PCSK9

Endocrine Reviews, 2022, Vol. 43, No.
3, 558–582
https://doi.org/10.1210/endrev/bnab035
Review
Review
The Multifaceted Biology of PCSK9

Nabil G. Seidah,1 and Annik Prat1
1
Laboratory of Biochemical Neuroendocrinology, Montreal Clinical Research Institute (IRCM, affiliated to
Downloaded from https://academic.oup.com/edrv/article/43/3/558/6385885 by guest on 25 August 2022

the University of Montreal), Montreal, QC, H2W 1R7, Canada
ORCiD numbers: 0000-0001-6503-9342 (N. G. Seidah); 0000-0002-8007-5277 (A. Prat).
Abbreviations: aa, amino acids; CAP1, cyclase-associated protein 1; CHRD, Cys/His-rich domain; DAA, direct-acting
antiviral; DENV, Dengue virus; E2, 17β-estradiol; ER, endoplasmic reticulum; FH, familial hypercholesterolemia; GOF, gain-
of-function; HCV, hepatitis C virus; KO, knockout; IFN, interferon; LDLc, low-density lipoprotein cholesterol; LDLR, LDL
receptor; LNP, lipid nanoparticle; LOF, loss-of-function; MHC, major histocompatibility complex; PC, proprotein convertase;
PD, programmed death; PTM, post-translational modification; SK, subtilisin/kexin; SRE, sterol regulatory element; TCR,
T-cell receptor; wild type, WT.
Received: 2 July 2021; Editorial Decision: 4 October 2021; First Published Online: 9 October 2021; Corrected and Typeset:
2 November 2021.
Abstract
This article reviews the discovery of PCSK9, its structure–function characteristics, and
its presently known and proposed novel biological functions. The major critical func-
tion of PCSK9 deduced from human and mouse studies, as well as cellular and struc-
tural analyses, is its role in increasing the levels of circulating low-density lipoprotein
(LDL)-cholesterol (LDLc), via its ability to enhance the sorting and escort of the cell sur-
face LDL receptor (LDLR) to lysosomes. This implicates the binding of the catalytic do-
main of PCSK9 to the EGF-A domain of the LDLR. This also requires the presence of
the C-terminal Cys/His-rich domain, its binding to the secreted cytosolic cyclase associ-
ated protein 1, and possibly another membrane-bound “protein X”. Curiously, in PCSK9-
deficient mice, an alternative to the downregulation of the surface levels of the LDLR by
PCSK9 is taking place in the liver of female mice in a 17β-estradiol-dependent manner
by still an unknown mechanism. Recent studies have extended our understanding of the
biological functions of PCSK9, namely its implication in septic shock, vascular inflamma-
tion, viral infections (Dengue; SARS-CoV-2) or immune checkpoint modulation in cancer
via the regulation of the cell surface levels of the T-cell receptor and MHC-I, which govern
the antitumoral activity of CD8+ T cells. Because PCSK9 inhibition may be advantageous
in these processes, the availability of injectable safe PCSK9 inhibitors that reduces by
50% to 60% LDLc above the effect of statins is highly valuable. Indeed, injectable PCSK9
monoclonal antibody or small interfering RNA could be added to current immunother-
apies in cancer/metastasis.
Key Words: β-cells, cancer/metastases, hypercholesterolemia, major histocompatibility complex I, sepsis
ISSN Print: 0163-769X

ISSN Online: 1945-7189
Printed: in USA
© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-
558 https://academic.oup.com/edrv NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction
and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and
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Endocrine Reviews, 2022, Vol. 43, No. 3 559
Graphical Abstract
By modulating the trafficking of key secretory proteins, PCSK9 is implicated in the regulation of major diseases. Secreted
PCSK9 shortens the half-life of cell surface receptors, such as LDLR and MHC-I, by escorting them into the lysosomal
pathway. The functional consequences of PCSK9 activity in different diseases is indicated.

ESSENTIAL POINTS
• PCSK9 is the third gene implicated in familial hypercholesterolemia, after LDLR and APOB genes.
• PCSK9 mAbs or siRNA over statin treatment reduce plasma LDL-cholesterol levels by a further 60%.
• PCSK9 is highly expressed and secreted by hepatocytes and β-cells.
• Circulating (liver) or locally secreted PCSK9 shortens the half-life of cell surface receptors, such as the LDLR,
VLDLR, or MHC-I, by directing them to the lysosomal degradation pathway.
• Thereby, PCSK9 modulates major functions, such as liver and peripheral lipoprotein uptake or antitumor immunity.
The first critical connection between cholesterol and heart to 25% to 50% reduction of low-density lipoprotein chol-
disease was initially reported in 1913 in St. Petersburg by esterol (LDLc) (5). Up to 2003, the reported major func-
Nikolai Nikolajewitsch Anitschkow based on his seminal tion of statins was to decrease LDLc levels by modulating
observation of the accelerated development of athero- cholesterol homeostasis in the liver. Mechanistically, by re-
sclerosis in the arterial intima of rabbits fed a high chol- ducing the synthesis of cholesterol, treatment with statins
esterol diet [reviewed in (1)]. Key epidemiological studies results in the activation of the sterol regulatory element
(2) and the similarity of atherosclerosis to the human path- binding protein SREBP-2 in the endoplasmic reticulum
ology of heart disease catalyzed a 60-year-long search for (ER) (6), whereupon its N-terminal domain reaches the
cholesterol-lowering agents. In 1973, Akira Endo and his nucleus and enhances the synthesis of the LDL receptor
team screened 3800 fungal strains to discover a potent (LDLR) following its association with sterol regulatory
β-hydroxy β-methylglutaryl-CoA (HMG-CoA) reductase elements (SREs) in the LDLR promoter (7). This in turn
reversible inhibitor, mevastatin (also called compactin) (3). activates cholesterol biosynthetic genes, leading to an in-
In 1987, a team at Merck Pharmaceutical Co. developed creased LDLc uptake from circulation. This exquisite feed-
a chemically related compound, lovastatin (trademark back mechanism balances cellular cholesterol levels and
Mevacor), which became the first statin to reach patients decreases the amount of circulating LDLc (for excellent
(4). This was followed by a successive improvement in reviews see (8, 9)).
the potency and specificity of statins: Zocor (simvastatin), The level of plasma cholesterol is influenced not only
Lipitor (atorvastatin), and Crestor (rosuvastatin), which led by its de novo biosynthesis, but also by the absorption of
560 Endocrine Reviews, 2022, Vol. 43, No. 3
dietary cholesterol and the removal of cholesterol from the paired basic amino acids (aa). Such a concept of limited
blood. Blocking the absorption of dietary cholesterol by the proteolysis of prohormones by cellular proteases was later
gut through inhibition of Niemann-Pick C1-Like 1 protein extended to many secretory proteins and even to infec-
on small intestinal epithelial cells and hepatocytes is the tious pathogens, and shown to occur at single and paired
basis of the mechanism of action of ezetimibe, a drug first basic residues within the motif: (K/R)-Xn-(K/R)↓, or
described by Schering–Plough (10). When combined with a more commonly (K/R)-Xn-(R)↓, where Xn are spacer res-
statin, ezetimibe further reduces LDLc by 15% to 20% (11) idues comprising 0, 2, 4, or 6 aa (27). It took more than
and improves cardiovascular outcomes (12). 15 years to identify the cognate processing enzymes that
In this review, we will introduce the family of proprotein recognize these motifs. They constitute a family of 7 basic
convertases (PCs) and the implication of some members in aa-specific serine proteases, the PCs, related to subtilisin/
cholesterol and fatty acid metabolism. We will then concen- kexin (SK), and their genes were mostly given the name
trate on PCSK9 implicated in the enhanced degradation of PCSKs (for extensive reviews see (27, 28)). In essence, PC1

the LDLR, and hence clearance of LDLc from circulation. (gene PCSK1) and PC2 (gene PCSK2)/7B2 (7B2: chap-
We will then present some of the multifaceted functions erone of PC2) are the principal convertases responsible for
of this fascinating protein and highlight knowledge gaps the generation of regulated bioactive hormones stored in
that remain to be addressed. We do not aim to exhaustively dense-core secretory granules, such as insulin; ACTH/α-
cover all information regarding the various tissue-specific MSH/β-endorphin, glucagon/GLP1,2; gastrin; enkephalins;
functions of PCSK9 in health and disease, but rather our TRH, etc. The other members are either ubiquitously ex-
focus has been placed on specific novel avenues, which we pressed (type-I membrane-bound Furin (gene Fur) and PC7
believe deserve more extensive investigations. (gene PCSK7)), widely expressed (PC5 (gene PCSK5) and
PACE4 (gene PCSK6)), or synthesized only in gonadal tis-
sues (PC4 (gene PCSK4)). All PCs are activated following
The Family of Proprotein Convertases 1 or 2 autocatalytic cleavages that remove their intramo-
Secretory proteins undergo various post-translational lecular N-terminal chaperone-like prodomain, starting in
modifications (PTMs) before reaching their intra/extra- the ER (except for PC2) and continuing in the trans-Golgi
cellular destination(s), thereby expanding and diversifying network (Furin), immature secretory granules (PC1 and
their functions (13). N- and O-glycosylation are major PC2), at the cell surface (PC5, PACE4, and possibly PC4)
irreversible modifications that result in substantial struc- or in early endosomes (PC7). Thus, no basic aa-specific PC
tural rearrangements that affect many functions and fates is active in the ER or early Golgi compartments, as they
of secretory proteins (14, 15). Reversible phosphorylation remain noncovalently associated with their respective in-
at specific Ser, Thr, and Tyr residues in secretory proteins hibitory prodomain until they reach their destination(s),
is performed by a limited number of secretory kinases, whereupon the cellular conditions (pH, calcium, etc.) are
FAM20C being the best studied one for Ser/Thr phosphor- suitable for prodomain removal either by further autocata-
ylation (16, 17). One of the major irreversible PTMs is the lytic cleavage (PC1, PC2, Furin, PC5, PACE4) or physical
limited proteolysis of selected peptide bonds (18), which separation (PC4, PC7). Although most PC cleavages result
depends on the specificity of processing enzyme(s) and in the activation of precursor proteins or generation of
the accessibility of scissile peptide bonds to the attacking novel functions, some cleavages lead to substrate inactiva-
protease (19). Inactive precursor protein processing, often tion (29). For example, N-cadherin is activated by Furin,
occurring in response to a stimulus, results in a prompt but inactivated by PC5 (29), and as discussed later PCSK9
physiological response including but not limited to blood is inactivated by Furin (30).
coagulation, fibrinolysis, active hormone production, fertil- It was also realized that processing of precursor proteins
ization, metamorphosis, and digestion. While some PTMs can also occur by cleavage at nonbasic residues within the
occur after the protein is secreted, precursor processing early secretory pathway. Typical examples are the sterol
within the secretory pathway plays a prominent role in the regulatory element binding proteins 1 and 2 (SREBP-1,
activation of a wide variety of secretory proteins such as 2) (31) and the surface glycoproteins of hemorrhagic fever
polypeptide hormones, growth factors, receptors, enzymes, viruses of Lassa virus (32) or Crimean Congo hemorrhagic
and surface glycoproteins (19, 20). fever virus (33) that are activated in the cis/medial Golgi.
Since the 1960s, it was realized that polypeptide hor- Using degenerate oligonucleotides hybridizing the active
mones such as insulin (21, 22), melanotropins (α- and site encoding mRNA of convertases and amplification by
β-melanotropin stimulating hormone [MSH]) (23) and polymerase chain reaction, we identified in 1999 the eighth
β-endorphin (24-26) are produced from longer, primarily member of the PCSK family and named it subtilisin-kexin
inactive, precursor proteins via successive cleavages at isozyme 1 (SKI-1) (34). This enzyme cleaves pro-Brain
Derived Neurotrophic Factor at R-G-LT↓S-L, where the nearby to the 1p34.1p32 locus identified in large French fam-
bold and italic residues are critical for cleavage by SKI-1 ilies potentially encoding a third gene for autosomal dominant
(34). The latter turned out to be the same protease (called site familial hypercholesterolemia (FH3), where the LDLR and
1 protease; S1P) identified in 1998 that activates SREBP-1, apolipoprotein B (APOB) genes were excluded. This locus was
-2 (35) and later on the ER stress factor ATF6 (36). Indeed, associated with increased hepatic secretion of cholesterol asso-
in all subsequent substrates of SKI-1/S1P (gene MBPTS1) ciated with very low–density lipoprotein (VLDL), which after
(37) the general consensus cleavage motif was found to be its secretion is converted into LDLc (53, 54). Armed with this
R-X-Aliphatic-Z↓, where X is any residue except Pro and information and the demonstration of the major PCSK9 ex-
Cys, and Z is any aa (best Leu) except Val, Pro, Cys, or pression in the liver (51), we contacted the French team led
Glu (34, 37-40). For an in-depth analysis of the multiple by Catherine Boileau, who was keenly interested in this re-
functions of SKI-1/S1P, including cholesterol and fatty acid gion of chromosome 1. Intensive genetic analyses in 23 French
regulation, neuronal development, lysosomal homeostasis, families, with no LDLR or APOB variations, by a positional

hypertension, activation of membrane-bound transcription cloning approach using genetic mapping, physical mapping,
factors and viral infections the reader is referred to recent and sequencing of several genes led to their identification of a
reviews on this ever-expanding subject (28, 41-49). missense variant, S127R, in the 22-kb PCSK9 gene in 2 large
families and another variant, F216L, in a third family (52).
This fruitful and joyful collaboration between a Canadian
The Discovery of PCSK9 team working on PCs and a French one deciphering the gen-
The identification of SKI-1/S1P as a convertase that etics of hypercholesterolemia culminated in the first report that
cleaves precursor proteins in the early cis/medial Golgi appeared in June 2003, with first author Marianne Abifadel
and regulate important physiological functions, such as revealing that human PCSK9 was the FH3 gene critical for
cholesterol and fatty acid metabolism (35), ER stress LDLc regulation (52).
(36), as well as the phosphorylation of mannose residues Analysis of PCSK9 biosynthesis in cells expressing
of proteins destined for lysosomal sorting (50), suggested human, rat or mouse orthologues revealed that, like other
to us that other homologous members of the PCSK PCSKs, this enzyme also undergoes autocatalytic pro-
family could also exist. Here again, we used polymerase cessing of its N-terminal prodomain in the ER (51) at the
chain reaction to amplify mRNAs that may encode a VFAQ152↓ site (55, 56), where the italic bold residues are
SKI-1/S1P homolog. This approach led us to clone the critical for processing (57). However, PCSK9 is the only
cDNA and analyze the biosynthesis of the 9th and last PCSK that always remains noncovalently associated with
member of the PC family, a putative convertase origin- its prodomain (58), even when secreted (51). This was
ally called neural apoptosis-regulated convertase-1 (51). confirmed by 3D-analysis (59, 60) of circulating PCSK9
Upon screening databases, we noticed that Millennium secreted from liver (61). Thus, PCSK9 acts as a protease
Pharmaceuticals and Eli Lilly had released part of its only once, upon its autocatalytic processing in the ER, sug-
cDNA, either in a patent database of a group of genes gesting that secreted PCSK9 regulates LDLc levels by a
upregulated upon induction of apoptosis in primary nonenzymatic mechanism, and rationalizing the existence
cerebellar neurons by serum withdrawal (patent no. WO of gain-of-function (GOF) variants, unusual for an enzyme.
01/57081 A2) or via the global cloning of secretory pro-
teins (related LP251 sequence; patent no. WO 02/14358
A2). We completed the human, rat and mouse cDNA PCSK9 Promotes the Degradation of the
sequences and renamed the ortologous soluble protein LDLR and Other Family Members by a
as PCSK9 and its gene PCSK9 (52). PCSK9 belongs to Nonenzymatic Mechanism
the proteinase K family of subtilases, and its catalytic do- The first critical clues on the mechanism of action of
main exhibits 25% of protein sequence identity to that PCSK9 came from 2 reports (early 2004 and late 2005) led
of its closest family member SKI-1/S1P (51). The human by Maxwell and Breslow, who demonstrated that PCSK9
PCSK9 mRNA (NM_174936.3) spans 3710 bp over 12 overexpression dramatically reduced the protein, but
exons encoding a 692-aa protein (NP_777596.2). In situ not mRNA, levels of the LDLR by inducing its degrad-
hybridization and tissue/cell lines analyses by Northern ation (62, 63) within the acidic endosomal/lysosomal
blots revealed that liver and small intestine are the major pathway (56, 62, 64). The same authors had shown a few
sources of PCSK9 synthesis in the adult mouse, rat, and months earlier by microarray analysis that PCSK9 was
human. This report first appeared in February 2003 (51). strongly downregulated by dietary cholesterol and highly
In a serendipitous twist of fate, the mapping of PCSK9 on upregulated by SREBP-1a and SREBP-2, providing evi-
the short arm of chromosome 1p32 (51) revealed that it was dence that PCSK9 is a cholesterol-regulated gene (65). This
conclusion was confirmed in a similar study (66) and by

the ability of statins to enhance PCSK9 transcription (67). PCSK9
Thus, although PCSK9 and LDLR mRNA levels were both
pr
o
CA
positively regulated by the lack of cholesterol and statin
P1
ca
treatment, PCSK9 induced LDLR protein degradation (68).
t
LDLR
CH M3
M1
This clarified the mechanism behind the reported human
RD M2
mutations leading to hypercholesterolemia (52, 69). Thus,
PCSK9 GOF result in exacerbated PCSK9-induced degrad- Protein X
(MHC-I...)
ation of the LDLR.
A definite further support for PCSK9 function came with 2
elegant studies by Cohen et al., who sequenced PCSK9 in in-
dividuals exhibiting very low cholesterol levels, thereby unam-

biguously associating 2 prevalent heterozygote loss-of-function
(LOF) variants Y142X and C679X in African Americans (2%
combined frequency) with ~40% reductions in LDLc (70), and
Figure 1. Schematic of PCSK9 and its potential partners. PCSK9 under-
amazingly with an 88% lower incidence of coronary heart dis-
goes an autocatalytic cleavage between its catalytic and N-terminal
ease during a 15-year follow-up period (71). This was the first prodomains, but the latter remains associated to the protein that has
strong evidence that PCSK9 may be acting stoichiometrically no other substrates. The C-terminal domain, CHRD, is composed of 3
on the LDLR, rather than as a protease, as enzymes usually tandem repeats (M1, M2, and M3) rich in Cys and His residues (Cys/His-
rich domain). The LDLR binds the catalytic domain of PCSK9, while the
require >90% loss activity to have a noticeable effect on their
MHC-I complex interacts with the M2 repeat of the CHRD. On the other
function (72). In 2005, Pcsk9 inactivation in mouse confirmed hand, CAP1 binds the M1 and M3 domains of the CHRD and enhances
that the loss of PCSK9 expression was associated with ~3-fold PCSK9 activity.
higher LDLR levels in the liver and with a dramatic reduction
of plasma LDLc (73). The viability of these knockout (KO) 31-152), the catalytic subunit (aa 152-421), and the
mice as well as the discovery of the first individuals completely C-terminal Cys/His-rich domain (CHRD; aa 453-692) (59,
lacking functional PCSK9 (74, 75) established this protein as 60), each playing critical roles in the regulation of PCSK9
an attractive therapeutic target for LDLc reduction. and its intracellular traffic.
In 2007, another key step was achieved by revealing that
PCSK9 does not carry any protease activity in trans. The first
reported crystal structures of PCSK9 (59, 60) showed that the The Prodomain (aa 31-152)
C-terminus of the autocatalytically excised prodomain was sol- Following cleavage of the signal peptide, the precursor
idly embedded in the substrate-binding groove, likely blocking of PCSK9 (proPCSK9) is autocatalytically cleaved at
access to any substrate. These structural data confirmed our VFAQ152↓SI very early in the ER (55, 56), but different
original observation of the secretion of mature PCSK9 as a from the other PCSKs, PCSK9 remains associated to its
noncovalent complex with its inhibitory prodomain (51). In prodomain, even after its secretion (51). The prodomain
agreement, co-expression of the prodomain of PCSK9 with a and its cleavage are necessary for the exit of PCSK9 from
catalytically dead mutant of mature PCSK9 in which the active the ER (51, 76, 77). Any variant that blocks ER exit,
site Ser386 was mutated to Ala (S386A) led to a reconstituted such as Q152H (57, 78), prevents its secretion and re-
fully functional and secreted PCSK9 that can mediate LDLR sults in a LOF and hypocholesterolemia (79). The X-ray
degradation, similar to wild-type (WT) PCSK9 (76). This con- crystallography analyses of PCSK9 at both neutral and
clusion was later confirmed in another study using a similar ap- acidic pH (59, 60, 80, 81) revealed that the structure of
proach with a PCSK9 mutant of the active site His226 (H226A) the N-terminal Gln31–Thr60 segment is not resolved, sug-
that also resulted in a similar degradation pathway of other gesting a disordered, mobile region. Interestingly, PCSK9
family members VLDLR and ApoER2 (77). Thus, PCSK9 acts lacking most of this segment composed of 25% of Glu and
as a protease only once during its autocatalytic zymogen pro- Asp residues (PCSK9-Δ33-53) enhanced by >7-fold the
cessing in the ER. affinity of PCSK9 for the LDLR (81) and by ~3-fold the
degradation of the LDLR in endosomes/lysosomes of hep-
atocytes (82, 83). This suggests that the acidic N-terminal
Insights From the Structure–Function of segment of PCSK9 may bind an important ligand/partner
PCSK9, LDLR, and Their Complex (84). More than 40 PCSK9 GOF or LOF variations were
The 3D structures of PCSK9 revealed the existence of 3 reported in this sequence (84), including the common LOF
distinct structural domains (Fig. 1): the prodomain (aa R46L (71, 85) associated with protection against heart
disease (71, 84), as well as Tyr38-sulphation (30) and Ser47- forming 3 disulfide bridges connecting Cys 1 to Cys 6, Cys
phosphorylation (86). 2 to Cys 5, and Cys 3 to Cys 4 within each repeat (59, 60).
Interestingly, 9 out the 14 His residues in the CHRD are
present within the M2 module, suggesting that they may
The Catalytic Domain (aa 153-421) exert a pH-dependent role, especially at the acidic pH of
Degradation of the LDLR by PCSK9 was shown to depend endosomes. The M1–M2–M3 modules exhibit a struc-
on the internalization of the PCSK9-LDLR complex into tural homology to the homotrimer resistin (111), a small
clathrin-coated acidic endosomes (87-89). Through its cata- cytokine associated with obesity and diabetes, and whose
lytic subunit, secreted/plasma PCSK9 (90) binds the EGF-A plasma levels have been linked to inflammation, cancer,
domain of the LDLR (80, 81, 91). This stable complex is atherosclerosis, and cardiovascular disease (112). Most of
directed toward endosomes/lysosomes for degradation by the reported PCSK9 variations in the C-terminal domains
a yet unknown mechanism(s), preventing LDLR recycling are within the M1 and M3 modules. The hinge region and

to the cell surface (56, 87, 88, 90). PCSK9 may also escort M2 module exhibit a lower rate of genetic variations, but
LDLR for degradation directly from the trans-Golgi net- a greater structural mobility (59, 60), suggesting that they
work, although this intracellular pathway does not fully are subject to a stronger selection pressure to interact with
resemble the extracellular route (61, 92-94). However, for putative partners.
still obscure reasons, the pathway utilizing extracellular/
circulating PCSK9 is the primary functional pathway in
hepatocytes (90, 95, 96), small intestine (97), and pancreas Complete LOF Variants of PCSK9 Exhibit a
(98, 99). In agreement, serum LDLc levels correlate dir- Healthy Profile
ectly with those of circulating PCSK9 levels (100-104), and Since 2003, a large number of substitutions/deletions have
statins regulate the levels of LDLc in part by upregulating been reported in each of the PCSK9 domains (for reviews
those of circulating PCSK9 in mice and human (73, 104, see (84, 95, 113, 114)), some having major effects on PCSK9
105). Within the catalytic domain, 3 reported PCSK9 LOF activity through various underlying mechanisms (57, 114-
variants F216L (52), R218S (106) and R215H (107) led 117). Interestingly, 2 unrelated Canadian FH patients who
to the demonstration that Furin inactivates PCSK9 by are refractory to intensive statin therapy exhibit a whole
cleavage at RFHR218↓ (30, 108). The latter site lays in an ex- gene duplication of PCSK9 (118). They presented severely
posed mobile loop not resolved in any crystal structure (59, elevated LDLc levels and premature cardiovascular events,
60), in agreement with its accessibility to proteases such as and one of them had PCSK9 levels of ~5000 ng/mL, thus
Furin. Among the GOF variants, the most powerful is the >20-fold higher than normal ones.
Anglo-Saxon D374Y (69), which exhibits a 10- to 20-fold The few individuals lacking functional PCSK9, namely
higher binding affinity for the LDLR (59, 109) and resist- ΔR97/Y142X (74), C679X/C679X (75), and the monoallelic
ance to Furin cleavage (30). Reciprocally, an LDLR-H306Y double variant R110C+V114A (116), seem healthy and ex-
variant within the EGF-A domain enhances PCSK9 binding hibit circulating levels of LDLc of ~14 to 16 ng/mL, a value
to the LDLR and hence is associated with hypercholester- that is ~8-fold lower than the normal levels (~110-150 ng/
olemia (110). The crystal structure of the PCSK9-EGF- mL) (71, 75, 116). In the latter case, a double mutation in
A(H306Y) complex revealed that indeed, Tyr306 forms a the prodomain abolishes the autocatalytic processing of
hydrogen bond with Asp374 in PCSK9 at neutral pH, which PCSK9, generating an unfolded proPCSK9 remaining in
strengthens the extracellular complex (110). the ER that acts as a dominant negative by preventing the
exit from the ER of the processed WT PCSK9 that is en-
coded by the intact allele (116). Similarly, French Canadian
The Hinge-CHRD Domain (aa 422-692) individuals carrying a Q152H substitution at the auto-
The first crystal structures of PCSK9 (59, 60) and its align- catalytic VFAQ152↓ cleavage site present an almost com-
ment with other PCSKs revealed that the catalytic domain plete loss of circulating PCSK9, due to a dominant negative
is followed by a relatively disordered hinge region (aa effect of proPCSK9-Q152H (57, 78), resulting in ~3-fold
422-452), and an ordered C-terminal 240 aa CHRD. The lower levels of LDLc (~40-50 ng/mL) (78, 79). Even
latter is composed of 3 tandem repeats tightly packed into though the Q152H substitution leads to the accumulation
structurally similar modules named M1 (aa 453-529), M2 of proPCSK9 in the ER, the 30 heterozygote and 3 homo-
(aa 530-603), and M3 (aa 604-692), whose frontiers are zygote individuals identified seem to be protected against
delimited by their β-sheet structures (Fig. 1). Each module ER-stress (79). Indeed, the ER-retained proPCSK9-Q152H
exhibits a 6-bladed domain containing 6 Cys within the may act as a co-chaperone by increasing the levels of the
motif: (X)4 C (X)17-19 C (X)8-9 C (X)18-25 C (X)11-23 C (X)2-13 ER-resident chaperones GRP94 (119) and GRP78, which
play central roles in the regulation of protein folding and positive charge was more important than specific sequences
the unfolded-protein response (120). This results in a pro- within the CHRD. However, this may not be the whole
tection against ER stress-induced apoptosis, as also reported story. Indeed, monoclonal antibodies (mAbs) (127, 132)
for other proteins that increase the stability of GRP78, such and single domain antibodies (133, 134) that target the
as Bag5 (121) and GALNT6 (122). These data suggested M1 and/or M3 domains of the CHRD inhibit the PCSK9-
that inhibition of the autocatalytic cleavage of proPCSK9 induced LDLR degradation without blocking the binding
in the ER may represent a safe and unique approach for the of PCSK9 to the LDLR. Although Sortilin and APLP2 can
management of hyperlipidemia, with the added benefit of bind the CHRD, they were shown not to be credible “pro-
preventing liver dysfunction (79). The above human genetic tein X” candidates (135). To date, none of the studies that
studies suggested that humans lacking functional PCSK9 investigated the trafficking of the PCSK9-LDLR complex
can live normal lives, and heterozygote complete LOF vari- (87, 131, 135-137), have identified all the critical partners
ants largely (88%) protect individuals from the development needed to escort the complex into late endosomes/lyso-

of cardiovascular complications and coronary heart disease somes for degradation.
over their lifetime (71). The unusually large contribution of A recent study proposed that the cytosolic adenyl
some heterozygote LOF variants (C679X, R110C+V114A, cyclase-associated protein 1 (CAP1) is a candidate “pro-
Q152H) seems to be in part related to a dominant negative tein X” (95, 135). The authors were inspired by its ability
effect of the protein carrying the mutant allele on the traf- to bind resistin (138), a protein that bears a structural
ficking of the WT PCSK9, preventing its exit from the ER homology to the CHRD (111), as we mentioned above.
(57, 78, 116). CAP1 seems to bind the M1 and M3 domains and en-
hances degradation of the PCSK9-LDLR complex in lyso-
somes (138). They also showed that the PCSK9 E670G and
The CHRD Is Critical for the PCSK9-Induced S668R LOF variations in M3 considerably reduced the
Degradation of the LDLR ability of PCSK9 to bind CAP1, likely due to the loss of
PCSK9 and the LDLR primarily interact through their cata- phosphorylation at both Ser666 and Ser668 (86). These Ser
lytic and EGF-A domains, respectively (80, 91). A minor phosphorylations are performed by FAM20C at Ser-X-Glu
interaction between Leu108 of the PCSK9 prodomain and or Ser-X-phosphoserine sites (16). In that context, we pre-
Leu626 in the β-propeller domain of the LDLR was also re- viously reported that PCSK9 phosphorylation at these Ser
ported (80, 123). Notably, the PCSK9 GOF L108R variant residues located within the M3 domain enhanced LDLR
may reinforce the interaction of these 2 domains by trans- degradation (86), suggesting that CAP1 may best bind
forming the initial hydrophobic bond into an electrostatic phosphorylated PCSK9. CAP1 is a cytosolic protein, but
one between Arg108 in the PCSK9 variant and Glu605 of the “protein X” was predicted to be a secretory membrane–
β-propeller domain of the LDLR (80, 123). bound protein (96). The authors proposed that CAP1 can
Cellular data revealed that the CHRD of PCSK9 is re- somehow flip from the inner-leaflet to the other side of the
quired to trigger LDLR degradation (87, 124-127), but hepatocyte plasma membrane for exposure to the extra-
its absence in PCSK9-ΔCHRD still allows the binding of cellular matrix (138), thereby binding extracellular PCSK9
PCSK9 (or proPCSK9) to the LDLR (87, 126). On the other to mediate caveolae-dependent endocytosis and lysosomal
hand, the PCSK9-D374Y variant was able to degrade the degradation of the PCSK9-LDLR complex.
LDLR in autosomal recessive hypercholesterolemia pro- However, because some cytosolic proteins can be se-
tein–independent manner (128), as well as the LDLR mu- creted via an unconventional secretory pathway (139),
tant K811X that lacks the cytosolic tail (129) or a chimeric we cannot exclude that this could also apply to CAP1, as
LDLR carrying the C-terminal domain of the transferrin re- for the cytosolic Annexin A2 that also binds the CHRD
ceptor (130). These data indicated that a third partner is re- (140). Note that the PCSK9-Annexin A2 complex is intern-
quired for the efficient LDLR degradation by PCSK9 (129, alized via clathrin-coated vesicles, but not caveolae (138).
131). It was hypothesized that this “protein X” binds the Nevertheless, CAP1 seems to be an important positive
CHRD and allows the sorting of the PCSK9-LDLR com- regulator of PCSK9 function on the LDLR, possibly by ex-
plex to acidic degradation compartments (96). Intriguingly, posing a domain in the CHRD (eg, M2 domain) that may
it was reported that the CHRD could be substituted by the best bind the elusive membrane-bound “protein X” (141).
C-terminal domain of DsRed-Express fluorescent protein,
an unrelated protein of comparable size and overall posi-
tive charge, and promote LDLR degradation (126). Because PCSK9 Mouse Models
mutations of multiple histidines in the CHRD are needed We originally showed that statins upregulate the expres-
to affect its activity, the authors suggested that the overall sion of both LDLR and PCSK9 mRNA and protein (67),
revealing a conundrum in LDLc regulation since PCSK9- livers exhibited necrotic lesions that were prevented by a
mediated LDLR degradation will blunt the contribution of high-cholesterol diet, suggesting that the absence of PCSK9
higher levels of LDLR transcripts (68). This effect explains severely reduces tissue cholesterol levels and confers re-
the higher sensitivity of apoB levels to statins in PCSK9 KO sistance to liver steatosis (143). Indeed, despite the 4-fold
mice (73). Indeed, statins have a more prominent effect in higher levels of LDLR at the hepatocyte surface of male
KO than in WT mice, where hepatic LDLR protein levels mice, as measured by immunohistochemistry or liver frac-
accumulate in the absence of PCSK9. This has paved the tionation, there is no accumulation of cholesterol in their
way for the combined statin/PCSK9 mAb therapy. In agree- liver (73, 143, 149). This is likely due to the 5-fold lower
ment, LDLR labeling of primary mouse hepatocytes lacking circulating LDLc levels, preventing any significant upsurge
PCSK9 (142), and of liver sections from mice lacking of cholesterol in hepatocytes. In the β-cells of the same
PCSK9 in the 2 first Pcsk9 KO models (73, 143), especially KO mice, the low levels of circulating LDLc resulted in in-
in male mice (61), revealed a strong accumulation of the creased LDLR mRNA and protein levels, indicating that

LDLR protein at the hepatocyte cell surface. a cholesterol deficit was not fulfilled by the higher LDLR
Analysis of mouse and rat PCSK9 mRNA expression levels in these cells, as reflected by an upregulated the
showed that it is restricted to discrete areas of the brain SREBP-2 pathway (98). Thus, even though both hepato-
during development (51, 95, 144), and that its antisense cytes and β-cells of male PCSK9 KO mice express higher
morpholino-oligonucleotide knockdown in zebrafish leads levels of LDLR, they do not accumulate cholesterol because
to embryonic death with major neuronal deficits (145), al- of the low circulating levels of LDLc. Early data showed
though CRISPR/Cas9 deletion of its gene in zebrafish did that like insulin, circulating PCSK9 is dramatically reduced
not (146). Based on the previously observed embryonic le- (>95%) in fasted in mice (150). In addition, circulating
thality of Furin, PC5 and SKI-1/S1P KO in mice (28), we PCSK9 strongly reduces the surface levels of the VLDLR
opted to develop a conditional mouse Pcsk9 KO strategy. protein in adipose tissue (61), and those of CD36 (151).
This long and tedious approach led to both complete (143) Thus, PCSK9 may favor cholesterol uptake by peripheral
and tissue-specific Pcsk9 KO mice, namely in hepatocytes tissues via hepatic LDLR degradation and reduced visceral
(61, 143), enterocytes (97) and pancreatic β-cells (98). adipogenesis.
PCSK9-deficient mice are viable, with no apparent neur- The first indication that PCSK9 enhances the develop-
onal phenotype (73, 144). ment of atherosclerosis was the demonstration that mice
Two other KO models were reported but not widely fed a high cholesterol diet for 15 weeks and expressing
used thereafter. The first one was a whole-body KO the GOF PCSK9-D374Y at physiological levels exhib-
model even though it derived from a conditional strategy. ited extensive atherosclerotic plaques compared with WT
Comparison of the generated KO mice with transgenic mice (152). This was further confirmed following a single
mice overexpressing PCSK9 in liver demonstrated that injection of recombinant adeno-associated viral vectors
overexpressed PCSK9 did not regulate LDLR, VLDLR, encoding PCSK9-D374Y that rapidly induced atheroscler-
and apoER2 in the brain, likely because it does not cross osis in mice and eliminated the need for germ-line genetic
the blood–brain barrier (147). The second KO model, modifications (153). These data suggested that high levels
homozygous for the Y119X mutation, was generated by or activity of PCSK9 may exacerbate the development of
N-ethyl N-nitrosourea mutagenesis and backcrossed into atherosclerosis and possibly enhance inflammation. To
the C57BL/6J background using speed congenics (148). prove that the reverse is true in mice lacking PCSK9, we
Thorough plasma, liver, and bile acid analyses in WT and used mouse models of accelerated atherosclerosis con-
KO mice treated or not with atorvastatin revealed that sisting of either WT mice, PCSK9 KO or transgenic mice in
cholesterol metabolism and hepatic homeostasis were well ApoE or LDLR KO backgrounds. The data demonstrated
maintained in the absence of PCSK9. a direct relationship between PCSK9 and atherosclerosis,
Mice lacking PCSK9 exhibit 40% to 50% lower cir- whereby PCSK9 overexpression is proatherogenic and its
culating cholesterol, ~80% less LDLc, and ~3- to 4-fold absence is protective (154). A later report demonstrated
higher levels of total liver LDLR protein (73, 143). Whole that PCSK9 mRNA silencing suppressed atherosclerosis
body and hepatocyte-specific PCSK9 KO mice revealed directly through inhibiting the TLR4/NF-κB signaling,
that, in the liver, PCSK9 is exclusively expressed in hepato- which induces the expression of pro-inflammatory medi-
cytes, which also represent the only source of circulating ators, without affecting plasma cholesterol levels in high
PCSK9 (61, 98, 143). Single LDLR or double LDLR/PCSK9 fat diet-fed ApoE KO mice (155). It was suggested that this
KO mice exhibit similar cholesterol profiles, indicating that may implicate the intermediary oxidized-LDL, a potent
PCSK9 regulates plasma cholesterol homeostasis essen- proinflammatory mediator of atherosclerosis (156), which
tially through the LDLR. Finally, PCSK9 KO regenerating induces PCSK9 expression in macrophages via activation
of TLR4/NF-κB signaling. Indeed, PCSK9 seems to interact 4-fold higher in male than female fractions (149). Thus, in
with the oxidized-LDL receptor-1 (LOX-1) in a mutually PCSK9-deficient female mice, E2 seems to take control over
facilitative fashion (157). Finally, a PCSK9-specific vac- LDLR density at the hepatocyte surface. Whether E2 in-
cine reduced total cholesterol, vascular inflammation, and hibits the targeting of the LDLR to the cell surface or favors
atherosclerosis in the atherogenic APOE*3Leiden.CETP its elimination from the cell surface remains to be deter-
mouse (158). In this context, in an inflammatory milieu, mined. Intriguingly, despite the E2-mediated modulation of
elevated levels of PCSK9 potently stimulate the expres- surface LDLR, male and female mice exhibit similar circu-
sion of LOX-1 and oxidized-LDL uptake in macrophages, lating levels of LDLc, indicating that the higher density of
and thus contribute to the process of atherogenesis (159). surface LDLR in male hepatocytes does not have a major
Accordingly, there may be additional clinical benefits of effect on the LDLc clearance.
PCSK9 inhibition on mechanisms unrelated to increased
LDLR activity, but rather to a reduction of vascular
Major Cardiovascular Outcome Trials Using

inflammation.
PCSK9 is expressed in β-cell-derived cell lines (51), as PCSK9 Inhibitors
well as in pancreatic islets (160). According to single-cell Lipid-modifying agents are often prescribed to reduce
transcriptomics performed on dissociated cells from 20 atherogenic lipid levels and to prevent atherosclerotic
murine tissues and compiled in the Tabula Muris data- cardiovascular disease (162). In view of the major effects
base (161), β-cells constitute the cell type richest in PCSK9 of the lack of PCSK9 on the substantial reduction of cir-
expression, closely followed by hepatocytes. Two re- culating LDLc, and the overall safety of PCSK9 LOF or
cent studies, including our data on β-cell-specific PCSK9 its complete absence in animal models (73, 143) and hu-
KO mice (98), show that endogenous PCSK9 in β-cells is mans (71, 74, 75, 116), it was likely that potent PCSK9
well secreted and induces LDLR degradation, but does inhibitors (PCSK9is) could represent a viable strategy to
not affect glucose-stimulated insulin secretion (98, 99). reduce LDLc. Various approaches have been proposed to
Although β-cell PCSK9 cannot contribute significantly to reduce the circulating PCSK9 activity via mAbs or protein/
the circulating pool of PCSK9 (98), its absence leads to peptide inhibitors, or through silencing its mRNA expres-
an adaptative downregulation of the pancreatic SREBP-2 sion or translation in liver (163, 164). So far, major efforts
pathway. This reflects a higher rate of cholesterol intern- by pharmaceutical companies led to the successful devel-
alization by unchanged LDLR protein levels and indicates opment of 2 distinct, robust, safe, and long-term PCSK9i
that endogenous PCSK9 synthesized in the β-cell does in- (Fig. 2): (1) PCSK9 mAbs (evolocumab/Repatha and
deed target the LDLR for degradation. However, in mice alirocumab/Praluent) that inhibit the interaction of circu-
completely lacking PCSK9, pancreatic LDLR protein levels lating PCSK9 with the LDLR, thereby silencing its extra-
are increased by 2-fold, showing that the 5-fold lower cir- cellular activity; (2) PCSK9 small interfering RNA (siRNA)
culating LDLc in these mice cannot satisfy the high require- in lipid nanoparticles (Inclisiran) specifically delivered to
ment that β-cells have for cholesterol (98). Altogether, these the liver that prevents PCSK9 translation (165), thereby
data are reassuring in relation to the concern of a possible abrogating both intracellular and extracellular functions
toxic effect of the absence of PCSK9 via excessive choles- of PCSK9 (92) in hepatocytes. Both approaches result in
terol internalization in β-cells (98, 99). a ~50% to 60% reduction in LDLc above that obtained
Even though the absence of PCSK9 results in 3- to with statins alone. The reader is referred to very recent re-
4-fold higher levels of the LDLR protein in the liver of both views on the history, efficacy, safety, and potential clinical
sexes, the cell surface levels of the receptor increased in
male livers, as previously reported (73, 143), but not in fe- Monoclonal Ab siRNA
male ones (149). This dimorphism was not restricted to the
liver. Pancreatic islets also exhibited a stronger cell surface
density of the LDLR in PCSK9 KO male mice vs female PCSK9
ones, while the opposite was observed for the intestinal
epithelium, with PCSK9 KO female mice presenting the
Vaccine CRISPR-Cas
strongest surface LDLR labeling (149). Supplementation Orally active inhibitors
of ovariectomized mice with placebo or 17β-estradiol
(E2) generated typical male and female labeling patterns, Figure 2. PCSK9 inhibition. Monoclonal Abs and siRNAs are safe and
temporary inhibitors of PCSK9. Irreversible inhibition may be achieved
respectively. These observations were confirmed biochem-
through vaccination or PCSK9 gene modification via CRISPR-Cas.
ically by Western blot analysis of liver fractions enriched in Finally, a more affordable inhibition based on orally active inhibitors is
plasma membrane, in which LDLR protein levels were 3- to in development. See text for references.
uses and outcomes of these PCSK9i (163, 165-174). Below, for homozygous FH (HoFH) patients who exhibit residual
we briefly describe salient features that emanated from the LDLR activity due to incomplete silencing of its function
major clinical trials using either mAb or siRNA approaches, (186).
the targeted populations, and their outcomes. In 2017 and 2018, 2 landmark outcome trials were re-
ported on the use of evolocumab (187, 188) and alirocumab
(189) as therapeutic mAb inhibitors of circulating PCSK9
The mAb Approach to Inhibit Circulating PCSK9 activity in combination with statins.
Activity on the LDLR
The liver is the primary source of circulating PCSK9, and • In the FOURIER trial a total of 27 564 patients with
the latter is a target of mAbs that recognize the catalytic a mean age of 62.5 years (25% women) and receiving
subunit and sterically prevent the binding of PCSK9 to at least 20 mg of atorvastatin were randomized to
the EGF-A domain of the LDLR in hepatocytes and other subcutaneous doses of evolocumab (140 mg every 2

tissues (175). A single subcutaneous injection of a neu- weeks or 420 mg monthly) or placebo. After 48 months,
tralizing PCSK9 mAb to monkeys resulted in an ~80% re- evolocumab treatment effectively lowered LDLc to
duction of LDLc after a single injection (176). Following a mean value of 30 mg/dL (maintained in follow up
this initial proof-of-principle and impressive efficacy, fully studies), and significantly decreased the risk of recurrent
humanized mAb were developed. evolocumab (Repatha; cardiovascular events in patients with established
Amgen) and alirocumab (Praluent; Sanofi) are presently the atherosclerotic cardiovascular disease (187) and/or
only 2 humanized mAb that successfully sailed through all type 2 diabetes (188). Evolocumab reduced the relative
the multiple clinical trials in hypercholesterolemic patients risk of the primary endpoint by 15% with a hazard ratio
exhibiting or not FH mutations. Starting from 2015/2016, of 0.85 (95% CI 0.79-0.92) during a median follow-up
these mAb have been prescribed to patients in more than 30 of 2.2 years. The risk of the key secondary endpoint,
countries as they generate a robust and sustained ~50% to a composite of cardiovascular death, myocardial
60% reduction in LDLc that has been consistently observed infarction, or stroke, had a relative reduction of 20%.
over the last 3 to 5 years. Their safety and tolerability pro- Effectively, this means that the number of patients to
file are excellent, especially for hypercholesterolemic pa- treat to have a highly significant prevention outcome is
tients or those who do not optimally respond to or tolerate between 5 and 7 patients. Interestingly, LDLc lowering
statin treatment. Only few patients exhibited mild injection with evolocumab was also highly effective in reducing
site reactions. Below, we describe some outcome data that the risk of myocardial infarcts (190). In general, it was
provide concrete evidence for the long-term clinical benefits observed that patients with higher cardiovascular risk
of reducing circulating PCSK9 activity. due to the presence of multiple cardiovascular risk
In patients with LDLc levels between 100 and 190 mg/ factors in their genome or those suffering from more
dL, mAb monotherapy reduced LDLc levels by 50% to severe atherosclerotic disease had the largest clinical
55% after 12 to 24 weeks of treatment (177, 178), and benefit as measured by the absolute risk reduction.
similar LDLc reduction was observed in statin-intolerant Treatment with evolocumab for 2.2 years did not result
patients (179). Thus, it became apparent that PCSK9 mAb in an increased risk of developing diabetes, or worsening
treatment was efficacious in a broad range of patients with glycemia (188), in agreement with recent studies on
similar outcomes with respect to LDLc reductions (180). complete and β-cell specific Pcsk9 KO mice (98), and
The combination of these mAbs with other lipid-lowering on β-cells isolated from human pancreas treated with
agents such as statins or the ezetimibe/fenofibrate resulted alirocumab or siRNA silencing of PCSK9 expression
in a modest (≤15%) further decrease in LDLc (181, 182). (99). Patients with metabolic syndrome, having a
Interestingly, all studies showed that treatment with PCSK9 higher event rate than patients without, exhibited
mAb leads to significantly higher reductions in LDLc in similar reductions in LDLc and risk for primary and
the presence of a statin treatment, even though statins key secondary endpoints, without increasing new-onset
enhance the expression of PCSK9 via activation of the diabetes, worsening glycemic control, or other major
SREBP-2 pathway (67, 105). The increased levels of circu- safety events (191). Overall, within the follow-up period
lating PCSK9 in response to statins and/or ezetimibe may of FOURIER, treatment with evolocumab resulted in
in part rationalize why PCSK9-antibodies are highly ef- very low LDLc and was not accompanied by any adverse
fective in reducing LDLc as an add-on therapy (183). Since medical events. It was also observed that treatment with
PCSK9 primarily reduces LDLR levels in hepatocytes, the evolocumab resulted in a median reduction of ~27% in
mAb approach works very well for normal and hetero- Lp(a), an independent cardiovascular risk factor (192,
zygote FH (HeFH) patients (184, 185) and only partially 193). Whether this is due to the higher levels of LDLR
in liver induced by evolocumab (194, 195) or to a lower assemble the RNA-induced silencing complex (RISC) re-
rate of Lp(a) secretion (196, 197) is still under debate. sulting in the PCSK9 mRNA degradation over a prolonged
• The ODYSSEY outcomes study included 18 924 period of time (171). Accordingly, Inclisiran subcutane-
patients with a mean age of 59 years (25% women), and ously injected twice a year substantially reduces by 50%
alirocumab treatment targeted patients with a history to 60% the levels of LDLc (168, 170). Various ORION
of an acute coronary syndrome within 1 to 12 months clinical trials were designed to test the efficacy and safety
before randomization (189). Atorvastatin 40 mg or of Inclisiran in the presence of statins. Currently, the phase
rosuvastatin 20 mg were included, and patients were III cardiovascular outcome study ORION-4 is ongoing
randomized to alirocumab 75 mg every 2 weeks or with completion expected in 2024 (https://clinicaltrials.
placebo subcutaneously in a 1:1 ratio, with dose gov/ct2/show/NCT03705234). Another study involves
adjustment allowed during treatment. In contrast to patients with atherosclerotic cardiovascular disease and
the FOURIER trial in which no dose adjustments were elevated LDLc (≥70 mg/dL) despite receiving maximally

allowed, at 48 months of alirocumab + statin treatments, tolerated statin therapy, with completion expected in 2023
LDLc was reduced to a mean of 66 mg/dL. The primary (https://clinicaltrials.gov/ct2/show/NCT04929249?term=in
endpoint was a composite of coronary death, myocardial clisiran&draw=2&rank=1). So far, it looks that Inclisiran
infarction, ischemic stroke, and unstable angina administration in various subgroups of patients generates
requiring hospitalization. After a median follow-up of a consistent and sustained ≥50% LDLc reduction with no
2.8 years, the relative risk for the primary endpoint was safety concern, except for some mild/moderate injection
reduced by 15% with a hazard ratio of 0.85 (95% CI site effects that are not persistent. These overall positive
0.78-0.93), although death from coronary artery disease data should encourage patient compliance with this siRNA
or from cardiovascular causes was not. These values treatment.
are comparable to those obtained in the FOURIER trial It should be underlined that the mAb approach requires
using evolocumab + statin. Notably, treatment with repeated injections at 2- to 4-week intervals (13 or 26 in-
alirocumab resulted in an absolute 16.2% reduction jections/year), whereas the siRNA treatment is administered
in death risk in patients suffering from a composite twice a year. Inclisiran received marketing authorization in
coronary artery, peripheral artery, and cerebrovascular the European Union in December 2020 for use in adults with
disease (198). Finally, extensions of this trial revealed primary hypercholesterolemia or mixed dyslipidemia, but its
that longer PCSK9 inhibition has the potential to reduce authorization in the United States still awaits the Food and
mortality after an acute coronary syndrome, especially Drug Administration approval. So far, both approaches seem
in patients with elevated risk due to high LDLc. safe following 2 to 5 years of clinical use, but we will have to
await longer treatments for a more thorough assessment of
the long-term effects of reducing liver vs circulating PCSK9.
The siRNA Approach to Silence Liver PCSK9 From 2008 to 2018, the consumption of cholesterol-
mRNA Expression lowering agents defined as standard units consumed per
Since loss of PCSK9 expression in liver resulted in substan- 1000 inhabitants increased by ~4.1%/year, with statins rep-
tial reduction of LDLc, without apparent negative con- resenting the lion share of a market that presently targets
sequences in human (71, 74, 75, 116) or mice (73, 143), >170 million people worldwide (201). However, PCSK9i
silencing of its hepatic expression was tested using an in- are very useful when maximally tolerated statin therapy
jectable lipid nanoparticle formulation carrying an anti- does not reduce LDLc sufficiently and in statin-intolerant
sense siRNA to PCSK9. Accordingly, an optimized siRNA patients. Compared to 2015, the year when PCSK9 mAbs
version (Inclisiran) is composed of complementary 21 sense were first approved by the Food and Drug Administration,
and 23 antisense oligonucleotide sequences that have been prescriptions of PCSK9i increased by >5-fold during the
modified for durability and low immunogenicity. The sense years 2016-2018, especially in higher income countries
strand is conjugated to triantennary N-acetylgalactosamine (201). Over the last 6 years the beneficial effects of PCSK9i
(GalNAc) to facilitate siRNA uptake by the liver via binding administration have been widely recognized, encouraging
to the asialoglycoprotein receptor 1 (ASGR1) (199), which their prescription both in the primary and secondary pre-
is highly expressed in hepatocytes (200). This approach en- vention of cardiovascular incidents. However, the major
hances the level of the liver-specific delivery of the antisense stumbling block preventing their wider use has been their
strand and its efficacy to silence PCSK9 expression. After exorbitant cost (recently reduced to ~5800$/year in the
endosomal uptake, a small part of the siRNA is released United States). It is likely that prescriptions of PCSK9i
into the cytoplasm, where the dissociated antisense strand would be much more widely adopted if the cost was sub-
binds the PCSK9 mRNA and recruits several proteins that stantially further reduced (202, 203).
Future Strategies Beyond mAb and siRNA PCSK9 KO mice were protected from septic shock induced
by lipopolysaccharide (LPS) injections. Subsequently,
PCSK9-vaccination is a permanent approach proposed to
PCSK9 LOF variants were reported to exhibit less frequent
silence PCSK9. Peptide-based vaccines have so far shown
septic shocks and organ failures (220, 221), whereas the
promising results in mice or macaques, including the use
reverse is true in transgenic mice overexpressing PCSK9
of bacteriophage virus-like particles displaying PCSK9-
(222). The LDLR clears, in an LDL-dependent mechanism,
derived peptides (204, 205). Whether vaccination against
gram-positive lipoteichoic acid and gram-negative LPS,
PCSK9 using modified lipid nanoparticles (206) or effective
known to exacerbate sepsis pathophysiology (223). Because
capsid-like particles (207) expressing full-length PCSK9
the protective effect of the lack of PCSK9 is abrogated in
will be approved to treat severe pathologies will have to be
LDLR KO mice, it was proposed that the lack of PCSK9,
carefully evaluated for any harmful side effects of such an
or the use of PCSK9 inhibitors (224, 225), would enhance
irreversible therapy (163).
pathogen lipid clearance via the LDLR. Using a cecal liga-
In 2012, the advent of clustered regularly interspaced

tion and puncture model of sepsis, PCSK9 KO mice were
short palindromic repeats (CRISPR)-associated (Cas)
indeed reported to have lower bacterial concentrations in
system of gene deletion/editing (208) has revolutionized
the blood, lungs, and peritoneal cavity fluid than WT ani-
the field of molecular biology, leading to powerful ap-
mals, suggesting that loss of PCSK9 improves bacterial sup-
proaches relying on Cas9/sgRNA ribonucleoprotein com-
pression/clearance (222). In support of this notion, clinical
plexes to edit DNA. Multiple applications are thus made
observations revealed that patients carrying a combination
possible in various pathologies and gene replacement ther-
of 3 LOF variant alleles of PCSK9 (R46L, A53V, I474V),
apies (209-211). Very early, proof-of-concept Pcsk9 in-
but not each individually (226), exhibited a >50% higher
activation was reported using an adeno-associated viral
1-year survival rate and protection from recurrent infec-
vector-9 delivery of CRISPR-Cas9 to mouse liver (212),
tions, likely due to enhanced resolution of infection and/
or in utero injections of an adenoviral vector expressing
or bacterial clearance after the initial infection (227). This
the safer SpCas9 base editor 3, which enables single-base
likely reflects that strong LOF variants of PCSK9 may be
conversions without generating double-stranded breaks
beneficial but weak single LOF variants are not. PCSK9 is
(213). In both cases, cholesterol levels were significantly
also known to sort VLDLR, ApoER2 (77) and CD36 (151)
reduced. The small size of Cas12a also promoted its use
toward lysosomal degradation, some of which are highly
in gene editing that led to a ~48% reduction in the ex-
expressed in adipose tissue. LPS can be sequestered in the
pression of PCSK9 in mice (214). Finally, intravenous in-
latter tissue via the VLDLR. In that respect, a homozygote
jection of nonviral lipid nanoparticles (LNPs) that deliver
intronic GOF variant (SNP rs7852409; CC genotype) of
Cas9/sgRNA ribonucleoprotein complexes was reported
VLDLR has been associated with improved sepsis survival,
to significantly decrease serum PCSK9 levels in mice, even
especially in patients with a body mass index below 25
though the latter are not specifically delivered to the liver
(228).
(215). More recently, a single infusion of LNPs allowed
However, there are translational challenges between mouse
the delivery of the more advanced base-editing CRISPR-
studies to human applications (229), and these should be over-
ABE8.8 system that successfully modified the PCSK9 gene
come before the widespread prescription of PCSK9i to sepsis
of cynomolgus monkeys and achieved ~90% and ~60%
patients. Furthermore, the complex nature of sepsis is species
reductions of plasma PCSK9 and LDLc, respectively, for
specific, putting the widespread utility of the rodent models
more than 8 months (216). These impressive reductions of
of sepsis to question (230). Indeed, mice and rats have a sig-
LDLc should even be reinforced by using LNPs that specif-
nificantly higher resistance to sepsis than humans. However,
ically target the liver.
recently humanized mouse models of sepsis were reported
to mitigate somewhat this restriction (231). Nevertheless, al-
though PCSK9 inhibitors may increase survival of adult pa-
PCSK9 and Sepsis tients, they may not be beneficial for young children with
Septic shock is often a fatal complication following sepsis, sepsis, as LOF of PCSK9 was associated with decreased
a multiorgan disease caused by complex arrays of bacterial survival in juvenile mice (14 day old) and young children
and pathogenic molecules that trigger an uncontrolled sys- (1-6 years old) (232). The rationale behind this observation
temic inflammatory response and subsequent organ failure is not clear, though it may be related to age-dependent pleio-
(217). Beyond antibiotic therapy, there are still no effective tropic effects of PCSK9 on other receptors or inflammation.
treatments for septic shock (218). The first evidence that the Thus, until this paradoxical effect of PCSK9 LOF is clarified,
loss of PCSK9 may be beneficial was provided by Walley it was suggested that children should be excluded from sepsis
et al. in 2014, both in humans and mice (219). Notably, clinical trials involving PCSK9 inhibitors (232).
It should also be noted that patients with septic shock which is more dependent on SR-B1 (scavenger receptor
and lower PCSK9 levels on day 1 (within the first quar- class B type I that mediates the uptake of HDL) for viral
tile) showed the highest 28- and 90-day mortality rate entry (242). In contrast, 4 weeks after the start of DAA
compared with other quartiles (233), suggesting that low therapy primarily in HCV-G1 patients (76%), circulating
levels of circulating PCSK9 1 day after the onset of sepsis PCSK9 levels were reduced and yet LDLc was increased,
are not correlated with a favorable disease outcome (234, suggesting that these changes are not functionally related
235). However, this does not necessarily exclude the pre- (243). Whether circulating PCSK9 levels, usually meas-
ventive use of PCSK9 inhibitors, eg, before surgery, to neu- ured by enzyme-linked immunosorbent assay, represent a
tralize circulating PCSK9, and therefore increase LDLR good biomarker of HCV infection remains to be proven.
levels before the onset of sepsis. Moreover, PCSK9 does Indeed, mass spectrometry assessment of the phosphoryl-
not significantly affect HDL levels (52), known to be crit- ation state and the ratio of active vs Furin-cleaved inactive
ical, as patients with declining HDL during sepsis are at PCSK9 would better evaluate circulating PCSK9 activity

much greater risk of succumbing to organ failure and death (86). Nevertheless, caution should be exerted before pre-
(236). In conclusion, we have to wait for the data from scribing PCSK9i to patients infected by HCV (244, 245),
2 ongoing clinical trials (https://clinicaltrials.gov/ct2/show/ as this would raise LDLR levels in hepatocytes, which may
NCT03869073 and/NCT03634293) to know whether the enhance viral packaging and infectivity (239).
quick removal of bacterial components through PCSK9 Dengue virus (DENV) is a single positive-stranded RNA
neutralization with a mAb is beneficial. virus of the family Flaviviridae, transmitted to human by
the urban-adapted Aedes mosquitoes, which yearly infects
>400 million individuals worldwide, resulting in ~25 000
PCSK9 and Viral Infection death/year, mostly children from southeast Asia (246).
Proprotein convertases (PCs), especially Furin and SKI-1/ Recently, we showed that DENV infection induces ex-
S1P, are implicated in the processing of enveloped vir- pression of PCSK9 in hepatocytes (247), thereby reducing
uses resulting in enhanced viral entry into host cells and cell surface levels of LDLR and LDLc uptake, resulting in
widespread infectivity (47). There has also been a limited enhanced de novo cholesterol synthesis by the SREBP-2
number of cases where PCSK9 was linked to viral infect- pathway (248). DENV exploits this mechanism for viral
ivity, these include infections by hepatitis C virus (HCV), packaging. Interestingly, following DENV infection, in-
Dengue virus (DENV), and possibly SARS-CoV-2, the etio- creased cholesterol levels in the ER causes a significant re-
logical agent of COVID-19. duction in the antiviral type I interferon (IFN) response of
The LDLR has been proposed as one of the host factors the host liver hepatocytes due to the cholesterol-induced
implicated in HCV entry into hepatocytes (237), but its suppression of the phosphorylation and activation of
role remains controversial (238). Recently, induced pluri- STING. The latter is an ER-resident stimulator of the anti-
potent stem cells (iPSC) were isolated from an LDLR-null viral IFN-regulated genes. This cellular observation was
patient, allowing their induced-differentiation into hep- supported by the detection of elevated plasma PCSK9 levels
atocytes. Although cells lacking functional LDLR can be in patients infected with DENV resulting in high levels of
well infected by HCV, viral production was significantly in- viremia and increased severity of plasma leakage (247). This
creased upon re-expression of functional LDLR, indicating unexpected role of PCSK9 in Dengue pathogenesis led us to
a role of the receptor in HCV packaging, which is tightly test the effect of blocking PCSK9 function by an inhibi-
linked to the lipid metabolism of the producing cell, rather tory mAb alirocumab. Befittingly, this treatment resulted
than to virus entry (239). in higher LDLR levels and lower viremia. Thus, different
Recently, direct-acting antivirals (DAAs; interferon-free) from HCV, our data suggested that PCSK9 inhibitors could
revolutionized HCV therapy by providing a highly sus- be beneficial for patients with DENV via increased levels of
tained virological response rate (240). It was shown that the antiviral IFN-response genes (247). Double-blind clin-
plasma PCSK9 was significantly increased in patients re- ical studies are now needed to support this proposal.
sponding well to an interferon-based therapy with added In a separate study, it was reported that proPCSK9 (eg,
first generation DAAs, (241). The anticipated protective proPCSK9-Q152H) in the ER can, in some way, reduce cel-
effect of circulating PCSK9 was confirmed by its ability to lular IFN-β expression by inhibiting the functional activity of
inhibit Huh-7.5.1 cell infection by HCV (241). In agree- the cytosolic/nuclear activating transcription factor 2 (ATF2)
ment, lower levels of circulating PCSK9 and LDLc were (249). However, whether the cytosolic ATF2 communicates
found in untreated patients chronically infected with HCV with the ER-localized proPCSK9 via another intermediate
genotype 3 (HCV-G3), likely resulting from upregulated factor has not been addressed. Nevertheless, if confirmed by
LDLR activity, than in patients infected with HCV-G1, other studies, this anti-IFN-β effect of proPCSK9 via ATF2
inhibition, combined to the anti-IFN effect of mature PCSK9 Other FPTase inhibitors have since been evaluated in clin-
via cholesterol upregulation in the ER and inhibition of STING ical trials implicating various cancers (https://clinicaltrials.
(see above for DENV), would solidify PCSK9’s role as a key in- gov/ct2/results?term=farnesyl-protein+transferase+inhibito
hibitor of IFN expression, and thus a target of choice to inhibit r+&Search=Search).
for combating viral infection. The roles of SREBP-2 activation and/or circulating
In the present COVID-19 pandemic, statins have been vs tumor-derived PCSK9 in tumor LDLR regulation are
shown to increase the expression of the SARS-CoV-2 receptor not clear. Individuals with high tumoral PCSK9 mRNA
angiotensin converting enzyme 2 (ACE2) in animal models expression exhibit worse overall survival than those
(250, 251), but they were also reported to significantly improve with low expression in different patient cohorts (266).
the prognosis of COVID-19 patients >65 year-old (252-254). Interestingly, PCSK9 mAbs that raise LDLR levels in the
This likely reflects the ability of statins to improve endothelial liver and other tissues but reduce circulating LDLc were
function and to reduce blood coagulation and inflammation not associated with a significant change in cancer inci-

via cholesterol-dependent and cholesterol-independent mech- dence, at least in the short term (162, 187). However,
anisms (255). Because PCSK9 mAb further reduces LDLc by upon PCSK9 neutralization, extremely low circulating
~60% and cardiovascular events in statin-treated patients, it LDLc levels (originating from liver VLDL) will not result
was suggested to also treat selected COVID-19 patients with in excessive internalization by supernumerary LDLR (98),
PCSK9i, especially those who would benefit most from a and may rather reduce tumor growth (267, 268). Indeed,
boost in their IFN levels (256). This is especially relevant for it was reported that PCSK9 deficiency reduces melanoma
patients presenting LOF variants in the Toll-like receptor 3 metastasis in mouse liver (269) and that LOF and GOF
(TLR3)- and interferon regulatory factor 7 (IRF7)-dependent variants are associated with lower and higher incidence
IFN immunity (257). of human breast cancer, respectively (270). Accordingly,
Time will tell whether PCSK9 inhibition coupled with PCSK9 expression could be a valuable biomarker for the
statins also alleviates the symptoms of patients carrying clinical prognostic outcome of certain types of malignan-
other viruses, such as the human cytomegalovirus (HCMV) cies, including hepatic, gastric, kidney, pancreatic and
that, like DENV, is more infectious when cellular choles- breast cancers (271).
terol levels are upregulated in absence of LRP1 (258), an- Early reports showed that PCSK9 is well expressed in the
other PCSK9 target (131). spleen and thymus (51, 95). Until very recently, its function(s)
in these regulatory immune tissues remained obscure. A recent
report shows that in CD8+ T cells the LDLR makes a com-
PCSK9 and Cancer/Metastasis plex with the T-cell receptor (TCR), which is activated upon
The 2 leading causes of death in industrialized countries binding antigenic peptides that are presented on tumor cells
are cardiovascular diseases and cancer (https://www.cdc. by the major histocompatibility complex (MHC). Because the
gov/nchs/fastats/leading-causes-of-death.htm). LDLs not LDLR association with TCR promotes its recycling to the cell
only promote atherosclerosis in a dose-dependent manner surface and enhances CD8+ T cell antitumor activity (272),
but also provide cholesterol to peripheral cells, including combining PCSK9 inhibition (163) with other immune modu-
cancer cells, which have higher cholesterol requirements to lating therapies, such as immune checkpoint inhibitors, may be
sustain tumor growth (259). Cholesterol plays a key role in quite promising (273, 274).
a plethora of cellular metabolic processes, particularly for Independently from the LDLR, PCSK9 was also reported
highly demanding anabolic steps such as cell growth and to bind MHC-I, but not MHC-II receptors and to target
division (260). In agreement, upregulation of the LDLR in the former to endosomes/lysosomes for degradation (266).
tumors is associated with cancer progression (260-262). In However, while the catalytic domain of PCSK9 is responsible
the 1980s it was suggested that suppression of the SREBP for LDLR binding, it is the CHRD M2 domain that interacts
pathway, would result in lower mevalonate synthesis (263). with an exposed R-X-E70 motif in the N-terminal region of
In turn this would lead to lower levels of isoprenylating MHC-I α-chain (HLA-C; https://www.uniprot.org/uniprot/
intermediates that are needed for farnesylated-RAS inser- P10321). Alignment of α-chain sequences of several species re-
tion into the plasma membrane, resulting in reduced cell fined the motif to R-(Y, M, E, G)-E. (266). In this context, it will
proliferation (264). Thus, it was proposed that specific be worth verifying whether the Furin-cleaved PCSK9 (aa 219-
farnesyl-protein transferase (FPTase) inhibitors may reduce 692) (30) detected in human (275) and mouse (108) plasma
the growth of certain Ras-dependent tumors (263). Indeed, competes with intact PCSK9 for MHC-I receptor binding.
it was shown that combined treatment of HeLa cells with The hemochromatosis protein HFE (high Fe2+; https://
cisplatin and an oral inhibitor of FPTase (L-744 832) sig- www.uniprot.org/uniprot/Q30201), which is mutated
nificantly enhances the cytostatic potency of cisplatin (265). in hereditary haemochromatosis, binds to the transferrin
receptor 1 (hTfR1), and somehow reduces cell surface LDLc in patients with metastatic pancreatic adenocarcinoma
LDLR levels. HFE LOF variants, such as C282Y, are as- combined with Folfirinox, a chemotherapy regimen for ad-
sociated with higher levels of the LDLR (276). Since HFE vanced pancreatic cancer (283), is presently being evaluated
shares a strong homology with some MHC-I α-chain (eg, in a phase I clinical trial (https://clinicaltrials.gov/ct2/show/
HLA-C) and the presence of an exposed R-X-E69 motif, we NCT04862260). Whether irreversible silencing of PCSK9,
recently hypothesized that it may interact with PCSK9 to such as gene editing using CRISPR-Cas, or vaccination will
modulate the levels of the LDLR. Ongoing modeling and have future applications in cancer/metastasis depends first on
site-directed mutagenesis should define more precisely the their careful evaluation for any potential harmful side effects
interaction between the M2 domain of PCSK9 and the R- (163).
X-E motif of MHC-I α-chain, and possibly HFE. Thus,
whether the type I transmembrane MHC-I, HFE or family
members, which are highly expressed in hepatocytes, are Knowledge Gaps to Address Going Forward

candidates for “protein X” remains to be defined. Over the last 18 years, our understanding of the PCSK9
Recently, Liu et al. (266) demonstrated that PCSK9- biology and its implications in various physiological and
deficient tumor cells that were inoculated in syngeneic mice pathophysiological processes have tremendously advanced,
had reduced abilities to form tumors (breast 4T1 and colon in large part due to its robust upregulation of LDLc and
CT26 cancer cells in Balb/c, or melanoma B16F10 and the clinically proven safety of mAb and siRNA inhibitors
colon MC38 cancer cells in C57BL/6 mice). Moreover, in- of PCSK9 to reduce by >50% LDLc consistently and reli-
jection of a programmed death 1 (PD-1) mAb, an immune ably in variety of patients. However, gaps do remain in our
checkpoint inhibitor widely used in immunotherapy (277), mechanistic understanding of the various PCSK9 functions
synergized with PCSK9 deficiency in all models to further during fetal development and in adults. Below we briefly
suppress tumor growth, indicating that inhibition of tumor- touch on some of the questions remaining to have a more
derived PCSK9 overcomes tumor resistance to anti-PD-1 comprehensive view of the PCSK9 biology and its clinical
therapy. Amazingly, a similar synergistic effect of anti-PD-1 applications:
and PCSK9 mAbs was observed in WT mice, an astonishing
result since PCSK9 mAbs target the catalytic domain (176, 1. What are the roles of PCSK9 in extrahepatic tissues such
278), whereas MHC-I binds the M2 domain of PCSK9 (266). as gut, kidney, immune system, and brain?
Whether PCSK9 mAbs sterically hinder the M2 domain and 2. What are the functions of PCSK9 (possibly independent
prevent its ability to bind MHC-I or a putative “protein X” of the LDLR) during development in liver and other
remains to be elucidated. In conclusion, inhibition of tumor- tissues?
derived and/or circulating PCSK9, which contribute to tumor 3. Is there any sex specificity in PCSK9-mediated effects?
growth in mice, synergizes with that of immune checkpoint 4. What are the domains of PCSK9 that interact with
receptors. This unexpected role for PCSK9, aside from its CD36 and receptors other than the LDLR?
canonical LDLR regulation, shows that the role of PCSK9 in 5. Is there any evidence of an intracellular degradation
immunity and/or in inflammation (279) remains to be better pathway of PCSK9 targets in vivo?
elucidated. In this respect, the generation of humanized mice 6. What are the long-term effects of PCSK9i and irreversible
with patient-derived xenografts for cancer immunotherapy silencing of PCSK9?
studies provides an exciting new approach to achieve more 7. Can the cost of PCSK9i be reduced (oral inhibitor) for
robust mouse to human translational applications and pre- worldwide affordability (Fig. 2)?
clinical evaluation of specific immunotherapies (280, 281). 8. Can PCSK9i reduce
Even though a strong body of evidence indicates that chol- a. human cancer/metastasis, alone or in combination
esterol plays a role in cancer development, the evaluation of with other therapies?
the impact of extremely low cholesterol levels (LDLc < 25 mg/ b. some viral infections and/or the severity of human
dL) on human tumorigenesis, cancer progression and/or me- sepsis?
tastasis should be addressed. Namely, the combination of c. allergies and inflammation?
cholesterol lowering drugs (PCSK9i, statins, ezetimibe) that
drastically reduce LDLc (282) and enhance surface LDLR
levels may reduce tumor growth and/or metastasis via chol- Concluding Remarks
esterol depletion and increased TCR and MHC-I activities. The discovery of PCSK9 in 2003 led to an astonishingly
Pairing this approach with chemotherapy/immunotherapy fast progress in our deeper understanding of cholesterol
has been proposed (268). In this vein, the drastic reduction of homeostasis and the implication of PCSK9 in various
pathologies. These include hypercholesterolemia, athero- applications deduced from animal models will surely need
sclerosis, inflammation, sepsis, viral infections, cancer/ rigorous validations before they can be safely and success-
metastasis, and likely many others. PCSK9 protein acts as fully applied to human pathologies.
a chaperone to escort selected surface receptors toward
endosomes/lysosomes for degradation. While the catalytic
domain of PCSK9 binds to certain receptors (eg, LDLR, Acknowledgments
VLDLR, ApoER2, LRP1), the CHRD is implicated in the The authors would like to acknowledge the expert secretarial
binding of other ones (eg, MHC class I receptor) (51, 95, 96, help of Habiba Oueslati in the preparation of this manuscript.
Financial Support: This work was funded thanks to grants
266). Since 2003, a barrage of biological, genetic, and epi-
to N.G.S. including a Canadian Institutes of Health Research
demiological studies in mouse and/or human has resulted Foundation Scheme grant (# 148363) and a Canada Chair in
in the development of the first potent PCSK9i as mAbs in Precursor Proteolysis (# 950-231335).
2015. Such PCSK9 “revolution” starting from discovery to Conflict of Interest Statement: The authors declare no conflict of
interest.

the safe clinical applications of inhibitors to a host target
protein within a period of 12 years is exemplary (284), and
may in part be related to the limited targets of PCSK9 and
its major expression in hepatocytes. Additional Information
The reason why the lack of PCSK9 activity has not yet Correspondence: Nabil G. Seidah, Laboratory of Biochem-
been associated with defined negative outcomes is sur- ical Neuroendocrinology, Montreal Clinical Research Institute
prising and begs the question of why PCSK9 was kept (IRCM, affiliated to the University of Montreal), 110 Pine Ave
West, Montreal, QC, H2W 1R7, Canada. Email: seidahn@ircm.
during mammalian evolution, but lost in some species,
qc.ca.
and very rarely in human. PCSK9 LOF variations re- Disclosure Summary: Nothing to disclose.
ducing circulating LDLc levels might have been prob-
lematic for our ancestors, such as the hunter-gatherer
Denisovans (285), who probably had a cholesterol- References
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Endocrine Reviews, 2022, Vol. 43, No.

The Multifaceted Biology of PCSK9

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ISSN Print: 0163-769X

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conclusion was confirmed in a similar study (66) and by

Thus, although PCSK9 and LDLR mRNA levels were both

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143. Zaid A, Roubtsova A, Essalmani R, et al. Proprotein

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