REVIEW Subtilase-Like Pro-Protein Convertases: from Molecular

1 REVIEW

Subtilase-like pro-protein convertases: from molecular specificity to therapeutic applications

F Bergeron, R Leduc and R Day Département de Pharmacologie, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, 3001, 12e Avenue Nord Sherbrooke, Québec, Canada J1H 5N4 (Requests for offprints should be addressed to R Day; Email: [email protected])

ABSTRACT Limited proteolysis of most large protein precursors structural analysis of the proteases, the mechanisms is carried out in vivo by the subtilisin-like of enzyme/substrate specificity, their interaction pro-protein convertases. Many important biological with other proteins such as 7B2, and the compara- processes such as peptide hormone synthesis, viral tive tissue and cellular distribution of the enzymes protein processing and receptor maturation involve and their substrates. These data are then used as a proteolytic processing by these enzymes, making background for the review of the known biological them potential targets for the development of novel functions of subtilisin-like pro-protein convertases, therapeutic agents. However, the efficient develop- the reported clinical cases involving proteolytic ment of such molecules requires a better under- processing defects and, finally, the ongoing develop- standing of the molecular mechanisms of proteolytic ment of new therapeutic inhibitor molecules based protein processing. Herein, we review the most on this knowledge. recent findings on the molecular aspects of Journal of Molecular Endocrinology (2000) 24, 1–22 subtilisin-like convertase activity, such as the

INTRODUCTION family of subtilisin-related pro-protein convertases, known as the SPCs. Proteolytic processing is a post-translational The discovery of the SPCs was based on initial modification by which the cell can diversify and observations made in the mid-80s using yeast regulate the products of its genes. Just as a single genetics. A mutation in the Kex2 gene in polycistronic bacterial gene can encode many Saccharomyces cerevisiae prevented conjugation proteins with different functions, a mammalian because it did not permit proteolytic processing of precursor protein can give rise, by hydrolysis of the alpha-mating factor precursor. Genetic com- selective peptide bonds, to several molecules with plementation with Kex2 revealed a deficiency in the different biological activities. Proteolytic processing expression of a peptidase with specificity for within the secretory pathway is crucial for the cleaving on the carboxyl (C) side of paired basic activation or inactivation of many proteins and the residues (Julius et al. 1984). The Kex2 peptidase, regulation of their cellular localization. Indeed, now known as kexin, is a serine protease of the processing is important in zymogen activation, subtilisin family (Wells et al. 1983, Mizuno et al. peptide hormone processing, complement acti- 1988) that has been shown to be calcium-dependent vation, clot formation and lysis, angiogenesis and (Fuller et al. 1989a, Mizuno et al. 1989) and to tissue remodeling. Many precursors involved in cleave correctly the pro-hormone precursors in these important biological functions require cleav- mammalian cells (Thomas et al. 1988). It was thus age at specific pairs of basic residues, such a logical to assume that a mammalian homolog could Lys-Arg. In mammalian species, this catalytic exist. Shortly after the discovery of the yeast function is now understood to be carried out by a enzyme, a sequence homology analysis (Fuller et al.

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1989b) through the human genome database positively charged amino acids, usually to produce identified a mammalian Kex2 homolog that was biologically active products. Other serine proteases, encoded by the fur gene, on chromosome 15 (van such as trypsin or chymotrypsin, are far less den Ouweland et al. 1989, 1990). Subsequently, selective, often playing a role in protein degra- seven kexin-related mammalian enzymes were dation. Peptide processing events occur in the identified. Although each enzyme has been secretory pathway of eukaryotes at the carboxyl independently named by its discoverers, a simplified terminal of Lys or Arg residues (occupying the P1 nomenclature for the group of mammalian position). (The cleavage site of proteases is processing proteases has been proposed (Chan et al. described according to a nomenclature proposed by 1992), using the term SPC: SPC1 (furin or paired Schechter & Berger (1967), whereby P1 is the first amino acid converting enzyme (PACE)), SPC2 residue on the amino (N)-terminal side of the (PC2), SPC3 (PC1 or PC3), SPC4 (PACE4), SPC5 cleaved peptide bond, P2 the second residue, and so (PC4) SPC6 (PC5 or PC6-A) and SPC7 (LPC, PC7 on. The amino acids located on the C-terminal side or PC8). of the cleavage site are named P1,P2,P3, etc.) The large number of mammalian SPCs now Typically, SPC cleavage sites feature Arg-Arg and discovered raises several intriguing questions as to Lys-Arg motifs, but some are composed of their potential overlapping or distinct functions. Arg-Lys, Lys-Lys, or even single Arg residues. Functional specificity could be determined by 1) a The substrate specificity of SPC1 has been the high degree of substrate specificity by each enzyme, object of numerous studies and is the best and 2) a distinct cellular expression or specific understood, revealing the consensus recognition intracellular localization of each SPC. The future sequence Arg-Xaa-Arg/Lys-Arg?, of which the P4 development and successful application of SPC arginine is critical for cleavage (Hosaka et al. 1991, inhibitors as potential therapeutic agents is highly Molloy et al. 1999). Analysis of a repertoire of more dependent on the availablility of this information. than 40 precursor molecules cleaved at furin sites Within the context of the intracellular environment, also showed that 50% of the pro-proteins possessed will SPC inhibitors have to be designed that target a serine residue in the P1 position. The mimi- all SPC activities simultaneously, or will inhibitors mal recognition sequence, Arg-Xaa-Xaa-Arg?,is that target specific SPC activities be more effective? observed in all substrates found to be cleaved by Thus the knowledge gained from defining whether SPC1. This sequence was later confirmed in studies SPCs have redundant or distinct functions, at the using random substrate phage display. In these level either of expression or of activity, will be experiments, virtually all clones coding for SPC1 extremely useful. One approach to this problem is substrates had an RxxR? motif, and many had Lys, to define the cellular localization of SPCs – that is, Arg, or Pro in the P2 position (Matthews et al. to identify precisely the ‘cocktail’ of SPCs expressed 1994a). The presence of a P4 Arg residue is not in any cell of interest. Indeed, this is an important mandatory for the activity of all convertases but, criterion, as the specificity of processing function is generally, the presence of an Arg residue in P6, P4 not only dictated by the cleavage specificity of an or P2 has been shown to enhance the cleavage SPC, but also by its distinct localization. A second efficiency by SPCs (Hosaka et al. 1991). approach is to define better the cleavage specificity The general features of intracellular precursor of each SPC and to define how SPCs are regulated, processing are shown schematically in Fig. 1. for example at different levels including transcrip- Precursor processing can occur in both the tional, translational, and through protein–protein constitutive and the regulated pathways. It is interactions (e.g. SPC2/7B2, see later). Finally, believed that SPC1 is important in the cleavage of the ability to ‘delete’ a specific SPC from the precursors in the constitutive pathway, whereas cellular environment, such as has been accom- SPC2 and SPC3 are important in the cleavage of plished with the creation of null mice, is especially precursors in the regulated pathway. After SPC useful to define the unique functions of that SPC cleavage, the newly exposed basic residues of the and the possible compensatory contributions of released N-terminal peptide are removed by other co-localized SPCs. carboxypeptidases specific for basic residues (Kemmler et al. 1973). Recently, it has been shown that this role can be carried out, not only by MECHANISM OF SPC PRO-PROTEIN carboxypeptidase E (CPE), but also by a structural PROCESSING homolog known as carboxypeptidase D (CPD) (Xin et al. 1997). CPE has been shown to be targeted to In general, SPCs appear to be highly specific secretory granules and appears to play this role in a enzymes, cleaving pro-protein precursors at specific relatively endocrine-specific manner (Fricker 1991).

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 1. Processing of inactive precursors into bioactive peptides in constitutive and regulated secretory pathways.

In contrast, CPD appears to have a role in accumulation of C-terminally extended peptides processing of proteins negotiating the constitutive (Naggert et al. 1995). secretory pathway (Dong et al. 1999). Finally, after removal of the basic residues, peptides having a C-terminal glycine are often amidated by the action of peptidyl-glycine-
-amidating mono-oxygenase. STRUCTURAL ANALYSIS OF SPC ENZYMES This modification enhances the binding of neuro- endocrine peptides to their receptor and increases Substrate specificity, catalytic efficiency and intra- peptide stability in vivo (Mains et al. 1990). cellular localization of each SPC are conferred by Interestingly, the steps of this enzymatic cascade the unique structural components of each molecule. involving the SPCs and carboxypeptidases appear to All the members of this enzymatic family have be functionally linked. It has been observed that similar N-terminal structures with common func- carboxypeptidase activity enhances SPC2 process- tional domains, whereas their C-terminal domains ing of pro-dynorphin, suggesting that the removal are variable. The more conserved N-terminal of C-terminal basic residues prevents product domains are the signal peptide, the pro-segment, inhibition (Day et al. 1998). Hence, CPE activity the catalytic domain and the P domain, which could play a regulatory role on SPC activity in vivo. includes a conserved RGD motif (Fig. 2). The more This notion is supported by studies on CPE-null variable C-terminal domains include Cys-rich mice (fat/fat), which show generally reduced SPC regions, transmembrane and cytosolic domains, processing activity that could be due to the amphipathic helices, and sorting domains. www.endocrinology.org Journal of Molecular Endocrinology (2000) 24, 1–22 4   and others · SPCs: molecular specificity to therapeutic applications

 2. Structural comparison of the mammalian SPCs and their related yeast kexin (yKexin) and bacterial subtilisin (Subt) BPN proteases.

Like other protein precursors, SPCs are first reticulum (ER) membrane. The second processing synthesized as large inactive pro-proteins that event is the removal of the 80–90 amino acid undergo proteolytic maturation in their transit pro-segment. This domain has two important through the secretory pathway. The first domain to functions: as an intramolecular chaperone and as a be removed is the N-terminal signal peptide that is competitive inhibitor. Study of the bacterial required for the entry of the protein into the subtilisin model and other pro-proteins suggests secretory pathway. This peptide is cleaved by a that N-terminal pro-peptides act as intramolecular signal peptidase after the translocation of the chaperones and are essential for proper folding of nascent polypeptide chain through the endoplasmic the proteins to which they are attached (Eder et al.

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1993, Shinde & Inouye 1993). It has been ing that negatively charged residues of the catalytic demonstrated that these pro-peptides are cleaved by pocket interact with positively charged residues of an intramolecular autocatalytic mechanism at the the substrate (Creemers et al. 1993b, Roebroek et al. ER level (Leduc et al. 1992, Creemers et al. 1993b, 1994, Rockwell & Fuller 1998). Interestingly, SPC Goodman & Gorman 1994, Matthews et al. 1994b, active site residues can be engineered to modify Lamango et al. 1999), and this cleavage is important substrate specificity (Rheinnecker et al. 1993), as is for the correct sorting of the enzymes out of the ER the case, for example with furilisin (Ballinger et al. (Creemers et al. 1995, Zhou et al. 1995). Two 1996), a bacterial subtilisin mutant that has been clusters of pairs of basic residues are found in the re-designed to induce a substrate recognition pro-segment of each convertase. The excision of the similar to that of mammalian SPC1. The active site pro-peptide in the ER occurs at the C-terminal site, of SPCs also contains a conserved Asn residue for which the consensus sequence is Arg-Xaa-Arg/ needed for the stabilization of the oxyanion hole Lys-Arg?. After this first cleavage, the pro-segment transiently formed during the hydrolysis reaction. most probably occupies the active site, acting as a Curiously, SPC2 differs from the other SPCs, as the competitive inhibitor until it is released. In the case Asn residue is replaced by an Asp. The significance of SPC1, a correct calcium and pH environment of this special feature of SPC2 is not clear, as an found in the trans-Golgi network (TGN) enables Aspcatalytic triad of the P domain of SPC3 demonstrate the importance subtilisin-related serine proteases, including the of this region in regulating the stability, calcium Asp, His and Ser active site residues. SPCs use the dependence, and optimal pH of these enzymes same enzymatic mechanism (i.e. the same catalytic (Zhou et al. 1998). The importance of this domain triad) as the trypsin-related enzymes, although no for the structural cohesion of the enzyme is also sequence homologies exist between these two demonstrated in human colon carcinoma LoVo families. Hence, SPCs are believed to have arisen cells, in which a single nucleotide deletion in the from a convergent evolutionary mechanism. Under- region covering the P domain of SPC1 completely standing the conformation of the catalytic domain of abolishes its activity (Takahashi et al. 1993, 1995b). SPCs is important, because this structure is The P domain of SPCs also contains a conserved primarily responsible for substrate selectivity. RGD motif, the role of which remains unclear. Although efforts are in progress to obtain three- RGD motifs are typically found in extracellular dimensional structures of SPCs, the only catalytic matrix proteins, such as fibrinogen, and are domain model available at present is based on the involved in cellular adhesion via integrins. How- structure of bacterial subtilisin (Siezen et al. 1994, ever, there is at present no evidence that the RGD Lipkind et al. 1995). Furthermore, various residues motif in SPCs is involved with integrin attachment, that play a role in the substrate recognition of SPCs either intracellularly or extracellularly (Seidah et al. have been identified by mutational analysis confirm- 1994, Rovere et al. 1999). Mutational analysis of the www.endocrinology.org Journal of Molecular Endocrinology (2000) 24, 1–22 6   and others · SPCs: molecular specificity to therapeutic applications

RGD motif in SPC3 produces confusing results, protein related to clathrin-associated molecules with effects on substrate processing (pro- (Wan et al. 1998). Other important cytoplasmic tail opiomelanocortin, POMC), precursor processing sorting signals have been identified, namely a (pro-segment removal and C-terminal cleavage), tyrosine-based motif important for the interaction enzyme stability and intracellular sorting (Lusson with AP-1 Golgi-specific assembly proteins et al. 1997). From these same studies, it also appears (Teuchert et al. 1999) and, recently, hydrophobic that modification of the P domain (i.e., within the based motifs that could mediate internalization RGD motif) probably results in a structural (Stroh et al. 1999). destabilization that could explain all other effects Other SPCs, such as SPC3 and SPC2, do not observed. Other experiments with kexin P domain have an integral membrane domain, but are believed mutants support the notion that such modifications to have an amphiphatic helix that has been can have drastic effects on enzyme trafficking, hypothesized to mediate hydrophobic interactions resulting in a failure to exit from the ER with membranes (Seidah et al. 1990). However, an (Gluschankof & Fuller 1994). SPC2 deletion mutant lacking the putative The regions located C-terminal to the P domain C-terminal amphipathic helix was nevertheless are highly variable for each convertase. For membrane associated, suggesting that this domain example, cysteine-rich regions are found in SPC1 was not essential for attachment of SPC2 to (van de Ven et al. 1990), SPC4 (Kiefer et al. 1991), membranes (Shennan et al. 1991). and SPC6 (Lusson et al. 1993). The isoform of SPC6, known as SPC6-B (see Fig. 2) has a particularly extended Cys-rich region. Interest- A COMPLEX CASE: SPC2 BIOSYNTHESIS ingly, this domain has some homology with receptor protein tyrosine kinase precursors. However, few As for other SPCs, during its biosynthesis, SPC2 studies have examined the role of this domain, proceeds through a series of intermediate forms which remains unknown. In contrast, the (Guest et al. 1992, Lamango et al. 1999). After C-terminal domain of SPC7 displays a unique pro-peptide removal by an autocatalytic mechanism serine/threonine-rich region of unknown function (Matthews et al. 1994b), the enzyme is further (Bruzzaniti et al. 1996, Meerabux et al. 1996, processed by a truncation of its C-terminal domain Seidah et al. 1996c), reminiscent of the yeast Kex2 and glycosylation to yield a final product of 64 kDa enzyme. SPC2 (Shen et al. 1993). It is noteworthy that SPC2 Other regions in the C-terminal part of SPCs and SPC3 pro-segment removal occurs at different have been identified for which the functions are pH and calcium requirements, suggesting different better understood, such as those regions that intracellular locations for these events (Shennan et interact with lipidic membranes. Some SPCs al. 1995). Finally, SPC2 is sorted to the regulated (SPC1, SPC7 and the variant isoforms, SPC4E and secretory pathway (Guest et al. 1992, Taylor et al. SPC6B) have an integral transmembrane domain 1998), where it fulfills its role in the production of followed by a cytoplasmic tail. This domain affects bioactive peptide hormones. cellular sorting and has been well studied for SPC1 The biosynthesis of active SPC2 is tightly linked (Molloy et al. 1999). As a constitutive pathway- to the expression of the neuroendocrine poly- associated protease, SPC1 is a resident TGN peptide, 7B2 (Jeannotte et al. 1997, Seidel et al. protein (Shapiro et al. 1997) that can enter a 1998). This protein was first isolated from pituitary recycling pathway through the plasma membrane extracts (Hsi et al. 1982, Seidah et al. 1983) and its and the endosomes (Molloy et al. 1994). This cDNA was later cloned (Martens 1988, Mbikay et particular sorting pattern is made possible by its al. 1989). It is a sulfated, non-glycosylated peptide transmembrane domain and the interactions of its (Ayoubi et al. 1990, Paquet et al. 1994) with a C-terminal tail with cytoplasmic proteins. An acidic strictly neuroendocrine distribution. 7B2 expression amino acid cluster important for cell sorting has is required for the efficient transport, folding and been identified within the SPC1 56 amino acid activation of SPC2 (Braks & Martens 1994, Martens cytosolic domain (Schafer et al. 1995). This motif et al. 1994, Zhu & Lindberg 1995, Lamango et al. contains phosphorylated serine residues (Takahashi 1996). The two molecules are bound together at et al. 1995a) that regulate the SPC1 intracellular their entry in the ER and are later cleaved by an traffic by casein kinase II (Jones et al. 1995). SPC1-like activity (Paquet et al. 1994) in their Dephosphorylation is carried out by PP2A phos- transport through the secretory pathway (Braks & phatase (Molloy et al. 1998). When phosphorylated, Martens 1994). The SPC2 pro-segment is required, the SPC1 cytoplasmic tail interacts with phospho- but is not sufficient to confer 7B2 binding (Zhu furin acidic cluster sorting protein 1, a novel sorting et al. 1998), and the interactions between these

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 3. Hypothetical model of 7B2 and SPC2 interactions. (A) Shortly after biosynthesis, pro-SPC2 (75 kDa) specifically associates with pro-7B2 (27 kDa) within the ER. (B) The complex exits from the ER and, within the TGN, SPC1 (scissors) cleaves the C-terminal (C-T) peptide from pro-7B2 and also cleaves pro-SPC2 (75 kDa to 71 kDa form). (C) By an as yet unexplained mechanism, the 21 kDa 7B2 facilitates the conversion of pro-SPC2 to fully active SPC2 (68 kDa form). (D) The C-T peptide inhibits the activity of SPC2 in late compartments (TGN or immature secretory granules). Inhibition of SPC2 (68 kDa form) is released by cleavage of the C-T peptide by SPC2 at KK residues (and after C-terminal basic residue trimming by carboxypeptidases). molecules have been used to explain the relatively In vivo studies have attempted to establish the slow exit of SPC2 from the ER (Muller et al. 1997). role of 7B2. Whereas 7B2 and SPC2 are clearly 7B2 is considered to be a bifunctional molecule, co-localized in rat brain neurons, 7B2 has also been with its N-terminal domain behaving as a shown to be more widely distributed than SPC2 chaperone-like protein, while its C-terminal peptide (Seidel et al. 1998), with many 7B2-positive neurons domain is a specific SPC2 inhibitor (Martens et al. showing a lack of SPC2 expression. What is the 1994, Lindberg et al. 1995, Zhu & Lindberg 1995) function of 7B2 in these SPC2-negative neurons? It having no effect on SPC3 activity (van Horssen remains to be established whether 7B2 specifically et al. 1995). This C-terminal region appears not to binds another protein, maybe even an ‘SPC2-like’ be responsible for the observed binding of pro-7B2 molecule, in these neurons. These potential with pro-SPC2 in the ER. A working model of 7B2 additional functions of 7B2 have been highlighted and SPC2 interactions based on these results is by recent gene knockout experiments (Westphal summarized in Fig. 3. et al. 1999). Although gene disruption of 7B2 www.endocrinology.org Journal of Molecular Endocrinology (2000) 24, 1–22 8   and others · SPCs: molecular specificity to therapeutic applications

 1. Overall distribution profile of the SPCs

Tissue distribution Intracellular localization Name SPC1 Ubiquitous, but expressed at variable levels TGN, endosomes, cell surface SPC2 Neuroendocrine cells Secretory granules SPC3 Neuroendocrine cells Secretory granules, TGN SPC4 Endocrine and non-endocrine, widespread tissue expression TGN, ? SPC5 Testicular germ cells ? SPC6-A Endocrine and non-endocrine, widespread tissue expression Secretory granules, TGN SPC6-B Digestive system and adrenal cortex TGN SPC7 Widespread, with higher levels in lymphoid-associated tissues TGN

prevents activation of SPC2, other important effects this way, as they show that some SPCs (SPC2, have been observed. 7B2-null mice display hypo- SPC3 and SPC6) are exclusively expressed in glycemia, hyperproinsulinemia, and hypogluca- neurons, whereas others (SPC1, SPC4 and SPC7) gonemia. They also have increased circulating are expressed in both neurons and glial cells. In adrenocorticotrophic hormone (ACTH) and corti- contrast to glial cells, which possess only a costerone concentrations, with adrenocortical constitutive secretory pathway, neurons also have a expansion. 7B2-null mice do not survive more than regulated secretory pathway. These data suggest 9 weeks, because of severe Cushing’s syndrome. In an association of SPC2, SPC3 and SPC6 with these mice, pituitary intermediate lobe ACTH the neuroendocrine phenotype and specialized hypersecretion results in a far more severe functions within the regulated secretory pathway. endocrine disorder than is found in the SPC2-null Of the seven known mammalian SPCs, the mice (Furuta et al. 1997). Thus it is highly likely distribution of SPC5 is the most restricted, as it is that 7B2 has functions additional to that of not expressed elsewhere than in testicular germ cells activating SPC2. (Nakayama et al. 1992, Seidah et al. 1992). Although this unique expression pattern may reflect a highly specialized processing function, no SPC TISSUE DISTRIBUTION AND CELLULAR substrates have, as yet, been identified in germ cells. LOCALIZATION OF SPC ENZYMES The exclusive expression of SPC5 in germ cells also suggests unique transcriptional regulation of this Numerous studies have been carried out to examine gene. However, the germ cells also have the capacity the detailed tissue and cellular distribution of each to express other SPCs, including SPC1 and SPC7. SPC (Day et al. 1992, 1993, Schafer et al. 1993, A comparative in situ hybridization analysis of Seidah et al. 1994, Beaubien et al. 1995, Dong et al. adjacent sections of rat seminiferous tubules (Fig. 5) 1995, 1997, Seidel et al. 1998). Table 1 summarizes reveals that both SPC5 and SPC7 are expressed the overall distribution profile. In general, it can be within germ cells, but in a mutually exclusive stated that cells or cell lines do not express only one pattern, some tubules containing greater levels of SPC at a time, but rather express a distinct SPC5 mRNA, the others being rich in SPC7. These ‘cocktail’ of SPCs. Overlapping cellular expression patterns of expression suggest distinct roles of SPC5 of SPCs raises the question of possible redundancy and SPC7 in the different stages of germ cell of processing. This is best illustrated by studies maturation. carried out in the rat brain, which examined The extensive tissue distribution studies carried SPC mRNA distribution by in situ hybridization out on the SPCs have revealed that each SPC could (Fig. 4). Analysis of the brain localization shows have a distinct function in vivo, as no two SPCs that SPCs have distinct distribution patterns, have identical patterns. These patterns of expres- suggesting that they also have distinct functions. sion (whether at a cellular or intracellular level) However, overlapping regional expressions are also could compensate for some apparent common observed, which hints at some redundant functions cleavage specificities observed in vitro. A substrate in the brain. cleaved by an SPC in vitro may in fact never A summary of the analysis of the cellular encounter that SPC in vivo, because they either are distribution of SPCs in the rat brain is given in not co-expressed in the same cell or are simply Table 2. In situ hybridization studies of brain tissue sorted to different intracellular compartments. Thus at a cellular level are very useful when illustrated in overlapping functions of SPCs in vivo may in fact be

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 4. Photomicrographs of in situ hybridization of SPC mRNAs in adjacent rat brain sagittal sections. Distinct distribution patterns are observed for SPCs. 3V, third ventricle; 4V, fourth ventricle; Acb, accumbens nucleus; AD, anterodorsal thalamic nucleus; AO, anterior olfactory nucleus; CA3, field CA3 of Ammon’s horn; Ce, cerebellum; Cx, cerebral cortex; DG, dentate gyrus; Hy, hypothalamus; IC, inferior colliculus; ICj, islands of Calleja; LS, lateral septum; LV, lateral ventricle; Olf, olfactory bulb; PCL, Purkinje cell layer; PF, parafascicular thalamic nucleus; Pn, pontine nucleus; S, subiculum; SC, superior colliculus; Th, thalamus; Tu, olfactory tubercle. minimized as a result of different regulatory gene suggesting that other convertases are able to provide expression mechanisms of the enzyme and sub- some compensatory cleavage at SPC2-favored sites strate. Compensatory mechanisms have been in pro-insulin. In SPC2-null mice, pancreatic observed in SPC-null mice, but often represent only concentrations of insulin are still detectable, but a fraction of the processing activity of the SPC that represent only about 20% of normal insulin was inactivated. For example, in SPC2-null mice concentrations. Thus, whereas some substrates are (Furuta et al. 1997), the processing of pro-glucagon, in fact cleaved by more than one SPC (at the same pro-somatostatin, and pro-insulin in the pancreatic cleavage site), it appears that in vivo compensatory islets was shown to be severely impaired. However, mechanisms are mostly limited events. in the case of pro-insulin processing, a severe but incomplete block in processing was observed, BIOLOGICAL FUNCTIONS OF SPC ENZYMES

 2. Cellular distribution of SPCs in rat brain A major objective of investigations on SPCs has been to determine the specific biological functions Neuron Glia of each enzyme – that is, to establish specific target Enzyme substrates within their biological context. Many SPC1 + + studies have focused on the use of cellular  SPC2 + co-infections using Vaccinia virus recombinants SPC3 +  SPC4 + + (Thomas et al. 1988) to establish the cleavage SPC6 +  properties of SPCs on target substrates. This SPC7 + + approach has been useful in certain applications, for example in the rapid production of recombinant www.endocrinology.org Journal of Molecular Endocrinology (2000) 24, 1–22 10   and others · SPCs: molecular specificity to therapeutic applications

 5. Comparative distribution of SPC5 and SPC7 mRNAs by in situ hybridization in adjacent sections of rat testis. In the top panels, note the complementary distributions, which show seminiferous tubules with high levels of SPC7 (open arrows) or high levels of SPC5 (white arrows). In the bottom panels, numbers indicate identical seminiferous tubles in adjacent tissue sections. Tubules 1–5 express high levels of SPC7, whereas tubules 6–9 express high levels of SPC5.

SPCs for in vitro studies. Also, with the use of a transfection studies (Rouille et al. 1994, 1995, cellular co-infection approach (co-infection of a 1997a, Furuta et al. 1997) all showed the obvious target precursor substrate with a particular SPC), involvement of SPC2 in the formation of glucagon. interesting results have been obtained regarding the Thus only a partial assessment of SPC biological unique specificity of SPCs and their ability to cleave function is possible with co-expression or in vitro precursors differentially (Thomas et al. 1988, studies. To complete the elucidation of the Benjannet et al. 1991, Leduc et al. 1992). However, biological role of SPCs, complementary in vivo this method has its drawbacks, such as degradation approaches must be considered, such as the study of of the cellular environment induced by vaccinia naturally occuring mutants (e.g. the LoVo cell line) infection and the difficulty of carrying out compara- and SPC gene modulation using antisense (or gene tive kinetic studies of substrate cleavage. Taken knockout) technologies. Another interesting method together, these conditions may explain certain by which to investigate the role of SPCs in vivo is to ambiguities that have been observed with the use study the co-localization of the enzymes with their of co-infection, as for example in the cleavage of substrates at the tissue, cellular and intracellular pro-glucagon by SPC2. In this case, the Vaccinia level. An example of such an approach is illustrated virus approach was unable to demonstrate the in Fig. 6, showing the co-localization of POMC specific formation of glucagon by SPC2 (even in the mRNA with various SPC mRNAs in hypothalamic presence of 7B2) (Dhanvantari et al. 1996), whereas neurons. The data show that all POMC neurons SPC2 antisense studies, gene knockout and gene contain SPC2 and SPC3, that some POMC neurons

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 6. Co-localization of SPCs and POMC in the hypothalamic arcuate nucleus neurons, using double labeling in situ hybridization histochemistry. POMC neurons were labeled with a digoxygenin-labeled cRNA probe and appear in each panel as dark purple. SPC cRNA probes were radioactively labeled with [35S]UTP/[35S]CTP and appear in each panel as autoradiographic grains (white dots). (A) Co-localization of POMC and SPC3; all POMC neurons express SPC3 (black arrows). (B) Co-localization of POMC and SPC2; all POMC neurons express SPC2 (black arrows). (C) POMC neurons do not express SPC6 (open arrows). (D) Partial co-localization of POMC and SPC4; black arrows show examples of co-localization and open arrows show examples of POMC neurons that do not express SPC4. contain SPC4, but that none can be shown to (Mondino et al. 1991), pro-von Willebrand factor express SPC6. These data reflect the established (Wise et al. 1990), pro--nerve growth factor role of SPC2 and SPC3 on in vivo POMC (Bresnahan et al. 1990), pro-transforming growth processing, but also suggest that SPC4 could also factor 1 (Dubois et al. 1995) and pro-endothelin-1 play a role in POMC processing in a subset of (Denault et al. 1995a). SPC1 does not appear to be arcuate neurons. This type of co-localization essential for survival in established cell lines, as the approach is a powerful way to gather data on absence of active SPC1 in LoVo or CHO RPE.40 cellular enzyme–substrate relationships and cells is not lethal. Survival of these cells without potential SPC biological functions. functional SPC1 may be due to the expression of One of the SPCs with the best-defined biological other SPCs, such as SPC4, in LoVo cells, function is SPC1 (Denault & Leduc 1996, suggesting compensatory functions by other SPCs. Nakayama 1997, Molloy et al. 1999). In terms of However, the essential role of SPC1 has been cell metabolism, SPC1 is believed to be vital, as it is revealed by in vivo studies demonstrating that SPC1 considered to be a ‘housekeeping’ convertase, expression is essential for the normal development processing constitutively secreted proteins in all of the mouse embryo (Roebroek et al. 1998). cells. Its ubiquitous distribution, cellular localiz- Whereas SPC1 is ubiquitously expressed, other ation and substrate specificity support this notion. SPCs, such as SPC2 and SPC3, have well-defined In vitro and in vivo studies reveal that SPC1 is patterns of expression that are mostly restricted to able to cleave various constitutively secreted pro- the neuroendocrine system. This distribution is tein precursors, including insulin pro-receptor consistent with their accepted role in pro-hormone www.endocrinology.org Journal of Molecular Endocrinology (2000) 24, 1–22 12   and others · SPCs: molecular specificity to therapeutic applications

and pro-neuropeptide processing. Furthermore, magnocellular neurons express both SPC2 and SPC2 and SPC3 are co-localized with peptide SPC3, whereas anterior lobe gonadotropes express hormones within dense-core secretory granules. only SPC3. Thus complete processing of pro- The specific role of SPC2 and SPC3 has been dynorphin to biologically active opioid peptide shown in the processing of pro-insulin (Bennett products is dependent upon the expression of et al. 1992, Smeekens et al. 1992), pro-glucagon SPC2. Additional support comes from studies with (Rouille et al. 1995, 1997a,b) and POMC (Benjannet SPC2-null mice, which showed impaired pro- et al. 1991, Thomas et al. 1991). These cleavage dynorphin processing, especially in the formation of specificity studies have been supported by gene dynorphin A(1–8) (Day et al. 1998). knockout (Furuta et al. 1998) and antisense studies Compared with SPC1, SPC2 and SPC3, the other (Bloomquist et al. 1991, Rouille et al. 1994, members of the SPC family have been the object of Rothenberg et al. 1996, Johanning et al. 1998). fewer studies and, thus, their biological roles are not These substrates possess multiple basic residues as well defined. The first enzymatic studies on that are differentially cleaved by SPC2 and SPC3, SPC4 activity revealed characteristics similar to yielding different products depending on the those of SPC1, but with some differences in relative expression of SPC2 and SPC3. In general, specificity (Rehemtulla et al. 1993). On the basis of it appears that SPC3 activity results in a more preliminary studies on tissue distribution and limited precursor proteolysis, generating relatively enzymatic properties, SPC4 was first believed to high-molecular-weight species, whereas SPC2 be very similar to SPC1. Moreover, the close cleavage is more extensive, producing smaller forms proximity of the SPC1 and SPC4 genes on of mature products. chromosome 15 suggested that these genes evolved The issue of differential processing by SPC2 and from a common ancestor by gene duplication SPC3 can be exemplified with pro-dynorphin as a (Kiefer et al. 1991). However, more recent studies model precursor. The interest in pro-dynorphin lies on SPC1 and SPC4 in vitro activities have in the fact that it contains multiple apparently highlighted the differences between these enzymes. similar paired basic amino acid cleavage sites, in Compared with SPC1, SPC4 has a low sensitivity addition to two potential single Arg cleavage sites. for calcium chelators and dithiothreitol (Mains Pro-dynorphin is also, along with POMC and et al. 1997), and it is not affected by the SPC1 pro-enkephalin, one of three precursors involved inhibitor,
1-antitrypsin-portland (AT-PDX, see in the production of mammalian opioid peptides. below). In fact, several reports show that SPC4 is In vivo,differential processing of this 26 kDa closer to SPC6 than SPC1 in many respects. SPC6 pro-protein is observed in different tissues (Day & is the enzyme with the greatest degree of sequence Akil 1989). For example, pro-dynorphin is ex- homology with SPC4 (Lusson et al. 1993, pressed in anterior pituitary gonadotropes and in Nakagawa et al. 1993). In vitro enzymatic studies magnocellular neurons of the hypothalmus. In the of the recombinant enzymes revealed a close anterior pituitary, pro-dynorphin is processed to relationship between SPC4 and SPC6 (Creemers yield high-molecular intermediates of 8, 10 and et al. 1993a). Finally, a distinct distribution has 16 kDa, whereas, in the hypothalamus, final opioid been observed for the two enzymes, which is peptide products, including dynorphin A(1–17,) complementary in some tissues (Dong et al. 1995, dynorphin A(1–8), dynorphin B(1–13) and
-neo- Zheng et al. 1997). Despite the differences in the endorphin, are obtained (Fig. 7). In vitro studies in vitro properties of SPC1 and SPC4, all the demonstrated that SPC3 is primarily responsible for reported SPC4 substrates can also be cleaved by the production of the high-molecular-weight inter- SPC1 or other SPCs with an equal or superior mediates. The 8 kDa intermediate requires cleavage efficiency. Among the reported SPC4 substrates at a single Arg residue, a function that can be are pro-brain-derived neurotrophic factor, pro- carried out by SPC3 (Dupuy et al. 1994). In neurotrophin 3 (Seidah et al. 1996a), the insulin contrast, SPC2 was shown to generate the smaller receptor (Alarcon et al. 1994), pro-nerve growth opioid-active dynorphin A(1–17), dynorphin B(1– factor (Seidah et al. 1996b), pro-somatostatin 13), and
-neo-endorphin products (Day et al. (Brakch et al. 1995), human serum albumin (Mori 1998). In common with SPC3, SPC2 was also able et al. 1999) and the HIV viral coat protein, GP160 to cleave at single Arg residues, generating the (Hallenberger et al. 1992, Morikawa et al. 1993). C-peptide but, in addition, dynorphin A(1–8). The The cleavage of the GP160 precursor illustrates differential cleavage patterns of pro-dynorphin the need to confirm in vitro data with in vivo by SPC2 and SPC3 obtained in vitro reflect observations. Even though enzymatic assays with the processing patterns observed in vivo. Co- recombinant proteins suggest that GP160 is cleav- localization studies indicate that hypothalamic able by SPC1 and SPC4 (Decroly et al. 1994), this

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 7. Differential processing of pro-dynorphin by SPC2 and SPC3. Cleavage by SPC3 produces large intermediate molecules, whereas SPC2 generates smaller peptides. The processing pattern obtained with SPC3 corresponds to that observed in vivo within the anterior lobe gonadotropes. The processing pattern obtained with SPC2 corresponds to that observed in vivo within the hypothalamus (see text for discussion).
NE,
-neo-endorphin; Dyn, dynorphin. processing is probably not physiologically relevant, The cloning of SPC6 cDNA revealed the because it occurs with very low efficiency (Inocencio existence of two enzyme isoforms, named SPC6-A et al. 1997). Moreover, SPC1-deficient cell lines and SPC6-B, which differ only in their C-terminal process GP160 as efficiently as do normal cells end. These isoforms are believed to exert different (Ohnishi et al. 1994). Finally, it has been proposed functions, as SPC6-A has a soluble C-terminal that a Ca2+-independent protease may in fact be domain and is targeted towards the dense-core responsible for in vivo GP160 cleavage (Inocencio secretory granules in endocrine cells, whereas et al. 1997). The role of SPC4 in vivo remains SPC6-B has a transmembrane C-terminal domain unclear, as none of the above cited precursors and is located in the vesicles of the Golgi apparatus demonstrates a clear preferential susceptibility to (De Bie et al. 1996). The SPC6 isoforms are SPC4 activity. differentially expressed in the gastrointestinal tract, www.endocrinology.org Journal of Molecular Endocrinology (2000) 24, 1–22 14   and others · SPCs: molecular specificity to therapeutic applications

and the expression of SPC6-A is affected by dietary The first evidence of a metabolic disorder content (Udupi et al. 1997), suggesting a regulatory induced by a defective enzyme involving precursor role for the processing of gut peptides. Other cleavage came from the study of fat/fat mice. A potential biological functions of SPC6 have been spontaneous mutation in the CPE gene of this strain identified. It may play a role in the cleavage of induced a single amino acid substitution in a highly pro-neurotensin/neuromedin N in human colon conserved domain of the exopeptidase, yielding an cancer cells (Rovere et al. 1998). It may be also inactive enzyme. Biochemical analysis of insulin- involved in the process of sexual differentiation, by like peptides in these animals revealed a high activating pro-Müllerian substance (Nachtigal & content (approximately 80%) of unprocessed pro- Ingraham 1996). In addition, the transfection of insulin, whereas the processed insulin chains still SPC6 in COS cells induces cleavage of the had their C-terminal pairs of basic residues extracellular domains of the receptor protein untrimmed. However, an unexpected finding was tyrosine phosphatases (Campan et al. 1996). the detection of unprocessed pro-insulin. This Enzymatic characterization of the recombinant phenomenon may be explained by the observation SPC7 reveals a substrate specificity largely similar that SPC2 activity is inhibited by C-terminally to that of SPC1 (Munzer et al. 1997), with which extended peptides, possibly by a product inhibition it is highly homologous. Just like SPC1, SPC7 has mechanism (Day et al. 1998). a C-terminal transmembrane domain and a cyto- Recently, an isolated case of SPC3 deficiency has plasmic tail responsible for its subcellular localiz- been identified in a human patient. One allele of the ation at the TGN level, possibly with an SPC3 gene has a Glyobesity and pro-insulinemia, accompanied determine its role in vivo. by abnormal glucose homeostasis, hypogonado- tropic hypogonadism and hypocortisolism. Such inherited syndromes of normal proteolytic process- CLINICAL ASPECTS OF SPC ACTIVITY ing are uncommon; they arise from the occasional combination of two alleles containing random Theoretically, there are two ways of designing mutations of an SPC gene. Recent advances in therapeutic approaches targeting SPCs: restoring gene transfer therapy (Knoell & Yiu 1998) raise the the defective processing responsible for a disorder, hope that these rare inherited defects could be and blocking a processing event that is essential for corrected by the reintroduction of normal SPC a given disease state. genes. With regard to the restoration of defective The other field of therapeutic application involv- processing, there is, at present, little evidence of ing SPC activity is the inhibition of unwanted pathologies associated with altered protein process- proteolytic processing. Various pathogenic agents, ing. Some autosomal dominant syndromes have been including bacterial toxins and viral coat proteins, identified in which a mutated precursor leads to an have been found to require proteolytic processing incorrect processing pattern, inducing pathological by SPCs in order to produce their effects (Garten conditions. These diseases are related to the et al. 1994, Gordon et al. 1995, Rott et al. 1995). impaired processing of pro-insulin (Peters 1987), Blocking the proteolytic processing of such patho- pro-albumin (Brennan 1989), and blood coagulation genic proteins opens the way to the development of factor IX (Bentley et al. 1986). However, these new therapeutic treatments based on enzyme diseases are not related to an impaired activity of inhibition. SPCs, but are caused by structural modifications in Blocking SPC activity may be deleterious, as the substrate. One interesting application of SPCs these enzymes are essential for the normal expres- in the restoration of hormone production is the sion of important cellular proteins. Thus, for each re-engineering of rat prepro-insulin-1 to introduce disease state, the enzyme specificity of patho- SPC1 cleavage sites, thus allowing processing genic precursors must be well understood and into mature insulin by SPC1 in liver (Muzzin et al. the design of therapeutic SPC inhibitors should 1997). be made as specific as possible, in order to avoid

Journal of Molecular Endocrinology (2000) 24, 1–22 www.endocrinology.org SPCs: molecular specificity to therapeutic applications ·   and others 15 cross-inhibition of important cellular proteolytic synthesize compounds of higher specificity. One of processing. Nonetheless, it cannot be ruled out that, these is the availability of all SPCs, enabling re- in certain pathological states, wide-spectrum SPC searchers to compare the inhibitory properties of a inhibitors may also be useful. given molecule in vitro. The use of these isolated SPCs for the identification of specific SPC inhibitors with high throughput methods such as peptide com- SPC INHIBITION binatorial libraries is already under way, as in the case of SPC2 and SPC3 (Apletalina et al. 1998). Ideally, effective control of the production of biologi- Because most enzymes possess natural inhibitors, cal peptides involved in various disease states could be efforts have also been made to identify endogenous achieved by specifically inhibiting the enzymatic ac- molecules that could modulate SPC activity. As tivity of SPCs. Although antisense technologies aimed discussed above, one of these, the dual function 7B2 at decreasing the endogenous levels of SPC mRNA that facilitates pro-SPC2 maturation (Muller et al. have been considered, enzyme-based inhibition 1997), has also been found to be inhibitory. Indeed, achieved by designing a variety of molecular entities, within its C-terminal peptide, the Lys171-Lys172 ranging from non-peptidic or peptidomimetic site is particularly important for the ability of 7B2 molecules to small peptide substrates and to larger to inhibit SPC2 (van Horssen et al. 1995). proteins, would be the preferred choice. Pro-segments of SPCs can also be considered to be The initial characterization of the enzymatic modulators of enzymatic activity. Acting as a specificities of SPCs spearheaded efforts in the multifunctional domain, the pro-peptide not only design, conception and testing of compounds aimed acts as a steric chaperone, but also binds to catalytic at inhibiting these enzymes. A straightforward domains of SPCs and inhibits protease activity. approach has made use of the consensus cleavage This mechanism has been observed in vitro and site sequence of SPCs for the synthesis of peptides in vivo for SPC1 and SPC3, whereby pro-segments capable of acting as pseudosubstrates or transition inhibit the enzymes by a slow, tight-binding state analogues. For example, elucidation of the mechanism (Boudreault et al. 1998). In fact, the SPC1 recognition sequence, Arg-Xaa-Xaa-Arg, has pro-domains are quite potent inhibitors, as led to the design of molecules such as the family demonstrated for SPC1, with IC50 values of 14 nM. of chloromethylketones, which behave as tight- Serpins (SERine Protease INhibitors) are nat- binding irreversible inhibitors of SPC1 (Angliker urally occuring proteins that play a role in the 1995, Jean et al. 1995). regulation of serine proteases activity. Although Structure–activity studies using peptidyl sub- most serpins identified today specifically inhibit strates based on a region spanning the pro-region trypsin-like serine proteases, a recently described and catalytic domain of mouse SPC3 (Molloy et al. member of this family, called PI8, has been shown 1992, Basak et al. 1995) or the cleavage sequence of to inhibit SPC1 by a rapid, tight-binding mech- human pro-parathyroid hormone (Lazure et al. anism (Dahlen et al. 1998). PI8 is a 45 kDa 1998), showed that inhibition constants in the ovalbumin-type serpin that contains two SPC1 micromolar range can be attained. Some of the pepti- recognition sequences, Arg-Asn-Ser-Arg339 and domimetic agents irreversibly react with the active- Arg339-Cys-Lys-Arg342 in its reactive site loop. site serine, thus acting as suicide inhibitors. These Once this loop enters the catalytic pocket, it forms peptide-based molecules have proven quite useful in an SDS-stable serpin–enzyme complex, characteris- ascertaining the role of SPCs in processing precur- tic of physiological serpin–proteinase interactions. sors of viral glycoproteins (Hallenberger et al. 1992, Attempts at abolishing SPC activity that were Volchkov et al. 1998), hormones (Lazure et al. 1998), based on redesigning the reactive site of known enzymes and other pro-proteins (Denault et al. inhibitors of serine proteinases to fit SPC recog- 1995b, Jean et al. 1993, Ledgerwood et al. 1996). nition sequences were initially performed using They have the inherent advantage of being synthe- turkey ovomucoid third domain. Its reactive site sized on a large scale and having the propensity of was re-engineered by incorporating the SPC1 modified peptides to penetrate membranes, making cleavage site, Arg-Xaa-Lys-Arg, in its reactive  7 1 them well suited for therapeutic use as large- pocket, yielding a molecule of Ka 1·1 10 M spectrum, pharmacologically stable inhibitors. How- (Lu et al. 1993). Similar modifications of the bait ever, none of them has, as yet, demonstrated marked region of the general protease inhibitor,
2 specificity. It is clear that, because of the enzymatic macroglobulin (Gly-Phe-Tyr-Glu686-Ser-Asp< property of this family of proteinases of cleaving on Arg-Ser-Lys-Arg686-Ser-Leu), also led to the the C-terminal of basic residues, other consider- production of an intracellularly active SPC1 ations will have to be taken into account in order to inhibitor (Van Rompaey et al. 1997). www.endocrinology.org Journal of Molecular Endocrinology (2000) 24, 1–22 16   and others · SPCs: molecular specificity to therapeutic applications

Using a similar approach of site-directed muta- CONCLUSIONS genesis of reactive centers, other existing poly- peptides of the serpin family were targeted that The mammalian SPCs comprise a complex family led to the development of a potent inhibitor for of enzymes that carry out apparently similar SPC1. Indeed, changing residues corresponding to processing functions (cleavage at basic residues), yet the P4 and P1 residues of the reactive site loop of clearly have unique biological roles as a result of
1-antitrypsin to accommodate the specificity of their distinct catalytic specificities and localizations. SPC1 (Ala-Ile-Pro-Met drographis paniculata, that possess low ( 30 µM) We wish to thank the Medical Research Council of inhibitory potencies toward SPCs (Basak et al. Canada for supporting this work. R L and R D are 1999). both scholars (Chercheurs-Boursiers) of the Fonds Lately, much focus has been put on develop- de la Recherche en Santé du Québec. We also wish ing approaches to abolishing SPC1-mediated to thank Mr Eric Dufour for helpful discussion. cleavage of viral and bacterial pathogens. However, because not all these precursors are processed in the same cellular compartment, the physicochemi- cal properties of the inhibitory compounds must be REFERENCES considered. For example, it is believed that cleavage of the protective antigen of Anthrax is Abrami L, Fivaz M, Decroly E, Seidah NG, Jean F, Thomas G, Leppla SH, Buckley JT & van der Goot FG 1998 The performed by SPC1 found at the cell surface, that pore-forming toxin proaerolysin is activated by furin. Journal proteolysis of exotoxin A of Pseudomonas would of Biological Chemistry 273 32656–32661. take place in endosomal compartments, but that Alarcon C, Cheatham B, Lincoln B, Kahn CR, Siddle K & viral glycoprotein precursors such as the Ebola Rhodes CJ 1994 A Kex2-related endopeptidase activity present in rat liver specifically processes the insulin glycoprotein would occur in the TGN. This proreceptor. Biochemical Journal 301 257–265. signifies that inhibiting cell surface, and possibly Anderson ED, Thomas L, Hayflick JS & Thomas G 1993 endosomal, SPC1 may be facilitated by its Inhibition of HIV-1 gp160-dependent membrane fusion by a accessibility to hydrophilic molecules such as furin-directed alpha 1-antitrypsin variant. Journal of peptides and proteins. To reach the intracellular Biological Chemistry 268 24887–24891. Anderson ED, VanSlyke JK, Thulin CD, Jean F & Thomas G TGN, more lipophilic compounds would be 1997 Activation of the furin endoprotease is a multiple-step needed. These issues must also be considered for process: requirements for acidification and internal all other SPCs. propeptide cleavage. EMBO Journal 16 1508–1518.

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Angliker H 1995 Synthesis of tight binding inhibitors and their Campan M, Yoshizumi M, Seidah NG, Lee ME, Bianchi C & action on the proprotein-processing enzyme furin. Journal of Haber E 1996 Increased proteolytic processing of protein Medicinal Chemistry 38 4014–4018. tyrosine phosphatase mu in confluent vascular endothelial Apletalina E, Appel J, Lamango NS, Houghten RA & cells: the role of PC5, a member of the subtilisin family. Lindberg I 1998 Identification of inhibitors of prohormone Biochemistry 35 3797–3802. convertases 1 and 2 using a peptide combinatorial library. Chan SJ, Oliva AA Jr, LaMendola J, Grens A, Bode H & Journal of Biological Chemistry 273 26589–26595. Steiner DF 1992 Conservation of the prohormone convertase Ayoubi TA, van Duijnhoven HL, van de Ven WJ, Jenks BG, gene family in metazoa: analysis of cDNAs encoding a Roubos EW & Martens GJ 1990 The neuroendocrine PC3-like protein from hydra. Proceedings of the National polypeptide 7B2 is a precursor protein. Journal of Biological Academy of Sciences of the USA 89 6678–6682. Chemistry 265 15644–15647. Creemers JW, Groot Kormelink PJ, Roebroek AJ, Nakayama Ballinger MD, TomJ&Wells JA 1996 Furilisin: a variant of K&VandeVenWJ1993a Proprotein processing activity subtilisin BPN engineered for cleaving tribasic substrates. and cleavage site selectivity of the Kex2-like endoprotease Biochemistry 35 13579–13585. PACE4. FEBS Letters 336 65–69. Basak A, Schmidt C, Ismail AA, Seidah NG, Chretien M & Creemers JW, Siezen RJ, Roebroek AJ, Ayoubi TA, Lazure C 1995 Peptidyl substrates containing unnatural HuylebroeckD&VandeVenWJ1993b Modulation of amino acid at the P1 position are potent inhibitors of furin-mediated proprotein processing activity by site-directed prohormone convertases. International Journal of Peptide and mutagenesis. Journal of Biological Chemistry 268 Protein Research 46 228–237. 21826–21834. Basak A, Cooper S, Roberge AG, Banik UK, Chretien M & Creemers JW, Vey M, Schafer W, Ayoubi TA, Roebroek AJ, Seidah NG 1999 Inhibition of proprotein convertases-1, -7 Klenk HD, Garten W & Van de Ven WJ 1995 Endoproteolytic and furin by diterpines of Andrographis paniculata and their cleavage of its propeptide is a prerequisite for efficient transport succinoyl esters. Biochemical Journal 338 107–113. of furin out of the endoplasmic reticulum. Journal of Biological Beaubien G, Schafer MK, Weihe E, Dong W, Chretien M, Chemistry 270 2695–2702. Seidah NG & Day R 1995 The distinct gene expression of Cui Y, Jean F, Thomas G & Christian JL 1998 BMP-4 is the pro-hormone convertases in the rat heart suggests proteolytically activated by furin and/or PC6 during potential substrates. Cell Tissue Research 279 539–549. vertebrate embryonic development. EMBO Journal 17 Benjannet S, Rondeau N, Day R, Chretien M & Seidah NG 4735–4743. 1991 PC1 and PC2 are proprotein convertases capable of Dahlen JR, Jean F, Thomas G, Foster DC & Kisiel W 1998 cleaving proopiomelanocortin at distinct pairs of basic Inhibition of soluble recombinant furin by human residues. Proceedings of the National Academy of Sciences of proteinase inhibitor 8. Journal of Biological Chemistry 273 the USA 88 3564–3568. 1851–1854. Bennett DL, Bailyes EM, Nielsen E, Guest PC, Rutherford Day R & Akil H 1989 The posttranslational processing of NG, Arden SD & Hutton JC 1992 Identification of the type prodynorphin in the rat anterior pituitary. Endocrinology 124 2 proinsulin processing endopeptidase as PC2, a member of 2392–2405. the eukaryote subtilisin family. Journal of Biological Day R, Schafer MK, Watson SJ, Chretien M & Seidah NG Chemistry 267 15229–15236. 1992 Distribution and regulation of the prohormone Bentley AK, Rees DJ, RizzaC&Brownlee GG 1986 Defective convertases PC1 and PC2 in the rat pituitary. Molecular propeptide processing of blood clotting factor IX caused by Endocrinology 6 485–497. mutation of arginine to glutamine at position 4. Cell 45 Day R, Schafer MK, Cullinan WE, Watson SJ, Chretien M & 343–348. Seidah NG 1993 Region specific expression of furin mRNA Bloomquist BT, Eipper BA & Mains RE 1991 Prohormone- in the rat brain. Neuroscience Letters 149 27–30. converting enzymes: regulation and evaluation of function Day R, Lazure C, Basak A, Boudreault A, Limperis P, Dong using antisense RNA. Molecular Endocrinology 5 2014–2024. W & Lindberg I 1998 Prodynorphin processing by Boudreault A, GauthierD&LazureC1998Proprotein proprotein convertase 2. Cleavage at single basic residues and convertase PC1/3-related peptides are potent slow tight- enhanced processing in the presence of carboxypeptidase binding inhibitors of murine PC1/3 and Hfurin. Journal of activity. Journal of Biological Chemistry 273 829–836. Biological Chemistry 273 31574–31580. De Bie I, Marcinkiewicz M, Malide D, Lazure C, Nakayama Brakch N, Galanopoulou AS, Patel YC, BoileauG&Seidah K, Bendayan M & Seidah NG 1996 The isoforms of NG 1995 Comparative proteolytic processing of rat proprotein convertase PC5 are sorted to different sub- prosomatostatin by the convertases PC1, PC2, furin, PACE4 cellular compartments. Journal of Cellular Biology 135 and PC5 in constitutive and regulated secretory pathways. 1261–1275. FEBS Letters 362 143–146. Decroly E, Vandenbranden M, Ruysschaert JM, Cogniaux J, Braks JA & Martens GJ 1994 7B2 is a neuroendocrine Jacob GS, Howard SC, Marshall G, Kompelli A, Basak A, chaperone that transiently interacts with prohormone Jean F et al. 1994 The convertases furin and PC1 can both convertase PC2 in the secretory pathway. Cell 78 263–273. cleave the human immunodeficiency virus (HIV)-1 envelope Brennan SO 1989 Propeptide cleavage: evidence from human glycoprotein gp160 into gp120 (HIV-1 SU) and gp41 proalbumins. Molecular Biology and Medicine 6 87–92. (HIV-I TM). Journal of Biological Chemistry 269 Bresnahan PA, Leduc R, Thomas L, Thorner J, Gibson HL, 12240–12247. Brake AJ, Barr PJ & Thomas G 1990 Human fur gene Decroly E, Wouters S, Di Bello C, Lazure C, Ruysschaert JM encodes a yeast KEX2-like endoprotease that cleaves & Seidah NG 1996 Identification of the paired basic pro-beta-NGF in vivo. Journal of Cellular Biology 111 convertases implicated in HIV gp160 processing based on in 2851–2859. vitro assays and expression in CD4(+) cell lines [published Bruzzaniti A, Goodge K, Jay P, Taviaux SA, Lam MH, Berta erratum appears in Journal of Biological Chemistry 1997 272 P, Martin TJ, Moseley JM & Gillespie MT 1996 PC8 8836]. Journal of Biological Chemistry 271 30442–30450. [corrected], a new member of the convertase family Denault JB & Leduc R 1996 Furin/PACE/SPC1: a convertase [published erratum appears in Biochemical Journal 1996 316 involved in exocytic and endocytic processing of precursor 1007]. Biochemical Journal 314 727–731. proteins. FEBS Letters 379 113–116. www.endocrinology.org Journal of Molecular Endocrinology (2000) 24, 1–22 18   and others · SPCs: molecular specificity to therapeutic applications

Denault JB, Claing A, D’Orleans-Juste P, Sawamura T, Gluschankof P & Fuller RS 1994 A C-terminal domain Kido T, MasakiT&LeducR1995a Processing of conserved in precursor processing proteases is required for proendothelin-1 by human furin convertase. FEBS Letters intramolecular N-terminal maturation of pro-Kex2 protease. 362 276–280. EMBO Journal 13 2280–2288. Denault JB, D’Orleans-Juste P, MasakiT&LeducR1995b Goodman LJ & Gorman CM 1994 Autoproteolytic activation Inhibition of convertase-related processing of of the mouse prohormone convertase mPC1. Biochemical and proendothelin-1. Journal of Cardiovascular Pharmacology 26 Biophysical Research Communications 201 795–804. S47–S50. Gordon VM, Klimpel KR, Arora N, Henderson MA & Leppla Dhanvantari S, Seidah NG & Brubaker PL 1996 Role of SH 1995 Proteolytic activation of bacterial toxins by prohormone convertases in the tissue-specific processing of eukaryotic cells is performed by furin and by additional proglucagon. Molecular Endocrinology 10 342–355. cellular proteases. Infection and Immunity 63 82–87. Dong W, Marcinkiewicz M, Vieau D, Chretien M, Seidah NG Guest PC, Arden SD, Bennett DL, Clark A, Rutherford NG & & Day R 1995 Distinct mRNA expression of the highly Hutton JC 1992 The post-translational processing and homologous convertases PC5 and PACE4 in the rat brain intracellular sorting of PC2 in the islets of Langerhans. and pituitary. Journal of Neuroscience 15 1778–1796. Journal of Biological Chemistry 267 22401–22406. Dong W, Seidel B, Marcinkiewicz M, Chretien M, Seidah NG Hallenberger S, Bosch V, Angliker H, Shaw E, Klenk HD & & Day R 1997 Cellular localization of the prohormone Garten W 1992 Inhibition of furin-mediated cleavage convertases in the hypothalamic paraventricular and supraoptic activation of HIV-1 glycoprotein gp160. Nature 360 nuclei: selective regulation of PC1 in corticotrophin-releasing 358–361. hormone parvocellular neurons mediated by glucocorticoids. van Horssen AM, van den Hurk WH, Bailyes EM, Hutton JC, Journal of Neuroscience 17 563–575. Martens GJ & Lindberg I 1995 Identification of the region Dong W, Fricker LD & Day R 1999 Carboxypeptidase D is a within the neuroendocrine polypeptide 7B2 responsible for potential candidate to carry out redundant processing the inhibition of prohormone convertase PC2. Journal of functions of carboxypeptidase E based on comparative Biological Chemistry 270 14292–14296. distribution studies in the rat central nervous system. Hosaka M, Nagahama M, Kim WS, Watanabe T, Hatsuzawa Neuroscience 89 1301–1317. K, Ikemizu J, MurakamiK&NakayamaK1991 Dubois CM, Laprise MH, Blanchette F, Gentry LE & Leduc Arg-X-Lys/Arg-Arg motif as a signal for precursor cleavage R 1995 Processing of transforming growth factor beta 1 catalyzed by furin within the constitutive secretory pathway. precursor by human furin convertase. Journal of Biological Journal of Biological Chemistry 266 12127–12130. Chemistry 270 10618–10624. Hsi KL, Seidah NG, De Serres G & Chretien M 1982 Dufour EK, Denault JB, Hopkins PC & Leduc R 1998 Isolation and NH2-terminal sequence of a novel porcine Serpin-like properties of alpha1-antitrypsin Portland towards anterior pituitary polypeptide. Homology to proinsulin, furin convertase. FEBS Letters 426 41–46. secretin and Rous sarcoma virus transforming protein Dupuy A, Lindberg I, Zhou Y, Akil H, Lazure C, Chretien M, TVFV60. FEBS Letters 147 261–266. Seidah NG & Day R 1994 Processing of prodynorphin by Inocencio NM, Sucic JF, Moehring JM, Spence MJ & the prohormone convertase PC1 results in high molecular Moehring TJ 1997 Endoprotease activities other than furin weight intermediate forms. Cleavage at a single arginine and PACE4 with a role in processing of HIV-I gp160 residue. FEBS Letters 337 60–65. glycoproteins in CHO-K1 cells. Journal of Biological Eder J, Rheinnecker M & Fersht AR 1993 Folding of subtilisin Chemistry 272 1344–1348. BPN: role of the pro-sequence. Journal of Molecular Biology Jackson RS, Creemers JW, Ohagi S, Raffin-Sanson ML, 233 293–304. Sanders L, Montague CT, Hutton JC & O’Rahilly S 1997 Fricker LD 1991 Peptide processing exopeptidases: amino- and Obesity and impaired prohormone processing associated with carboxypeptidases involved in peptide biosynthesis. In mutations in the human prohormone convertase 1 gene [see Peptide Biosynthesis and Processing,pp199–229. Boca Raton: comments]. Nature Genetics 16 303–306. CRC Press. Jean F, Basak A, Rondeau N, Benjannet S, Hendy GN, Seidah Fuller RS, Brake A & Thorner J 1989a Yeast prohormone NG, Chretien M & Lazure C 1993 Enzymic characterization processing enzyme (KEX2 gene product) is a Ca2+- of murine and human prohormone convertase-1 (mPC1 and dependent serine protease. Proceedings of the National hPC1) expressed in mammalian GH4C1 cells. Biochemical Academy of Sciences of the USA 86 1434–1438. Journal 292 891–900. Fuller RS, Brake AJ & Thorner J 1989b Intracellular targeting Jean F, Boudreault A, Basak A, Seidah NG & Lazure C 1995 and structural conservation of a prohormone-processing Fluorescent peptidyl substrates as an aid in studying the endoprotease. Science 246 482–486. substrate specificity of human prohormone convertase PC1 Furuta M, Yano H, Zhou A, Rouille Y, Holst JJ, Carroll R, and human furin and designing a potent irreversible Ravazzola M, Orci L, FurutaH&Steiner DF 1997 inhibitor. Journal of Biological Chemistry 270 19225–19231. Defective prohormone processing and altered pancreatic Jean F, Stella K, Thomas L, Liu G, Xiang Y, Reason AJ & islet morphology in mice lacking active SPC2. Proceedings Thomas G 1998 alpha1-antitrypsin Portland, a bioengineered of the National Academy of Sciences of the USA 94 serpin highly selective for furin: application as an 6646–6651. antipathogenic agent. Proceedings of the National Academy of Furuta M, Carroll R, Martin S, Swift HH, Ravazzola M, Orci Sciences of the USA 95 7293–7298. L & Steiner DF 1998 Incomplete processing of proinsulin to Jeannotte R, Paquin J, Petit-TurcotteC&DayR1997 insulin accompanied by elevation of Des-31,32 proinsulin Convertase PC2 and the neuroendocrine polypeptide 7B2 are intermediates in islets of mice lacking active PC2. Journal of co-induced and processed during neuronal differentiation of Biological Chemistry 273 3431–3437. P19 embryonal carcinoma cells. DNA Cell Biology 16 Garten W, Hallenberger S, Ortmann D, Schafer W, Vey M, 1175–1187. AnglikerH,ShawE&KlenkHD1994 Processing of viral Johanning K, Juliano MA, Juliano L, Lazure C, Lamango NS, glycoproteins by the subtilisin-like endoprotease furin and its Steiner DF & Lindberg I 1998 Specificity of prohormone inhibition by specific peptidylchloroalkylketones. Biochimie convertase 2 on proenkephalin and proenkephalin-related 76 217–225. substrates. Journal of Biological Chemistry 273 22672–22680.

Journal of Molecular Endocrinology (2000) 24, 1–22 www.endocrinology.org SPCs: molecular specificity to therapeutic applications ·   and others 19

Jones BG, Thomas L, Molloy SS, Thulin CD, Fry MD, Lu W, Zhang W, Molloy SS, Thomas G, Ryan K, Chiang Y, Walsh KA & Thomas G 1995 Intracellular trafficking of AndersonS&Laskowski M Jr 1993 Arg15-Lys17-Arg18 furin is modulated by the phosphorylation state of a casein turkey ovomucoid third domain inhibits human furin. kinase II site in its cytoplasmic tail. EMBO Journal 14 Journal of Biological Chemistry 268 14583–14585. 5869–5883. Lusson J, Vieau D, Hamelin J, Day R, Chretien M & Seidah Julius D, Brake A, Blair L, Kunisawa R & Thorner J 1984 NG 1993 cDNA structure of the mouse and rat subtilisin/ Isolation of the putative structural gene for the lysine- kexin-like PC5: a candidate proprotein convertase expressed arginine-cleaving endopeptidase required for processing of in endocrine and nonendocrine cells. Proceedings of the yeast prepro-alpha-factor. Cell 37 1075–1089. National Academy of Sciences of the USA 90 6691– Kayo T, Sawada Y, Suda M, Konda Y, Izumi T, Tanaka S, 6695. ShibataH&TakeuchiT1997 Proprotein-processing Lusson J, Benjannet S, Hamelin J, Savaria D, Chretien M & endoprotease furin controls growth of pancreatic beta-cells. Seidah NG 1997 The integrity of the RRGDL sequence of Diabetes 46 1296–1304. the proprotein convertase PC1 is critical for its zymogen and Kemmler W, Steiner DF & Borg J 1973 Studies on the C-terminal processing and for its cellular trafficking. conversion of proinsulin to insulin. 3. Studies in vitro with a Biochemical Journal 326 737–744. crude secretion granule fraction isolated from rat islets of Mains RE, Dickerson IM, May V, Stoffers DA, Perkins SN, Langerhans. Journal of Biological Chemistry 248 4544–4551. Ouafik L, Husten EJ & Eipper BA 1990 Cellular and Kiefer MC, Tucker JE, Joh R, Landsberg KE, Saltman D & molecular aspects of peptide hormone biosynthesis. Frontiers Barr PJ 1991 Identification of a second human subtilisin-like in Neuroendocrinology 11 52–89. protease gene in the fes/fps region of chromosome 15. DNA Mains RE, Berard CA, Denault JB, Zhou A, Johnson RC & Cell Biology 10 757–769. Leduc R 1997 PACE4: a subtilisin-like endoprotease with Knoell DL & Yiu IM 1998 Human gene therapy for hereditary unique properties. Biochemical Journal 321 587–593. diseases: a review of trials. American Journal of Health Martens GJ 1988 Cloning and sequence analysis of human System Pharmacy 55 899–904. pituitary cDNA encoding the novel polypeptide 7B2. FEBS Lamango NS, Zhu X & Lindberg I 1996 Purification and enzymatic Letters 234 160–164. characterization of recombinant prohormone convertase 2: Martens GJ, Braks JA, Eib DW, Zhou Y & Lindberg I 1994 stabilization of activity by 21 kDa 7B2. Archives of The neuroendocrine polypeptide 7B2 is an endogenous Biochemistry and Biophysics 330 238–250. inhibitor of prohormone convertase PC2. Proceedings of Lamango NS, Apletalina E, LiuJ&Lindberg I 1999 The the National Academy of Sciences of the USA 91 5784– proteolytic maturation of prohormone convertase 2 (PC2) is a 5787. pH-driven process. Archives of Biochemistry and Biophysics Matthews DJ, Goodman LJ, Gorman CM & Wells JA 1994a A 362 275–282. survey of furin substrate specificity using substrate phage Lazure C, Gauthier D, Jean F, Boudreault A, Seidah NG, display. Protein Science 3 1197–1205. Bennett HP & Hendy GN 1998 In vitro cleavage of Matthews G, Shennan KI, Seal AJ, Taylor NA, Colman A & internally quenched fluorogenic human proparathyroid Docherty K 1994b Autocatalytic maturation of the hormone and proparathyroid-related peptide substrates by prohormone convertase PC2. Journal of Biological Chemistry furin. Generation of a potent inhibitor. Journal of Biological 269 588–592. Chemistry 273 8572–8580. Mbikay M, Grant SG, Sirois F, Tadros H, Skowronski J, Ledgerwood EC, Brennan SO, Birch NP & George PM 1996 Lazure C, Seidah NG, Hanahan D & Chretien M 1989 The specificity of the neuroendocrine convertase PC3 is cDNA sequence of neuroendocrine protein 7B2 expressed in determined by residues NH2- and COOH-terminal to the beta cell tumors of transgenic mice. International Journal of cleavage site. Biochemistry and Molecular Biology Peptide and Protein Research 33 39–45. International 39 1167–1176. Meerabux J, Yaspo ML, Roebroek AJ, Van de Ven WJ, Lister Leduc R, Molloy SS, Thorne BA & Thomas G 1992 TA & Young BD 1996 A new member of the proprotein Activation of human furin precursor processing endoprotease convertase gene family (LPC) is located at a chromosome occurs by an intramolecular autoproteolytic cleavage. Journal translocation breakpoint in lymphomas. Cancer Research 56 of Biological Chemistry 267 14304–14308. 448–451. LindbergI,vandenHurkWH,BuiC&Batie CJ 1995 MizunoK,NakamuraT,OhshimaT,TanakaS&Matsuo H Enzymatic characterization of immunopurified prohormone 1988 Yeast KEX2 genes encodes an endopeptidase convertase 2: potent inhibition by a 7B2 peptide fragment homologous to subtilisin-like serine proteases. Biochemical [published erratum appears in Biochemistry 1995 34 7780]. and Biophysical Research Communications 156 246–254. Biochemistry 34 5486–5493. MizunoK,NakamuraT,OhshimaT,TanakaS&Matsuo H LipkindG,GongQ&Steiner DF 1995 Molecular modeling of 1989 Characterization of KEX2-encoded endopeptidase from the substrate specificity of prohormone convertases SPC2 yeast Saccharomyces cerevisiae. Biochemical and Biophysical and SPC3. Journal of Biological Chemistry 270 13277–13284. Research Communications 159 305–311. LipkindGM,ZhouA&Steiner DF 1998 A model for the Molloy SS, Bresnahan PA, Leppla SH, Klimpel KR & structure of the P domains in the subtilisin-like prohormone Thomas G 1992 Human furin is a calcium-dependent serine convertases. Proceedings of the National Academy of Sciences endoprotease that recognizes the sequence Arg-X-X-Arg and of the USA 95 7310–7315. efficiently cleaves anthrax toxin protective antigen. Journal of Logeat F, Bessia C, Brou C, LeBail O, Jarriault S, Seidah NG Biological Chemistry 267 16396–16402. & Israel A 1998 The Notch1 receptor is cleaved Molloy SS, Thomas L, VanSlyke JK, Stenberg PE & Thomas constitutively by a furin-like convertase. Proceedings of the G 1994 Intracellular trafficking and activation of the furin National Academy of Sciences of the USA 95 8108–8112. proprotein convertase: localization to the TGN and recycling van de Loo JW, Creemers JW, Bright NA, Young BD, from the cell surface. EMBO Journal 13 18–33. Roebroek AJ & Van de Ven WJ 1997 Biosynthesis, distinct Molloy SS, Thomas L, Kamibayashi C, Mumby MC & post-translational modifications, and functional Thomas G 1998 Regulation of endosome sorting by a characterization of lymphoma proprotein convertase. Journal specific PP2A isoform. Journal of Cellular Biology 142 of Biological Chemistry 272 27116–27123. 1399–1411. www.endocrinology.org Journal of Molecular Endocrinology (2000) 24, 1–22 20   and others · SPCs: molecular specificity to therapeutic applications

Molloy SS, Anderson ED, Jean F & Thomas G 1999 Paquet L, Bergeron F, Boudreault A, Seidah NG, Chretien M, Bi-cycling the furin pathway: from TGN localization to Mbikay M & Lazure C 1994 The neuroendocrine precursor pathogen activation and embryogenesis. Trends in Cell 7B2 is a sulfated protein proteolytically processed by a Biology 9 28–35. ubiquitous furin-like convertase. Journal of Biological Mondino A, GiordanoS&ComoglioPM1991Defective Chemistry 269 19279–19285. posttranslational processing activates the tyrosine kinase Peters T Jr 1987 Intracellular precursor forms of plasma encoded by the MET proto-oncogene (hepatocyte growth proteins: their functions and possible occurrence in plasma. factor receptor). Molecular and Cellular Biology 11 Clinical Chemistry 33 1317–1325. 6084–6092. Rehemtulla A, Barr PJ, Rhodes CJ & Kaufman RJ 1993 Mori K, Imamaki A, Nagata K, Yonetomi Y, Kiyokage- PACE4 is a member of the mammalian propeptidase family Yoshimoto R, Martin TJ, Gillespie MT, Nagahama M, Tsuji that has overlapping but not identical substrate specificity to A & Matsuda Y 1999 Subtilisin-like proprotein convertases, PACE. Biochemistry 32 11586–11590. PACE4 and PC8, as well as furin, are endogenous proalbumin Rheinnecker M, Baker G, EderJ&Fersht AR 1993 Engineering a convertases in HepG2 cells. Journal of Biochemistry 125 627– novel specificity in subtilisin BPN. Biochemistry 32 1199–1203. 633. Rockwell NC & Fuller RS 1998 Interplay between S1 and S4 Morikawa Y, BarsovE&JonesI1993Legitimate and subsites in Kex2 protease: Kex2 exhibits dual specificity for illegitimate cleavage of human immunodeficiency virus the P4 side chain. Biochemistry 37 3386–3391. glycoproteins by furin. Journal of Virology 67 3601–3604. Roebroek AJ, Creemers JW, Ayoubi TA & Van de Ven WJ Muller L, ZhuX&Lindberg I 1997 Mechanism of the 1994 Furin-mediated proprotein processing activity: facilitation of PC2 maturation by 7B2: involvement in involvement of negatively charged amino acid residues in the ProPC2 transport and activation but not folding. Journal of substrate binding region. Biochimie 76 210–216. Cellular Biology 139 625–638. Roebroek AJ, Umans L, Pauli IG, Robertson EJ, van Leuven Munzer JS, Basak A, Zhong M, Mamarbachi A, Hamelin J, F, Van de Ven WJ & Constam DB 1998 Failure of ventral Savaria D, Lazure C, Benjannet S, Chretien M & Seidah closure and axial rotation in embryos lacking the proprotein NG 1997 In vitro characterization of the novel proprotein convertase furin. Development 125 4863–4876. convertase PC7. Journal of Biological Chemistry 272 Rothenberg ME, Eilertson CD, Klein K, Mackin RB & Noe 19672–19681. BD 1996 Evidence for redundancy in propeptide/ Muzzin P, Eisensmith RC, Copeland KC & Woo SL 1997 prohormone convertase activities in processing proglucagon: Hepatic insulin gene expression as treatment for type 1 an antisense study. Molecular Endocrinology 10 331–341. diabetes mellitus in rats. Molecular Endocrinology 11 833– Rott R, Klenk HD, Nagai Y & Tashiro M 1995 Influenza 837. viruses, cell enzymes, and pathogenicity. American Journal of Nachtigal MW & Ingraham HA 1996 Bioactivation of Respiratory and Critical Care Medicine 152 S16–S19. Mullerian inhibiting substance during gonadal develop- Rouille Y, Westermark G, Martin SK & Steiner DF 1994 ment by a kex2/subtilisin-like endoprotease. Proceedings of Proglucagon is processed to glucagon by prohormone the National Academy of Sciences of the USA 93 7711– convertase PC2 in alpha TC1–6 cells. Proceedings of the 7716. National Academy of Sciences of the USA 91 3242–3246. Naggert JK, Fricker LD, Varlamov O, Nishina PM, Rouille Y, Rouille Y, MartinS&Steiner DF 1995 Differential processing Steiner DF, Carroll RJ, Paigen BJ & Leiter EH 1995 of proglucagon by the subtilisin-like prohormone convertases Hyperproinsulinaemia in obese fat/fat mice associated with a PC2 and PC3 to generate either glucagon or glucagon-like carboxypeptidase E mutation which reduces enzyme activity. peptide. Journal of Biological Chemistry 270 26488–26496. Nature Genetics 10 135–142. Rouille Y, Bianchi M, Irminger JC & Halban PA 1997a Role Nakagawa T, Hosaka M, Torii S, Watanabe T, Murakami K & of the prohormone convertase PC2 in the processing of Nakayama K 1993 Identification and functional expression of proglucagon to glucagon. FEBS Letters 413 119–123. a new member of the mammalian Kex2-like processing endo- Rouille Y, Kantengwa S, Irminger JC & Halban PA 1997b protease family: its striking structural similarity to PACE4. Role of the prohormone convertase PC3 in the processing of Journal of Biochemistry 113 132–135. proglucagon to glucagon-like peptide 1. Journal of Biological Nakayama K 1997 Furin: a mammalian subtilisin/Kex2p-like Chemistry 272 32810–32816. endoprotease involved in processing of a wide variety of Rovere C, Barbero P, Maoret JJ, Laburthe M & Kitabgi P precursor proteins. Biochemical Journal 327 625–635. 1998 Pro-neurotensin/neuromedin N expression and Nakayama K, Kim WS, Torii S, Hosaka M, Nakagawa T, processing in human colon cancer cell lines. Biochemical and Ikemizu J, Baba T & Murakami K 1992 Identification of the Biophysical Research Communications 246 155–159. fourth member of the mammalian endoprotease family Rovere C, Luis J, Lissitzky JC, Basak A, Marvaldi J, Chretien homologous to the yeast Kex2 protease. Its testis-specific M & Seidah NG 1999 The RGD motif and the C-terminal expression. Journal of Biological Chemistry 267 5897–5900. segment of proprotein convertase 1 are critical for its cellular Ohnishi Y, Shioda T, Nakayama K, Iwata S, Gotoh B, trafficking but not for its intracellular binding to integrin Hamaguchi M & Nagai Y 1994 A furin-defective cell line is able alpha5beta1. Journal of Biological Chemistry 274 12461–12467. to process correctly the gp160 of human immunodeficiency Schafer MK, Day R, Cullinan WE, Chretien M, Seidah NG & virus type 1. Journal of Virology 68 4075–4079. Watson SJ 1993 Gene expression of prohormone and van den Ouweland AM, Van Groningen JJ, Roebroek AJ, proprotein convertases in the rat CNS: a comparative in situ OnnekinkC&vandeVenWJ1989Nucleotide sequence hybridization analysis. Journal of Neuroscience 13 1258–1279. analysis of the human fur gene. Nucleic Acids Research 17 Schafer W, Stroh A, Berghofer S, Seiler J, Vey M, Kruse ML, 7101–7102. Kern HF, Klenk HD & Garten W 1995 Two independent van den Ouweland AM, van Duijnhoven HL, Keizer GD, targeting signals in the cytoplasmic domain determine trans- Dorssers LC & van de Ven WJ 1990 Structural homology Golgi network localization and endosomal trafficking of the between the human fur gene product and the subtilisin-like proprotein convertase furin. EMBO Journal 14 2424–2435. protease encoded by yeast KEX2 [published erratum appears SchechterI&BergerA1967 On the size of the active site in in Nucleic Acids Research 1990 18 1332]. Nucleic Acids proteases. I. Papain. Biochemical and Biophysical Research Research 18 664. Communications 27 157–162.

Journal of Molecular Endocrinology (2000) 24, 1–22 www.endocrinology.org SPCs: molecular specificity to therapeutic applications ·   and others 21

Scougall K, Taylor NA, Jermany JL, Docherty K & Shennan Smeekens SP, Montag AG, Thomas G, Albiges-Rizo C, Carroll KI 1998 Differences in the autocatalytic cleavage of pro-PC2 R, Benig M, Phillips LA, Martin S, Ohagi S, Gardner P et al. and pro-PC3 can be attributed to sequences within the 1992 Proinsulin processing by the subtilisin-related pro- propeptide and Asp310 of pro-PC2. Biochemical Journal 334 protein convertases furin, PC2, and PC3. Proceedings of the 531–537. National Academy of Sciences of the USA 89 8822–8826. Seidah NG, Hsi KL, De Serres G, Rochemont J, Hamelin J, Stroh A, Schafer W, Berghoter S, Eickmann M, Teuchert M, Antakly T, Cantin M & Chretien M 1983 Isolation and Burger I, Klenk HD & Garten W 1999 A mono NH2-terminal sequence of a highly conserved human and phenylalanine-based motif (F790) and a leucine-dependent porcine pituitary protein belonging to a new superfamily. motif (LI760) mediate internalization of furin. European Immunocytochemical localization in pars distalis and pars Journal of Cellular Biology 78 151–160. nervosa of the pituitary and in the supraoptic nucleus of the Takahashi S, Kasai K, Hatsuzawa K, Kitamura N, Misumi Y, hypothalamus. Archives of Biochemistry and Biophysics 225 IkeharaY,MurakamiK&Nakayama K 1993 A mutation of 525–534. furin causes the lack of precursor-processing activity in Seidah NG, Gaspar L, Mion P, Marcinkiewicz M, Mbikay M & human colon carcinoma LoVo cells. Biochemical and Chretien M 1990 cDNA sequence of two distinct pituitary Biophysical Research Communications 195 1019–1026. proteins homologous to Kex2 and furin gene products: tissue- Takahashi S, Nakagawa T, Banno T, Watanabe T, Murakami specific mRNAs encoding candidates for pro-hormone K & Nakayama K 1995a Localization of furin to the trans- processing proteinases. DNA Cell Biology 9 415–424. Golgi network and recycling from the cell surface involves Seidah NG, Day R, Hamelin J, Gaspar A, Collard MW & Ser and Tyr residues within the cytoplasmic domain. Journal Chretien M 1992 Testicular expression of PC4 in the rat: of Biological Chemistry 270 28397–28401. molecular diversity of a novel germ cell-specific Takahashi S, Nakagawa T, Kasai K, Banno T, Duguay SJ, Kex2/subtilisin-like proprotein convertase. Molecular Van de Ven WJ, MurakamiK&NakayamaK1995b A Endocrinology 6 1559–1570. second mutant allele of furin in the processing-incompetent Seidah NG, Chretien M & Day R 1994 The family of subtilisin/ cell line, LoVo. Evidence for involvement of the homo B kexin like pro-protein and pro-hormone convertases: divergent domain in autocatalytic activation. Journal of Biological or shared functions. Biochimie 76 197–209. Chemistry 270 26565–26569. Seidah NG, Benjannet S, Pareek S, Chretien M & Murphy RA Taylor NA, Jan G, Scougall KT, Docherty K & Shennan KI 1996a Cellular processing of the neurotrophin precursors of 1998 Sorting of PC2 to the regulated secretory pathway NT3 and BDNF by the mammalian proprotein convertases. in AtT20 cells. Journal of Molecular Endocrinology 21 FEBS Letters 379 247–250. 209–216. Seidah NG, Benjannet S, Pareek S, Savaria D, Hamelin J, Teuchert M, Schafer W, Berghofer S, Hoflack B, Klenk HD & Goulet B, Laliberte J, Lazure C, Chretien M & Murphy RA Garten W 1999 Sorting of furin at the trans-Golgi network. 1996b Cellular processing of the nerve growth factor precursor Interaction of the cytoplasmic tail sorting signals with AP-1 by the mammalian pro-protein convertases. Biochemical Golgi-specific assembly proteins. Journal of Biological Journal 314 951–960. Chemistry 274 8199–8207. Seidah NG, Hamelin J, Mamarbachi M, Dong W, Tardos H, Thomas G, Thorne BA, Thomas L, Allen RG, Hruby DE, Mbikay M, Chretien M & Day R 1996c cDNA structure, Fuller R & Thorner J 1988 Yeast KEX2 endopeptidase tissue distribution, and chromosomal localization of rat PC7, correctly cleaves a neuroendocrine prohormone in a novel mammalian proprotein convertase closest to yeast mammalian cells. Science 241 226–230. kexin-like proteinases. Proceedings of the National Academy of Thomas L, Leduc R, Thorne BA, Smeekens SP, Steiner DF & Sciences of the USA 93 3388–3893. Thomas G 1991 Kex2-like endoproteases PC2 and PC3 Seidel B, Dong W, Savaria D, Zheng M, Pintar JE & Day R accurately cleave a model prohormone in mammalian cells: 1998 Neuroendocrine protein 7B2 is essential for proteolytic evidence for a common core of neuroendocrine processing conversion and activation of proprotein convertase 2 in vivo. enzymes. Proceedings of the National Academy of Sciences of DNA Cell Biology 17 1017–1029. the USA 88 5297–5301. Shapiro J, Sciaky N, Lee J, Bosshart H, Angeletti RH & Udupi V, Gomez G, Lambertz I, Qi X, ReedJ&Greeley GH Bonifacino JS 1997. Localization of endogenous furin in Jr 1997 Distribution of prohormone convertase-6 expression cultured cell lines. Journal of Histochemistry and in the gastrointestinal tract and effects of a fasting-refeeding Cytochemistry 45 3–12. regimen and a high-fat diet on ileal prohormone convertase-6 Shen FS, Seidah NG & Lindberg I 1993 Biosynthesis of the expression. Endocrine 7 9–13. prohormone convertase PC2 in Chinese hamster ovary cells Van Rompaey L, Ayoubi T, Van De Ven W & Marynen P and in rat insulinoma cells. Journal of Biological Chemistry 1997 Inhibition of intracellular proteolytic processing of 268 24910–24915. soluble proproteins by an engineered alpha 2-macroglobulin Shennan KI, Seal AJ, Smeekens SP, Steiner DF & Docherty containing a furin recognition sequence in the bait region. K 1991 Site-directed mutagenesis and expression of PC2 in Biochemical Journal 326 507–514. microinjected Xenopus oocytes. Journal of Biological van de Ven WJ, Voorberg J, Fontijn R, Pannekoek H, van den Chemistry 266 24011–24017. Ouweland AM, van Duijnhoven HL, Roebroek AJ & Siezen Shennan KI, Taylor NA, Jermany JL, Matthews G & RJ 1990 Furin is a subtilisin-like proprotein processing Docherty K 1995 Differences in pH optima and calcium enzyme in higher eukaryotes. Molecular Biology Reports 14 requirements for maturation of the prohormone convertases 265–275. PC2 and PC3 indicates different intracellular locations for Volchkov VE, Feldmann H, Volchkova VA & Klenk HD 1998 these events. Journal of Biological Chemistry 270 1402–1407. Processing of the Ebola virus glycoprotein by the proprotein ShindeU&Inouye M 1993 Intramolecular chaperones and convertase furin. Proceedings of the National Academy of protein folding. Trends in Biochemical Sciences 18 442–446. Sciences of the USA 95 5762–5767. Siezen RJ, Creemers JW & Van de Ven WJ 1994 Homology Wan L, Molloy SS, Thomas L, Liu G, Xiang Y, Rybak SL & modelling of the catalytic domain of human furin. A model Thomas G 1998 PACS-1 defines a novel gene family of for the eukaryotic subtilisin-like proprotein convertases. cytosolic sorting proteins required for trans-Golgi network European Journal of Biochemistry 222 255–266. localization. Cell 94 205–216. www.endocrinology.org Journal of Molecular Endocrinology (2000) 24, 1–22 22   and others · SPCs: molecular specificity to therapeutic applications

Watanabe M, Hirano A, Stenglein S, Nelson J, Thomas G & leukemia virus envelope glycoprotein gp72 by subtilisin/ Wong TC 1995 Engineered serine protease inhibitor kexin-like mammalian convertases. FEBS Letters 406 prevents furin-catalyzed activation of the fusion glycoprotein 205–210. and production of infectious measles virus. Journal of Zheng M, Seidah NG & Pintar JE 1997 The developmental Virology 69 3206–3210. expression in the rat CNS and peripheral tissues of Wells JA, Ferrari E, Henner DJ, Estell DA & Chen EY 1983 proteases PC5 and PACE4 mRNAs: comparison with other Cloning, sequencing, and secretion of Bacillus proprotein processing enzymes. Developmental Biology 181 amyloliquefaciens subtilisin in Bacillus subtilis. Nucleic Acids 268–283. Research 11 7911–7925. Zhou A, PaquetL&MainsRE1995Structuralelements that Westphal CH, Muller L, Zhou A, Zhu X, Bonner-Weir S, direct specific processing of different mammalian subtilisin- Schambelan M, Steiner DF, LindbergI&LederP1999 like prohormone convertases. Journal of Biological Chemistry The neuroendocrine protein 7B2 is required for peptide 270 21509–21516. hormone processing in vivo and provides a novel mechanism Zhou A, Martin S, Lipkind G, LaMendolaJ&Steiner DF for pituitary Cushing’s disease. Cell 96 689–700. 1998 Regulatory roles of the P domain of the subtilisin-like Wise RJ, Barr PJ, Wong PA, Kiefer MC, Brake AJ & prohormone convertases. Journal of Biological Chemistry 273 Kaufman RJ 1990 Expression of a human proprotein 11107–11114. processing enzyme: correct cleavage of the von Willebrand ZhuX&Lindberg I 1995 7B2 facilitates the maturation of factor precursor at a paired basic amino acid site. Proceedings proPC2 in neuroendocrine cells and is required for the of the National Academy of Sciences of the USA 87 expression of enzymatic activity. Journal of Cellular Biology 9378–9382. 129 1641–1650. Xin X, Varlamov O, Day R, Dong W, Bridgett MM, Leiter Zhu X, Muller L, Mains RE & Lindberg I 1998 Structural EH & Fricker LD 1997 Cloning and sequence analysis of elements of PC2 required for interaction with its helper cDNA encoding rat carboxypeptidase D. DNA Cell Biology protein 7B2. Journal of Biological Chemistry 273 1158–1164. 16 897–909. Zarkik S, Decroly E, Wattiez R, Seidah NG, Burny A & Ruysschaert JM 1997 Comparative processing of bovine  23 August 1999

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