Nabil G. Seidah

Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal
110 Pine Ave. West, Montreal, QC H2W 1R7, Canada
Tel: (514) 987-5609; E-mail: seidahn@ircm.qc.ca

Abstract

Limited proteolysis of secretory proteins is performed by one or more of the 9-membered proprotein convertase (PC) family known as: PC1/3, PC2, Furin, PC4, PC5/6, PACE4, PC7, SKI-1/S1P and PCSK9. The first 7 proteinases cleave proproteins at single or pairs of basic residues in the Golgi, secretory granules, cell surface or endosomes. These include neuronal and endocrine hormones and their release/inhibiting factors, growth factors and their receptors, adhesion molecules, etc. The regulated neural and endocrine PC1/3 and PC2 generate multiple endocrine peptide hormones and neuropeptides implicated in a variety of biological functions including the family of hypothalamic releasing/inhibiting factors. The ubiquitously expressed Furin is the principal PC that processes constitutively secreted proteins in most tissues and cells. PC4 controls reproduction via the regulation of testicular and ovarian physiology. PC5/6 and PACE4 bind heparin sulfate proteoglycans, are active at the cell surface and extracellular matrix and play critical roles during development by regulating body axis and polarity determinants. PC7 exerts unique functions in the brain and behavior. The members SKI-1/S1P and PCSK9 do not require a basic residue at the cleavage site and play major roles in the regulation of cholesterol/lipid homeostasis. In vivo studies in mice and human demonstrated that PCs play major roles in health and disease states, including cancer/metastasis, viral infections, neurodegeneration and dyslipidemia/atherosclerosis. PCSK9 regulates plasma LDL-cholesterol by enhancing the degradation of liver LDLR. Clinical trials are already underway to test the effects of inhibiting PCSK9 function on human dyslipidemias.

Introduction

The recent sequencing of many genomes led to the amazing realization that the predicted number of encoded proteins was not correlated with the size and/or developmental organization of advanced and more complex species. A major revision of the current catalogs, estimates the number of human protein-coding genes to ≈20,500. In comparison, the genomes of the fruit fly Drosophila melanogaster and that of a house mouse predict ≈13,600 and ≈24,200 protein-coding genes. It is clear that aside from the exact number of proteins coded by a given genome, a number that can vary somewhat between individuals and cell types, other mechanisms must exist to provide diversity between species and increase the effective number of protein/peptide products of a given genome. It is also likely that such mechanisms and their ramifications are continuously evolving, thus providing further diversification and specialization pathways. Among the many pathways used to enhance the diversity and generation of combinatorial products from a given protein, post-translational modifications (PTMs) of the primary protein product represent a major mechanism. These modifications invariably influence some structural aspect and/or functional role of the affected protein or peptide. Most PTMs are introduced into target proteins or peptides via specific enzymatic steps and/or pathways. Several hundreds of these PTMs are now known. Among many others, some of the more common ones that covalently and/or reversibly/irreversibly add a structural element to the protein include Asn- and Ser/Thrglycosylation, Ser/Thr/Tyr-phosphorylation, Tyr-sulfation, protein lipidation, cholesterol attachment, Cys-palmitoylation, Ser/Thr-octanoylation, N-terminal acetylation, N-terminal pyroglutamate formation, and C-terminal amidation. Another most common irreversible mechanism involves the limited proteolysis of proteins at specific sites to generate multiple products. This implicates one or more proteases cleaving at specific sites to release protein or peptide products that can be further covalently modified to shape the usually final bioactive entities. Analysis of the human and mouse genomes revealed the presence of ~600 distinct proteases belonging to one of the 5 major classes of proteolytic enzymes1. Among these, the serine proteases are the most abundant proteases and can be divided into two major families: those related to the trypsin/chymotrypsin fold and those closer to the bacterial subtilisin-clan.

For secretory proteins (the main subject of this review), such processing events occur in distinct subcellular locations such as the trans Golgi network (TGN), cell surface, endosomes, or immature secretory granules. The “hormonal theory” was based on the discovery that specific polypeptide hormones, including γ-LPH/γ-MSH and Insulin derive from larger inactive precursor proteins (POMC and proInsulin) by cleavage at specific paired basic residues2,3. The later discovery that many polypeptide hormones also derive from larger inactive precursor proteins by cleavage at specific single or paired basic residues was a major focal point that led to the long and arduous 22-year search for the cognate processing enzymes responsible for such exquisite cleavages. Indeed, many of the ~3500 mammalian secretory proteins are subjected to limited proteolysis by one or more members of the proprotein convertase (PC) family of serine proteinases of the subtilisin-kexin type identified and characterized during the years 1989-2003: PC1/3, PC2, Furin, PC4, PC5/6, PACE4, PC7, SKI-1/S1P and PCSK9, and their genes known respectfully as PCSK1 to PCSK9 (Figure 1).

Figure 1. Schematic primary structures of the 9 PCs

Extensive biochemical and in vivo analyses expanded our understanding of some of the physiological functions of these PCs, and their role in embryonic development and in the adult. The first seven enzymes PC1/3, PC2, Furin, PC4, PC5/6, PACE4, and PC7 cleave at pairs or single of basic amino acids (aa) within the motif (R/K)-(X)n-R (where n = 0,2,4 or 6 aa) , and the last two known as SKI-1/S1P (cleavage motif R-X-aliphatic aa-X) and PCSK9 do not require a basic residue at the site of cleavage and PCSK9 has only one substrate, itself, and its activity does not implicate its catalytic function4. The tissue distribution (Figure 2) and subcellular localization (Figure 3) of these enzymes revealed that PC1/3 and PC2 are mostly localized within immature and dense core secretory granules of neural and endocrine cells, and as such are poised to process most polypeptide prohormones within the regulated secretory pathway.

Figure 2. Tissue distribution of the PCs.

Figure 3. Schematic representation of trafficking of PCs within the secretory pathway

In contrast, Furin and PC7 are ubiquitously expressed and are implicated in major constitutive processing events of multiple precursors including growth factors and their receptors, proteases, adhesion molecules and even surface glycoproteins of infectious viruses and parasites (Figure 4). The germ-cell specific PC4 is mostly involved in the processing of proproteins regulating gonadal functions, sperm motility and species-specific reproduction. The widely expressed PC5/6 and PACE4 play major roles at the cell surface and in the cleavage of extracellular matrix proteins, including the activation of specific TGF•-like precursors implicated in homeostatic balance and body axis development4 and inactivation of specific lipoprotein lipases regulating the levels of free fatty acids and triglycerides5. The ubiquitously expressed SKI-1/S1P is mostly involved in the initial steps of activation of membrane-bound transcription factors such as those that regulate lipid/cholesterol synthesis, cellular stress response, extracellular matrix organization and bone development5,6. Finally, PCSK9 is a major regulator of the plasma levels of circulating low density lipoprotein (LDL) via enhancing the degradation of the LDL receptor (LDLR) by resident endosomal/lysosomal proteases7. The genetic, molecular, and cellular characterization of the PCs opened wide the field of investigation towards the definition of their specific substrates and their implication in health and disease states8-10. In this review we will present a summary of the biology and diverse functions of the PCs, concentrating on some of their neuroendocrinological functions, with special emphasis on what we have learned so far during the last 20 years since the first members were identified. The realization that some pathological states implicate the overexpression or genetic variants of some of these PCs led to the first clinical trials in 2010 to test the relevance of the last member PCSK9 in human cardiovascular diseases associated with hypercholesterolemia, atherosclerosis, and the metabolic syndrome.

Figure 4. Diversity of the substrates of the basic aa-specific PCs

The neural and endocrine convertases PC1/3 and PC2

PC1/3 and PC2 are the principal activators of prohormones and proneuropeptides within the regulated secretory pathway of neural and endocrine cells. PC1/3 and PC2 process many precursors into their functional hormones: pro-opiomelanocortin (POMC; precursor of ACTH and β-endorphin), proInsulin (Figure 5), proMelanin Concentrating Hormone (MCH), Secretogranin II, proEnkephalin, proDynorphin, proSomatostatin, Chromogranins A and B, proPACAP, proEnkephalin and the neurotrophic factor proBDNF. The functional activation of the convertases PC2 is regulated by a ~30 kDa neuroendocrine PC2-binding protein 7B2 that is co-regulated with it11,12. Both proteases are calcium dependent enzymes that function within the confines of immature and dense core secretory granules13, where they exert their maximal activity. The NMR structure of proInsulin was finally solved and showed that the flexible connecting C-peptide (Figure 5) contributes the presentation and selection of the cleavage sites by PC1/3 and PC214.

Figure 5. Processing of POMC and proInsulin

The physiological importance of PC1/3 and PC2 was deduced from studies of the phenotypes of their gene knockout in mice15,16 and the discovery of some human patients with defects in PC1/317-19. In all cases mice were viable, thus suggesting that individually the genes of these convertases are not essential for life and that these enzymes show some degree of redundancy. However, mice lacking both PC1/3 and PC2 are not viable and die during embryogenesis (Steiner DF, personal communication).

Nevertheless, even though PC2 null mice appear normal at birth, they exhibit retarded growth. Analysis of these mice reveals chronic fasting, hypoglycemia and a deficiency in circulating glucagon16,20. PC2 is known to process various neuroendocrine precursors, and many of these were not fully processed in PC2-null mice20-23, including proSomatostatin, neuronal proCCK, Neurotensin, Neuromedin N, proDynorphin, proOrphanin FQ/Nociceptin and POMC-derived peptides.

Contrary to PC2, PC1/3 gene disruption results in severe developmental abnormalities and a much reduced number of pups born. The PC1/3-null mice exhibit growth retardation and dwarfism15, likely related to the requirement of PC1/3 to process the C-terminal end of the growth hormone releasing hormone (GHRH) precursor at 4 RARLSR73QE 24,25. The adult mutant mice are ~60% of the normal size and phenotypically resemble mice that have mutant growth hormone-releasing hormone (GHRH) receptor. Interestingly, insulin growth factor 1 (IGF-1) and GHRH levels were significantly reduced along with pituitary GH mRNA levels, suggesting that this reduction contributes to the growth retardation observed in these mice. Similarly, analysis of several protein precursors known to be processed by PC1/3 revealed that these mice, like PC2 mutant mice, exhibit multiple defects in multiple hormone precursor processing events26,27. These include the hypothalamic GHRH, pituitary POMC, proInsulin and intestinal proGlucagon. In contrast to PC2-null mice, PC1/3-null mice process normally pituitary POMC to adrenocorticotropic hormone (ACTH), and have normal levels of blood corticosterone. The compensatory enzyme responsible for the production of ACTH in absence of PC1/328 is not known, but is likely to be PACE429. Like PC2-null mice, they also developed hyperproinsulinemia.

We reported that PC2 was a candidate enzyme implicated in the activation of the gonadotrophin releasing factor precursor proGnRH via cleavage at the RPGGKR73DAE, which is then followed by the C-terminal amidation and N-terminal pyroglutamate formation of the GnRH peptide resulting in the active decapeptide pEHWSYGLRPG-amide30. Since then, it became apparent that the synthesis and release of GnRH itself is under the control of multiple factors regulating the sex steroid effects on brain control of GnRH synthesis and release. These include a positive regulation of secretion by the C-terminally amidated Kisspeptin-5431, and a negative regulation by gonadotrophin inhibiting hormone (GnIH, also known as RFRP-3)32,33, both of which belonging the RFamide family of neural and endocrine peptides33. The enzymes implicated in the complex processing of proKisspeptin and proGnIH have not yet been identified, but the implication of PC1/3 and PC2 is quite probable, at least for the production of the C-terminally amidated 54 residues Kisspeptin-54 and the 37 amino acid GnIH. While the former implicates processing at pairs of basic residues, the latter only requires cleavage at single Arg residues at P1, with a P4 Lys or Arg. Based on the reduced number of pups born from PC1 KO mice15, and proteomics studies recently reported in the brain of knockout mice23,27, it is likely that PC1/3 is implicated in the processing of mammalian proGnIH into GnIH, while both PC1/3 and PC2 may process proKisspeptin. This has yet to be demonstrated experimentally in mammals.

Since PC2 is the major convertase that cleaves POMC and proEnkephalin to generate the morphinomimetic peptides β-Endorphin28 and Met-and Leu-Enkephalins34, respectively, it was important to investigate the role of PC2 in pain perception. Unexpectedly, after a short forced swim in warm water, PC2-null mice were significantly less (rather than more) responsive to the stimuli than wild-type mice, an indication of increased opioid-mediated stress-induced analgesia35. The enhanced analgesia in PC2null mice may be caused by an accumulation of opioid precursor processing intermediates with potent analgesic effects, or by loss of anti-opioid peptides. Thus, the presence of abnormal cocktails of pain neuropeptides in the brain of PC2 KO mice are likely to disturb pain perception mechanisms in ways that remain to be fully elucidated.

PC1/3 deficiency in a female patient compound heterozygote for both splicing and non-synonymous mutations resulted in very low expression of the protein. This subject exhibited neonatal massive obesity, abnormal glucose homeostasis, moderate adrenal insufficiency and infertility of hypothalamic origin. These were caused by high circulating levels of misprocessed proInsulin, low circulating levels of active ACTH due to abnormal POMC cleavage and dysfunctional hypothalamic-pituitary-gonadal axis17. Another PC1/3 deficiency female subject presented severe diarrhea, which started on the third postnatal day. Clinical investigations revealed a defect in the absorption of monosaccharides and fat, revealing the role of PC1/3 in the small-intestinal absorptive function18. More recently, hyperphagia and early-onset obesity was reported in a 6-year old boy offspring of a consanguineous union of parents of North African origin, who presented a novel homozygous complete loss of function missense mutation S307L in PC1/319. Although the phenotypes of the PC1/3-null mice differ from those observed in these patients (PC1/3-null mice are not obese), the findings confirmed the importance of PC1/3 as a key neuroendocrine convertase in mammals.

Interestingly, obesity, hyperphagia and increased metabolic efficiency was recently identified in PC1/3 mutant mice exhibiting a homozygote mutation N222D/N222D that results in ~60% decrease in PC1/3 activity, suggesting that it is the dose of PC1/3 and possibly reduced hypothalamic αMSH that may define the obesity phenotype36. Genome wide screens of genes of human chromosome 5q implicated in the development of monogenic obesity, and often associated type-2 diabetes, singled out the PC1/3 gene (PCSK1)37,38. Amazingly, these authors discovered a nonsynonymous variant rs6232, encoding N221D that was associated with the development of obesity in humans37. In human and mouse PC1/3, Asn221 is just adjacent to Asn222 of which mutation to Asp222 was, as noted above, also reported to decrease the activity of mouse PC1/3 and to be associated with obesity36. In another study, the SNP variant rs6235 resulting in a T690S mutation was also associated with reduced postprandial fat oxidation in obese patients38. This established a possible functional role of PC1/3 in the development of a form of monogenic obesity both in human and mouse. The mechanism(s) behind these observations and the cognate substrates that are affected are still not adequately defined.

One possible PC1/3 substrate related to the development of obesity is the gut proGhrelin39, which is post-translationally processed in the stomach into at least two secretable hormones the Ghrelin and Obestatin. These two circulating peptides seem to exhibit opposite functions on food intake. Thus, while Ser3-octanoylated Ghrelin is an orexigenenic appetite-inducing hormone, the C-terminally amidated Obestatin suppresses food intake due to its anorexigenic activity40. From knockout studies in mice, it was proposed that in the stomach, proGhrelin processing into Ghrelin requires PC1/3 but not PC2, and that Ser3-octanoylation of Ghrelin by the acyltransferase GOAT41 requires prior cleavage by PC1/3 at the single Arg51 within the conserved sequence KLQPR5142. However, the processing enzymes that result in the production of Obestatin at the unusual, conserved, single Arg75 and Lys100 residues are not known.

Furthermore, although suggestive and important for binding of Obestatin to its receptor GPR3940 in vitro, it is not absolutely proven that naturally occurring human circulating Obestatin ends with a C-terminal Leu98-amide. If the processing enzyme(s) of Obestatin are different from PC1/3, as was suggested from knockout mice studies42, then the secretion of Ghrelin and Obsetatin may be controlled by different enzymes and their opposing effects on food intake regulated in a differential fashion. The therapeutic potential of Ghrelin and/or Obestatin agonists or antagonists for the treatment of gastrointestinal (GI) motility disorders and the regulation of the hypothalamic control of feeding may thus represent a novel approach to regulate food intake, and possibly type2 diabetes, with potential clinical implications.

Implication of PC4 in reproductive biology

PC4 is expressed exclusively in male testicular germline pachytene spermatocytes and round spermatids, suggesting that it may play a specific physiological function in reproduction. In agreement, PC4 was detected in the acrosomal granules of round spermatids, in the acrosomal ridges of elongated spermatids, and on the sperm plasma membrane overlying the acrosome43. In female mice, PC4 was expressed in macrophage-like cells of the ovary and that its levels are downregulated in activated macrophages, such as in inflammation44. Later on, PC4 was also shown to be expressed in human placenta45.

The in vivo fertility of homozygous mutant males that lack PC446 was severely impaired, without any evident spermatogenic abnormality. Sperm physiologic anomalies likely contribute to the severe subfertility of PC4-deficient male mice43. These results suggested that PC4 in the male may be important for achieving fertilization and for supporting early embryonic development in mice.

So far, one of the identified specific substrates of PC4 in the testis is pituitary adenylate cyclase-activating polypeptide (PACAP) and PC4 is its sole processing enzyme in the testis and ovary of mice47. In vitro studies with purified enzyme concluded that the most probable sequence motif for recognition by PC4 is KXKXXRor KXXR, where X is any amino acid other than cysteine and that it prefers proline at P3, P5 and/or P2′ positions. It was also revealed that PC4 is a good candidate processing enzyme for the growth factors IGF-1 and -2, and several ADAM proteins such as ADAM-1, -2, -3 and -548. Recent work unraveled an unusual property of PC4 in the processing of IGF-II, which has been shown to be an important regulator of fetoplacental growth. Thus, PC4 cleaves pro-IGF-II to generate the intermediate processed form, IGF-II (1-102) and, subsequently, mature IGF-II (1-67), thereby regulating fetoplacental growth45. Specific inhibitors of PC4, such as those recently reported in flavonoids49, may one day serve as male contraceptives.

The ubiquitous membrane-bound convertases Furin, PC7 and SKI-1/S1P

The proper functioning of a cell requires numerous proteins, including growth factors and their receptors, adhesion molecules, properly folded extracellular matrix proteins, lipids and sterols as well as various enzymes such as metallo- and serine-proteases. Such housekeeping processes are controlled at many levels, including posttranslational processing of secretory precursor proteins.

Furin, a ubiquitous type-I membrane-bound proteinase, process a wide variety of proproteins at the general recognition motif R-X-(R/K)R50, in the trans-Golgi network, cell surface or in recycling endosomes51. In the CNS, Furin is responsible for the processing of a number of growth factors including the neurotrophins proNGF52 and neural cell adhesion and cueing proteins such as L1 CAM53 and Semaphorins54. For example, Neuropilin is an essential cell surface receptor that functions in both semaphorin-dependent axon guidance and vascular endothelial growth factor (VEGF)dependent angiogenesis. Semaphorin 3F (Sema3F) is proteolytically processed by Furin at its C-terminal RXRR site, thereby potently and competitively inhibiting the binding of VEGF to neuropilin55, resulting in an anti-angiogenic effect. By exposing a fusiogenic site in the surface glycoproteins of infectious viruses and parasites, Furin plays a critical role in regulating the infectivity of various retroviruses, e.g., HIV56,57 and influenza viruses58,59, as well as neurotropic viruses such as the influenza virus serotypes H1N160 and H5N161.

Inactivation of the mouse fur gene (Pcsk3) causes embryonic death at about embryonic day 11 (E11), due to hemodynamic insufficiency and cardiac ventral closure defects62. Mutant embryos failed to develop large vessels despite the presence of endothelial cell precursors. A conditional KO in liver, resulted in viable Pcsk3flox/flox Tg(Mx1-cre) mice with almost no phenotype63. This demonstrated some redundancy with other PCs, since some typical Furin substrates were cleaved to a lesser extent.

Different from the other PC-null mice, our multiple studies on PC7-null mice embryos revealed no apparent abnormal phenotype under resting conditions64. This may be explained by the fact that PC7 expression extensively overlaps with that of Furin. Many reports show that PC7 and Furin process the same substrates, such as PDGF-AA, PDGF-BB65, VEGF-C66, BMP467 and others. Alternatively, the most conserved convertase PC7 may be involved in the processing of a panel of non-essential substrates, and its absence may result in a subtle phenotype. We have recently found that PC7 is unique among the PCs in its ability to reach the cell surface either directly from the endoplasmic reticulum or through the conventional secretory pathway68. Furthermore, PC7 is the only convertase to process pro-epidermal growth factor (proEGF) into an intermediate 115 kDa product with enhanced EGF-like activity69. Furthermore, our preliminary data demonstrated that PC7 KO mice exhibit an anxiolytic phenotype, likely related to an amygdala and/or hippocampal dysfunction (Seidah NG et al., unpublished observations). Double KOs of Furin and PC7 may also help resolve the issue of the possible critical functions of PC7 during embryogenesis in a Furin-null background.

SKI-1/S1P5,70 is a key enzyme in the regulation of lipid metabolism and cholesterol homeostasis that cleaves the transcription factors sterol regulatory element binding proteins (SREBP-1 and SREBP-2). A two-step proteolytic process starting with SKI1/S1P at the consensus sequence R-X-V-L71 and then site 2 protease (S2P) releases the cytosolic N-terminal segments of SREBPs from cell membranes, allowing their translocation to the nucleus (nSREBP), where they activate transcription of more than 35 mRNAs coding for proteins/enzymes required for the biosynthesis and uptake of cholesterol and fatty acids, as well as the LDL receptor (LDLR)72-74 .

Other type-II membrane-bound substrates include ATF6, at least 6 CREB-like basic leucine zipper transcription factors5,6,71. Brain-derived neurotrophic factor (BDNF) is a soluble substrate and the study of its processing led to the initial cloning of SKI-1/S1P70. We also showed that the soluble pro-Somatostatin is cleaved by SKI-1/S1P to release the N-terminal peptide Antrin75. SKI-1/S1P is also essential for the activation of the surface glycoproteins of a number hemorrhagic fever viruses resulting in fusion competent viruses76,77.

The essential roles played by SKI-1/S1P are evident from the fact that deletion of its gene results in embryonic death at the earliest stages of cell division, i.e., from one to two-cell stage, and hence preventing blastocyst formation73. The importance of this enzyme in the CNS is yet to be unraveled using tissue-specific knockout mice. When this was done in liver, the levels of circulating total cholesterol and triglycerides were decreased by ~50%, clearly emphasizing its critical control of cholesterol and fatty acid synthesis and uptake73. Cholesterol metabolism has been implicated in the pathogenesis of several neurodegenerative diseases, including the abnormal accumulation of β-amyloid, one of the pathological hallmarks of Alzheimer’s disease (AD). Accordingly, we have very recently delineated the importance of 24(S)hydroxycholesterol, the major form of cholesterol in brain, in the development of AD, and the use of cholesterol-ester transferase ACAT-1 inhibitors as a viable therapeutic approach for treating certain forms of AD78.

The widely expressed convertases PC5/6 and PACE4

The convertases PC5/6 and PACE4 form a class of their own based on their primary structures and their ability to bind the cell surface via their C-terminal Cys-rich domains which bind tissue inhibitors of metalloproteases (TIMPs) and heparin sulfate proteoglycans (HSPGs), and in many cases inactivate HSPG-bound proteins such as endothelial and lipoprotein lipases, and possibly adhesion molecules4. Since in cellular experiments and in vitro many of the substrates processed by either enzyme can also be cleaved by Furin and/or PC7, the identities of possibly unique, specific physiological substrates of PC5/6 and PACE4 need to be unraveled. In the CNS, it was shown that PC5/6 can process the neural adhesion molecule L1 assisting in neuronal repair and migration53. Ontogeny and tissue distribution analysis of these convertases showed that PC5/6 expression is detected early during embryonic development, appearing first in extra-embryonic tissues. By E9, it is also specifically expressed in cells of the maternalembryonic junction, where no other convertase is expressed79. Present data strongly suggest unique tissue-specific functions of PC5/6 and PACE480. Thus, PC5/6 mRNA was detected only in neuronal cells, whereas PACE4 mRNA was expressed in both neuronal and glial cells. In areas that are rich in neuropeptides such as cortex, hippocampus, and hypothalamus, mRNA levels of PC5/6 were high but PACE4 were low or undetectable. In regions, such as the amygdaloid body and thalamus, distinct but complementary distributions of PC5/6 and PACE4 mRNAs were observed. The medial habenular and cerebellar Purkinje cells expressed very high levels of PACE4 mRNA.

The knockout (KO) of PACE4 and PC5/6 genes in mice resulted in different phenotypes. Thus, while the PACE4 KO results in a 75% viable phenotype with bone morphogenesis defects and cyclopia81, that of PC5/6 causes death at birth with multiple CNS defects, bone morphogenic malformations and heart ventricular-septal defects82,83. So far, in PACE4 KO mice the identity of the substrate(s), which if left unprocessed would result in the observed complex craniofacial malformations, including cyclopia81, is/are yet to be defined. In contrast, we identified one of the major substrates of PC5/6 as the developmentally regulated growth differentiating factor 11 (GDF11), a secreted member of the TGF-β superfamily that participates in the establishment of the anterior– posterior axis by controlling the spatiotemporal expression of Hox genes. Amazingly, the distinguishing feature of this substrate is the presence of an Asn residue just after the GDF11 cleavage site within the unusual motif RSRR296NL82. Furthermore, we identified nonsynonymous mutations in the B-isoform of PC5/6 (PC5/6B in Figure 1) in patients with VACTERL (vertebral, anorectal, cardiac, tracheoesophageal, renal, limb malformation) and caudal regression syndrome, the phenotypic features of which resemble the mouse mutation. Accordingly, we proposed that PC5/6, at least in part via GDF11, coordinately regulates caudal Hox paralogs, to control anteroposterior patterning, nephrogenesis, skeletal, and anorectal development83.

PC5/6 is a protective gene, since its specific absence in gut leads to increased tumorigenesis in intestinal duodenum84, and results in a decreased extracellular inactivation of the HIV-1 accessory protein Vpr85. However, this does not exclude that under certain pathological conditions (e.g., metastastatic cancer), overexpression of PC5/6 may result in enhanced pathology, e.g., via inactivation of adhesion molecules. A similar type of situation may exist in osteoarthritis, where PACE4 is overexpressed, and its inactivation may relieve the associated pain symptoms. Indeed, proteolytic degradation of the major cartilage macromolecules, aggrecan and type II collagen, a key pathological event in osteoarthritis, is performed by ADAMTS-4,5, matrix metalloproteases that are proteolytically activated by PACE4 processing of their inhibitory pro-domains in cartilage86,87.

Finally genome wide screens identified specific single nucleotide polymorphism in PCSK5 (coding for PC5/6) and PCSK6 (coding for PACE4) genes relating the former to enhanced bone formation possibly through processing of FGF2388, and the latter to handedness in individuals with a dyslexia syndrome89, likely due to the ability of PACE4 to process specific TGFβ-like precursors.

PCSK9 and its implication in hypercholesterolemia

PCSK9 was first characterized by our group in 2003, is highly expressed in adult liver, gut and kidney90. During development PCSK9 mRNA levels are transiently high in telencephalon (E12-E15), in the rostral extension of the olfactory peduncle and in cerebellar neurons. Under resting conditions, the expression of PCSK9 in adult brain is restricted to the olfactory peduncle and cerebellum. Overexpression of PCSK9 in telencephalic progenitor cells enhanced proliferation and neuronal differentiation90. PCSK9 expression in liver hepatocytes is now known to be upregulated by SREBP-2 and downregulated by cholesterol. We established the first association between single-point mutations in PCSK9 and autosomal dominant hypercholesterolemia (ADH) in two French families91. Later, it was shown that non-sense PCSK9 mutations, likely resulting in a loss of function, are associated with hypocholesterolemia in ~2% of black subjects92. Accordingly, mutations associated with hypercholesterolemia result in a gain of function of PCSK9 that triggers the degradation of LDLR in acidic compartments, likely endosomes93. By an as yet unknown mechanism, high levels of PCSK9 lead to a faster rate of degradation of cell surface LDLR, resulting in increased circulating LDLcholesterol, as LDL uptake in hepatocytes by LDLR is diminished7. In agreement, Pcsk9 null mice exhibit increased LDLR protein, but not mRNA, and a ~2-fold drop in circulating cholesterol94,95, whereas mice overexpressing PCSK9 following recombinant adenoviral infections exhibit high levels of circulating cholesterol95. A 32-year-old female aerobic instructor with two compound heterozygote mutations leading to complete loss of PCSK9 expression was recently identified96. Although healthy, fertile, normotensive, with normal liver and renal functions, her LDL-cholesterol is remarkably low (14 mg/dL). This and the observation that loss-of-function nonsense mutations could lead to 88% reduction in the risk of development of cardiovascular artery disease (CAD) over a 15year period97, indicate that inhibition of PCSK9 may represent a safe and effective strategy for the primary prevention of CAD7,98. However, the paucity of humans lacking PCSK9 suggests that caution is advised upon implementing PCSK9-directed therapies, especially if these result in almost complete loss of its expression7,95. The role of PCSK9 in the brain is less clear, and may only become apparent under stressful or pathogenic conditions.

Even though we described Annnexin A2 as an endogenous inhibitor of PCSK9 activity in extra-hepatic tissues99, so far, no potent small molecule inhibitor of PCSK9 has been reported. In view of the activity of secreted PCSK9 on LDLR and because it can be easily measured in human plasma100, a number of pharmaceutical companies developed blocking monoclonal antibodies to inhibit PCSK9’s interaction with the LDLR. Indeed, Amgen developed a clinically relevant monoclonal antibody (mAb) that results in a ~80% reduction of LDL-cholesterol that lasted for 2-weeks in monkey101. Since then, other companies indicated that they are also starting phase 1 clinical trials using a similar mAb approach, including Merck, SA-Regeneron, Novartis, and Pfizer-Rinat. Using another approach aimed at silencing the mRNA of PCSK9 using injectable antisense oligonucleotides102, RNAi103 or Locked Nucleic Acid (LNA)104 approaches, BMS-ISIS, Alnylam and Santaris pharmaceuticals are also pursuing preclinical/phase 1 trials. It remains to be seen which of these varied strategies will bear fruit in clinic and will result in a safe, an economically viable and patient-compliant approach to treat hypercholesteromia and the associated metabolic syndrome. The selected anti-PCSK9 therapy will either be used alone or, more likely, in combination with already available cholesterol lowering therapies, such as the use of HMG-CoA reductase inhibitors known as statins7,105.

Conclusions and future perspectives

The 9-membered family of the proprotein convertases (PCs) comprises 7 basic amino acid specific subtilisin-like serine proteinases, related to yeast kexin, known as PC1/3, PC2, Furin, PC4, PC5/6, PACE4 and PC7, and two other subtilases that cleave at non basic residues called SKI-1/S1P and PCSK9. Except for the testicular PC4, all the other convertases are expressed in brain and play a critical role in various neuronal functions including the production of diverse neuropeptides as well as neural growth factors and receptors, the regulation of cellular adhesion/migration, cholesterol and fatty acid homeostasis and repair mechanisms and growth/differentiation of progenitor cells.

Some of these convertases process proteins implicated in neuropathologies, such as Alzheimer’s disease β-secretase (BACE1), neural growth such as NGF, as well as adhesion molecules such as L1 and N-CAM, important for cancer malignancies and neuronal regeneration and repair following injury.

The field has now matured enough that it is now the ripe time to define the physiological functions and secretome pathways affected by each PC, their substrates and partners, and to devise specific therapies aimed at controlling their activities. Regulating the levels of specific PCs may find future applications in the control of some pathology, e.g., hypercholesterolemia, atherosclerosis, obesity, diabetes, reproduction, neurodegenerative diseases, endocrine dysfunctions, cancer/metastasis, and viral infections. The development of specific inhibitors/modulators of convertases may well find clinically relevant applications in the control of some of these pathologies.

Acknowledgements: This work was supported by a CIHR Canada Research Chair on precursor proteolysis # 216684. The multiple discussions and contributions of the various members of the Seidah lab over the years are acknowledged and the secretarial help of Brigitte Mary is highly appreciated.

FIGURE LEGENDS

Figure 1. Schematic primary structures of the 9 PCs. The basic-aa-specific PCs together with bacterial subtilisin, yeast kexin, SKI-1/S1P and PCSK9 are individually boxed to emphasize their distinct subclasses. Their genes PCSK1 to PCSK9 are emphasized. PC5/6 exists as 2 alternatively spliced isoforms, soluble PC5/6A and membrane-bound PC5/6B. The various domains and the catalytic triad residues Asp, His, and Ser and the oxyanion hole Asn are indicated. The three different motifs generally recognized by the 7 basic-aa specific convertases, SKI-1/S1P and PCSK9 are also shown, with the downward arrow indicating the cleavage site. Here X = variable amino acid

Figure 2. Tissue distribution of the PCs. The expression profiles of the various PCs in different tissues are shown.

Figure 3. Schematic representation of trafficking of PCs within the secretory pathway. The convertase PC7 is the only one that can reach the cell surface by the conventional and unconventional pathway directly from the endoplasmic reticulum68. the constitutive (all others) and regulated (PC/3, PC2) secretion of the other PCs are also emphasized.

Figure 4. Diversity of the substrates of the basic aa-specific PCs. The recognition motifs by the basic aa-specific PCs are emphasized.

Figure 5. Processing of POMC and proInsulin. Schematic representation of the sitespecific processing of POMC and proInsulin by PC1/3 and PC2 followed by the trimming action of carboxypeptidase E (CPE) to release the final bioactive products are emphasized. The NMR structure of Yang Y et al.14 is emphasized.

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