are either statin-resistant or statin-intolerant to report that appeared in June 2003, revealing that human reduce their LDL-c levels (Stein and Raal, 2014; Seidah, PCSK9 was the FH3 gene critical for LDL-c regulation 2015). Before this drug was approved by the health (Abifadel et al., 2003). care authorities worldwide, a large number of phase 1–3 This discovery was soon followed by the identification clinical trials was performed for safety, tolerability, and of the devastating PCSK9 D374Y mutation in a large efficacy. Utah pedigree (Timms et al., 2004) and 1 month later in three Norwegian subjects (Leren, 2004). Other ADH- Role of PCSK9 in Lipoprotein (a) Regulation causing PCSK9 mutations were discovered in the follow- Unexpectedly, not only does reduction of plasma PCSK9 ing years (Abifadel et al., 2009, 2014), and, more recently, levels result in ;60% lowering of LDL-c, it also reduced 16 different PCSK9 mutations were found in patients by ;30–35% the levels of the highly atherogenic lipopro- from eight countries (Hopkins et al., 2015). tein (a) [Lp(a)] particles (Stein, 2013; Seidah et al., 2014; It was evident from the analysis of the first identified Stein and Raal, 2014). This result opened a new potential mutants S127R and F216L that PCSK9 plays a major therapeutic application because Lp(a) has long been role in the regulation of LDL-c, but not TG (Abifadel known to be an independent risk factor for atherosclero- et al., 2003). This was confirmed by the seminal exper- sis for which no known therapeutic lowering agent had iments in mice done by Maxwell and Breslow (2004), been identified. The reduction in Lp(a) levels was re- who demonstrated that PCSK9 promotes the degrada- cently rationalized by the fact that, under supraphysio- tion of the LDLR. Adenoviral-mediated expression of logic levels of the LDLR resulting from PCSK9 mAb PCSK9 led to high plasma LDL-c levels in a LDLR- treatments, the LDLR becomes a functional receptor dependent manner and resulted in the quasi-absence of of Lp(a) both in cells (Romagnuolo et al., 2015) and in the LDLR in the liver. As the mouse gene Pcsk9 was humans (Raal et al., 2016). It was also shown that strongly downregulated by dietary cholesterol (Maxwell PCSK9 binds the apoB component of Lp(a), but not to et al., 2003), they hypothesized that PCSK9 was apo(a) (Romagnuolo et al., 2015). Interestingly, a recent coregulated with the LDLR to rapidly modulate lipo- report suggests that PCSK9 is associated with Lp(a) in protein clearance. Because the two first loci related to the plasma of patients with very high circulating Lp(a) FH were that of the LDLR and its ligand apolipoprotein concentrations, and that the Lp(a)-associated PCSK9 B (apoB), it was very gratifying that a strong func- levels seem to be directly correlated with plasma Lp(a) tional link could be established between the LDLR and levels, but not with total plasma PCSK9 levels (Tavori PCSK9. Thus, gain-of-function (GOF) mutations result- et al., 2016), suggesting that polymorphic variations in ing in higher activity or levels of PCSK9 were expected Lp(a) and its levels may affect its affinity to PCSK9. to lower liver LDLR levels and hence raise circulating Whether the reported association of Lp(a) with PCSK9 in LDL-c, due its reduced clearance by the remaining plasma samples is direct or requires a bridging protein is LDLR. The demonstration that, conversely, LOF muta- not clear, because we have shown in vitro that PCSK9 tions lead to higher LDLR concentrations and hence does not directly bind Lp(a), whereas it binds LDL with a reduced LDL-c levels was first achieved by Cohen et al. KD of ;130 nM (Romagnuolo et al., 2015). (2005) 2 years later. By screening 32 subjects from the Dallas Heart study that had the lowest LDL-c levels, IV. The Absence of PCSK9 Leads to a Sex- and they discovered two heterozygote nonsense mutations Tissue-Specific Subcellular Distribution of present in ;2% of the African American population, the LDLR Y142X and C679X. The latter are responsible for a ;40% drop in circulating LDL-c and associated with a In mice, PCSK9 is highly expressed in liver, pancre- ;88% reduction in the risk of CVD (Cohen et al., 2006). atic islets, intestine, and kidney (Seidah et al., 2003; Polymorphisms, such as R46L, resulting in a partial Zaid et al., 2008; Langhi et al., 2009). PCSK9 knockout PCSK9 LOF, were also associated with lower LDL-c (KO) mice, as well as mice lacking specifically PCSK9 in Roles of PCSKs in Cholesterol and Triglyceride Metabolism 39 the liver [hepatocyte-specific KO (HepKO)], were gen- hepatocytes was evidenced by Western blot analysis of erated (Rashid et al., 2005; Zaid et al., 2008; Parker plasma membrane preparations (Roubtsova et al., et al., 2013). In these mice, the lack of PCSK9 leads to a 2015). This sex-specific distribution is dictated by two- to threefold increase of the LDLR content in the estrogens, as illustrated by the accumulation of the liver, pancreas, or intestine, as estimated by Western LDLR at the hepatocyte surface in ovariectomized blotting, with no change in LDLR mRNA levels (Rashid female mice supplemented with placebo, whereas those et al., 2005; Zaid et al., 2008; Roubtsova et al., 2015). supplemented with 17b-estradiol exhibited low surface GOF mutations in human PCSK9 or PCSK9 over- levels of the LDLR (Roubtsova et al., 2015). expression in mice stimulate apoB-containing lipopro- The physiologic relevance of the above regulation by tein secretion from the liver (Ouguerram et al., 2004; 17b-estradiol of the LDLR subcellular distribution is of Park et al., 2004; Sun et al., 2005; Lambert et al., 2006). the highest interest if it has a counterpart in humans. In contrast, PCSK9 deficiency in mice leads to lower The sex-dependent efficacy of the PCSK9 mAbs has not levels of apoB-containing lipoproteins with two- and yet been extensively examined. The long-term study fivefold less total cholesterol and LDL-c, respectively, LTS11717 conducted on 1438 men and 872 women (pre- and to a fivefold higher rate of 125I-LDL clearance dominantly postmenopausal) resulted in 65.5% and 53.4% (Rashid et al., 2005; Zaid et al., 2008). Unexpectedly, drops in LDL-c in men and women, respectively, thus although the liver expresses ;80% of the whole-body showing a 22% higher efficacy in men (see Supplemental LDLR, the absence of PCSK9 does not promote choles- Fig. S4 in Robinson et al., 2015). However, a subgroup terol accumulation or higher bile acid production in this analysis revealed that postmenopausal women responded tissue (Rashid et al., 2005; Zaid et al., 2008; Parker with a 16% higher efficacy than premenopausal women et al., 2013). Rather, PCSK9 KO, but not wild-type mice (54.8% versus 47.3%). Thus, men (65.5% drop) responded that underwent partial hepatectomy had higher levels with 38.5% and 19.5% higher efficacies than premeno- of SREBP-2 and 3-hydroxy-3-methylglutaryl–CoA re- pausal (47.3% drop) and postmenopausal (54.8% drop) ductase mRNA levels in their regenerating liver, sug- women, respectively (http://www.fda.gov/downloads/ gesting the sensing of a lack of intracellular cholesterol. AdvisoryCommittees/CommitteesMeetingMaterials/Drugs/ How elevated levels of LDLR do not lead to cholesterol EndocrinologicandMetabolicDrugsAdvisoryCommittee/ accumulation in the liver or pancreatic islets (Langhi UCM449865.pdf). Clearance experiments as well as et al., 2009) remains an enigma. A possibility is that extensive analyses of phase 3 clinical trials will be more substantial amounts of cholesterol are excreted needed to confirm the existence in women of a lower via transintestinal cholesterol excretion (Le May et al., response to PCSK9 inhibition. 2013). Whether transintestinal cholesterol excretion increases in individuals lacking PCSK9 or treated with V. Role of PCSK9 in Inflammation, Sepsis, and mAbs remains to be verified. Viral Infections PCSK9 is undetectable in the plasma of HepKO mice, demonstrating that the circulating protein originates Inflammation and infection induce marked changes exclusively from hepatocytes (Zaid et al., 2008). Circu- in lipid and lipoprotein metabolism, and administration lating PCSK9 can also regulate peripheral receptors. of bacteria endotoxins induces hyperlipidemia in ani- For example, in HepKO or complete KO mice, very mals (Khovidhunkit et al., 2004). Lipopolysaccharide LDLR (VLDLR) immunolabeling increases at the cell (LPS) administration, a model for Gram-negative bac- surface of visceral adipocytes that do not express terial infection, increases LDL and VLDL levels, most PCSK9 locally. Intriguingly, this increase was ;10-fold likely via decreased LDLR and VLDLR protein levels in higher in female than in male KO mice (Roubtsova the liver (Liao et al., 1996), possibly caused by LPS- et al., 2011), providing the first evidence for a sex- stimulated expression of PCSK9 in liver (Feingold et al., dependent cell surface accumulation of a PCSK9- 2008). Sepsis is a complex disease characterized by targeted receptor in PCSK9 KO mice. In liver, pancreas, systemic activation of inflammation and coagulation, and small intestine of KO male and female mice, the most commonly in response to bacterial infection. Severe absence of PCSK9-triggered LDLR degradation led to sepsis, accompanied by dysfunction of at least one organ, similar two- to threefold increases in the total protein affects 0.3–1% of individuals/yr in the United States with levels of LDLR. However, the subcellular distribution of mortality rates of ;30% (Gaieski et al., 2013). Therapies the receptor differed in a sex- and tissue-dependent include antibiotic treatments and hemoperfusion filtra- manner. LDLR cell surface immunolabeling was dra- tion that specifically remove LPS from circulation (Cruz matically increased in the liver and pancreatic islets of et al., 2009). KO males, but not females. Conversely, LDLR cell Mice lacking LDLR exhibit increased mortality in a surface labeling was higher in the small intestine of cecal ligation and puncture (CLP) model (Lanza-Jacoby females, similar to the VLDLR in perigonadal fat depots et al., 2003), suggesting that LDLR plays a notable role (Roubtsova et al., 2015). Moreover, the accumulation of in sepsis. In that context, Walley et al. (2014) recently the LDLR at the cell surface of male but not female showed that exogenous human PCSK9 reduces LPS 40 Seidah et al. uptake in vitro by HepG2 cells, and that inhibitory exacerbates the phenotype (Awan et al., 2011). This is PCSK9 antibodies reduced plasma endotoxin levels in in part due to an inflammatory response to the forma- mice subjected to CLP, blunted the cytokine response, tion of cholesterol crystals in hypercholesterolemic and doubled survival rates. In humans, PCSK9 LOF conditions, which are reversed by the administration (R46L) polymorphisms were associated with better of anti-inflammatory mAbs to interleukin-1b (Awan survival of septic patients and reduced cytokine levels, et al., 2016). whereas GOF (E670G) polymorphisms had the oppo- A possible drawback of PCSK9 inhibition is to trigger site effect. Furthermore, i.v. injection of endotoxin to higher levels of hepatitis C virus receptors, that is, LDLR, healthy volunteers generated lower cytokine levels in VLDLR, and possibly CD81, and to promote viral infec- R46L individuals (Walley et al., 2014). Finally, it was tivity (Labonté et al., 2009; Le et al., 2015), especially for recently shown that LPS binds plasma lipids to be genotype 3 hepatitis C virus (Bridge et al., 2015). In internalized in human HepG2 cells and mouse primary contrast, patients affected by the human immunodefi- hepatocytes by the LDLR, but also other receptors, ciency virus–associated neurocognitive disorder, resulting including the VLDLR (Topchiy et al., 2016). It is from enhanced neuroinflammation and neurodegenera- important to note that, in septic patients, the median tion (Kim et al., 2015), may benefit from PCSK9 inhibition levels of PCSK9 were positively correlated with the that would reduce inflammation. extent of the respiratory and cardiovascular failure (Boyd et al., 2016), leading to the hypothesis that VI. Other Physiologic Roles of PCSK9 reducing PCSK9 levels, and hence increasing LDLR- mediated hepatic clearance of pathogen lipids, may be We investigated by in situ hybridization the expres- protective (Walley et al., 2014). Indeed, lower circulat- sion of mouse PCSK9 at embryonic day 14.5 (Fig. 4). ing levels of endotoxins may reduce their binding to High signals were observed in the hepatic primordium, Toll-like receptors that leads to nuclear factor kB embryonic membranes, midgut and umbilical artery, activation and subsequent proinflammatory cytokine and lower signals in the telencephalon (Fig. 4B). At this secretion by leukocytes (Savva and Roger, 2013). In developmental stage, the mouse placenta did not show agreement, we have recently shown that, in a mouse PCSK9 expression. In embryonic membranes (Fig. 4, C– CLP model of sepsis, PCSK9 deficiency confers pro- F), PCSK9 mRNA was present within the smooth tection against systemic bacterial dissemination, organ muscle cell layer, but not in endothelial or embryonic pathology, and tissue inflammation, particularly in the blood cells. The role of PCSK9 in these tissues is not lungs and liver, whereas PCSK9 overexpression exac- known and maybe vary during development. Interest- erbates early hypercoagulable and proinflammatory ingly, PCSK9 mRNA and protein are expressed in states and multiorgan pathology (Dwivedi et al., human placenta at full-term in patients with gesta- 2016). Future studies, including clinical trials, are tional diabetes mellitus, a common complication of needed to assess the promising therapeutic efficacy of pregnancy aggravated by obesity (Dubé et al., 2013). inhibitory PCSK9 mAbs in sepsis (dos Santos and In situ hybridization analyses at embryonic day 17 (Fig. Marshall, 2014). 5, upper panel) and postpartum day 10 (Fig. 5, lower Interestingly, berberine inhibits LPS-induced in- panels) in mouse revealed that PCSK9 is mostly creases in LDL-c and TG in mice (Xiao et al., 2012), expressed in liver, small intestine, colon, cerebellum, most likely via the downregulation of the HNF1a pro- and kidney. At postpartum day 10, PCSK9 and LDLR tein (Dong et al., 2015). The latter is known to strongly colocalize especially in the liver and intestine, but also stimulate PCSK9 transcription, but not that of the in thymus (Seidah et al., 2003; Zaid et al., 2008). The LDLR (Li et al., 2009; Dong et al., 2010), whereas function of PCSK9 in extrahepatic tissues is still poorly SREBP-2 upregulates the expression of both (Dubuc understood (Seidah, 2016). et al., 2004). Importantly, statins upregulate more Multiple studies revealed that PCSK9 can escort not efficiently PCSK9 mRNA levels than those of the LDLR only the LDLR to lysosomal degradation compartments, (Dubuc et al., 2004), rationalizing the poor incremental but also its family members VLDLR, apolipoprotein E LDL-c decreases with increasing statin doses. Mediter- receptor 2 (ApoER2), and LDLR-related protein-1 ranean diet (Richard et al., 2012) and exercise (Kamani (LRP1) (Poirier et al., 2008; Roubtsova et al., 2011; et al., 2015) reduce circulating levels of PCSK9, whereas Canuel et al., 2013), best seen in livers lacking LDLR high-fat or fructose-rich diets (Cariou et al., 2013) and PCSK9 (unpublished data). This suggests that, in increase the levels of PCSK9 (for a general review see FH patients, especially those with very low levels of Cui et al., 2015). functional LDLR, PCSK9 may also regulate LRP1 In another manifestation of inflammation in relation levels. In addition, the fatty acid transporter CD36 to PCSK9 regulation of the LDLR, we reproduced in seems to be another target of PCSK9 (Demers et al., LDLR KO mice the aorta and vascular calcification 2015), although the exact binding site has not yet observed in FH patients and reported that PCSK9 KO been determined. This is in line with the observed mice are protected, whereas PCSK9 overexpression hypertrophy of visceral adipocytes in PCSK9 KO mice Roles of PCSKs in Cholesterol and Triglyceride Metabolism 41

Fig. 4. PCSK9 mRNA is highly expressed in the liver, midgut, embryonic membranes, and umbilical artery at embryonic day 14.5. The same sagittal section through mouse embryo and attached placenta is shown in (A–D) and (F). (A) Staining with cresyl violet. (B) Autoradiography after in situ hybridization with a PCSK9 antisense cRNA probe. (C) Light-field illumination at medium magnification. The arrow points to the umbilical artery. (D) The same field as in (C) under dark-field illumination after incubation in autoradiography emulsion. (E) Adjacent section at higher magnification after in situ hybridization with a control sense PCSK9 cRNA. (F) Higher magnification of the umbilical artery shown in (D). EBC, embryonic blood cells; EM, embryonic membranes; Emb., embryo; End, endothelium; Li, liver primordium; MG, midgut; Plac., placenta; SMC, smooth muscle cells; UA, umbilical artery.

(Roubtsova et al., 2011) that indicates higher binding of VII. Cellular Studies: Mechanism of Action TG-rich lipoproteins and fatty acid uptake via increased of PCSK9 VLDLR and CD36. Elucidation of the role(s) of PCSK9 Surprisingly, 13 years after the discovery of PCSK9, as an intracellular and/or paracrine regulator of LDLR no sorting molecular mechanism(s) can yet explain and/or VLDLR protein levels in gut, kidney, and its ability to induce the degradation of the LDLR pancreas will require tissue-specific KOs, as was done (Seidah et al., 2014) in late endosomes/lysosomes in hepatocytes (Zaid et al., 2008). Although expressed in (Nassoury et al., 2007) (Fig. 6). Our data revealed that kidney, PCSK9 is not secreted into the urine (Dubuc the C-terminal Cys-His–rich domain (CHRD) of PCSK9, et al., 2004), and it does not seem to play a role in which contains three repeat structures called M1, M2, controlling blood pressure even under induced hyper- and M3 (Cunningham et al., 2007), is needed for the tensive conditions (Berger et al., 2015). However, PCSK9-induced degradation of the PCSK9–LDLR com- patients with chronic kidney disease exhibit higher plex (Nassoury et al., 2007), including the repeat liver expression and circulating levels of PCSK9 domain M2 (Saavedra et al., 2012), which has 14 His (Konarzewski et al., 2014), most likely due to enhanced (Cunningham et al., 2007) and a number of natural expression of HNF1a in hepatocytes (Sucajtys-Szulc mutations associated with it (Seidah et al., 2014). This et al., 2016). Finally, although PCSK9 does not cross the critical importance of the CHRD in regulating the blood brain barrier, little is known about the function of ability of PCSK9 to induce the degradation of the LDLR PCSK9 in the cerebellum, or other neuronal cells was confirmed in two independent studies (Zhang et al., (Seidah et al., 2003). PCSK9 has been shown to reduce 2008; Poirier et al., 2016) and was shown to be inhibited LDLR levels, and possibly those of VLDLR and by mAbs (Ni et al., 2010; Schiele et al., 2014) or single- ApoER2, during brain development and after ischemic domain antibodies that target the CHRD, without stroke in adult mice, although poststroke behavior and inhibiting the PCSK9–LDLR complex formation (Weider lesion volume were not affected by the lack of PCSK9 et al., 2016). (Rousselet et al., 2011). The possible role of PCSK9 in We suggested that a putative protein Xp binds the some neurodegenerative diseases is yet to be conclu- CHRD of extracellular PCSK9 in the PCSK9–LDLR sively shown and validated in humans. complex and escorts it to lysosomes for degradation by 42 Seidah et al.

other Xp candidates proposed in the literature, that is, sortilin (Gustafsen et al., 2014) and amyloid precursor- like protein 2 (DeVay et al., 2013), as neither protein is critical for the in vivo activity of PCSK9 on LDLR in hepatocytes (Butkinaree et al., 2015), and mice lacking both proteins do not exhibit significantly different LDLR levels in the liver (Fig. 7). Thus, the identity and necessity of Xp are yet to be defined and validated in cells and invivo,fortheabilityofPCSK9toenhancethedegrada- tion of the LDLR and possibly other targets. A genetic approach showed that the cytosolic adaptor protein Sec24A implicated in the ER–Golgi trafficking of the coat protein complex COPII vesicles was critical for the secretion of PCSK9 from the ER into the medium (Chen et al., 2013). This facilitation is thought to be mediated by a membrane-bound protein that binds PCSK9 in the ER lumen and Sec24A in the cytosol via its CT. It was also recently reported that the LDLR does not bind efficiently PCSK9 in the ER because of the presence of a competitive chaperone like-protein GRP94 that prevents such ER interaction (Poirier et al., 2015). Finally, Leren (2014) hypothesized that the membrane- bound PCSK9–LDLR complex is first shed into a soluble form upon entry into acidic endosomes via a cathepsin- like cysteine protease inhibited by E64, which may facilitate its efficacious degradation in lysosomes. Although it was reported that in mice the cytosolic Fig. 5. PCSK9 and LDLR mRNA expression at embryonic day 17 (E17) adaptor protein ARH is essential for the ability of and postnatal day 10 (P10). At E17, in situ hybridization of a sagittal section with an antisense cRNA probe reveals the high expression of extracellular PCSK9 to enhance the degradation of PCSK9 in the liver, small intestine, cortex of the kidney, and cerebellum. the LDLR in liver hepatocytes (Lagace et al., 2006), At P10, PCSK9 expression is high in the liver, small intestine, brain olfactory tract, cerebellum, spleen, thymus, colon, caudate putamen, and recent clinical trials using an inhibitory PCSK9 mAb eye retina inner nuclear layer. LDLR expression is also high in the liver, Alirocumab revealed that absence of ARH in some small intestine, and thymus, and is specifically abundant in the autosomal recessive hypercholesterolemic patients, al- trigeminal and cranial ganglia. Cb, cerebellum; CG, cranial ganglia; Co, colon; CPu, caudate putamen; Ki, kidney; Li, liver; Mo, molar; OT, though reducing the activity of PCSK9, still left room for olfactory tract; Sin, small intestine; Sk, skin; Sp, spleen; Re, retina; Tg, an ARH-independent pathway, at least as ascertained trigeminal ganglion; Th, thymus. *, **: nonspecific signal. in lymphocytes (Thedrez et al., 2016). Similar results were also observed in ARH-negative lymphocytes (Fasano unknown resident hydrolases (Canuel et al., 2013; et al., 2009), revealing the presence of an ARH-independent Seidah et al., 2014; Butkinaree et al., 2015). In view of pathway for PCSK9 activity on LDLR. The presumed the importance of the cytosolic adaptor protein, autoso- relatively lower contribution of this pathway in human mal recessive hypercholesterolemia (ARH), which con- liver (Thedrez et al., 2016) is yet to be unambiguously tains a critical NP-X-Y motif for entry into clathrin-coated validated. endosomes (Lagace et al., 2006), it was thought that the cytosolic tail (CT) of the LDLR (containing an NP-X-Y VIII. What Have We Learned from Key Natural motif) controls this ARH-dependent internalization. Mutations on the Functions of PCSK9 and However, we and others realized that PCSK9 can Its Partners? enhance the degradation of LDLR lacking this CT (LDLR-DCT) or replaced with another one lacking an Since the discovery of PCSK9 and its relationship to LDL-c, NP-X-Y motif (Holla et al., 2010; Canuel et al., 2013). .100 PCSK9 SNPs were reported (http://www.ncbi.nlm. Thus, we proposed that the putative Xp may be a nih.gov/projects/SNP/snp_ref.cgi?geneId=255738) with var- membrane-bound protein with an NP-X-Y motif in its ied consequences (http://www.ucl.ac.uk/ldlr/LOVDv.1.1.0/ CT. Proteomic analyses of livers of mice lacking or not search.php?select_db=PCSK9&srch=all). Many of these PCSK9 suggested that the LDLR family member LRP1 is are silent, others are suspected to be damaging, and some also upregulated in the absence of PCSK9 in liver (Canuel have been validated for their functional effects. The et al., 2013). However, the implication of the PCSK9 structure of the 692-aa human PCSK9 protein (Fig. 6A) target LRP1 as a putative Xp was soon eliminated contains a prodomain (aa 31–152) that is noncovalently (Canuel et al., 2013). We also recently discarded two associated with the mature protease chain (aa 153–692), Roles of PCSKs in Cholesterol and Triglyceride Metabolism 43

Fig. 6. A. Schematic representation of proPCSK9 (75 kDa, 692 aa) and its processed form PCSK9 (62 kDa) that is noncovalently bound to the autocatalytically processed prodomain (pro; 15 kDa) at VFAQ152-SIP. Notice the active site residues Asp, His, and Ser and the oxyanion hole Asn, as well as the single N-glycosylation site (in blue). B. Schematic of the extracellular pathway of PCSK9-induced degradation of the LDLR. High PCSK9 levels (left part of the graph) or GOF forms enhance the degradation of the LDLR following the endocytosis of the PCSK9–LDLR complex in clathrin- coated vesicles, together with the LDL, leading to the degradation of the PCSK9–LDLR complex in lysosomes. This results in low levels of the LDLR at the cell surface and increased levels of circulating LDL-c. Low levels of PCSK9 or LOF forms lead to increased levels of cell surface LDLR because the latter can be recycled back to the surface after delivery of LDL particles to acidic endosomes/lysosomes. PCSK9 mAbs target the extracellular pathway by sequestering extracellular PCSK9 and hence preventing its binding to the LDLR. keeping it in an enzymatically inactive state. The catalytic 6A), composed of three repeats called M1, M2, and M3 subunit (aa 153–404) is followed by a short hinge domain (Cunningham et al., 2007). Mutations can be found in all (Hi; aa 405–452) and the C-terminal CHRD (aa 453–692) four structural components of PCSK9. The catalytic subunit (Seidah et al., 2003, 2014; Seidah and Prat, 2012) (Fig. of PCSK9 directly binds the epidermal growth factor-like

Fig. 7. The lack of amyloid precursor-like protein 2 and/or sortilin has no effect on total cholesterol, PCSK9, and LDLR levels. Mice deficient for amyloid precursor-like protein 2 (Aplp22/2) and/or sortilin (Sort1+/+ or Sort12/2) were analyzed for their level of circulating cholesterol and PCSK9 and those of LDLR protein in liver extracts. No changes were observed. Note that, individually, amyloid precursor-like protein 2 or sortilin deficiency does not affect these parameters (Butkinaree et al., 2015). 44 Seidah et al. domain-A domain of the LDLR with nM potency (Zhang Mutations within the hinge domain and the CHRD et al., 2007) and is critical for the ability of PCSK9 to domain also resulted in either GOF or LOF of PCSK9 enhance the degradation of the LDLR (Zhang et al., 2008; (Tables 1 and 2) (Abifadel et al., 2009). The function of Saavedra et al., 2012). these domains relates to their regulation of the ability of Interestingly, the prodomain of PCSK9 also makes the PCSK9–LDLR complex to be directed to late contact with the b-barrel domain of the LDLR (Lo Surdo endosomes/lysosomes for degradation (Nassoury et al., et al., 2011), as also evidenced by specific mutations 2007; Zhang et al., 2008; Poirier et al., 2016) (Fig. 6B, therein, for example, L108R, that enhance the func- left part). However, the underlying mechanism and the tional activity of PCSK9 in enhancing the degradation importance of post-translational modifications, for ex- of the LDLR in endosomes/lysosomes (Abifadel et al., ample, Asn533 glycosylation (Seidah et al., 2003) or Ser688 2012). However, the various crystal structures of phosphorylation (Gauthier et al., 2015; Tagliabracci PCSK9 reported at both neural and acidic pHs with or et al., 2015) in this process are still unknown. In vitro without the ectodomain of the LDLR could not detect a biochemical experiments suggested that the CHRD may specific structure for N-terminal aa 31–59 of the interact with the ligand binding domain of the LDLR at prodomain (Cunningham et al., 2007; Lo Surdo et al., acidic pHs (Yamamoto et al., 2011), but this is not evident 2011). This suggests that alone this 29-aa sequence of from the available PCSK9–LDLR crystal structures (Lo the prodomain may be quite mobile, most likely predict- Surdo et al., 2011). We suggested that the CHRD may ing that a partner may be needed to stabilize in space bind an as yet unknown protein X that would then direct this highly acidic segment, or that it may bind, albeit the PCSK9–LDLR complex toward late endosomes/ weakly, to another segment of PCSK9. It has been lysosomes (Seidah et al., 2014; Butkinaree et al., 2015). suggested that the prodomain may interact with Alternatively, the CHRD may displace a protein required apoB100 on LDL (Kosenko et al., 2013), but not VLDL for the default recycling of the LDLR to the cell surface (Lagace, 2014) particles, although the importance of the found in most tissues (Fig. 6B, right part), thereby aa 31–59 segment of the prodomain in this interaction allowing the PCSK9–LDLR complex to reach the degra- has yet to be defined. Interestingly, deletion of the acidic dation compartments. It is thus conceivable that, in the aa 31–51 sequence of the prodomain enhances the future, specific PCSK9 and/or LDLR mutations may be affinity of PCSK9-D31–51 for the LDLR by .sevenfold identified that would disrupt or enhance the interaction (Kwon et al., 2008), suggesting that, if apoB binds this with such putative regulator and hence favor the recy- region, it results in a dampening of PCSK9-binding cling or degradation pathways of the LDLR. affinity for the LDLR. The physiologic importance of the 122–aa prodomain (aa 31–152) is exemplified by muta- IX. Ongoing Outcome Clinical Trials tions that result in LOF (e.g., R46L, D97, G106R, Q152H) or GOF (e.g., E32K, D35Y, L108R, S127R) of PCSK9. Several pharmaceutical strategies were and are being Japanese patients that are E32K homozygotes have very developed to reduce PCSK9 levels/activity on LDLR high LDL-c, but still lower than FH patients that degradation and were reviewed (Seidah et al., 2014; completely lack functional LDLR (Mabuchi et al., 2014). Elbitar et al., 2016; Stein, 2016). In essence, an Although many natural mutations within the encapsulated in nanoparticles antisense RNA interfer- PCSK9’s catalytic domain (aa 153–452) result in either ence oligonucleotide when i.v. injected once at 0.4 mg/kg GOF (Table 1) or LOF (Table 2) (Abifadel et al., 2009), resulted in ;70% and ;40% drops in PCSK9 and LDL-c one of the most damaging GOF mutations is the Anglo- levels, respectively (Fitzgerald et al., 2014). It is now Saxon D374Y mutation (Timms et al., 2004), which being tested in phase 2 clinical trials. However, the enhances the binding affinity of PCSK9 to the LDLR present, most prevalent approach consists in blocking by .10-fold (Cunningham et al., 2007). A corresponding mAbs that inhibit the binding of extracellular or mutation H306Y in LDLR has a similar enhancing circulating PCSK9 to the LDLR (Stein and Raal, effect on the PCSK9–LDLR complex formation (McNutt 2014). Three human mAbs are now in multiple phase et al., 2009). Interestingly, the GOF F216L and R218S 3 clinical trials. Two of them, Evolocumab (Repatha; mutations reduce or eliminate the ability of furin to Amgen; Thousand Oaks, CA) and Alirocumab (Praluent; cleave and inactivate PCSK9 (Benjannet et al., 2006). Sanofi; Bridgewater, NJ), are already prescribed to That furin processing of PCSK9 at Arg218↓ in vivo FH patients worldwide, whereas the Bococizumab (Essalmani et al., 2011) does inactivate PCSK9 was (Pfizer; Groton, CT) is not yet available commercially recently confirmed by functional analysis of human (Ito and Santos, 2016). However, very recently Pfizer plasma PCSK9 (Han et al., 2014). Furthermore, such discontinued the global development of Bococizumab, processing was shown to extend the half-life of a distinct as the efficacy of this noncompletely humanized mAb anti-PCSK9 mAb that recognizes the N-terminal se- decreased with time (http://www.pfizer.com/news/press- quence aa 168–181, which, different from Evolocumab release/press-release-detail/pfizer_discontinues_global_ or Alirocumab, does not sterically impede the inactivat- development_of_bococizumab_its_investigational_pcsk9_ ing furin cleavage at Arg218↓ (Schroeder et al., 2015). inhibitor). In all cases, the s.c. injection of either 150 mg Roles of PCSKs in Cholesterol and Triglyceride Metabolism 45

TABLE 1 PCSK9 gene GOF mutations causing autosomal dominant hypercholesterolemia

Nucleotide Geographical Protein Position Position Exon Origin of Patients Clinical Features References p.Val4Ile c.10G . A 1 Japan CAD, PVD, arcus, xanthomas Hopkins et al., 2015 p.Glu32Lys c.94G . A 1 Japan CAD, stroke, arcus, xanthoma Miyake et al., 2008 pAsp35Tyr 103G . T 1 France Mild dyslipidemia Abifadel et al., 2012 p.Glu48Lys 142G . A 1 Netherlands Mild dyslipidemia Hopkins et al., 2015 p.Pro71Leu 212C . T 2 Netherlands CAD, stroke Hopkins et al., 2015 p.Arg96Cys 286C . T 2 Netherlands CAD Hopkins et al., 2015 p.Leu108Arg 323T . G 2 France: Republic of CAD Abifadel et al., 2012 Mauritius p.Ser127Arg c.381T . A 2 2 French families Tendon xanthomas Abifadel et al., 2003; Cameron 1 South African patient CAD, early MI et al., 2006; Nassoury et al., 2007 1 Norwegian patient Stroke, arcus p.Asp129Gly c.386A . G 2 1 New Zealand family MI strong family history of CVD Homer et al., 2008 p.Asp129Asn c.385G . A 2 United Kingdom CAD Hopkins et al., 2015 p.Arg215His c.644G . A 4 2 Norwegian families CAD Cameron et al., 2006 p.Phe216Leu c.646T . C 4 1 French family Early MI Abifadel et al., 2003; Benjannet et al., 2004 p.Arg218Ser c.654A . T 4 1 French family Tendinous xanthomas, arcus cornea Benjannet et al., 2004; Allard et al., 2005 p.Arg357His c.1070G . A 7 1 French proband Family history of CVD Allard et al., 2005 p.Asp374Tyr c.1120G . T 7 1 Utah family Achilles tendon xanthomas, arcus, Benjannet et al., 2004; Leren, 3 Norwegian families Premature CAD 2004; Timms et al., 2004; 3 English families More stringent treatment Naoumova et al., 2005 United Kingdom, United States p.Asp374His c.1120G . C 7 2 Portuguese probands Severe phenotype. Premature Bourbon et al., 2008 + 1 relative CAD, arcus, xanthomas France p.Ser465Leu c.1394 C . T 9 Netherlands CAD Hopkins et al., 2015 p.Arg496Trp c.1486C . T 9 Italy; Netherlands Higher total cholesterol Pisciotta et al., 2006; Hopkins et al., 2015

CAD, coronary artery disease; CVD, cardiovascular disease; MI, myocardial infarction; PVD, peripheral vascular disease. every 2 weeks or 420 mg every 4 weeks results in a significant ;50% reduction in major adverse cardiovas- consistent and sustained ;50–60% reduction in LDL-c. cular events following more than 1 year of treatment. The safety and tolerability profile is very good, especially for hypercholesterolemia patients who fail to obtain an optimal clinical response to statin therapy, those who X. Other Genes and Loci Implicated in are statin-intolerant or have contraindications to LDL-c Regulation statin therapy. The combination of these mAbs with other lipid-lowering agents such as statins or the About 50 years after the pioneering work of nonstatin ezetimibe/fenofibrate resulted in a modest Khachadurian (1964) on the genetics of FH in Lebanese (#10%) further decrease in LDL-c (Ito and Santos, families, and ;40 years after the discovery by Brown 2016; Rey et al., 2016). and Goldstein (1974) of the LDL receptor that paved the Evolocumab is currently being tested in a large-scale way to statin therapy, our discovery of PCSK9 and its FOURIER study (27,500 patients) to evaluate its impact implication in FH opened the way to intensive studies on the occurrence of CVD death, nonfatal myocardial by researchers and pharmaceutical companies, leading infarction, or hospitalization for unstable angina, stroke, to a promising new class of lipid-lowering drugs: the or coronary revascularization (NCT01764633). The PCSK9 mAbs. The race to discover new genes impli- estimated completion date is 2018. The ODYSSEY cated in FH and its cardiovascular complications is still OUTCOMES study (;18,000), which should also be very vigorous. In 2001, the LDLRAP1 gene encoding completed in 2018, is designed to determine the impact ARH was identified in families suffering from autoso- of Alirocumab over a treatment period of 64 months mal recessive hypercholesterolemia (Garcia et al., (NCT01663402). The ongoing SPIRE-1/-2 trials will 2001). PCSK9 was the third gene implicated in ADH analyze the effect of Bococizumab (Ballantyne et al., after the LDLR and APOB genes (Innerarity et al., 2015) by 2017–2018, although the future of this study is 1987). Familial mutations in the APOE gene were now in doubt. Outcome data should provide concrete recently linked to ADH (Awan et al., 2013; Marduel evidence for the long-term clinical benefits of reducing et al., 2013). Other mutations have been reported in PCSK9 activity or its levels. A glimpse of what might be genes encoding proteins implicated in cholesterol me- expected was reported for patients treated with either tabolism or regulation, notably SREBP2 (Muller and Evolocumab (Sabatine et al., 2015) or Alirocumab Miserez, 2002) and CYP7A1, but they are limited to rare (Robinson et al., 2015), showing a very encouraging and cases (Pullinger et al., 2002). 46 Seidah et al.

TABLE 2 PCSK9 gene LOF mutations and polymorphisms causing hypocholesterolemia

Geographical Origin of Protein Position Nucleotide Position Exon Patients Clinical Features References Leucine stretch: c.dup61_63CTG also 1 Frequently found in Decrease of LDL-c levels of Abifadel et al., 2003; p.L21dup also designated several populations 10–15 mg/dL Chen et al., 2005; Yue designated c.43_44insCTG et al., 2006 p.L15_L16insL p.Arg46Leu c.137 G . T 1 Frequently found in Associated with a 15% reduction of Abifadel et al., 2003; Cameron several populations LDL-c and 47% of CHD in et al., 2006; Cohen et al., 2006; whites; reduction of the risk of Kotowski et al., 2006; Scartezini early-onset myocardial et al., 2007; Dewpura et al., infraction 2008; Kathiresan et al., 2008 p.A68fsL82X c.202delG 1 Sicily: 1 kindred Fatty liver in the proband and his Fasano et al., 2007 2 hypocholesterolemic father but not in blood donors blood donors from healthy carriers p.Thr77Ile c.230C . T 2 Sicily Hypocholesterolemic blood donors Fasano et al., 2007 p.Arg104Cys c.310C . T 2 French R104C/V114A segregates with Cariou et al., 2009 FHBL; dominant negative when associated with c.341T . C (p.Val114Ala) on the same allele: impairs processing and secretion of wild-type PCSK9 in liver; low plasma PCSK9 concentration, LDL clearance rate .200% of normal p.Gly106Arg c.316G . A 2 1 Norwegian family Hypocholesterolemic subjects; low Berge et al., 2006; Nassoury autocatalytic cleavage et al., 2007 p.Val114Ala c.341T . C 2 Sicily Hypocholesterolemic blood donors Fasano et al., 2007 p.Tyr142X c.426C . G 3 0.4% DHS1 Reduction of LDL-c levels of 40% Cohen et al., 2006; Kotowski 0.8% ARIC2 of African and CHD of 88% et al., 2006 Americans p.Q152H c.436G . C 3 French-Canadian Reduction of PCSK9 levels by 79% Mayne et al., 2011 and LDL-c by 48% p.Leu253Phe c.757C . T 5 Black American and Hypocholesterolemic Kotowski et al., 2006 Hispanic p.Trp428X c.1284G . A 8 1 Japanese subject Hypocholesterolemic: complete Miyake et al., 2008 LOF due to early termination p.Ala522Thr c.1564G . A 10 Sicily Hypocholesterolemic blood donors Fasano et al., 2007 p.Gln554Glu c.1660C . G 10 Black American, Mild reduction in LDL-c levels Kotowski et al., 2006 Hispanic p.Pro616Leu c.1847C . T 11 Sicily Hypocholesterolemic blood donors Fasano et al., 2007 p.Cys679X c.2037C . A 12 1.4% (DHS); 1.8% Reduction of LDL-c levels of 40% Cohen et al., 2006; Kotowski (ARIC) and CHD of 88% for et al., 2006 Blacks. ,0.1% of White heterozygous carriers Americans

CHD, coronary heart disease; FHBL, familial hypobetalipoproteinemia; LDL-c, low-density lipoprotein cholesterol; LOF, loss-of-function.

Using classic approaches based on positional cloning previously (Taylor et al., 2003; Seidah and Prat, 2007, and linkage analysis, presumptive new loci for ADH were 2012; Seidah et al., 2008, 2013; Turpeinen et al., 2013; mapped to 8q24.22 (Cenarro et al., 2011) and to 16q22.1 Ramos-Molina et al., 2016). In this work, we will only (Marques-Pinheiro et al., 2010). In the next-generation briefly summarize our present understanding of their sequencing era, parametric linkage analysis combined physiologic and pathologic roles in both health and in with exome sequencing in an ADH family resulted in the CVD. identification of the variant p.Glu97Asp in signal- transducing adaptor family member 1 (Fouchier et al., A. PC1/3 and PC2 in Diabetes and Obesity 2014). Other approaches using exome sequencing (Stitziel PC1/3 and PC2 activate by cleavage most of the et al., 2015) or genome-wide association studies (GWAS) polypeptide hormone precursors in endocrine and neu- (Patel et al., 2016), however, did not identify any major ral tissues. The lack of either convertase results in new gene. This may be explained by the difficulty to diseases such as obesity or diabetes (Fig. 1). PC1/3 ; diagnose polygenic cases in ADH, estimated to 13% and PC2 sequentially cleave proinsulin into insulin (Talmud et al., 2013), a value close to the percentage of (Smeekens et al., 1992; Steiner, 2011). The two enzymes no LDLR,noAPOB, and no PCSK9 cases. also differentially cleave pro-opiomelanocortin in a tissue-specific manner to generate adrenocorticotropin hormone in corticotrophs of the pituitary gland or XI. Implication of the Enzymatically Active PCs a-melanotrophic hormone and b-endorphin in the hy- in CVD pothalamus and pituitary melanotrophs, respectively PCSK9 is not the only PC to be implicated in CVD, (Benjannet et al., 1991; Seidah et al., 2013). In fact, and relevant studies on other PCs were reviewed PC1/3 alone or in combination with PC2 can generate Roles of PCSKs in Cholesterol and Triglyceride Metabolism 47 both anorexigenic hormones (a-melanotrophic hor- C. PACE4 in Blood Pressure Regulation mone,insulin,cholecystokinin,andglucagon-like PACE4 is a cell surface enzyme bound to heparin peptide 1) and orexigenic peptides (neuropeptide Y, sulfate proteoglycans that activates a number of recep- cocaine- and amphetamine-regulated transcript, and tors and growth factors (Seidah et al., 2008). In cardio- orexin/hypocretin). It was thus not a surprise that myocytes, PACE4 activates the serine protease corin subjects lacking functional PC1/3 are obese (Jackson that produces atrial natriuretic peptide/atrial natriuretic ; et al., 1997). PCSK1 LOF mutations can be found in 1% factor (Chen et al., 2015). Moreover, the authors identi- of morbidly obese patients (Creemers et al., 2012), and fied in a hypertensive patient a dominant-negative PCSK1 represents the third most prevalent monogenic D282N mutation in PACE4 that impedes the intracellu- contributor to the risk of developing obesity (Choquet lar autoactivation of proPACE4 into PACE4. Mutated et al., 2013). To date, no human PC2 mutations associ- proPACE4 is retained in the ER in complex with normal ated with disease have been found. Interestingly, PACE4, thereby inhibiting corin activation (Chen et al., whereas PC1/3 and PC2 are critical for the generation 2015). Atrial natriuretic peptide is a hormone essential of insulin, the absence of functional PC1/3 in humans has for sodium homeostasis (Flynn et al., 1983; Seidah et al., not been associated with insulin resistance or diabetes 1984) and reduces blood pressure (Yan et al., 2000). (Stijnen et al., 2016), suggesting the presence of compen- In agreement, PACE4 KO mice were found to be hyper- satory pathways and/or modifier genes. tensive on a normal salt (0.3% NaCl) diet, and this B. Furin and SKI-1/S1P in Cardiovascular Diseases phenotype was exacerbated when the mice were placed on high-salt (4% and 8% NaCl) diets, indicating that SKI-1/S1P is expressed in all cells, as furin. The corin activation by PACE4 is essential for maintaining importance of SKI-1/S1P in lipid metabolism has been normal blood pressure. Consistent with these findings, discussed at the beginning of this review. It is encoded the PCSK6 gene was previously linked to hypertension by the gene MBTPS1 for which no missense mutations in humans (Li et al., 2004). were associated with disease, and a .90% reduction of SKI-1/S1P expression is required to decrease choles- terol and TG production in liver (Yang et al., 2001). The D. PC5/6 in Heart Development and Extracellular complete KO of this gene in mice leads to very early Matrix Remodeling embryonic lethality (Mitchell et al., 2001). Mice lacking PC5/6 die at birth and perfectly re- Furin is essential during development because de- capitulate the phenotypes of mice lacking the trans- ficient mouse embryos die at embryonic day 11 and forming growth factor-b–like growth differentiation exhibit multiple developmental abnormalities, particu- factor 11 (Gdf11, also known as bone morphogenetic larly defects related to the function of endothelial cells protein 11) (McPherron et al., 1999), including the (Roebroek et al., 1998). To define the role of furin in absence of kidneys and major defects in the antero- endothelial cells, a mouse endothelial cell–specific KO posterior axis with extra-thoracic and -lumbar verte- (ecKO) of the Furin gene was generated (Kim et al., brae, and a lack of tail (Essalmani et al., 2008; Szumska 2012). Newborns die shortly after birth, and magnetic et al., 2008). In agreement, Gdf11 is exclusively acti- resonance imaging revealed ventricular septal defects vated by PC5/6 cleavage. In that context, high Gdf11 and/or valve malformations. In addition, primary cultures levels were associated with frailty, susceptibility to of wild-type and ecKO lung endothelial cells revealed that diabetes, or cardiac complications in old adults with ecKO cells are unable to grow, but are rescued by severe aortic stenosis (Schafer et al., 2016). Thus, PC5/6 extracellular soluble furin. These cells are deficient for inhibition may reduce cardiac Gdf11 production. To endothelin-1, transforming growth factor-b1, adrenome- circumvent lethality, studies were done in tissue- dullin, and bone morphogenetic protein 4 processing. The specific ecKO. Old ecKO mice lacking PC5/6 exhibit a mature forms of the two last ones were reduced by ;90%. cardiovascular hypotrophy associated with decreased Thus, furin is a major regulator of cardiac functions, via extracellular matrix collagen deposition, decreased left the processing of multiple substrates. Recently, a GWAS ventricular diastolic function, and vascular stiffness, revealed that a nucleotide A to C substitution in intron suggesting a trophic role of the enzyme PC5/6 1oftheFURIN gene (rs17514846) was significantly most likely mediated by insulin-like growth factor 1/ associated with the development of the metabolic syn- protein kinase B, also known as Akt/mammalian target drome, with the protective minor A allele correlating with of rapamycin signaling and control of autophagy a 21% decreased serum TG (Ueyama et al., 2015). (Marchesi et al., 2011). Among the endothelial cell Interestingly, furin inactivates lipoprotein lipase, which surface precursors known to be cleaved by PC5/6 is is implicated in the hydrolysis of TG into fatty acids from the receptor protein tyrosine phosphatase receptor TG-rich lipoproteins (Jin et al., 2005; Lei et al., 2011). The protein tyrosine phosphatase m (Campan et al., 1996), protective A allele in rs17514846 may thus be a furin LOF of which KO in mice results in increased stiffness of mutation leading to increased lipoprotein lipase activity, mesenteric arteries (Koop et al., 2003), whereas our and hence to lower TG. ecKO mice show the opposite phenotype (Marchesi 48 Seidah et al. et al., 2011). Finally, human intronic PCSK5 SNPs who do not reach target levels of LDL-c with the correlate with low levels of HDL cholesterol (Iatan available medications, cannot tolerate statins, or who et al., 2009), a correlation modulated by dietary experience painful side effects with statins, such as polyunsaturated fatty acids (Jang et al., 2014). muscle pain, cramps, weakness to myopathy, and, rarely, rhabdomyolysis. Notably, homozygote FH pa- E. PC7 in HDL, small dense LDL, and TG tients who have minimal LDLR activity left can now be Human GWAS studies suggest that PCSK7 LOF treated with PCSK9 mAbs, resulting in a ;30% de- mutations result in favorable CVD parameters, that is, crease in circulating LDL-c (Raal et al., 2015). In these high HDL, low TG (Guillemot et al., 2014; Peloso et al., FH patients, switching from two to three times weekly 2014; Yao et al., 2015), low highly atherogenic small LDL apheresis to a PCSK9 mAb combined or not with dense LDL (Hoogeveen et al., 2014), and reduced insulin once per week LDL-c apheresis maintained the LDL-c- resistance (Huang et al., 2015). Understanding the PC7 lowering effect, thereby giving a much better quality of biology underlying these phenotypes should lead to life to these patients that is less dependent on the use of powerful novel therapies for cardiometabolic disorders. biweekly long sessions to clear LDL-c from their blood using special apheresis dialysis columns (Lappegård et al., 2016). Although the outcomes of the various XII. Conclusions and Future Perspectives ongoing phase 3 clinical trials using PCSK9 mAbs will The original hypothesis of the existence of secretory not be known until 2017–2018 (Elbitar et al., 2016), precursors, which upon limited proteolysis releases already early signs revealed that this treatment results bioactive peptides or proteins, has been validated in in a ;50% reduction in cumulative cardiovascular multiple precursor proteins from various species events within 1–2 years of treatment (Robinson et al., (Chrétien, 2011; Steiner, 2011). The arduous process of 2015; Sabatine et al., 2015). However, one should be hunting for the cognate proteinases responsible for such cautious, as these results are only partial and not powered processing led to the identification of nine PCs with enough to unambiguously assess cardiovascular out- varied physiologic functions, which are sometimes asso- comes of prespecified endpoints in cardiometabolic ciated with pathology (Seidah and Prat, 2012). The diseases. Finally, the fact that PCSK9 is inactivated cumulative knowledge over the last 26 years led us to by some proteases such as furin (Benjannet et al., 2006; propose that inhibiting some of these convertases may in Essalmani et al., 2011) might open new strategies to some cases be favorable to specific patients (Fig. 1). The enhance this inactivation mechanism, and thus lower most advanced target is PCSK9, as injectable inhibitory the levels of active PCSK9. Indeed, recently a new mAb mAbs are now prescribed in clinics for the treatment of was reported that inhibits PCSK9 activity but still hypercholesterolemia, and will most likely be used for allows furin to inactivate it, resulting in a longer half- the prevention of septic shock. In the future, it is hoped life of the antibody (Schroeder et al., 2015). The future that modulation of some of the other PCs for specific will tell which strategies targeting PCSK9, including conditions may have new clinical applications, for exam- longer lasting mAbs, RNA interference (Fitzgerald et al., ple, PC7 in anxiety regulation (Wetsel et al., 2013), and 2014), and small-molecule inhibitor approaches (Seidah, possibly in hypertriglyceridemia (Peloso et al., 2014). 2013), will find their way in dyslipidemia, cardiology, The present review exposed the many new facets of and sepsis in clinics worldwide, which would result in PCSK9 and its biology, concentrating only on its ability affordable and safe treatments and/or prevention of life- to enhance the degradation of the LDLR and VLDLR. threatening conditions. However, PCSK9 has been shown to target for degra- dation other members of the LDLR family, including Acknowledgments ApoER2 (Poirier et al., 2008), the fatty acid transporter The authors thank Brigitte Mary for efficacious editorial assistance. CD36 (Demers et al., 2015), in liver and other tissues, such as small intestine, pancreas, brain, and adipocytes Authorship Contributions (Seidah et al., 2014). A lot more remains to be unraveled Participated in research design: Seidah, Abifadel, Prost, Boileau, Prat. regarding the cellular trafficking of PCSK9 together Conducted experiments: Seidah, Prost, Prat. with its targeted receptors, its complex web of interact- Performed data analysis: Seidah, Prost, Prat. ing proteins, and its influence on the fate or levels of Wrote or contributed to the writing of the manuscript: Seidah, other proteins. Furthermore, PCSK9 has been shown to Abifadel, Prost, Boileau, Prat. have a regulatory role of inflammatory processes in- dependent from its effects on LDL-c (Leander et al., References Abifadel M, Elbitar S, El Khoury P, Ghaleb Y, Chémaly M, Moussalli ML, Rabès JP, 2016). This is clearly a very exciting period in the field of Varret M, and Boileau C (2014) Living the PCSK9 adventure: from the identifi- dyslipidemia, where, thanks to new PCSK9-silencing cation of a new gene in familial hypercholesterolemia towards a potential new class of anticholesterol drugs. Curr Atheroscler Rep 16:439. therapies, LDL-c levels were lowered to unprecedented Abifadel M, Guerin M, Benjannet S, Rabès JP, Le Goff W, Julia Z, Hamelin J, low levels, reaching 0.4 mM and lower. This is de- Carreau V, Varret M, Bruckert E, et al. (2012) Identification and characterization of new gain-of-function mutations in the PCSK9 gene responsible for autosomal finitively good news for hypercholesterolemic patients dominant hypercholesterolemia. Atherosclerosis 223:394–400. Roles of PCSKs in Cholesterol and Triglyceride Metabolism 49

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