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

ASSOCIATE EDITOR: RHIAN M. TOUYZ

The Proprotein Convertases in Hypercholesterolemiaand Cardiovascular Diseases: Emphasis on Proprotein

Convertase Subtilisin/Kexin 9Nabil 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 ofIntegrative 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, andUniversité Paris Diderot, Paris, France (C.B.)

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33I. The Proprotein Convertases and Their Functions in Health and Disease . . . . . . . . . . . . . . . . . . . . . 34II. Phylogenetic and Haplotype Analyses of PCSK9 in Human Species . . . . . . . . . . . . . . . . . . . . . . . . . . 36III. The Enzymatically Inactive Mature PCSK9 Regulates Circulating LDL Cholesterol Levels . . . 37

Role of PCSK9 in Lipoprotein (a) Regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38IV. The Absence of PCSK9 Leads to a Sex- and Tissue-Specific Subcellular Distribution

of the LDLR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38V. Role of PCSK9 in Inflammation, Sepsis, and Viral Infections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39VI. Other Physiologic Roles of PCSK9. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40VII. Cellular Studies: Mechanism of Action of PCSK9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41VIII. What Have We Learned from Key Natural Mutations on the Functions of PCSK9

and Its Partners? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42IX. Ongoing Outcome Clinical Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44X. Other Genes and Loci Implicated in LDL-c Regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45XI. Implication of the Enzymatically Active PCs in CVD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

A. PC1/3 and PC2 in Diabetes and Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46B. Furin and SKI-1/S1P in Cardiovascular Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47C. PACE4 in Blood Pressure Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47D. PC5/6 in Heart Development and Extracellular Matrix Remodeling . . . . . . . . . . . . . . . . . . . . . . 47E. PC7 in HDL, small dense LDL, and TG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

XII. Conclusions and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Abstract——The secretory proprotein convertase (PC)family comprises nine members, as follows: PC1/3, PC2,furin, PC4, PC5/6, paired basic amino acid cleavingenzyme 4, PC7, subtilisin kexin isozyme 1/site 1 protease(SKI-1/S1P), and PC subtilisin/kexin type 9 (PCSK9). Thefirst seven PCs cleave their substrates at single/pairedbasic residues and exhibit specific and often essentialfunctions during development and/or in adulthood. Theessential SKI-1/S1P cleaves membrane-bound transcription

factors at nonbasic residues. In contrast, PCSK9 cleavesitself once, and the secreted inactive protease drags thelow-density lipoprotein receptors (LDLR) and veryLDLR (VLDLR) to endosomal/lysosomal degradation.Inhibitory PCSK9 monoclonal antibodies are nowprescribed to treat hypercholesterolemia. This reviewfocuses on the implication of PCs in cardiovascularfunctions and diseases, with a major emphasis onPCSK9. 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 Chair216684; and Leducq Foundation [Grant 13CVD03].

Address correspondence to: Dr. Nabil G. Seidah, Institut de Recherches Cliniques de Montréal, 110 Pine Avenue West, Montreal, QCH2W1R7, Canada. E-mail: seidahn@ircm.qc.ca

dx.doi.org/10.1124/pr.116.012989.

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analysis of PCSK9 haplotypes in modern and archaichuman species. The absence of PCSK9 in mice led tothe discovery of a sex- and tissue-specific subcellu-lar distribution of the LDLR and VLDLR. PCSK9inhibition may have other applications because itreduces inflammation and sepsis in a LDLR-dependentmanner. Our present understanding of the cellular

mechanism(s) that enables PCSK9 to induce thedegradation of receptors is reviewed, as well as theconsequences of its key natural mutations. The PCSK9ongoing clinical trials are reviewed. Finally, how theother PCs may impact cardiovascular disease and themetabolic syndrome, and become relevant targets, isdiscussed.

I. The Proprotein Convertases and TheirFunctions in Health and Disease

Post-translational modifications of secretory proteinsfashion the final bioactive forms of primary proteinproducts in various tissues and cells during developmentand in adulthood (Seidah and Guillemot, 2016). Of allpost-translational modifications of proteins known, pep-tide bond cleavage represents one of themost drastic andirreversible reactions leading to two or more fragmentsof distinct, but sometimes complementary, biologicfunctions. From the time it was realized that secretorypolypeptide hormones such as insulin (Steiner, 2011)and adrenocorticotropic hormone/b-endorphin (Chrétien,2011) were synthesized from inactive precursors, pro-insulin and pro-opiomelanocortin, respectively, it took23 years to identify the cognate proteases that generatevarious peptide hormones and protein products (Seidahand Chrétien, 1999; Seidah and Prat, 2012).The mammalian genome encodes nine proprotein

convertases (PCs). These serine proteases share sequenceidentity with bacterial subtilisin and yeast kexin (Juliuset al., 1984; Mizuno et al., 1988), and their genes arereferred as proprotein convertases subtilisin kexin types1–9 (PCSK1 toPCSK9), with two exceptions formembers3 and 8, respectively, named FURIN and MBTPS1. Thenine corresponding proteins are known as follows: PC1/3,PC2, furin, PC4, PC5/6, paired basic amino acid cleavingenzyme 4 (PACE4), PC7, subtilisin kexin isozyme 1/site1 protease (SKI-1/S1P), and PCSK9. Except for PCSK9,they all cleave secretory proteins, such as polypeptidehormones, receptors, growth factors, adhesion molecules,membrane-bound transcription factors, and neuronalregulators, during embryonic development and in adult-hood (Seidah and Prat, 2012). Such processing regulatesessential cellular functions, and their dysregulation canlead to the metabolic syndrome, obesity, diabetes, hyper-tension, dyslipidemia, inflammation, cancer/metastasis,pain, memory deficits, anxiety/depression, neurodegen-eration, iron dysregulation, and even viral and parasiticinfections.Accordingly, a number of ongoing clinical trialsare investigating the plausible roles of PCs in diseases.

Potentially the most advanced/promising clinical trialsare for inhibitors of furin in cancer and viral infections,PACE4 in arthritis pain and prostate cancer, and PCSK9in hypercholesterolemia and septic shock (Fig. 1).

The first seven basic amino acid–specific PCs werediscovered between 1990 and 1996 (Seidah and Prat,2012; Seidah, 2015). However, other precursors, such asbrain-derived neurotrophic factor, cell surface glycopro-teins of hemorrhagic fever viruses, andmembrane-boundtranscription factors [sterol regulatory element-bindingproteins (SREBPs); endoplasmic reticulum (ER)–stressprotein activating transcription factor 6; or cAMP-response element-binding proteins], are cleaved afternonbasic residues, predicting the implication of a distinctprotease(s). This led to the simultaneous discovery andcDNA cloning of site 1 protease (S1P) (Cheng et al., 1999;Espenshade et al., 1999), also known as subtilisin-kexinisozyme 1 (SKI-1) (Seidah et al., 1999).

The transcription factors SREBP-1 and SREBP-2 aremajor regulators of cholesterol/low-density lipoprotein(LDL) receptor (LDLR) and fatty acid synthesis, re-spectively. They are anchored in the secretory pathwayvia a luminal loop separating two transmembrane–cytosolic domains. The initial cleavage by SKI-1/S1P inthe luminal loop within the cis/medialGolgi is followedby an intramembrane cleavage in the first transmem-brane domain by the metalloprotease site-2 proteasein the medial Golgi (Rawson et al., 1997). The releasedcytosolic N-terminal basic helix-loop-helix leucinezipper-containing domains of both factors are thentranslocated to the nucleus, where they activate theirrespective target genes. SKI-1/S1P was the first PCimplicated in the regulation of lipid metabolism, as itsinactivation in the liver decreased by 75% lipid synthe-sis (Yang et al., 2001). However, the complete Mbtps1gene inactivation in mouse resulted in an early embry-onic death with no epiblast formation (Yang et al.,2001), thus hampering clinical applications of SKI-1/S1P inhibitors. Only short treatments with the SKI-1/S1P small-molecule inhibitor PF-429242 (Hawkinset al., 2008) were suggested for persistent arenavirus

ABBREVIATIONS: aa, amino acid; ADH, autosomal dominant hypercholesterolemia; apoB, apolipoprotein B; ApoER2, apolipoprotein Ereceptor 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 acidcleaving 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.

34 Seidah et al.

(Pasquato et al., 2012; Pasquato and Kunz, 2016),hepatitis C (Blanchet et al., 2015), and Dengue viralinfections (Uchida et al., 2016). Mbtps1 inactivation inosteoblasts further showed that SKI-1/S1P is critical forextracellular matrix signaling and axial elongationduring somitogenesis and vertebral development, andpossibly linked to the caudal regression syndrome(Achilleos et al., 2015). In addition,Mbtps1 inactivationin osteocytes stimulates soleusmuscle regeneration andincreased muscle size and contractile force with age(Gorski et al., 2016). Understanding bone–muscle cross-talk may provide a novel approach to prevention andtreatment of age-related muscle loss.Although the first seven PCs cleave their substrates

after basic residues often organized in pairs within themotif K/R-2Xn-K/R↓ [where Xn = 0, 1, 2, or 3 amino acids(aa)], SKI-1/S1P recognizes the motif R-X-(aliphaticresidue)-X↓. In contrast, the last convertase, PCSK9(Seidah et al., 2003), which is abundantly synthesizedby the liver and released into the circulation, is onlyfound in some vertebrates. Interestingly, because thebovine PCSK9 genome exhibits a premature stopcodon in exon 10 (Ding et al., 2007; Cameron et al.,2008), this revealed that fetal bovine serum, widelyused in cell biology, does not contain a PCSK9 protein.Like all other PCs, PCSK9 undergoes in the ER anautocatalytic cleavage between its pro and catalyticdomains at the site VFAQ152↓ (Naureckiene et al.,2003; Benjannet et al., 2004), where Gln152 is critical(Benjannet et al., 2012). Note that, different from theeight other members of the family, it does not require

the presence of any basic amino acid at the vicinity of thecleavage site.

The most amazing and key feature that distinguishesPCSK9 from the other PCs is its inability to get rid of itsprodomain, leading to a secreted inactive protease. In-deed, after autocatalytic cleavage of the prodomain,which acts as an intramolecular chaperone and potentbut transient inhibitor in the other PCs (Seidah andPrat, 2012), in mature PCSK9 it remains noncovalentlyattached to its catalytic pocket (Seidah et al., 2003),thereby preventing any in trans catalytic activity of theenzyme (Cunningham et al., 2007). Thus, PCSK9 acts asa protease only once in its lifetime, whereas the other PCscleave and activate/inactivate secretory precursor pro-teins either in the cis/medial (SKI-1/S1P) or trans Golginetwork, cell surface or endosomes (furin, PC4, PC5/6,PACE4 and PC7), or immature secretory granules (PC1/3and PC2) (Seidah and Prat, 2007, 2012; Creemers andKhatib, 2008; Seidah et al., 2008, 2013; Turpeinen et al.,2013). In contrast, PCSK9 binds various receptors andescorts them to endosomes/lysosomes for degradation(Seidah and Prat, 2012; Seidah et al., 2014). Interest-ingly, from 1990 to the present, a literature statisticalanalysis of the interest in PCs, and especially in PCSK9,revealed that, since its discovery in 2003, it has stimu-lated wide interest, as exemplified by the .1850 manu-scripts published on PCSK9, compared with a totalof .4100 manuscripts reported on all PCs (Fig. 2).

In this review, we will focus on the newly describedaspects of the biology of PCSK9, its mechanism ofaction, as well as its validated and possibly new targets.

Fig. 1. Proprotein convertases in health and diseases. The clinical fields relevant to each PC are indicated. To date, only PCSK9 is clinically targetedusing mAbs. These mAbs are used for the treatment of hypercholesterolemia and are very promising for the prevention of septic shock. In thisschematic, overlaps between PC clinical fields are only qualitative. Diabetes is related to both PC1/3 and PC2 through insulin processing. Cancers aremainly regulated by furin, PC5/6, and PACE4 (prostate) via processing of growth factors and their receptors. Most viral surface glycoproteins areprocessed 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 byPCSK9. The convertase PC4 has a unique role in fertility, and PC7 in iron metabolism and anxiety/depression. Finally, in humans, PC1/3, PC5/6, andPACE4 were genetically linked to obesity, developmental defects, and osteoarthritis pain, respectively.

Roles of PCSKs in Cholesterol and Triglyceride Metabolism 35

We will also discuss the consequences of PCSK9 naturalmutations on themetabolism of LDL cholesterol (LDL-c),as well as the clinical applications of its inhibitors inpathologies. Finally, we will summarize the implicationof other PCs in cardiovascular diseases (CVD) and howsome of them can regulate PCSK9.

II. Phylogenetic and Haplotype Analyses ofPCSK9 in Human Species

A phylogenetic tree based on the amino acid sequencesof the PCs revealed that SKI-1/S1P is the most ancientmember of the family (Seidah et al., 1999) and is foundin parasites and the plant kingdom (Barale et al., 1999).Although the closest members form couples, that is,PC1/PC2; furin/PC4, PC5/PACE4; and SKI-1/PCSK9,the highly conserved PC7 (Seidah et al., 1996) is difficultto place on the phylogenetic tree (Fig. 3A).Alignment of PCSK9 protein sequences of modern and

archaic human species revealed only three variationsbetween themodern human reference sequence and eitherthe denisovan individual,H449L,V474I, andG670E, or theAltai neandertal individual, A53V, V474I, and G670E.Interestingly, the benign polymorphic variants A53V andV474I were also detected in modern humans before. Also,G670E is a major common variant in modern humans,

with only ;13% carrying a Gly670 in the Chinese Hanpopulation (He et al., 2016). We have consistently found aGlu670 in all of our DNA sequences of PCSK9 to date. Thecommon His449 in modern humans and neandertal isreplaced by Leu449 in the denisova individual (H449L).The functional consequence of this replacement in thehinge domain (aa 422–452) (Cunningham et al., 2007;Saavedra et al., 2012) is the loss of a positive charge at theacidic pHs of endosomes/lysosomes and begs for moredetailed cellular analyses. In that context, a similar lossof positive charge, R434W, in the same domain resulted ina loss-of-function (LOF) associated with lower levels ofcirculating PCSK9 and LDL-c (Dubuc et al., 2010).

Given the possible LOF phenotype of the denisovanPCSK9 protein (H449L variation), we investigatedwhether its genomic haplotype could have been adaptivelyintrogressed into modern humans. Recent studies havedemonstrated that several important genes show signs ofadaptive introgression from neandertal and/or denisovansto modern humans, such as the EPAS1 gene important forhigh altitude adaptation in Tibetans (introgressed fromdenisovans to modern humans) (Huerta-Sánchez et al.,2014) orhumanToll-like receptors important for our innateimmunity (introgressed from both neandertals and deni-sovans into modern humans) (Dannemann et al., 2016).However, PCSK9 does not show a pattern consistent with

Fig. 2. Research interest of the nine PCs. Literature search revealed .1850 manuscripts published on PCSK9, compared with a total of .4100manuscripts on all PCs. The discovery of PCSK9 in 2003, and especially the benefit of its inhibition in hypercholesterolemia, boosted the number of PCpublications. 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.

36 Seidah et al.

adaptive introgression for the denisovanhaplotype (H449Lvariation, Fig. 3B). In case of adaptive introgression, wewould have expected this haplotype to be present in high(or higher) frequency within certain populations in ourdata, aswell as genetic distance to the archaic haplotype toincrease with a sharp incline.In conclusion, only minor changes in PCSK9 primary

protein sequence occurred within the years that sepa-ratemodern humans from either denisovans (estimatedseparation ;380,000–473,000 years ago) (Prüfer et al.,2014) or neandertals (estimated separation ;550,000–765,000 years ago) (Prüfer et al., 2014). However, wefound one interesting amino acid change (H449L) indenisovans that may result in a LOF of PCSK9. Incontrast to the conserved protein sequence of PCSK9between hominids, their genomic sequences are quitevariable in individuals outside of East Asia. Further-more, it does not show any pattern consistent withadaptive introgression. More in-depth analyses (includ-ing all nine PCs and more extensive sampling) will beneeded to get amore detailed picture of the evolutionaryhistory of PCSK9 and the other eight PCs.

III. The Enzymatically Inactive Mature PCSK9Regulates Circulating LDL Cholesterol Levels

Screening of nucleotide databases with conservedsequences encoding the active site residues of S1P/SKI-1

as baits led us to report in February 2003 (Seidah et al.,2003) the cDNA cloning and protein analysis of the ninthand lastmember of the PC-family, a putative convertase,originally called neural apoptosis-regulated convertase-1. Millennium Pharmaceuticals had released part of itscDNA in a patent database as belonging to a group ofgenes upregulated upon induction of apoptosis in pri-mary cerebellar neurons by serum withdrawal. Neuralapoptosis-regulated convertase-1, nowknown as PCSK9,belongs to the proteinase K family of subtilases, and itscatalytic domain exhibits;25% of sequence identity withthat of its closest familymember SKI-1/S1P (Seidah et al.,2003) (Fig. 3A). Human PCSK9 mRNA (NM_174936.3)spans 3710 bp over 12 exons encoding a 692 amino-acidprotein (NP_777596.2).

Chromosomal mapping of the PCSK9 gene revealed itto be located on chromosome 1p32 (Seidah et al., 2003),close to a predicted third major gene locus [familialhypercholesterolemia (FH)3] for autosomal dominantfamilial hypercholesterolemia (ADH) located at 1p34.1-p32 in a large French family in whom the two genesknown to be implicated in this disease, the LDLR andAPOB genes, had been excluded (Varret et al., 1999). Thislocus was previously associated with an increase in thehepatic secretion of LDL, but not high-density lipoprotein(HDL) cholesterol, or triglycerides (TG) (Varret et al.,1999; Hunt et al., 2000). Armedwith this information andthe major expression of PCSK9 in liver (Seidah et al.,

Fig. 3. Phylogenetic reconstruction of the nine PCs and haplotype structure of PCSK9 in modern and extinct human species. (A) Phylogenetic treedisplaying the relationship among the nine PCs. The phylogeny was inferred using maximum likelihood (indicated numbers) with 100 bootstrapreplicates. (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 haplotypesequence (sites). Black rectangles represent sites with derived mutations, and white rectangles indicate sites with the ancestral nucleotide. The colorson the y-axis show which population the respective haplotype originates from among the 683 individuals in total.

Roles of PCSKs in Cholesterol and Triglyceride Metabolism 37

2003), we contacted a French team that was activelystudying this region of chromosome 1. Intensive geneticanalyses in 23French non-LDLR/non-APOB families hadallowed the identification of a new multiplex family thatwas crucial in refining the boundaries of the geneticregion on chromosome1. Thispositional cloning approachusing genetic mapping, physical mapping, and sequenc-ing of several genes led to the identification of a commonmutation, S127R, in the 22-kb PCSK9 gene in two largefamilies and another mutation, F216L, in a third family.The very fruitful collaboration between the Canadianteam working on PCs and the French one working on thegenetics of hypercholesterolemia culminated in the firstreport that appeared in June 2003, revealing that humanPCSK9 was the FH3 gene critical for LDL-c regulation(Abifadel et al., 2003).This discovery was soon followed by the identification

of the devastating PCSK9 D374Y mutation in a largeUtah pedigree (Timms et al., 2004) and 1 month later inthree Norwegian subjects (Leren, 2004). Other ADH-causing PCSK9mutations were discovered in the follow-ing years (Abifadel et al., 2009, 2014), and,more recently,16 different PCSK9 mutations were found in patientsfrom eight countries (Hopkins et al., 2015).It was evident from the analysis of the first identified

mutants S127R and F216L that PCSK9 plays a majorrole in the regulation of LDL-c, but not TG (Abifadelet al., 2003). This was confirmed by the seminal exper-iments in mice done by Maxwell and Breslow (2004),who demonstrated that PCSK9 promotes the degrada-tion of the LDLR. Adenoviral-mediated expression ofPCSK9 led to high plasma LDL-c levels in a LDLR-dependent manner and resulted in the quasi-absence ofthe LDLR in the liver. As the mouse gene Pcsk9 wasstrongly downregulated by dietary cholesterol (Maxwellet al., 2003), they hypothesized that PCSK9 wascoregulated with the LDLR to rapidly modulate lipo-protein clearance. Because the two first loci related toFH were that of the LDLR and its ligand apolipoproteinB (apoB), it was very gratifying that a strong func-tional link could be established between the LDLR andPCSK9. Thus, gain-of-function (GOF) mutations result-ing in higher activity or levels of PCSK9 were expectedto lower liver LDLR levels and hence raise circulatingLDL-c, due its reduced clearance by the remainingLDLR. The demonstration that, conversely, LOF muta-tions lead to higher LDLR concentrations and hencereduced LDL-c levels was first achieved by Cohen et al.(2005) 2 years later. By screening 32 subjects from theDallas Heart study that had the lowest LDL-c levels,they discovered two heterozygote nonsense mutationspresent in ;2% of the African American population,Y142X and C679X. The latter are responsible for a;40% drop in circulating LDL-c and associated with a;88% reduction in the risk of CVD (Cohen et al., 2006).Polymorphisms, such as R46L, resulting in a partialPCSK9 LOF, were also associated with lower LDL-c

levels (Kotowski et al., 2006). Finally, four individualswith complete LOF of PCSK9 have undetectable circu-latingPCSK9andLDL-c values of;0.4mM(;15mg/dL)(Zhao et al., 2006; Hooper et al., 2007; Cariou et al., 2009;Mayne et al., 2011).

The fact that complete LOF of human PCSK9 resultedin an unprecedented large decrease in LDL-c levelswithout seemingly adverse effects encouraged pharma-ceutical companies to develop the first inhibitor approachusing PCSK9 monoclonal antibodies (mAbs) to reducethe levels of circulating PCSK9. As will be discussedlater, thesemAbsarenows.c. administered toFHpatientsthat are either statin-resistant or statin-intolerant toreduce their LDL-c levels (Stein and Raal, 2014; Seidah,2015). Before this drug was approved by the healthcare authorities worldwide, a large number of phase 1–3clinical trials was performed for safety, tolerability, andefficacy.

Role of PCSK9 in Lipoprotein (a) Regulation

Unexpectedly, not only does reduction of plasma PCSK9levels result in ;60% lowering of LDL-c, it also reducedby;30–35% the levels of the highly atherogenic lipopro-tein (a) [Lp(a)] particles (Stein, 2013; Seidah et al., 2014;Stein andRaal, 2014). This result opened a newpotentialtherapeutic application because Lp(a) has long beenknown to be an independent risk factor for atherosclero-sis for which no known therapeutic lowering agent hadbeen identified. The reduction in Lp(a) levels was re-cently rationalized by the fact that, under supraphysio-logic levels of the LDLR resulting from PCSK9 mAbtreatments, the LDLR becomes a functional receptorof Lp(a) both in cells (Romagnuolo et al., 2015) and inhumans (Raal et al., 2016). It was also shown thatPCSK9 binds the apoB component of Lp(a), but not toapo(a) (Romagnuolo et al., 2015). Interestingly, a recentreport suggests that PCSK9 is associated with Lp(a) inthe plasma of patients with very high circulating Lp(a)concentrations, and that the Lp(a)-associated PCSK9levels seem to be directly correlated with plasma Lp(a)levels, but not with total plasma PCSK9 levels (Tavoriet al., 2016), suggesting that polymorphic variations inLp(a) and its levels may affect its affinity to PCSK9.Whether the reported association of Lp(a) withPCSK9 inplasma samples is direct or requires a bridging protein isnot clear, because we have shown in vitro that PCSK9does not directly bind Lp(a), whereas it binds LDLwith aKD of ;130 nM (Romagnuolo et al., 2015).

IV. The Absence of PCSK9 Leads to a Sex- andTissue-Specific Subcellular Distribution of

the LDLR

In mice, PCSK9 is highly expressed in liver, pancre-atic islets, intestine, and kidney (Seidah et al., 2003;Zaid et al., 2008; Langhi et al., 2009). PCSK9 knockout(KO) mice, as well as mice lacking specifically PCSK9 in

38 Seidah et al.

the liver [hepatocyte-specific KO (HepKO)], were gen-erated (Rashid et al., 2005; Zaid et al., 2008; Parkeret al., 2013). In these mice, the lack of PCSK9 leads to atwo- to threefold increase of the LDLR content in theliver, pancreas, or intestine, as estimated by Westernblotting, with no change in LDLRmRNA levels (Rashidet al., 2005; Zaid et al., 2008; Roubtsova et al., 2015).GOF mutations in human PCSK9 or PCSK9 over-

expression in mice stimulate apoB-containing lipopro-tein secretion from the liver (Ouguerram et al., 2004;Park et al., 2004; Sun et al., 2005; Lambert et al., 2006).In contrast, PCSK9 deficiency in mice leads to lowerlevels of apoB-containing lipoproteins with two- andfivefold less total cholesterol and LDL-c, respectively,and to a fivefold higher rate of 125I-LDL clearance(Rashid et al., 2005; Zaid et al., 2008). Unexpectedly,although the liver expresses ;80% of the whole-bodyLDLR, the absence of PCSK9 does not promote choles-terol accumulation or higher bile acid production in thistissue (Rashid et al., 2005; Zaid et al., 2008; Parkeret al., 2013). Rather, PCSK9 KO, but not wild-type micethat underwent partial hepatectomy had higher levelsof SREBP-2 and 3-hydroxy-3-methylglutaryl–CoA re-ductase mRNA levels in their regenerating liver, sug-gesting the sensing of a lack of intracellular cholesterol.How elevated levels of LDLR do not lead to cholesterolaccumulation in the liver or pancreatic islets (Langhiet al., 2009) remains an enigma. A possibility is thatmore substantial amounts of cholesterol are excretedvia transintestinal cholesterol excretion (Le May et al.,2013). Whether transintestinal cholesterol excretionincreases in individuals lacking PCSK9 or treated withmAbs remains to be verified.PCSK9 is undetectable in the plasma of HepKOmice,

demonstrating that the circulating protein originatesexclusively from hepatocytes (Zaid et al., 2008). Circu-lating PCSK9 can also regulate peripheral receptors.For example, in HepKO or complete KO mice, veryLDLR (VLDLR) immunolabeling increases at the cellsurface of visceral adipocytes that do not expressPCSK9 locally. Intriguingly, this increase was;10-foldhigher in female than in male KO mice (Roubtsovaet al., 2011), providing the first evidence for a sex-dependent cell surface accumulation of a PCSK9-targeted receptor in PCSK9KOmice. In liver, pancreas,and small intestine of KO male and female mice, theabsence of PCSK9-triggered LDLR degradation led tosimilar two- to threefold increases in the total proteinlevels of LDLR. However, the subcellular distribution ofthe receptor differed in a sex- and tissue-dependentmanner. LDLR cell surface immunolabeling was dra-matically increased in the liver and pancreatic islets ofKO males, but not females. Conversely, LDLR cellsurface labeling was higher in the small intestine offemales, similar to the VLDLR in perigonadal fat depots(Roubtsova et al., 2015). Moreover, the accumulation ofthe LDLR at the cell surface of male but not female

hepatocytes was evidenced by Western blot analysis ofplasma membrane preparations (Roubtsova et al.,2015). This sex-specific distribution is dictated byestrogens, as illustrated by the accumulation of theLDLR at the hepatocyte surface in ovariectomizedfemale mice supplemented with placebo, whereas thosesupplemented with 17b-estradiol exhibited low surfacelevels of the LDLR (Roubtsova et al., 2015).

The physiologic relevance of the above regulation by17b-estradiol of the LDLR subcellular distribution is ofthe highest interest if it has a counterpart in humans.The sex-dependent efficacy of the PCSK9 mAbs has notyet been extensively examined. The long-term studyLTS11717 conducted on 1438 men and 872 women (pre-dominantly postmenopausal) resulted in 65.5% and 53.4%drops in LDL-c in men and women, respectively, thusshowing a 22% higher efficacy in men (see SupplementalFig. S4 in Robinson et al., 2015). However, a subgroupanalysis revealed that postmenopausal women respondedwith a 16% higher efficacy than premenopausal women(54.8% versus 47.3%). Thus, men (65.5% drop) respondedwith 38.5% and 19.5% higher efficacies than premeno-pausal (47.3% drop) and postmenopausal (54.8% drop)women, respectively (http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/Drugs/EndocrinologicandMetabolicDrugsAdvisoryCommittee/UCM449865.pdf). Clearance experiments as well asextensive analyses of phase 3 clinical trials will beneeded to confirm the existence in women of a lowerresponse to PCSK9 inhibition.

V. Role of PCSK9 in Inflammation, Sepsis, andViral Infections

Inflammation and infection induce marked changesin lipid and lipoprotein metabolism, and administrationof bacteria endotoxins induces hyperlipidemia in ani-mals (Khovidhunkit et al., 2004). Lipopolysaccharide(LPS) administration, a model for Gram-negative bac-terial infection, increases LDL and VLDL levels, mostlikely via decreased LDLR and VLDLR protein levels inthe liver (Liao et al., 1996), possibly caused by LPS-stimulated expression of PCSK9 in liver (Feingold et al.,2008). Sepsis is a complex disease characterized bysystemic activation of inflammation and coagulation,most commonly in response to bacterial infection. Severesepsis, accompanied by dysfunction of at least one organ,affects 0.3–1% of individuals/yr in theUnited Stateswithmortality rates of;30% (Gaieski et al., 2013). Therapiesinclude antibiotic treatments and hemoperfusion filtra-tion that specifically remove LPS from circulation (Cruzet al., 2009).

Mice lacking LDLR exhibit increased mortality in acecal ligation and puncture (CLP) model (Lanza-Jacobyet al., 2003), suggesting that LDLR plays a notable rolein sepsis. In that context, Walley et al. (2014) recentlyshowed that exogenous human PCSK9 reduces LPS

Roles of PCSKs in Cholesterol and Triglyceride Metabolism 39

uptake in vitro by HepG2 cells, and that inhibitoryPCSK9 antibodies reduced plasma endotoxin levels inmice subjected to CLP, blunted the cytokine response,and doubled survival rates. In humans, PCSK9 LOF(R46L) polymorphisms were associated with bettersurvival of septic patients and reduced cytokine levels,whereas GOF (E670G) polymorphisms had the oppo-site effect. Furthermore, i.v. injection of endotoxin tohealthy volunteers generated lower cytokine levels inR46L individuals (Walley et al., 2014). Finally, it wasrecently shown that LPS binds plasma lipids to beinternalized in human HepG2 cells and mouse primaryhepatocytes by the LDLR, but also other receptors,including the VLDLR (Topchiy et al., 2016). It isimportant to note that, in septic patients, the medianlevels of PCSK9 were positively correlated with theextent of the respiratory and cardiovascular failure(Boyd et al., 2016), leading to the hypothesis thatreducing PCSK9 levels, and hence increasing LDLR-mediated hepatic clearance of pathogen lipids, may beprotective (Walley et al., 2014). Indeed, lower circulat-ing levels of endotoxins may reduce their binding toToll-like receptors that leads to nuclear factor kBactivation and subsequent proinflammatory cytokinesecretion by leukocytes (Savva and Roger, 2013). Inagreement, we have recently shown that, in a mouseCLP model of sepsis, PCSK9 deficiency confers pro-tection against systemic bacterial dissemination, organpathology, and tissue inflammation, particularly in thelungs and liver, whereas PCSK9 overexpression exac-erbates early hypercoagulable and proinflammatorystates and multiorgan pathology (Dwivedi et al.,2016). Future studies, including clinical trials, areneeded to assess the promising therapeutic efficacy ofinhibitory PCSK9 mAbs in sepsis (dos Santos andMarshall, 2014).Interestingly, berberine inhibits LPS-induced in-

creases in LDL-c and TG in mice (Xiao et al., 2012),most likely via the downregulation of the HNF1a pro-tein (Dong et al., 2015). The latter is known to stronglystimulate PCSK9 transcription, but not that of theLDLR (Li et al., 2009; Dong et al., 2010), whereasSREBP-2 upregulates the expression of both (Dubucet al., 2004). Importantly, statins upregulate moreefficiently PCSK9 mRNA levels than those of the LDLR(Dubuc et al., 2004), rationalizing the poor incrementalLDL-c decreases with increasing statin doses. Mediter-ranean diet (Richard et al., 2012) and exercise (Kamaniet al., 2015) reduce circulating levels of PCSK9, whereashigh-fat or fructose-rich diets (Cariou et al., 2013)increase the levels of PCSK9 (for a general review seeCui et al., 2015).In another manifestation of inflammation in relation

to PCSK9 regulation of the LDLR, we reproduced inLDLR KO mice the aorta and vascular calcificationobserved in FH patients and reported that PCSK9 KOmice are protected, whereas PCSK9 overexpression

exacerbates the phenotype (Awan et al., 2011). This isin part due to an inflammatory response to the forma-tion of cholesterol crystals in hypercholesterolemicconditions, which are reversed by the administrationof anti-inflammatory mAbs to interleukin-1b (Awanet al., 2016).

A possible drawback of PCSK9 inhibition is to triggerhigher levels of hepatitis C virus receptors, that is, LDLR,VLDLR, and possibly CD81, and to promote viral infec-tivity (Labonté et al., 2009; Le et al., 2015), especially forgenotype 3 hepatitis C virus (Bridge et al., 2015). Incontrast, patients affected by the human immunodefi-ciency virus–associated neurocognitive disorder, resultingfrom enhanced neuroinflammation and neurodegenera-tion (Kim et al., 2015),may benefit fromPCSK9 inhibitionthat would reduce inflammation.

VI. Other Physiologic Roles of PCSK9

We investigated by in situ hybridization the expres-sion of mouse PCSK9 at embryonic day 14.5 (Fig. 4).High signals were observed in the hepatic primordium,embryonic membranes, midgut and umbilical artery,and lower signals in the telencephalon (Fig. 4B). At thisdevelopmental stage, the mouse placenta did not showPCSK9 expression. In embryonicmembranes (Fig. 4, C–F), PCSK9 mRNA was present within the smoothmuscle cell layer, but not in endothelial or embryonicblood cells. The role of PCSK9 in these tissues is notknown and maybe vary during development. Interest-ingly, PCSK9 mRNA and protein are expressed inhuman placenta at full-term in patients with gesta-tional diabetes mellitus, a common complication ofpregnancy aggravated by obesity (Dubé et al., 2013).In situ hybridization analyses at embryonic day 17 (Fig.5, upper panel) and postpartum day 10 (Fig. 5, lowerpanels) in mouse revealed that PCSK9 is mostlyexpressed in liver, small intestine, colon, cerebellum,and kidney. At postpartum day 10, PCSK9 and LDLRcolocalize especially in the liver and intestine, but alsoin thymus (Seidah et al., 2003; Zaid et al., 2008). Thefunction of PCSK9 in extrahepatic tissues is still poorlyunderstood (Seidah, 2016).

Multiple studies revealed that PCSK9 can escort notonly the LDLR to lysosomal degradation compartments,but also its family members VLDLR, apolipoprotein Ereceptor 2 (ApoER2), and LDLR-related protein-1(LRP1) (Poirier et al., 2008; Roubtsova et al., 2011;Canuel et al., 2013), best seen in livers lacking LDLRand PCSK9 (unpublished data). This suggests that, inFH patients, especially those with very low levels offunctional LDLR, PCSK9 may also regulate LRP1levels. In addition, the fatty acid transporter CD36seems to be another target of PCSK9 (Demers et al.,2015), although the exact binding site has not yetbeen determined. This is in line with the observedhypertrophy of visceral adipocytes in PCSK9 KO mice

40 Seidah et al.

(Roubtsova et al., 2011) that indicates higher binding ofTG-rich lipoproteins and fatty acid uptake via increasedVLDLR and CD36. Elucidation of the role(s) of PCSK9as an intracellular and/or paracrine regulator of LDLRand/or VLDLR protein levels in gut, kidney, andpancreas will require tissue-specific KOs, as was donein hepatocytes (Zaid et al., 2008). Although expressed inkidney, PCSK9 is not secreted into the urine (Dubucet al., 2004), and it does not seem to play a role incontrolling blood pressure even under induced hyper-tensive conditions (Berger et al., 2015). However,patients with chronic kidney disease exhibit higherliver expression and circulating levels of PCSK9(Konarzewski et al., 2014), most likely due to enhancedexpression of HNF1a in hepatocytes (Sucajtys-Szulcet al., 2016). Finally, although PCSK9 does not cross theblood brain barrier, little is known about the function ofPCSK9 in the cerebellum, or other neuronal cells(Seidah et al., 2003). PCSK9 has been shown to reduceLDLR levels, and possibly those of VLDLR andApoER2, during brain development and after ischemicstroke in adult mice, although poststroke behavior andlesion volume were not affected by the lack of PCSK9(Rousselet et al., 2011). The possible role of PCSK9 insome neurodegenerative diseases is yet to be conclu-sively shown and validated in humans.

VII. Cellular Studies: Mechanism of Actionof PCSK9

Surprisingly, 13 years after the discovery of PCSK9,no sorting molecular mechanism(s) can yet explainits ability to induce the degradation of the LDLR(Seidah et al., 2014) in late endosomes/lysosomes(Nassoury et al., 2007) (Fig. 6). Our data revealed thatthe C-terminal Cys-His–rich domain (CHRD) of PCSK9,which contains three repeat structures called M1, M2,and M3 (Cunningham et al., 2007), is needed for thePCSK9-induced degradation of the PCSK9–LDLR com-plex (Nassoury et al., 2007), including the repeatdomain M2 (Saavedra et al., 2012), which has 14 His(Cunningham et al., 2007) and a number of naturalmutations associated with it (Seidah et al., 2014). Thiscritical importance of the CHRD in regulating theability of PCSK9 to induce the degradation of the LDLRwas confirmed in two independent studies (Zhang et al.,2008; Poirier et al., 2016) and was shown to be inhibitedby mAbs (Ni et al., 2010; Schiele et al., 2014) or single-domain antibodies that target the CHRD, withoutinhibiting the PCSK9–LDLR complex formation (Weideret al., 2016).

We suggested that a putative protein Xp binds theCHRD of extracellular PCSK9 in the PCSK9–LDLRcomplex and escorts it to lysosomes for degradation by

Fig. 4. PCSK9 mRNA is highly expressed in the liver, midgut, embryonic membranes, and umbilical artery at embryonic day 14.5. The same sagittalsection through mouse embryo and attached placenta is shown in (A–D) and (F). (A) Staining with cresyl violet. (B) Autoradiography after in situhybridization 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 afterin 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, umbilicalartery.

Roles of PCSKs in Cholesterol and Triglyceride Metabolism 41

unknown resident hydrolases (Canuel et al., 2013;Seidah et al., 2014; Butkinaree et al., 2015). In view ofthe importance of the cytosolic adaptor protein, autoso-mal recessive hypercholesterolemia (ARH), which con-tains a criticalNP-X-Ymotif for entry into clathrin-coatedendosomes (Lagace et al., 2006), it was thought that thecytosolic tail (CT) of the LDLR (containing an NP-X-Ymotif) controls this ARH-dependent internalization.However, we and others realized that PCSK9 canenhance the degradation of LDLR lacking this CT(LDLR-DCT) or replaced with another one lacking anNP-X-Y motif (Holla et al., 2010; Canuel et al., 2013).Thus, we proposed that the putative Xp may be amembrane-bound protein with an NP-X-Y motif in itsCT. Proteomic analyses of livers of mice lacking or notPCSK9 suggested that the LDLR familymemberLRP1 isalso upregulated in the absence of PCSK9 in liver (Canuelet al., 2013). However, the implication of the PCSK9target LRP1 as a putative Xp was soon eliminated(Canuel et al., 2013). We also recently discarded two

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 iscritical for the in vivo activity of PCSK9 on LDLR inhepatocytes (Butkinaree et al., 2015), and mice lackingboth proteins do not exhibit significantly different LDLRlevels in the liver (Fig. 7). Thus, the identity and necessityof Xp are yet to be defined and validated in cells andin vivo, for the ability of PCSK9 to enhance the degrada-tion of the LDLR and possibly other targets.

A genetic approach showed that the cytosolic adaptorprotein Sec24A implicated in the ER–Golgi traffickingof the coat protein complex COPII vesicles was criticalfor the secretion of PCSK9 from the ER into themedium(Chen et al., 2013). This facilitation is thought to bemediated by a membrane-bound protein that bindsPCSK9 in the ER lumen and Sec24A in the cytosol viaits CT. It was also recently reported that the LDLR doesnot bind efficiently PCSK9 in the ER because of thepresence of a competitive chaperone like-proteinGRP94that prevents such ER interaction (Poirier et al., 2015).Finally, Leren (2014) hypothesized that the membrane-bound PCSK9–LDLR complex is first shed into a solubleform upon entry into acidic endosomes via a cathepsin-like cysteine protease inhibited by E64, which mayfacilitate its efficacious degradation in lysosomes.

Although it was reported that in mice the cytosolicadaptor protein ARH is essential for the ability ofextracellular PCSK9 to enhance the degradation ofthe LDLR in liver hepatocytes (Lagace et al., 2006),recent clinical trials using an inhibitory PCSK9 mAbAlirocumab revealed that absence of ARH in someautosomal recessive hypercholesterolemic patients, al-though reducing the activity of PCSK9, still left room foran ARH-independent pathway, at least as ascertainedin lymphocytes (Thedrez et al., 2016). Similar resultswere also observed in ARH-negative lymphocytes (Fasanoet al., 2009), revealing thepresence of anARH-independentpathway for PCSK9 activity on LDLR. The presumedrelatively lower contribution of this pathway in humanliver (Thedrez et al., 2016) is yet to be unambiguouslyvalidated.

VIII. What Have We Learned from Key NaturalMutations on the Functions of PCSK9 and

Its Partners?

Since the discovery of PCSK9 and its relationship to LDL-c,.100 PCSK9 SNPs were reported (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?geneId=255738) with var-ied consequences (http://www.ucl.ac.uk/ldlr/LOVDv.1.1.0/search.php?select_db=PCSK9&srch=all). Many of theseare silent, others are suspected to be damaging, and somehave been validated for their functional effects. Thestructure of the 692-aa human PCSK9 protein (Fig. 6A)contains a prodomain (aa 31–152) that is noncovalentlyassociated with the mature protease chain (aa 153–692),

Fig. 5. PCSK9 and LDLR mRNA expression at embryonic day 17 (E17)and postnatal day 10 (P10). At E17, in situ hybridization of a sagittalsection with an antisense cRNA probe reveals the high expression ofPCSK9 in the liver, small intestine, cortex of the kidney, and cerebellum.At P10, PCSK9 expression is high in the liver, small intestine, brainolfactory tract, cerebellum, spleen, thymus, colon, caudate putamen, andeye retina inner nuclear layer. LDLR expression is also high in the liver,small intestine, and thymus, and is specifically abundant in thetrigeminal and cranial ganglia. Cb, cerebellum; CG, cranial ganglia; Co,colon; CPu, caudate putamen; Ki, kidney; Li, liver; Mo, molar; OT,olfactory tract; Sin, small intestine; Sk, skin; Sp, spleen; Re, retina; Tg,trigeminal ganglion; Th, thymus. *, **: nonspecific signal.

42 Seidah et al.

keeping it in an enzymatically inactive state. The catalyticsubunit (aa 153–404) is followed by a short hinge domain(Hi; aa 405–452) and the C-terminal CHRD (aa 453–692)(Seidah et al., 2003, 2014; Seidah and Prat, 2012) (Fig.

6A), composed of three repeats called M1, M2, and M3(Cunningham et al., 2007).Mutations can be found in allfour structural componentsofPCSK9.Thecatalytic subunitof PCSK9 directly binds the epidermal growth factor-like

Fig. 6. A. Schematic representation of proPCSK9 (75 kDa, 692 aa) and its processed form PCSK9 (62 kDa) that is noncovalently bound to theautocatalytically processed prodomain (pro; 15 kDa) at VFAQ152-SIP. Notice the active site residues Asp, His, and Ser and the oxyanion hole Asn, aswell as the single N-glycosylation site (in blue). B. Schematic of the extracellular pathway of PCSK9-induced degradation of the LDLR. High PCSK9levels (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 atthe 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 thelatter can be recycled back to the surface after delivery of LDL particles to acidic endosomes/lysosomes. PCSK9 mAbs target the extracellular pathwayby sequestering extracellular PCSK9 and hence preventing its binding to the LDLR.

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 foramyloid precursor-like protein 2 (Aplp22/2) and/or sortilin (Sort1+/+ or Sort12/2) were analyzed for their level of circulating cholesterol and PCSK9 andthose of LDLR protein in liver extracts. No changes were observed. Note that, individually, amyloid precursor-like protein 2 or sortilin deficiency doesnot affect these parameters (Butkinaree et al., 2015).

Roles of PCSKs in Cholesterol and Triglyceride Metabolism 43

domain-A domain of the LDLR with nM potency (Zhanget al., 2007) and is critical for the ability of PCSK9 toenhance the degradation of the LDLR (Zhang et al., 2008;Saavedra et al., 2012).Interestingly, the prodomain of PCSK9 also makes

contact with the b-barrel domain of the LDLR (Lo Surdoet al., 2011), as also evidenced by specific mutationstherein, for example, L108R, that enhance the func-tional activity of PCSK9 in enhancing the degradationof the LDLR in endosomes/lysosomes (Abifadel et al.,2012). However, the various crystal structures ofPCSK9 reported at both neural and acidic pHs with orwithout the ectodomain of the LDLR could not detect aspecific structure for N-terminal aa 31–59 of theprodomain (Cunningham et al., 2007; Lo Surdo et al.,2011). This suggests that alone this 29-aa sequence ofthe prodomain may be quite mobile, most likely predict-ing that a partner may be needed to stabilize in spacethis highly acidic segment, or that it may bind, albeitweakly, to another segment of PCSK9. It has beensuggested that the prodomain may interact withapoB100 on LDL (Kosenko et al., 2013), but not VLDL(Lagace, 2014) particles, although the importance of theaa 31–59 segment of the prodomain in this interactionhas yet to be defined. Interestingly, deletion of the acidicaa 31–51 sequence of the prodomain enhances theaffinity of PCSK9-D31–51 for the LDLR by .sevenfold(Kwon et al., 2008), suggesting that, if apoB binds thisregion, it results in a dampening of PCSK9-bindingaffinity for the LDLR. The physiologic importance of the122–aa prodomain (aa 31–152) is exemplified by muta-tions that result in LOF (e.g., R46L,D97, G106R,Q152H)or GOF (e.g., E32K, D35Y, L108R, S127R) of PCSK9.Japanese patients that are E32K homozygotes have veryhigh LDL-c, but still lower than FH patients thatcompletely lack functional LDLR (Mabuchi et al., 2014).Although many natural mutations within the

PCSK9’s catalytic domain (aa 153–452) result in eitherGOF (Table 1) or LOF (Table 2) (Abifadel et al., 2009),one of the most damaging GOF mutations is the Anglo-Saxon D374Y mutation (Timms et al., 2004), whichenhances the binding affinity of PCSK9 to the LDLRby.10-fold (Cunningham et al., 2007). A correspondingmutation H306Y in LDLR has a similar enhancingeffect on the PCSK9–LDLR complex formation (McNuttet al., 2009). Interestingly, the GOF F216L and R218Smutations reduce or eliminate the ability of furin tocleave and inactivate PCSK9 (Benjannet et al., 2006).That furin processing of PCSK9 at Arg218↓ in vivo(Essalmani et al., 2011) does inactivate PCSK9 wasrecently confirmed by functional analysis of humanplasma PCSK9 (Han et al., 2014). Furthermore, suchprocessing was shown to extend the half-life of a distinctanti-PCSK9 mAb that recognizes the N-terminal se-quence aa 168–181, which, different from Evolocumabor Alirocumab, does not sterically impede the inactivat-ing furin cleavage at Arg218↓ (Schroeder et al., 2015).

Mutations within the hinge domain and the CHRDdomain also resulted in either GOF or LOF of PCSK9(Tables 1 and 2) (Abifadel et al., 2009). The function ofthese domains relates to their regulation of the ability ofthe PCSK9–LDLR complex to be directed to lateendosomes/lysosomes for degradation (Nassoury et al.,2007; Zhang et al., 2008; Poirier et al., 2016) (Fig. 6B,left part). However, the underlying mechanism and theimportance of post-translational modifications, for ex-ample, Asn533 glycosylation (Seidah et al., 2003) or Ser688phosphorylation (Gauthier et al., 2015; Tagliabracciet al., 2015) in this process are still unknown. In vitrobiochemical experiments suggested that the CHRDmayinteract with the ligand binding domain of the LDLR atacidic pHs (Yamamoto et al., 2011), but this is not evidentfrom the available PCSK9–LDLR crystal structures (LoSurdo et al., 2011). We suggested that the CHRD maybind an as yet unknown protein X that would then directthe PCSK9–LDLR complex toward late endosomes/lysosomes (Seidah et al., 2014; Butkinaree et al., 2015).Alternatively, theCHRDmaydisplace a protein requiredfor the default recycling of the LDLR to the cell surfacefound in most tissues (Fig. 6B, right part), therebyallowing the PCSK9–LDLR complex to reach the degra-dation compartments. It is thus conceivable that, in thefuture, specific PCSK9 and/or LDLR mutations may beidentified that would disrupt or enhance the interactionwith such putative regulator and hence favor the recy-cling or degradation pathways of the LDLR.

IX. Ongoing Outcome Clinical Trials

Several pharmaceutical strategies were and are beingdeveloped to reduce PCSK9 levels/activity on LDLRdegradation and were reviewed (Seidah et al., 2014;Elbitar et al., 2016; Stein, 2016). In essence, anencapsulated in nanoparticles antisense RNA interfer-ence oligonucleotide when i.v. injected once at 0.4mg/kgresulted in;70% and;40% drops in PCSK9 and LDL-clevels, respectively (Fitzgerald et al., 2014). It is nowbeing tested in phase 2 clinical trials. However, thepresent, most prevalent approach consists in blockingmAbs that inhibit the binding of extracellular orcirculating PCSK9 to the LDLR (Stein and Raal,2014). Three human mAbs are now in multiple phase3 clinical trials. Two of them, Evolocumab (Repatha;Amgen; Thousand Oaks, CA) and Alirocumab (Praluent;Sanofi; Bridgewater, NJ), are already prescribed toFH patients worldwide, whereas the Bococizumab(Pfizer; Groton, CT) is not yet available commercially(Ito and Santos, 2016). However, very recently Pfizerdiscontinued the global development of Bococizumab,as the efficacy of this noncompletely humanized mAbdecreased with time (http://www.pfizer.com/news/press-release/press-release-detail/pfizer_discontinues_global_development_of_bococizumab_its_investigational_pcsk9_inhibitor). In all cases, the s.c. injection of either 150 mg

44 Seidah et al.

every 2 weeks or 420 mg every 4 weeks results in aconsistent and sustained ;50–60% reduction in LDL-c.The safety and tolerability profile is very good, especiallyfor hypercholesterolemia patients who fail to obtain anoptimal clinical response to statin therapy, those whoare statin-intolerant or have contraindications tostatin therapy. The combination of these mAbs withother lipid-lowering agents such as statins or thenonstatin ezetimibe/fenofibrate resulted in a modest(#10%) further decrease in LDL-c (Ito and Santos,2016; Rey et al., 2016).Evolocumab is currently being tested in a large-scale

FOURIER study (27,500 patients) to evaluate its impacton the occurrence of CVD death, nonfatal myocardialinfarction, or hospitalization for unstable angina, stroke,or coronary revascularization (NCT01764633). Theestimated completion date is 2018. The ODYSSEYOUTCOMES study (;18,000), which should also becompleted in 2018, is designed to determine the impactof Alirocumab over a treatment period of 64 months(NCT01663402). The ongoing SPIRE-1/-2 trials willanalyze the effect of Bococizumab (Ballantyne et al.,2015) by 2017–2018, although the future of this study isnow in doubt. Outcome data should provide concreteevidence for the long-term clinical benefits of reducingPCSK9 activity or its levels. A glimpse of what might beexpected was reported for patients treated with eitherEvolocumab (Sabatine et al., 2015) or Alirocumab(Robinson et al., 2015), showing a very encouraging and

significant;50% reduction in major adverse cardiovas-cular events following more than 1 year of treatment.

X. Other Genes and Loci Implicated inLDL-c Regulation

About 50 years after the pioneering work ofKhachadurian (1964) on the genetics of FH in Lebanesefamilies, and ;40 years after the discovery by Brownand Goldstein (1974) of the LDL receptor that paved theway to statin therapy, our discovery of PCSK9 and itsimplication in FH opened the way to intensive studiesby researchers and pharmaceutical companies, leadingto a promising new class of lipid-lowering drugs: thePCSK9 mAbs. The race to discover new genes impli-cated in FH and its cardiovascular complications is stillvery vigorous. In 2001, the LDLRAP1 gene encodingARH was identified in families suffering from autoso-mal recessive hypercholesterolemia (Garcia et al.,2001). PCSK9 was the third gene implicated in ADHafter the LDLR and APOB genes (Innerarity et al.,1987). Familial mutations in the APOE gene wererecently linked to ADH (Awan et al., 2013; Marduelet al., 2013). Other mutations have been reported ingenes encoding proteins implicated in cholesterol me-tabolism or regulation, notably SREBP2 (Muller andMiserez, 2002) andCYP7A1, but they are limited to rarecases (Pullinger et al., 2002).

TABLE 1PCSK9 gene GOF mutations causing autosomal dominant hypercholesterolemia

Protein Position NucleotidePosition Exon Geographical

Origin of Patients Clinical Features References

p.Val4Ile c.10G . A 1 Japan CAD, PVD, arcus, xanthomas Hopkins et al., 2015p.Glu32Lys c.94G . A 1 Japan CAD, stroke, arcus, xanthoma Miyake et al., 2008pAsp35Tyr 103G . T 1 France Mild dyslipidemia Abifadel et al., 2012p.Glu48Lys 142G . A 1 Netherlands Mild dyslipidemia Hopkins et al., 2015p.Pro71Leu 212C . T 2 Netherlands CAD, stroke Hopkins et al., 2015p.Arg96Cys 286C . T 2 Netherlands CAD Hopkins et al., 2015p.Leu108Arg 323T . G 2 France: Republic of

MauritiusCAD Abifadel et al., 2012

p.Ser127Arg c.381T . A 2 2 French families Tendon xanthomas Abifadel et al., 2003; Cameronet al., 2006; Nassoury et al., 20071 South African patient CAD, early MI

1 Norwegian patient Stroke, arcusp.Asp129Gly c.386A . G 2 1 New Zealand family MI strong family history of CVD Homer et al., 2008p.Asp129Asn c.385G . A 2 United Kingdom CAD Hopkins et al., 2015p.Arg215His c.644G . A 4 2 Norwegian families CAD Cameron et al., 2006p.Phe216Leu c.646T . C 4 1 French family Early MI Abifadel et al., 2003; Benjannet

et al., 2004p.Arg218Ser c.654A . T 4 1 French family Tendinous xanthomas, arcus cornea Benjannet et al., 2004; Allard

et al., 2005p.Arg357His c.1070G . A 7 1 French proband Family history of CVD Allard et al., 2005p.Asp374Tyr c.1120G . T 7 1 Utah family Achilles tendon xanthomas, arcus, Benjannet et al., 2004; Leren,

2004; Timms et al., 2004;Naoumova et al., 2005

3 Norwegian families Premature CAD3 English families

United Kingdom,United States

More stringent treatment

p.Asp374His c.1120G . C 7 2 Portuguese probands+ 1 relative

Severe phenotype. PrematureCAD, arcus, xanthomas

Bourbon et al., 2008

Francep.Ser465Leu c.1394 C . T 9 Netherlands CAD Hopkins et al., 2015p.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.

Roles of PCSKs in Cholesterol and Triglyceride Metabolism 45

Using classic approaches based on positional cloningand linkage analysis, presumptive new loci for ADHweremapped to 8q24.22 (Cenarro et al., 2011) and to 16q22.1(Marques-Pinheiro et al., 2010). In the next-generationsequencing era, parametric linkage analysis combinedwith exome sequencing in anADH family resulted in theidentification of the variant p.Glu97Asp in signal-transducing adaptor family member 1 (Fouchier et al.,2014). Other approaches using exome sequencing (Stitzielet al., 2015) or genome-wide association studies (GWAS)(Patel et al., 2016), however, did not identify any majornew gene. This may be explained by the difficulty todiagnose polygenic cases in ADH, estimated to ;13%(Talmud et al., 2013), a value close to the percentage ofno LDLR, no APOB, and no PCSK9 cases.

XI. Implication of the Enzymatically Active PCsin CVD

PCSK9 is not the only PC to be implicated in CVD,and relevant studies on other PCs were reviewed

previously (Taylor et al., 2003; Seidah and Prat, 2007,2012; Seidah et al., 2008, 2013; Turpeinen et al., 2013;Ramos-Molina et al., 2016). In this work, we will onlybriefly summarize our present understanding of theirphysiologic and pathologic roles in both health and inCVD.

A. PC1/3 and PC2 in Diabetes and Obesity

PC1/3 and PC2 activate by cleavage most of thepolypeptide hormone precursors in endocrine and neu-ral tissues. The lack of either convertase results indiseases such as obesity or diabetes (Fig. 1). PC1/3and PC2 sequentially cleave proinsulin into insulin(Smeekens et al., 1992; Steiner, 2011). The two enzymesalso differentially cleave pro-opiomelanocortin in atissue-specific manner to generate adrenocorticotropinhormone in corticotrophs of the pituitary gland ora-melanotrophic hormone and b-endorphin in the hy-pothalamus and pituitary melanotrophs, respectively(Benjannet et al., 1991; Seidah et al., 2013). In fact,PC1/3 alone or in combination with PC2 can generate

TABLE 2PCSK9 gene LOF mutations and polymorphisms causing hypocholesterolemia

Protein Position Nucleotide Position Exon Geographical Origin ofPatients Clinical Features References

Leucine stretch:p.L21dup alsodesignatedp.L15_L16insL

c.dup61_63CTG alsodesignatedc.43_44insCTG

1 Frequently found inseveral populations

Decrease of LDL-c levels of10–15 mg/dL

Abifadel et al., 2003;Chen et al., 2005; Yueet al., 2006

p.Arg46Leu c.137 G . T 1 Frequently found inseveral populations

Associated with a 15% reduction ofLDL-c and 47% of CHD inwhites; reduction of the risk ofearly-onset myocardialinfraction

Abifadel et al., 2003; Cameronet al., 2006; Cohen et al., 2006;Kotowski et al., 2006; Scarteziniet al., 2007; Dewpura et al.,2008; Kathiresan et al., 2008

p.A68fsL82X c.202delG 1 Sicily: 1 kindred Fatty liver in the proband and hisfather but not in blood donorsfrom healthy carriers

Fasano et al., 20072 hypocholesterolemic

blood donorsp.Thr77Ile c.230C . T 2 Sicily Hypocholesterolemic blood donors Fasano et al., 2007p.Arg104Cys c.310C . T 2 French R104C/V114A segregates with

FHBL; dominant negative whenassociated with c.341T . C(p.Val114Ala) on the sameallele: impairs processing andsecretion of wild-type PCSK9 inliver; low plasma PCSK9concentration, LDL clearancerate .200% of normal

Cariou et al., 2009

p.Gly106Arg c.316G . A 2 1 Norwegian family Hypocholesterolemic subjects; lowautocatalytic cleavage

Berge et al., 2006; Nassouryet al., 2007

p.Val114Ala c.341T . C 2 Sicily Hypocholesterolemic blood donors Fasano et al., 2007p.Tyr142X c.426C . G 3 0.4% DHS1 Reduction of LDL-c levels of 40%

and CHD of 88%Cohen et al., 2006; Kotowski

et al., 20060.8% ARIC2 of AfricanAmericans

p.Q152H c.436G . C 3 French-Canadian Reduction of PCSK9 levels by 79%and LDL-c by 48%

Mayne et al., 2011

p.Leu253Phe c.757C . T 5 Black American andHispanic

Hypocholesterolemic Kotowski et al., 2006

p.Trp428X c.1284G . A 8 1 Japanese subject Hypocholesterolemic: completeLOF due to early termination

Miyake et al., 2008

p.Ala522Thr c.1564G . A 10 Sicily Hypocholesterolemic blood donors Fasano et al., 2007p.Gln554Glu c.1660C . G 10 Black American,

HispanicMild reduction in LDL-c levels Kotowski et al., 2006

p.Pro616Leu c.1847C . T 11 Sicily Hypocholesterolemic blood donors Fasano et al., 2007p.Cys679X c.2037C . A 12 1.4% (DHS); 1.8%

(ARIC)Reduction of LDL-c levels of 40%

and CHD of 88% forheterozygous carriers

Cohen et al., 2006; Kotowskiet al., 2006

Blacks. ,0.1% of WhiteAmericans

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

46 Seidah et al.

both anorexigenic hormones (a-melanotrophic hor-mone, insulin, cholecystokinin, and glucagon-likepeptide 1) and orexigenic peptides (neuropeptide Y,cocaine- and amphetamine-regulated transcript, andorexin/hypocretin). It was thus not a surprise thatsubjects lacking functional PC1/3 are obese (Jacksonet al., 1997).PCSK1LOFmutations can be found in;1%of morbidly obese patients (Creemers et al., 2012), andPCSK1 represents the third most prevalent monogeniccontributor to the risk of developing obesity (Choquetet al., 2013). To date, no human PC2 mutations associ-ated with disease have been found. Interestingly,whereas PC1/3 and PC2 are critical for the generationof insulin, the absence of functional PC1/3 in humans hasnot been associated with insulin resistance or diabetes(Stijnen et al., 2016), suggesting the presence of compen-satory pathways and/or modifier genes.

B. Furin and SKI-1/S1P in Cardiovascular Diseases

SKI-1/S1P is expressed in all cells, as furin. Theimportance of SKI-1/S1P in lipid metabolism has beendiscussed at the beginning of this review. It is encodedby the gene MBTPS1 for which no missense mutationswere associated with disease, and a .90% reduction ofSKI-1/S1P expression is required to decrease choles-terol and TG production in liver (Yang et al., 2001). Thecomplete KO of this gene in mice leads to very earlyembryonic lethality (Mitchell et al., 2001).Furin is essential during development because de-

ficient mouse embryos die at embryonic day 11 andexhibit multiple developmental abnormalities, particu-larly defects related to the function of endothelial cells(Roebroek et al., 1998). To define the role of furin inendothelial cells, a mouse endothelial cell–specific KO(ecKO) of the Furin gene was generated (Kim et al.,2012). Newborns die shortly after birth, and magneticresonance imaging revealed ventricular septal defectsand/or valvemalformations. In addition, primary culturesof wild-type and ecKO lung endothelial cells revealed thatecKO cells are unable to grow, but are rescued byextracellular soluble furin. These cells are deficient forendothelin-1, transforming growth factor-b1, adrenome-dullin, and bone morphogenetic protein 4 processing. Themature forms of the two last ones were reduced by;90%.Thus, furin is a major regulator of cardiac functions, viathe processing of multiple substrates. Recently, a GWASrevealed that a nucleotide A to C substitution in intron1 of the FURIN gene (rs17514846) was significantlyassociated with the development of the metabolic syn-drome, with the protectiveminor A allele correlating witha 21% decreased serum TG (Ueyama et al., 2015).Interestingly, furin inactivates lipoprotein lipase, whichis implicated in the hydrolysis of TG into fatty acids fromTG-rich lipoproteins (Jin et al., 2005; Lei et al., 2011). Theprotective A allele in rs17514846may thus be a furin LOFmutation leading to increased lipoprotein lipase activity,and hence to lower TG.

C. PACE4 in Blood Pressure Regulation

PACE4 is a cell surface enzyme bound to heparinsulfate proteoglycans that activates a number of recep-tors and growth factors (Seidah et al., 2008). In cardio-myocytes, PACE4 activates the serine protease corinthat produces atrial natriuretic peptide/atrial natriureticfactor (Chen et al., 2015). Moreover, the authors identi-fied in a hypertensive patient a dominant-negativeD282N mutation in PACE4 that impedes the intracellu-lar autoactivation of proPACE4 into PACE4. MutatedproPACE4 is retained in the ER in complex with normalPACE4, thereby inhibiting corin activation (Chen et al.,2015). Atrial natriuretic peptide is a hormone essentialfor sodium homeostasis (Flynn et al., 1983; Seidah et al.,1984) and reduces blood pressure (Yan et al., 2000).In agreement, PACE4 KO mice were found to be hyper-tensive on a normal salt (0.3% NaCl) diet, and thisphenotype was exacerbated when the mice were placedon high-salt (4% and 8% NaCl) diets, indicating thatcorin activation by PACE4 is essential for maintainingnormal blood pressure. Consistent with these findings,the PCSK6 gene was previously linked to hypertensionin humans (Li et al., 2004).

D. PC5/6 in Heart Development and ExtracellularMatrix Remodeling

Mice lacking PC5/6 die at birth and perfectly re-capitulate the phenotypes of mice lacking the trans-forming growth factor-b–like growth differentiationfactor 11 (Gdf11, also known as bone morphogeneticprotein 11) (McPherron et al., 1999), including theabsence of kidneys and major defects in the antero-posterior axis with extra-thoracic and -lumbar verte-brae, and a lack of tail (Essalmani et al., 2008; Szumskaet al., 2008). In agreement, Gdf11 is exclusively acti-vated by PC5/6 cleavage. In that context, high Gdf11levels were associated with frailty, susceptibility todiabetes, or cardiac complications in old adults withsevere aortic stenosis (Schafer et al., 2016). Thus, PC5/6inhibition may reduce cardiac Gdf11 production. Tocircumvent lethality, studies were done in tissue-specific ecKO. Old ecKO mice lacking PC5/6 exhibit acardiovascular hypotrophy associated with decreasedextracellular matrix collagen deposition, decreased leftventricular diastolic function, and vascular stiffness,suggesting a trophic role of the enzyme PC5/6most likely mediated by insulin-like growth factor 1/protein kinase B, also known as Akt/mammalian targetof rapamycin signaling and control of autophagy(Marchesi et al., 2011). Among the endothelial cellsurface precursors known to be cleaved by PC5/6 isthe receptor protein tyrosine phosphatase receptorprotein tyrosine phosphatase m (Campan et al., 1996),of which KO in mice results in increased stiffness ofmesenteric arteries (Koop et al., 2003), whereas ourecKO mice show the opposite phenotype (Marchesi

Roles of PCSKs in Cholesterol and Triglyceride Metabolism 47

et al., 2011). Finally, human intronic PCSK5 SNPscorrelate with low levels of HDL cholesterol (Iatanet al., 2009), a correlation modulated by dietarypolyunsaturated fatty acids (Jang et al., 2014).

E. PC7 in HDL, small dense LDL, and TG

Human GWAS studies suggest that PCSK7 LOFmutations result in favorable CVD parameters, that is,high HDL, low TG (Guillemot et al., 2014; Peloso et al.,2014; Yao et al., 2015), low highly atherogenic smalldense LDL (Hoogeveen et al., 2014), and reduced insulinresistance (Huang et al., 2015). Understanding the PC7biology underlying these phenotypes should lead topowerful novel therapies for cardiometabolic disorders.

XII. Conclusions and Future Perspectives

The original hypothesis of the existence of secretoryprecursors, which upon limited proteolysis releasesbioactive peptides or proteins, has been validated inmultiple precursor proteins from various species(Chrétien, 2011; Steiner, 2011). The arduous process ofhunting for the cognate proteinases responsible for suchprocessing led to the identification of nine PCs withvaried physiologic functions, which are sometimes asso-ciated with pathology (Seidah and Prat, 2012). Thecumulative knowledge over the last 26 years led us topropose that inhibiting some of these convertases may insome cases be favorable to specific patients (Fig. 1). Themost advanced target is PCSK9, as injectable inhibitorymAbs are now prescribed in clinics for the treatment ofhypercholesterolemia, and will most likely be used forthe prevention of septic shock. In the future, it is hopedthat modulation of some of the other PCs for specificconditions may have new clinical applications, for exam-ple, PC7 in anxiety regulation (Wetsel et al., 2013), andpossibly in hypertriglyceridemia (Peloso et al., 2014).The present review exposed the many new facets of

PCSK9 and its biology, concentrating only on its abilityto enhance the degradation of the LDLR and VLDLR.However, PCSK9 has been shown to target for degra-dation other members of the LDLR family, includingApoER2 (Poirier et al., 2008), the fatty acid transporterCD36 (Demers et al., 2015), in liver and other tissues,such as small intestine, pancreas, brain, and adipocytes(Seidah et al., 2014). A lot more remains to be unraveledregarding the cellular trafficking of PCSK9 togetherwith its targeted receptors, its complex web of interact-ing proteins, and its influence on the fate or levels ofother proteins. Furthermore, PCSK9 has been shown tohave a regulatory role of inflammatory processes in-dependent from its effects on LDL-c (Leander et al.,2016). This is clearly a very exciting period in the field ofdyslipidemia, where, thanks to new PCSK9-silencingtherapies, LDL-c levels were lowered to unprecedentedlow levels, reaching 0.4 mM and lower. This is de-finitively good news for hypercholesterolemic patients

who do not reach target levels of LDL-c with theavailable medications, cannot tolerate statins, or whoexperience painful side effects with statins, such asmuscle pain, cramps, weakness to myopathy, and,rarely, rhabdomyolysis. Notably, homozygote FH pa-tients who have minimal LDLR activity left can now betreated with PCSK9 mAbs, resulting in a ;30% de-crease in circulating LDL-c (Raal et al., 2015). In theseFH patients, switching from two to three times weeklyLDL apheresis to a PCSK9 mAb combined or not withonce per week LDL-c apheresis maintained the LDL-c-lowering effect, thereby giving a much better quality oflife to these patients that is less dependent on the use ofbiweekly long sessions to clear LDL-c from their bloodusing special apheresis dialysis columns (Lappegårdet al., 2016). Although the outcomes of the variousongoing phase 3 clinical trials using PCSK9 mAbs willnot be known until 2017–2018 (Elbitar et al., 2016),already early signs revealed that this treatment resultsin a ;50% reduction in cumulative cardiovascularevents within 1–2 years of treatment (Robinson et al.,2015; Sabatine et al., 2015). However, one should becautious, as these results are only partial andnot poweredenough to unambiguously assess cardiovascular out-comes of prespecified endpoints in cardiometabolicdiseases. Finally, the fact that PCSK9 is inactivatedby some proteases such as furin (Benjannet et al., 2006;Essalmani et al., 2011) might open new strategies toenhance this inactivation mechanism, and thus lowerthe levels of active PCSK9. Indeed, recently a newmAbwas reported that inhibits PCSK9 activity but stillallows furin to inactivate it, resulting in a longer half-life of the antibody (Schroeder et al., 2015). The futurewill tell which strategies targeting PCSK9, includinglonger lasting mAbs, RNA interference (Fitzgerald et al.,2014), and small-molecule inhibitor approaches (Seidah,2013), will find their way in dyslipidemia, cardiology,and sepsis in clinics worldwide, which would result inaffordable and safe treatments and/or prevention of life-threatening conditions.

Acknowledgments

The authors thankBrigitteMary for efficacious editorial assistance.

Authorship Contributions

Participated in research design: Seidah, Abifadel, Prost, Boileau,Prat.Conducted experiments: Seidah, Prost, Prat.Performed data analysis: Seidah, Prost, Prat.Wrote or contributed to the writing of the manuscript: Seidah,

Abifadel, Prost, Boileau, Prat.

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