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The proprotein convertases (PCs) are responsible for the maturation of precursor proteins, and are involved in multiple and critical biological processes. Over the past 30 years, the PCs have had great translational applications, but the physiological roles of PC7, the seventh member of the family, are still obscure. Searching for new substrates of PC7, a quantitative proteomics screen for selective enrichment of N-glycosylated polypeptides secreted from hepatic HuH7 cells identified two human type-II transmembrane proteins of unknown function(s): Cancer Susceptibility Candidate 4 (CASC4) and Golgi Phosphoprotein of 130 kDa (GPP130/GOLIM4). Concentrating on CASC4, its mutagenesis characterized the PC7/Furin-shedding site to occur at KR 66 ↓NS, in HEK293 cells. We defined PC7 and Furin trafficking and activity, and demonstrated that CASC4 shedding occurs in acidic endosomes and/or in the trans-Golgi Network. Our data unraveled a cancer-protective role for CASC4, because siRNA silencing of endogenous CASC4 expression in the invasive triple-negative breast cancer human cell line MDA-MB-231 resulted in a significantly increased cellular migration and invasion. Conversely, MDA-MB-231 cells stably expressing CASC4 exhibited reduced migration and invasion, which can be explained by an increased number of paxillin-positive focal adhesions. This phenotypic cancer-protective role of CASC4 is reversed in cells overexpressing an optimally PC7/Furin-cleaved CASC4 mutant, or upon overexpression of the N-terminally convertase-generated membrane-bound segment. This phenotype was associated with increased formation of podosome-like structures, especially evident in cells overexpressing the N-terminal fragment. In accord, breast cancer patients' data sets show that high CASC4 and PCSK7 expression levels predict a significantly worse prognosis compared to high CASC4 but low PCSK7 levels. In conclusion, CASC4 shedding not only disrupts its anti-migratory/invasive role, but also generates a membrane-bound fragment that drastically modifies the actin cytoskeleton, resulting in an enhanced cellular migration and invasion. This phenotype might be clinically relevant in the prognosis of breast cancer patients.

The locus of the human proprotein convertase subtilisin-kexin type-7 (PC7) gene (PCSK7) is on chromosome 11q23.3 close to the gene cluster APOA5/APOA4/APOC3/APOA1, a region implicated in the regulation of lipoprotein metabolism. A GWAS reported the association of PCSK7 SNPs with plasma triglyceride (TG), and exome sequencing of African Americans revealed the association of a low-frequency coding variant of PC7 (R504H; SNP rs142953140) with a ~ 30% TG reduction. Another PCSK7 SNP rs508487 is in linkage disequilibrium with a promoter variant of the liver-derived apolipoprotein A-V (apoA-V), an indirect activator of the lipoprotein lipase (LpL), and is associated with elevated TG levels. We thus hypothesized that PC7 regulates the levels/activity of apoA-V. Studies in the human hepatic cell line HuH7 revealed that wild-type (WT) PC7 and its endoplasmic reticulum (ER)-retained forms bind to and enhance the degradation of human apoA-V in acidic lysosomes in a nonenzymatic fashion. PC7-induced degradation of apoA-V is inhibited by bafilomycin A1 and the alkalinizing agents: chloroquine and NH 4 Cl. Thus, the PC7-induced apoA-V degradation implicates an ER-lysosomal communication inhibited by bafilomycin A1. In vitro, the natural R504H mutant enhances PC7 Ser 505 phosphorylation at the structurally exposed Ser-X-Glu 507 motif recognized by the secretory kinase Fam20C. Co-expression of the phosphomimetic PC7-S505E with apoA-V resulted in lower degradation compared to WT, suggesting that Ser 505 phosphorylation of PC7 lowers TG levels via reduced apoA-V degradation. In agreement, in Pcsk7 -/- mice fed high-fat diet, plasma apoA-V levels and adipocyte LpL activity are increased, providing an in vivo mechanistic link for a role of liver PC7 in enhanced TG storage in adipocytes., (© 2020 Federation of European Biochemical Societies.)

Many secretory proteins are activated by cleavage at specific sites. The proprotein convertases (PCs) form a family of nine secretory subtilisin-like serine proteases, seven of which cleave at specific basic residues within the trans -Golgi network, granules, or at the cell surface/endosomes. The seventh member, PC7, is a type-I transmembrane (TM) protein with a 97-residue-long cytosolic tail (CT). PC7 sheds human transferrin receptor 1 (hTfR1) into soluble shTfR1 in endosomes. To better understand the physiological roles of PC7, here we focused on the relationship between the CT-regulated trafficking of PC7 and its ability to shed hTfR1. Deletion of the TMCT resulted in soluble PC7 and loss of its hTfR1 shedding activity. Extensive CT deletions and mutagenesis analyses helped us zoom in on three residues in the CT, namely Glu-719, Glu-721, and Leu-725, that are part of a novel motif, E X E XXX L 725 , critical for PC7 activity on hTfR1. NMR studies of two 14-mer peptides mimicking this region of the CT and its Ala variants revealed that the three exposed residues are on the same side of the molecule. This led to the identification of adaptor protein 2 (AP-2) as a protein that recognizes the E X E XXX L 725 motif, thus representing a potentially new regulator of PC7 trafficking and cleavage activity. Immunocytochemistry of the subcellular localization of PC7 and its Ala variants of Leu-725 and Glu-719 and Glu-721 revealed that Leu-725 enhances PC7 localization to early endosomes and that, together with Glu-719 and Glu-721, it increases the endosomal activity of PC7 on hTfR1., (© 2020 Durand et al.)

Endoproteases in the secretory pathway process pro-cholecystokinin (CCK) into the biologically active forms found in the tissues that express CCK mRNA. Thus far, the endoproteases involved in CCK processing include cathepsin L and the prohormone convertases (PC) 1, 2, and 5. This study finds that PC7 is also critical for normal production of CCK in specific areas of the brain. Loss of PC7 results in decreased levels of CCK in more brain regions than any other endoprotease studied to date. Substantial decreases in brain levels of CCK are found in the prefrontal, frontal, parietal-insular-pyriform, and temporal cortex, caudate-putamen, basal forebrain, thalamus, hippocampus, septum, and medulla of PC7 knock-out (KO) mice. A tissue-specific sexual dimorphism of PC7 activity was also identified. This is the first report that loss of PC7 alters levels of a neuropeptide in the brain. This loss of PC7 and CCK may independently contribute to the decrease in Brain Derived Neurotrophic Factor production and be partially responsible for the learning and memory defects observed in mice that lack PC7., (Copyright © 2016 Elsevier Inc. All rights reserved.)

PC7 belongs to the proprotein convertase family, whose members are implicated in the cleavage of secretory precursors. The in vivo function of PC7 is unknown. Herein, we find that the precursor proBDNF is processed into mature BDNF in COS-1 cells coexpressing proBDNF with either PC7 or Furin. Conversely, the processing of proBDNF into BDNF is markedly reduced in the absence of either Furin or PC7 in mouse primary hepatocytes. In vivo we observe that BDNF and PC7 mRNAs are colocalized in mouse hippocampus and amygdala and that mature BDNF protein levels are reduced in these brain areas in PC7 KO mice but not in the hippocampus of PC1/3 KO mice. Various behavioral tests reveal that in PC7 KO mice spatial memory is intact and plasticity of responding is mildly abnormal. Episodic and emotional memories are severely impaired, but both are rescued with the tyrosine receptor kinase B agonist 7,8-dihydroxyflavone. Altogether, these results support an in vivo role for PC7 in the regulation of certain types of cognitive performance, in part via proBDNF processing. Because polymorphic variants of human PC7 are being characterized, it will be important in future studies to determine their effects on additional physiological and behavioral processes.

Unlabelled: The first seven members of the proprotein convertase (PC) family activate protein precursors by cleavage after basic residues. While PC7 has no known specific substrates, it shows redundancy with other PCs. A genome-wide association study suggested that circulating levels of shed human transferrin receptor 1 (hTfR1) are regulated by PC7. We thus examined whether hTfR1 constitutes a specific substrate for PC7. Coexpression of hTfR1 with PCs in several cell lines indicated that PC7 is the only convertase that sheds this receptor into the medium. Site-directed mutagenesis showed that cleavage occurs at the unusual site KTECER100 ↓LA, in which the P1 Arg100 and P6 Lys95 are critical. Pharmacological treatments revealed that shedding of hTfR1 by PC7 requires endocytosis into acidic clathrin-coated vesicles. A PC7 chimera, in which the transmembrane domain and the cytosolic tail of PC7 were replaced by that of the convertase furin, lost its ability to cleave the receptor, demonstrating the importance of these domains in the regulation of PC7 function. Analysis of primary hepatocytes from mice lacking furin, PC5, PACE4, or PC7 revealed that hepcidin, which limits iron availability in the circulation, is specifically generated by furin and not by PC7. Finally, depletion of iron in the medium of hepatoma cell lines incubated with the iron chelator desferrioxamine resulted in PC7 down-regulation., Conclusion: Among the PC family members, only furin activates hepcidin in hepatocytes, and uniquely the full-length membrane-bound PC7 can directly shed hTfR1 by cleavage at Arg100 ↓. Our results support the notion that, when iron is limiting, hTfR1 levels increase at least in part by way of the down-regulation of PC7 expression. (HEPATOLOGY 2013;)., (Copyright © 2013 American Association for the Study of Liver Diseases.)

Although the processing profile of the membrane-bound epidermal growth factor precursor (pro-EGF) is tissue-specific, it has not been investigated at the cellular level nor have the cognate proteinases been defined. Among the proprotein convertases (PCs), only the membrane-bound PC7, the most ancient and conserved basic amino acid-specific PC family member, induces the processing of pro-EGF into an ∼115-kDa transmembrane form (EGF-115) at an unusual VHPR(290)↓A motif. Because site-directed mutagenesis revealed that Arg(290) is not critical, the generation of EGF-115 by PC7 is likely indirect. This was confirmed by testing a wide range of protease inhibitors, which revealed that the production of EGF-115 is most probably achieved via the activation by PC7 of a latent serine and/or cysteine protease(s). EGF-115 is more abundant at the cell surface than pro-EGF and is associated with a stronger EGF receptor (EGFR) activation, as evidenced by higher levels of phosphorylated ERK1/2. This suggests that the generation of EGF-115 represents a regulatory mechanism of juxtacrine EGFR activation. Thus, PC7 is distinct from the other PCs in its ability to enhance the activation of the cell surface EGFR.

The zymogen activation mechanism and physiological functions of the most ancient and highly conserved basic amino acid-specific proprotein convertase 7 (PC7) are not known. Herein, we characterized the biosynthesis, subcellular localization, and trafficking of the membrane-bound full-length rat and human PC7. The prosegment of PC7 is primarily secreted alone as a non-inhibitory protein via the conventional, Golgi-dependent, secretory pathway. Mature PC7 is partially sulfated and thus reaches the cell surface via the conventional route. However, a fraction of PC7 reaches the cell surface through a brefeldin A- and COPII-independent unconventional secretory pathway. The latter trafficking may explain the rapid (<10 min) transit of a fraction of PC7 from the ER to the cell surface. Electron microscopy further confirmed the localization of PC7 to the cell surface of HEK293 cells. Within the cytosolic tail, only two cysteines (Cys(699) and Cys(704)) are palmitoylated, but this modification does not affect the choice of trafficking pathway. Swapping the transmembrane-cytosolic tail (TMCT) sequences of the convertases Furin and PC7 revealed that PC7(TMCT-Furin) is much more sulfated and hence traffics more efficiently through the conventional secretory pathway. In contrast, the Furin(TMCT-PC7) is no longer sulfated and thus reaches the cell surface by the unconventional pathway. Because trafficking of PC7(CT-Furin) and Furin(CT-PC7) resemble their wild type counterparts, we deduce that the transmembrane domain of PC7 regulates the sorting of PC7 toward the unconventional secretory pathway. In conclusion, PC7 is distinct from other proprotein convertases in its zymogen activation, subcellular localization, and trafficking.

The secretory factor VEGF-C has been directly implicated in various physiological processes during embryogenesis and human cancers. However, the importance of the conversion of its precursor proVEGF-C to mature VEGF-C in tumorigenesis, and vessel formation and the identity of the protease(s) that regulate these processes is/are not known. The intracellular processing of proVEGF-C that occurs within the dibasic motif HSIIRR(227)SL suggests the involvement of the proprotein convertases (PCs) in this process. In addition, furin and VEGF-C were found to be coordinately expressed in adult mouse tissues. Cotransfection of the furin-deficient colon carcinoma cell line LoVo with proVEGF-C and different PC members revealed that furin, PC5, and PC7 are candidate VEGF-C convertases. This finding is consistent with the in vitro digestions of an internally quenched synthetic fluorogenic peptide mimicking the cleavage site of proVEGF-C ((220)Q-VHSIIRR downward arrow SLP(230)). The processing of proVEGF-C is blocked by the inhibitory prosegments of furin, PC5, and PACE4, as well as by furin-motif variants of alpha2-macroglobulin and alpha1-antitrypsin. Subcutaneous injection of CHO cells stably expressing VEGF-C into nude mice enhanced angiogenesis and lymphangiogenesis, but not tumor growth. In contrast, expression of proVEGF-C obtained following mutation of the cleavage site (HSIIRR(227)SL to HSIISS(227)SL) inhibits angiogenesis and lymphangiogenesis as well as tumor growth. Our findings demonstrate the processing of proVEGF-C by PCs and highlight the potential use of PC inhibitors as agents for inhibiting malignancies induced by VEGF-C.

Neurotensin (NT) and neuromedin N (NN) are generated by endoproteolytic cleavage of a common precursor molecule, pro-NT/NN. To gain insight into the role of prohormone convertases PC1, PC2, and PC7 in this process, we investigated the maturation of pro-NT/NN in the brain of PC7 (PC7-/-), PC2 (PC2-/-), and/or PC1 (PC1+/- and PC2-/-; PC1+/-) knock down mice. Inactivation of the PC7 gene was without effect, suggesting that this convertase is not involved in the processing of pro-NT/NN. By contrast, there was a 15% decrease in NT and a 50% decrease in NN levels, as measured by radioimmunoassay, in whole brain extracts from PC2 null as compared with wild type mice. Using immunohistochemistry, we found that this decrease in pro-NT/NN maturation products was uneven and that it was most pronounced in the medial preoptic area, lateral hypothalamus, and paraventricular hypothalamic nuclei. These results suggest that PC2 plays a critical role in the processing of pro-NT/NN in mouse brain and that its deficiency may be compensated to a regionally variable extent by other convertases. Previous data have suggested that PC1 might be subserving this role. However, there was no change in the maturation of pro-NT/NN in the brain of mice in which the PC1 gene had been partially inactivated, implying that complete PC1 knock down may be required for loss of function.

The proprotein convertase PC2 is primarily expressed in neuroendocrine cells where it mediates the proteolytic maturation of prohormones and proneuropeptides. We have identified in the upstream sequence of its gene a conserved domain partially homologous to the repressor element RE1/NRSE found in several genes for neuronal proteins. RE1/NRSE binds the silencing transcription factor REST/NRSF, a nuclear protein primarily found in nonneuronal cells. To determine the functionality of the PC2 gene RE1-like sequence (RE1-lk), we examined by electrophoretic mobility shift assays its ability to attach nuclear factors from PC2-expressing and nonexpressing cells. Specific binding factors were mostly detectable in PC2-non-expressing cells. These factors differ from REST/NRSF, as molar excess of competing RE1/NRSE could not prevent their binding to RE1-lk. Reciprocally, molar excess of RE1-lk could not prevent the binding of RE1/NRSE to the DNA-binding domain of a recombinant REST/NRSF. The presence of RE1-lk in cis reduced the ability of the PC2 promoter and the heterologous phosphoglycerate kinase promoter to drive expression of a green fluorescent protein reporter gene in transiently transfected PC2-nonexpressing cells, but not in PC2-expressing cells. These observations suggest that binding of transcription-silencing factors to the RE1-lk element may contribute to repression of the PC2 gene in nonneuroendocrine cells.

The mammalian subtilisin/kexin-like proprotein convertase (PC) family has been implicated in the activation of a wide spectrum of proteins. These proteins are usually synthesized as inactive precursors before their conversion to fully mature bioactive forms. A large majority of these active proteins such as matrix metalloproteases, growth factors, and adhesion molecules are crucial in the processes of cellular transformation, acquisition of the tumorigenic phenotype, and metastases formation. Inhibition of PCs significantly affects the malignant phenotype of various tumor cells. In addition to direct tumor cell proliferation and migration blockade, PC inhibitors can also be used to target tumor angiogenesis. In this Review article we discuss a number of recent findings on the clinical relevance of PCs in cancer patients, their implication in the regulation of multiple cellular functions that impact on the invasive/metastatic potential of cancer cells. Thus, PC inhibitors may constitute new promising agents for the treatment of multiple tumors and/or in adjuvant therapy to prevent recurrence.

Familial amyloidosis of the Finnish type (FAF) is caused by two proteolytic cleavages of mutant gelsolin leading to the accumulation of FAF amyloid in the patients' tissues. Here, we demonstrate that, in mouse pituitary corticotropic AtT20 cells, the enzyme responsible for the first cleavage of mutant secretory FAF gelsolin to FAF amyloid precursor is present in reasonable amounts. Furthermore, in At T20 cells stably expressing alpha1-PDX a potent inhibitor of most proprotein convertases, this cleavage was inhibited The present data provide strong evidence that proprotein convertases, possibly furin or PC5, are involved in the initialpathological cleavage of mutant secretory FAF gelsolin leading ultimately to the amyloid disease.

Mouse 3T3-L1 cells are widely used to study adipocyte differentiation in vitro. When treated with insulin, dexamethasone and isobutylmethylxanthine these fibroblastic cells differentiate into round triglyceride-rich adipocytes. Because several proteins implicated in adipocyte differentiation (e.g. type 1 IGF receptors) are proteolytically activated by endoproteinases of the proprotein convertase family, we sought to determine whether these endoproteinases are crucial for adipose conversion. In this study, we show that expression of the proprotein convertases PACE4, PC7 and furin increases when 3T3-L1 cells are induced to differentiate into adipocytes. The differentiation was blocked in transfected cells expressing alpha1-antitrypsin Portland or in normal cells pre-treated with the synthetic inhibitor decanoyl-RVKR-chloromethylketone. Both inhibitors are known to specifically inactivate proprotein convertases. The block was associated with impaired proteolytic activation of proIGF-1 receptor, absence of induction of the adipogenic transcriptional factor PPARgamma and marked reduction of the nuclear translocation of the C/EBPbeta factor. Taken together, these data constitute evidence that proprotein convertases are crucial mediators of adipogenesis.

Precursor proteins that transit through the secretory pathway often require processing at specific sites in order to release their bioactive entities. The most prevalent limited proteolysis occurs at single or paired basic residues, and is achieved by one or more of the seven subtilisin-like proprotein convertases (PCs); Furin, PC1, PC2, PACE4 (paired basic amino acid converting enzyme 4), PC4, PC5 and PC7. Other types of cleavages occur at hydophobic residues, some of which are performed by subtilisin/kexin-like isozyme-1 (SKI-1), which is also known as site-1 protease. Together, the PCs and SKI-1 regulate the activity of a large variety of cellular proteins, including growth factors, neuropeptides, receptors, enzymes and even toxins and glycoproteins from infectious retroviruses. These processing events are exquisitely regulated by multiple zymogen-activation steps, as well as by specific subcellular localization signals. The above mentioned convertases are implicated in a number of pathologies such as cancer, neurodegenerative diseases, endocrine disorders and inflammation. Recently, it was recognized that the metalloendopeptidase N-arginine dibasic convertase (NRDc; nardilysin), which cleaves at the N-terminus side of basic residues in dibasic pairs, is localized both in the cytosol and at the cell surface or in the extracellular milieu. While NRDc binds heparin-binding epidermal growth factor (HB-EGF) at the cell surface and potentiates its physiological effect, HB-EGF potently inhibits the NRDc's activity. NRDc could represent the equivalent of the PCs in the cytosol or the extracellular space.

Prohormone convertase 1 (PC1), mediating the proteolytic processing of neural and endocrine precursors, is thought to be regulated by the neuroendocrine protein proSAAS. The PC1 inhibitory sequence is mostly confined within a 10-12-amino acid segment near the C terminus of the conserved human proSAAS and contains the critical KR(244) dibasic motif. Our results show that the decapeptide proSAAS-(235-244)( 235)VLGALLRVKR(244) is the most potent reversible competitive PC1-inhibitor (K(i) approximately 9 nm). The C-terminally extended proSAAS-(235-246) exhibits a 5-6-fold higher K(i) ( approximately 51 nm). The additional LE sequence at P1'-P2', resulted in a competitive substrate cleaved by PC1 at KR(244) downward arrowLE(246). Systematic alanine scanning and in some cases lysine scanning tested the contribution of each residue within proSAAS-(235-246) toward the PC1-inhibition's specificity and potency. The amino acids P1 Arg, P2 Lys, and P4 Arg are all critical for inhibition. Moreover, the aliphatic P3 Val and P5, P6, and P1' Leu significantly affect the degree of enzyme inactivation and PC1 specificity. Interestingly, a much longer N- and C-terminally extended endogenous rat proSAAS-(221-254) called little PenLen, was found to be a 3-fold less potent PC1 inhibitor with reduced selectivity but a much better substrate than proSAAS-(235-246). Molecular modeling studies and circular dichroism analysis indicate an extended and poly-l-proline II type structural conformation for proSAAS-(235-244), the most potent PC1 inhibitor, a feature not present in poor PC1 inhibitors.

The endoprotease furin, which belongs to the family of mammalian proprotein convertase (PC), is synthesized as a zymogen with an N-terminal, 81-residue inhibitory prodomain. It has been shown that the proenzyme form of furin undergoes a multistep 'autocatalytic' removal of the prodomain at the C-terminal side of the two consensus sites, R(78)-T-K-R(81) approximately and R(44)-G-V-T-K-R(49) approximately. The furin-mediated cleavage at R(44)-G-V-T-K-R(49) approximately, in particular, is significantly accelerated in an 'acidic' environment. Here, we show that under neutral pH conditions, the inhibitory prodomain of furin is partially folded and undergoes conformational exchanges as indicated by extensive broadening of the NMR spectra. Presence of many ring-current shifted methyl resonances suggests that the partially folded state of the prodomain may still possess a 'semirigid' protein core with specific packing interactions among amino acid side chains. Measurements of the hydrodynamic radii and compaction factors indicate that this partially folded state is significantly more compact than a random chain. The conformational stability of the prodomain appears to be pH sensitive, in that the prodomain undergoes an unfolding transition towards acidic conditions. Our NMR analyses establish that the acid-induced unfolding is mainly experienced by the residues from the C-terminal half of the prodomain (residues R(44)-R(81)) that contains the two furin cleavage sites. A 38-residue peptide fragment derived from the entire pH-sensitive C-terminal region (residues R(44)-R(81)) does not exhibit any exchange-induced line broadening and adopts flexible conformations. We propose that at neutral pH, the cleavage site R(44)-G-V-T-K-R(49) approximately is buried within the protein core that is formed in part by residues from the N-terminal region, and that the cleavage site becomes exposed under acidic conditions, leading to a facile cleavage by the furin enzyme.

Fluorogenic peptides encompassing the processing sites of envelope glycoproteins of the infectious influenza A Hong Kong virus (HKV), Ebola virus (EBOV) and respiratory syncytial virus (RSV) were tested for cleavage by soluble recombinants of the proprotein convertases furin, PC5 and PC7. Kinetic studies with these intramolecularly quenched fluorogenic peptides revealed selective cleavages at the physiological dibasic sites. The HKV peptide is cleaved by both furin and PC5 with similar efficacy; in comparison, PC7 cleaves this substrate poorly. In contrast with the basic tetrapeptide insertion within the haemagglutinin sequence of HKV, two other dipeptide insertions revealed a poorer cleavage with a similar rank order of potency. These results demonstrate that the N-terminal RERR insertion to the wild-type avian RKKR downward arrow sequence is functionally significant, and suggest that the approx. 5-fold increase in cleavage efficacy contributes to the high infectivity of the H5N1 virus subtype. With regard to RSV peptide processing, PC7 is twice as effective as PC5 and furin. The EBOV peptide was processed with similar efficiency by the three enzymes. Our observations that all of these cleavages can be effectively inhibited by a plant andrographolide derivative at 250 microM or less might aid in the design of potent convertase inhibitors as alternative antiviral therapies.

Transforming growth factor (TGF)-beta1 plays an essential role in cell growth and differentiation. It is also considered as a gatekeeper of immune homeostasis with gene disruption leading to autoimmune and inflammatory diseases. TGF-beta1 is produced as an inactive precursor polypeptide that can be efficiently secreted but correct proteolytic cleavage is an essential step for its activation. Assessment of the cleavage site has revealed a unique R-H-R-R sequence reminiscent of proprotein convertase (PC) recognition motifs and has previously demonstrated that this PC-like cleavage site is correctly cleaved by furin, a member of the PC family. Here we report that among PC members, furin more closely satisfies the requirements needed to fulfill the role of a genuine TGF-beta1 convertase. Even though six members of the PC family have the ability to cleave TGF-beta1, ectopic expression of alpha(1)-antitrypsin Portland (alpha(1)-AT-PDX), a potent furin inhibitor, blocked 80% of TGF-beta1 processing mediated by endogenous enzymes as demonstrated in an in vitro digestion assay. Genetic complementation of a furin-deficient LoVo cell line with the wild-type gene restores the production of mature and bioactivable TGF-beta1. Moreover, both furin and TGF-beta are coordinately expressed and regulated in vitro and in vivo in the hematopoietic and immune system, an important tissue target. These results demonstrate for the first time that furin is an authentic and adaptive TGF-beta1-converting enzyme whereas other members of the PC family might substitute or supplement furin activity. Our study advances our comprehension of the complexity of the TGF-beta system and should facilitate the development of therapeutically useful TGF-beta inhibitors.

A general strategy is presented for the dominant negative reduction in the levels of heterodimeric soluble proteins within the secretory pathway through fusion of one of its partners C-terminal to the lysosomal enzyme cathepsin B (CB). Stable transfectants of CB-7B2 chimeras in AT20 cells result in a drastic reduction of the endogenous levels of its partner, the proprotein convertase PC2. This dominant negative suppressive effect requires active CB. It was partially reversed by NH(4)Cl, the cell-permeable CB inhibitor CA-074Me, but not by the proteasome inhibitor Lactacystin, suggesting the potential participation of the lysosomal/endosomal degradative pathway in this process., (Copyright 2000 Academic Press.)

The neuropeptides/neurotransmitters neurotensin (NT) and neuromedin (NN) are synthesized by endoproteolytic cleavage of a common inactive precursor, pro-NT/NN. In vitro studies have suggested that the prohormone convertases PC5A and PC2 might both be involved in this process. In the present study, we used dual immunohistochemical techniques to determine whether either one or both of these two convertases were co-localized with pro-NT/NN maturation products and could therefore be involved in the physiological processing of this propeptide in rat brain. PC2-immunoreactive neurons were present in all regions immunopositive for NT. All but three regions expressing NT were also immunopositive for PC5A. Dual localization of NT with either convertase revealed that NT was extensively co-localized with both PC5A and PC2, albeit with regional differences. These results strongly suggest that PC5A and PC2 may play a key role in the maturation of pro-NT/NN in mammalian brain. The regional variability in NT/PC co-localization patterns may account for the region-specific maturation profiles previously reported for pro-NT/NN. The high degree of overlap between PC5A and PC2 in most NT-rich areas further suggests that these two convertases may act jointly to process pro-NT/NN. At the subcellular level, PC5A was largely co-localized with the mid-cisternae Golgi marker MG-160. By contrast, PC2 was almost completely excluded from MG-160-immunoreactive compartments. These results suggest that PC5A, which is particularly efficient at cleaving the two C-terminal-most dibasics of pro-NT/NN, may be acting as early as in the Golgi apparatus to release NT, whereas PC2, which is considerably more active than PC5A in cleaving the third C-terminal doublet, may be predominantly involved further distally along the secretory pathway to release NN., (Copyright 2000 Wiley-Liss, Inc.)

PC1 and PC2 are two major enzymes involved in the processing of protein precursors directed to the regulated secretory pathway. Whereas transcripts encoding both enzymes are widely distributed in the central nervous system, information regarding the localization of proteins themselves is still lacking. In an attempt to gain insight into the neurobiologic roles of PC1 and PC2, both enzymes were immunolocalized in the rat brain by using C-terminally directed antibodies, which respectively recognize the 87-kDa PC1 and the 75 and 68-kDa PC2 forms. Adjacent sections immunoreacted with PC1 or PC2 antibodies exhibited selective patterns of immunostaining in regions well characterized with respect to their biosynthesis of multiple neuropeptides such as the cerebral cortex, hippocampus, and hypothalamus. PC1 signal intensity was generally weaker than that of PC2, although both enzymes displayed extensive overlapping patterns of expression. As assessed by double-labeling experiments at the cellular level, PC1 and PC2 immunoreactive signals were localized within the trans-Golgi network and nerve terminals, in keeping with the biosynthetic pathways of neuropeptides. Immunoreactive fibers were detected in many areas throughout the brain but were particularly densely distributed in the hypothalamus and the brainstem. Both enzymes were also localized within dendrites of numerous neurons, supporting the hypothesis that dendritic neuropeptide maturation and release may occur in a large number of brain regions. Taken together, our results provide new evidence that both convertases are efficiently targeted to the neuronal regulated secretory pathway and are well poised to process protein precursors in biologically active end-products within the mammalian brain., (Copyright 2000 Wiley-Liss, Inc.)

PC1 and PC2 (prohormone convertase) represent neuroendocrine members of the mammalian subtilisin-like family of proprotein convertases. The goal of this study was to compare the primary sequence motifs of bovine PC1 and PC2 with those of homologs from other mammalian species to establish the structural basis for PC1 and PC2 activities in bovine that resemble other mammalian homologs. Molecular cloning from bovine adrenal medulla resulted in the isolation of cDNAs for bovine PC1 and PC2 with highly conserved primary sequences with respect to signal sequence, prosegment, catalytic domain, and P domain. Bovine PC1 and PC2 contained the catalytic triad residues Asp, His, Ser, which are identical to the triads in PC1 and PC2 from other mammalian species. Bovine PCl contained Asn as the oxyanion hole residue; in contrast, bovine PC2 contained Asp as the oxyanion hole residue, which is identical to PC2 in other mammalian species. Bovine PC1 and PC2 possessed the P domain that contains the functional RRGDL motif. The cloned cDNAs detected expression of PC1 and PC2 mRNAs in bovine adrenal medulla. These results establish the defined structural domains of bovine PC1 and PC2 that are known to be essential for the activities of these enzymes in various species.

The Saccharomyces cerevisiae prohormone-processing enzyme Kex2p is biosynthesized as an inactive precursor extended by its N-terminal proregion. Here we show that deletion of the proregion renders Kex2p inactive both in vivo and in vitro. Absence of the proregion impaired glycosylation and stability and resulted in the retention of the enzyme in the endoplasmic reticulum. These phenotypes were partially complemented by expression of the proregion in trans. Trans complementation was specific to Kex2p proregion because expression of any of the seven mammalian prohormone convertase propeptides had no effect. These data are consistent with a model whereby Kex2p proregion functions as an intramolecular chaperone and indicate that covalent linkage to the protein is not an absolute requirement for proregion function. Furthermore, extensive mutagenesis revealed that, in addition to their function as proteolytic recognition sites, C-terminal basic residues play an active role in proregion-dependent Kex2p activation.

Mammalian proprotein convertases (PCs) belong to the family of recently discovered serine proteases responsible for the processing of a large number of precursor proteins into their active forms. The enzymatic activities of the convertases have been implicated in a variety of disease states, such as cancer and infectious and inflammatory diseases. Like many other proteases, PCs are also synthesized as inactive proenzymes with N-terminal extensions as their prosegments. Here, we present the inhibitory activities of a number of "putative" interfacial peptide fragments derived from the proregion of PC7. We found that a peptide fragment corresponding to the C-terminal region (residues 81p-104p, or C24: E(1)-A-V-L-A-K-H-E-A-V-R-W-H-S-E-Q-R-L-L-K-R-A-K-R(24)) of the PC7 prosegment displays a strong inhibition (K(i) = 7 nM) of the PC7 enzyme comparable to that of the full-length (104 residue) prosegment. The same 24 residue peptide shows significantly populated helical conformations in an aqueous solution close to the physiological condition. Structure calculations driven by NOE distance restraints revealed a slightly kinked helical conformation for the entire peptide, characterized by many side-chain/side-chain interactions including those involving charged residues E8-R11-E15 and hydrophobic residues W12 and L19. These results suggest that the C-terminal region of the prosegment of PC7 may play a dominant role in conferring the inhibitory potency to the cognate enzyme and this strong inhibitory activity may be a direct consequence of the folded conformation of the peptide fragment in solution. We surmise that such a structure-function correlation for an inhibitory peptide could lead to the design and discovery of molecules mimicking the specific interactions of the PC prosegments for their cognate proteases.

Recently, it has been shown that alpha-melanocyte stimulating hormone can directly activate tyrosinase by removing the allosteric regulator 6(R)-L-erythro 5,6,7,8 tetrahydrobiopterin resulting in a stable alpha-melanocyte stimulating hormone/6(R)-L-erythro 5,6,7,8 tetrahydrobiopterin complex. As melanin production occurs in the melanosome, a specific organelle of the melanocyte, it seemed important to investigate whether these organelles themselves actually produce pro-opiomelanocortin-related peptides in their acidic environment. The presence of alpha-melanocyte stimulating hormone and adrenocorticotropin in the epidermis and melanocytes has been shown by several investigators. In order to follow possible pro-opiomelanocortin processing in the melanosome, human melanocytes were established in MCDB 153 medium and utilized for immunohistochemistry, immunogold electron microscopy, and western blotting. For this purpose antibodies against alpha-melanocyte stimulating hormone, adrenocorticotropin, prohormone convertases 1 and 2 (PC1 and PC2) and the PC2 regulatory protein 7B2 were used. Our results demonstrated the presence of the entire system for pro-opiomelanocortin processing in the melanosome. Considering the pH optima of these convertases, the results are in agreement with an autocrine intramelanosomal production of pro- opiomelanocortin-related peptides and an autocrine production and recycling of the cofactor 6(R)-L- erythro 5,6,7,8 tetrahydrobiopterin in melanocytes. Based on these novel observations, we would like to propose that the pigmentation process may not necessarily involve a melanocortin-1 receptor-mediated mechanism.

Several integrin alpha subunits undergo post-translational endoproteolytic processing at pairs of basic amino acids that is mediated by the proprotein convertase furin. Here we ask whether other convertase family members can participate in these processing events. We therefore examined the endoproteolysis rate of the integrin subunits pro-alpha5, alpha6 and alphav by recombinant furin, proprotein convertase (PC)5A, paired basic amino acid converting enzyme (PACE)4, PC1, PC2 and PC7 in vitro and/or ex vivo after overexpression in LoVo cells that were deficient in furin activity. We found that 60-fold more PC1 than furin was needed to produce 50% cleavage of pro-alpha subunit substrates in vitro; the defective pro-alpha chain endoproteolysis in LoVo cells was not rescued by overexpression of PC1 or PC2. No endoproteolysis occurred with PC7 either in vitro or ex vivo, although similar primary sequences of the cleavage site are found in integrins and in proteins efficiently processed by PC7, which suggests that a particular conformation of the cleavage site is required for optimal convertase-substrate interactions. In vitro, 50% cleavage of pro-alpha subunits was obtained with one-third of the amount of PC5A and PACE4 than of furin. In LoVo cells, PC5A remained more active than furin, PACE4 activity was quite low, and PC5B, which differs from PC5A by a C-terminal extension containing a transmembrane domain, was very inefficient in processing integrin alpha-subunit precursors. In conclusion, these results indicate that integrin alpha-subunit endoproteolytic processing involves the redundant function of furin and PC5A and to a smaller extent PACE4, but not of PC1, PC2, PC5B or PC7.

Proproteins and prohormones are the fundamental units from which bioactive proteins and peptides as well as neuropeptides are derived by limited proteolysis within the secretory pathway. Precursors are usually cleaved at the general motif (K/R)--(X)n--(K/R)down arrow, where n=0, 2, 4 or 6 and X is any amino acid and usually is not a Cys. Seven mammalian precursor convertases (PCs) have been identified: PC1, PC2, furin, PC4, PC5, PACE4 and PC7. Each of these enzymes, either alone or in combination with others, is responsible for the tissue-specific processing of multiple polypeptide precursors both in the brain and in periphery. This combinatorial mechanism generates a large diversity of bioactive molecules in an exquisitively regulated manner. The production of null mice allowed the assessment of the critical role of convertases in vivo. Thus, male PC4 (-/-) mice are infertile, furin (-/-) and PC1(-/-) mice are embryonic lethal, and PC2 (-/-) mice are mildly diabetic and runted. Interestingly, animals deficient in 7B2, a PC2-specific binding protein, exhibit a Cushing-like syndrome and die soon after birth. Recently, the first member of a new class of subtilisin--kexin-like convertases, called SKI-1, was identified. Its structure is closer to pyrolysin than to mammalian PCs and it exhibits a specificity for cleavage at the motif (R/K)--X--X--(L,T) down arrow as deduced from its ability to process sterol regulatory element binding proteins and pro-brain derived neurotrophic factor. Thus, while PCs are responsible for the processing of neuropeptides, adhesion molecules, receptors, growth factors, cell surface glycoprotein and enzymes, SKI-1 cleaves proproteins that are critical for the control of cholesterol and fatty acid metabolism and for neuronal protection and growth.

All proprotein convertases (PCs) of the subtilisin/kexin family contain an N-terminal prosegment that is presumed to act both as an intramolecular chaperone and an inhibitor of its parent enzyme. In this work, we examined inhibition by purified, recombinant bacterial prosegments of furin and PC7 on the in vitro processing of either the fluorogenic peptide pERTKR-MCA or the human immunodeficiency virus envelope glycoprotein gp160. These propeptides are potent inhibitors that display measurable selectivity toward specific proprotein convertases. Small, synthetic decapeptides derived from the C termini of the prosegments are also potent inhibitors, albeit less so than the full-length proteins, and the C-terminal P1 arginine is essential for inhibition. The bacterial, recombinant prosegments were also used to generate specific antisera, allowing us to study the intracellular metabolic fate of the prosegments of furin and PC7 expressed via vaccinia virus constructs. These vaccinia virus recombinants, along with transient transfectants of the preprosegments of furin and PC7, efficiently inhibited the ex vivo processing of the neurotrophins nerve growth factor and brain-derived neurotrophic factor. Thus, we have demonstrated for the first time that PC prosegments, expressed ex vivo as independent domains, can act in trans to inhibit precursor maturation by intracellular PCs.

The physiological maturation of the beta-amyloid precursor protein (betaAPP) leads to the secretion of a fragment termed APPalpha, after cleavage by a proteolytic enzyme called-secretase. In Alzheimer's disease, betaAPP undergoes exacerbated proteolytic attacks by beta- and gamma-secretases, which liberate a readily aggregatable 40-42-amino acid peptide called AP. We show here that overexpression of the prohormone convertase PC7 triggers increased secretion of APPalpha and lowers both Abeta40 and Abeta42 recoveries. Overexpression of alpha1-antitrypsin Portland (alpha1-PDX), which blocks mammalian precursor convertases of the constitutive secretory pathway, reverses the PC7-induced APPalpha increase as well as the decrease of Abeta40/42 in HEK293 cells. It is interesting that alpha1-PDX also lowers the level of APPalpha endogenously produced by mock-transfected HEK293 cells. Finally, a Jurkat clone stably expressing alpha1-PDX produces noticeably lower amounts of APPalpha. Therefore, this serpin affects endogenous a-secretase activity/pathway in distinct cell types. By contrast, alpha1-PDX does not alter the processing of presenilin 1 or its mutated congeners linked to some familial forms of Alzheimer's disease. Altogether, we demonstrate that a prohormone convertase participates in the alpha-secretase pathway of betaAPP maturation in human cells and concomitantly contributes to slowing the pathogenic route leading to the formation of Abeta. Our data strongly suggest that PC7 could fulfill such a role.

Proopiomelanocortin (POMC) is a precursor to various, bioactive peptides including ACTH, beta LPH, alpha MSH, and beta endorphin (beta END). Processing of POMC at dibasic residues is tissue-specific and is performed by either PC1 alone (resulting in ACTH and beta LPH, anterior pituitary corticotrophes) or by a combination of PC1 and PC2 (yielding alpha MSH and beta END, pituitary neurointermediate lobe and hypothalamus). The PC2-specific binding protein 7B2 is intimately involved in the zymogen activation of proPC2 into PC2. Structure-function studies of these enzymes demonstrated the presence of N- and C-terminal domains, as well as specific amino acids within the catalytic segment that influence the degree of activity of each enzyme and the interaction of PC2 with 7B2. The tissue distribution, plasticity of expression, and the multiple precursors that are differentially cleaved by PC1 and/or PC2, predict a wide array of combinatorial activities of these convertases within the endocrine and neuroendocrine system. The phenotypic consequences of the absence of genetic expression of either PC1 or PC2 are now explored using knockout mice and in human patients suffering from obesity and diabetes.

We previously showed that the processing of proparathyroid hormone (proPTH) to PTH was accomplished most efficiently by furin (17). Colocalization studies demonstrated that furin is expressed in the parathyroid, whereas proprotein convertase (PC)1 and PC2 are not. Since that time, another member of the PC family, called PC7, has been identified. Here we show, using coinfection studies, that PC7, as well as furin, can appropriately cleave PTH from proPTH. ProPTH and PTH were purified from cell extracts by reversed-phase HPLC and were identified by Western blot analysis and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Colocalization studies, using Northern blot and reverse transcriptase-PCR analyses, showed that PC7 messenger RNA (mRNA) is expressed in the parathyroid gland. Therefore, PC7, like furin, has the potential to be involved in the physiological processing of proPTH to PTH. The two major regulators of parathyroid cell synthetic and secretory activity are the extracellular fluid calcium and 1,25-dihydroxyvitamin D [1,25(OH)2D] levels. We investigated whether either of these agents might modulate processing of proPTH to PTH by altering parathyroid convertase gene expression. In both in vitro and in vivo systems in which regulation of PTH mRNA levels were clearly apparent, there was no effect of either calcium or 1,25(OH)2D3 on parathyroid furin or PC7 mRNA levels. This is in contrast to the processing of proinsulin to insulin in the pancreatic beta-cell, which is up-regulated by glucose stimulation of PC1 and PC2 synthesis.

Human immunodeficiency virus (HIV) glycoprotein (gp) 160 processing by host cell proteinases is an essential step for viral fusion and infectivity. We have identified a rat liver subcellular fraction which specifically processes gp160 into gp120 and gp41. Using equilibration of microsomes in sucrose gradients, the gp160 cleavage activity was associated with particles equilibrating at low densities, well-separated from the endoplasmic reticulum, cis-Golgi network, Golgi stacks, lysosomes and plasma membrane. Its density distribution was compatible with light secretory vesicles derived from the trans-Golgi network (TGN) or to endosomes, but association with endosomes was not supported by free flow electrophoresis. Although furin and pro-protein convertase (PC) 7/LPC have been proposed as the major gp160 processing convertases, the rat liver microsomal gp160 processing activity was essentially resolved from furin and only partially overlapped PC7/LPC. These data suggest that proteinase(s) other than furin and PC7/LPC, presumably located in TGN-derived vesicles, may participate in the gp160 processing into gp120 and gp41.

Pituitary adenomas are members of the family of neuroendocrine cells and tumors which have secretory granules containing chromogranins/secretogranins and other proteins. Pituitary adenomas express the neuroendocrine specific proconvertases PC1 (also known as PC3) and PC2, which are important for the proteolytic processing of chromogranins/secretogranins molecules. We examined the distribution of PC1 and PC2 in primary cultures of 20 pituitary adenomas and analyzed the regulation of the proconvertase mRNAs and proteins by various secretagogues including hypothalamic hormones and phorbol ester to determine the role of PC1 and PC2 in CgA processing in pituitary adenomas. Although PC2 was present in all adenomas, there was a differential distribution of PC1 with PRL adenomas expressing lower levels of PC1 compared to other adenoma types by RT-PCR analysis, in situ hybridization and immunostaining. Treatment of primary cultures of pituitary adenomas with phorbol 12-myristrate 13-acetate (PMA) resulted in an increase in pancreastatin (PST) secretion in most pituitary adenomas and increased PC1 mRNA and protein expression in gonadotroph adenomas, but not in other types of adenomas. Analysis of a human pituitary adenoma cell line, immortalized by recombinant defective adenovirus (HP75), which expressed chromogranin A, FSH, PC1 and PC2 showed that PST was secreted by these immortalized cells. Treatment with TGF beta 1 resulted in an increase in PST secretion and in PC1 mRNA and protein. These results indicate that a) there is a differential distribution of PC1 in human pituitary adenomas with PRL adenomas expressing very little PC1 mRNA and protein and b) that PC1 expression in gonadotropin hormone-producing adenomas is regulated by PMA and TGF beta 1. These findings support the observation that chromogranin A is a substrate for the endoproteinase PC1 in human pituitary adenoma cells.

Using reverse transcriptase-PCR and degenerate oligonucleotides derived from the active-site residues of subtilisin/kexin-like serine proteinases, we have identified a highly conserved and phylogenetically ancestral human, rat, and mouse type I membrane-bound proteinase called subtilisin/kexin-isozyme-1 (SKI-1). Computer databank searches reveal that human SKI-1 was cloned previously but with no identified function. In situ hybridization demonstrates that SKI-1 mRNA is present in most tissues and cells. Cleavage specificity studies show that SKI-1 generates a 28-kDa product from the 32-kDa brain-derived neurotrophic factor precursor, cleaving at an RGLT downward arrowSL bond. In the endoplasmic reticulum of either LoVo or HK293 cells, proSKI-1 is processed into two membrane-bound forms of SKI-1 (120 and 106 kDa) differing by the nature of their N-glycosylation. Late along the secretory pathway some of the membrane-bound enzyme is shed into the medium as a 98-kDa form. Immunocytochemical analysis of stably transfected HK293 cells shows that SKI-1 is present in the Golgi apparatus and within small punctate structures reminiscent of endosomes. In vitro studies suggest that SKI-1 is a Ca2+-dependent serine proteinase exhibiting a wide pH optimum for cleavage of pro-brain-derived neurotrophic factor.

Studies were performed to investigate the prohormone/proprotein convertase (PC)-inhibitory properties of chemical constituents of the medicinally active plant Andrographis paniculata (AP; from the family Acanthaceae), also known as 'King of Bitters'. Among the individual components tested against the clinically important convertases, furin and PC1, neoandrographolide (a C3 O-glucoside derivative of the major constituent andrographolide) exhibited the highest inhibitory action with an IC50 of 53.5 microM against furin. The data further revealed that although andrographolide, the major bitter principle of AP, exhibited a relatively small enzyme inhibition (IC50=1.0 mM and Ki=200 microM against furin), upon succinoylation, its inhibitory action against the above convertases was enhanced significantly with a Ki in the low micromolar range (<30 microM), suggesting that a specific structural modification of the andrographolide skeleton may be exploited to develop a new class of non-peptide inhibitors of PCs. When tested against PC7, these succinoylated derivatives of andrographolide also displayed strong inhibitory action, with Ki values again in the low micromolar range. This potentially interesting observation may be attributed to the reported anti-HIV property of 14-dehydroandrographolide succinic acid monoester (DASM). It is suggested here that DASM, by virtue of this protease inhibitory property, possibly acts by suppressing the proteolytic cleavage of envelope glycoprotein gp160 of HIV, which is known to be PC-mediated, particularly by furin and PC7.

Aerolysin is secreted as an inactive dimeric precursor by the bacterium Aeromonas hydrophila. Proteolytic cleavage within a mobile loop near the C terminus of the protoxin is required for oligomerization and channel formation. This loop contains the sequence KVRRAR432, which should be recognized by mammalian proprotein convertases such as furin, PACE4, and PC5/6A. Here we show that these three proteases cleave proaerolysin after Arg-432 in vitro, yielding active toxin. We also investigated the potential role of these enzymes in the in vivo activation of the protoxin. We found that Chinese hamster ovary cells were able to convert the protoxin to aerolysin in the absence of exogenous proteases and that activation did not require internalization of the toxin. The furin inhibitor alpha1-antitrypsin Portland reduced the rate of proaerolysin activation in vivo, and proaerolysin processing was even further reduced in furin-deficient FD11 Chinese hamster ovary cells. The cells were also less sensitive to proaerolysin than wild type cells; however, transient transfection of FD11 cells with the cDNA encoding furin conferred normal sensitivity to the protoxin. Together these findings argue that furin catalyzes the cell-surface activation of proaerolysin in vivo.

The Notch receptor, which is involved in numerous cell fate decisions in invertebrates and vertebrates, is synthesized as a 300-kDa precursor molecule (p300). We show here that proteolytic processing of p300 is an essential step in the formation of the biologically active receptor because only the cleaved fragments are present at the cell surface. Our results confirm and extend recent reports indicating that the Notch receptor exists at the plasma membrane as a heterodimeric molecule, but disagree as to the nature of the protease that is responsible for the cleavage that takes place in the extracellular region. We report here that constitutive processing of murine Notch1 involves a furin-like convertase. We show that the calcium ionophore A23187 and the alpha1-antitrypsin variant, alpha 1-PDX, a known inhibitor of furin-like convertases, inhibit p300 processing. When expressed in the furin-deficient Lovo cell line, p300 is not processed. In vitro digestion of a recombinant Notch-derived substrate with purified furin allowed mapping of the processing site to the carboxyl side of the sequence RQRR (amino acids 1651-1654). Mutation of these four amino acids (and of two secondary dibasic furin sites located nearby) completely abolished processing of the Notch1 receptor.

The prohormone convertase PC2 is one of the major subtilisin/kexin-like enzymes responsible for the formation of small bioactive peptides in neural and endocrine cells. This convertase is unique among the members of the subtilisin/kexin-like mammalian serine proteinase family in that it undergoes zymogen processing of its inactive precursor proPC2 late along the secretory pathway and requires the help of a PC2-specific binding protein known as 7B2. We hypothesized that some of these unique properties of PC2 are dictated by the presence of PC2-specific amino acids, which in the six other known mammalian convertases are otherwise conserved but distinct. Accordingly, six sites were identified within the catalytic segment of PC2. Herein we report on the site-directed mutagenesis of Tyr194 and of the oxyanion hole Asp309 and the consequences of such mutations on the cellular expression and enzyme activity of PC2. The data show that the Y194D mutation markedly increases the ex vivo ability of PC2 to process proopiomelanocortin (POMC) into beta-endorphin in cells devoid of 7B2, e.g. BSC40 cells. In these cells, expression of native PC2 does not result in the secretion of measurable in vitro activity against a pentapeptide fluorogenic substrate. In contrast, secreted Y194D-PC2 exhibited significant enzymatic activity, even in the absence of 7B2. Based on co-immunoprecipitations and Western blots, binding assays indicate that Tyr194 participates in the interaction of PC2 with 7B2, and that the oxyanion hole Asp309 is critical for the binding of proPC2 with pro7B2.

The cleavage of parathyroid hormone (PTH) from its precursor proparathyroid hormone (pro-PTH) is accomplished efficiently by the proprotein convertase furin (Hendy, G. N., Bennett, H. P. J., Gibbs, B. F., Lazure, C., Day, R., and Seidah, N. G. (1995) J. Biol. Chem. 270, 9517-9525). We also showed that a synthetic peptide comprising the -6 to +7 sequence of human pro-PTH is appropriately cleaved by purified furin in vitro. The human pro-PTH processing site Lys-Ser-Val-Lys-Lys-Arg differs from the consensus furin site Arg-Xaa-(Lys/Arg)-Arg that is represented by Arg-Arg-Leu-Lys-Arg in the cleavage site of pro-PTH-related peptide (pro-PTHrP). An earlier study demonstrated that an internally quenched fluorogenic substrate bearing an O-aminobenzoyl fluorescent donor at the NH2 terminus and an acceptor 3-nitrotyrosine near the COOH terminus was appropriately cleaved by the convertases furin and PC1 (Jean, F., Basak, A., DiMaio, J., Seidah, N. G., and Lazure, C. (1995) Biochem. J. 307, 689-695). Here, we have synthesized a series of internally quenched fluorogenic substrates based upon the pro-PTH and pro-PTHrP sequences to determine which residues are important for furin cleavage. Purified recombinant furin and PC1 cleaved the human pro-PTH internally quenched substrate at the appropriate site in an identical manner to that observed with the nonfluorescent peptide. Several substitutions in the P6-P3 sequence were well tolerated; however, replacement of the Lys at the P6 position with Gly and replacement of the P3 Lys by an acidic residue led to markedly compromised cleavage by furin. Furin activity was very sensitive to substitution in P' positions. Replacement of Ser at P1' with Gly and Val at P2' with Ala generated substrates that were less well cleaved. Substitution at the P1' position of Val for Ser in conjunction with Ala for Val at P2', as well as a single substitution of Lys for Val at P2', generated specific inhibitors of furin cleavage. The findings of this study open the way to the rational design of inhibitors of furin with therapeutic potential.

The prohormone convertases PC1 and PC2 are subtilisin-related endopeptidases that process prohormone and neuropeptide precursors. Using different ultrastructural immunocytochemical approaches, we have investigated their intracellular distribution in a neuroendocrine cell type that has not been examined thus far, the rat anterior pituitary lactotrope. These cells secrete mainly prolactin and also express the neuroendocrine-specific protein secretogranin II, which is considered a peptide precursor. Our study provides evidence for the expression of PC1 and PC2 in rat lactotropes and provides new information on their subcellular localization. Apart from their presence in the secretory granules, PC1 and PC2 displayed different major localization along the secretory pathway. PC1 immunoreactivity was concentrated in the Golgi apparatus, whereas PC2 immunoreactivity was prominent in the rough endoplasmic reticulum (RER). These observations provide morphological support for previous biochemical analysis of proPC1 and proPC2 post-translational processing, which has demonstrated that PC1 exits very rapidly from the RER, whereas PC2 is retained much longer in this compartment. (J Histochem Cytochem 46:101-108, 1998)

Two experimental approaches were used to study the processing of chromogranin B and secretogranin II by prohormone convertases. In GH3 cells various prohormone convertases were overexpressed together with the substrate chromogranin B by use of a vaccinia virus infection system. PC1 appeared to be by far the most active enzyme and converted chromogranin B to several smaller molecules, including the peptide PE-11. In brain this peptide is cleaved physiologically from chromogranin B. Some processing of chromogranin B and formation of free PE-11 were also observed with PC2 and PACE4. Furin produced larger fragments, whereas PC5-A and PC5-B had negligible effects. As a second model, PC12 cells were stably transfected with PC1 or PC2 to investigate the processing of endogenous chromogranins. Both enzymes effectively cleaved chromogranin B and secretogranin II, liberating the peptides PE-11 and secretoneurin, respectively. However, in transfection experiments the ability to generate the free peptides was more pronounced with PC2 than with PC1. The extent of proprotein processing achieved by prohormone convertases apparently differed depending on the experimental system applied. This suggests that in vivo mechanisms to support and fine-tune the activity of the processing enzymes exist, which might be overlooked by using only one methodological approach.

The post-translational processing of prothyrotropin-releasing hormone (pro-TRH25-255) has been extensively studied in our laboratory, and the processing pathway to mature TRH has been elucidated. We have also demonstrated that recombinant PC1 and PC2 process partially purified pro-TRH to cryptic peptides in vitro and that pro-TRH and PC1 mRNAs are coexpressed in primary cultures of hypothalamic neurons. To further define the role of each convertase, and particularly PC1 and PC2, in pro-TRH processing, recombinant vaccinia viruses were used to coexpress the prohormone convertases PC1, PC2, PACE4, PC5-B, furin, or control dynorphin together with rat prepro-TRH in constitutively secreting LoVo cells or in the regulated endocrine GH4C1 cell line. Radioimmunoassays from LoVo-derived secreted products indicated that furin cleaves the precursor to generate both N- and C-terminal intermediates. PC1, PC2, and PACE4 only produced N-terminal intermediates, but less efficiently than furin. In GH4C1 cells, PC1, PC2, furin, PC5-B, and PACE4 produced both N-terminal and C-terminal forms. Significantly, TRH-Gly and TRH were mostly produced by PC1, PC2, and furin. Utilizing gel electrophoresis to further analyze the cleavage specificities of PC1 and PC2, we found that PC1 seems primarily responsible for cleavage to both intermediates and mature TRH, since it generated all products at significantly higher levels than PC2. The addition of 7B2 to the coinfection did not augment the ability of PC2 to cleave pro-TRH to either N- or C-terminal forms.

Biochemical and enzymatic characterization of the novel proprotein convertase rat PC7 (rPC7) was carried out using vaccinia virus recombinants overexpressed in mammalian BSC40 cells. Pro-PC7 is synthesized as a glycosylated zymogen (101 kDa) and processed into mature rPC7 (89 kDa) in the endoplasmic reticulum. No endogenously produced soluble forms of this membrane-anchored protein were detected. A deletion mutant (65 kDa), truncated well beyond the expected C-terminal boundary of the P-domain, produced soluble rPC7 in the culture medium. Enzymatic activity assays of rPC7 using fluorogenic peptidyl substrates indicated that the pH optimum, Ca2+ dependence, and cleavage specificity of this enzyme are largely similar to those of furin. However, with some substrates, cleavage specificity more closely resembled that of yeast kexin, suggesting differential processing of proprotein substrates by this novel convertase. We examined the rPC7- and human furin-mediated cleavage of synthetic peptides containing the processing sites of three proteins known to colocalize in situ with rPC7. Whereas both enzymes correctly processed the pro-parathyroid hormone tridecapeptide and the pro-PC4 heptadecapeptide, neither enzyme cleaved a pro-epidermal growth factor hexadecapeptide. Thus, this study establishes that rPC7 is an enzymatically functional subtilisin/kexin-like serine proteinase with a cleavage specificity resembling that of hfurin. In addition, we have demonstrated that rPC7 can correctly process peptide precursors that contain the processing sites of at least two potential physiological substrates.

1. Inhibition of gastric acid secretion by proton pump inhibitors like omeprazole increases the synthesis and secretion of the pyloric antral hormone gastrin. We report here how omeprazole influences the conversion of the gastrin precursor to its final products, and the abundance of mRNAs encoding proteins associated with progastrin processing in rat antral mucosa. 2. Progastrin processing was studied using a pulse-chase protocol in antral mucosa, incubated in vitro, from rats treated with omeprazole for up to 5 days. Labelled peptides were detected by on-line scintillation counting after immunoprecipitation and HPLC. The mRNAs encoding prohormone-processing enzymes were identified by Northern blot, polymerase chain reaction or ribonuclease protection assay, and their cellular origins identified by immunocytochemistry. 3. Cleavage of [3H]- and [35S]-labelled progastrins at Arg-94-95 or Arg-57-58, and amidation at Phe-92 were not influenced by pretreatment with omeprazole. In contrast, cleavage of G34 (the thirty-four amino acid amidated gastrin) at Lys-74-75 to give G17 (the seventeen amino acid amidated gastrin), and of G34-Gly to G17-Gly (G34 and G17 with COOH-terminal glycine), was increased 3-fold after treatment with omeprazole for either 1 or 5 days. 4. Approximately 20% of newly synthesized amidated and Gly-extended gastrins were secreted within 240 min of the labelling period in omeprazole-treated samples, but secretion of labelled gastrins from control tissue was undetectable over a comparable period. 5. The amidating enzyme, peptidyglycine alpha-amidating mono-oxygenase (PAM), the prohormone convertases PC1/3, PC2, PC5 and the PC2 chaperone 7B2 were localized to rat antral gastrin cells by immunocytochemistry. The relative abundance of mRNA species encoding 7B2, PC5 and PAM were unchanged after treatment with omeprazole for 5 days, whereas gastrin, PC1/3 and PC2 mRNAs are known to increase at this time. 6. The main consequence of increased cleavage at Lys-74-75 is the production of G17 and G17-Gly at the expense of G34 and G34-Gly, respectively. The latter have longer plasma half-lives, and so their increased cleavage may serve to limit the rise in plasma gastrin concentration after inhibition of acid secretion. Changes in the abundance of mRNAs encoding prohormone-processing enzymes cannot account for the rapidity of the changes in cleavage of progastrin at Lys residues after omeprazole.

PC1 and PC2 are subtilisin-like processing enzymes capable of cleaving thyrotropin releasing hormone (TRH) precursor (pro-TRH) at paired basic residues in vitro. In the paraventricular nucleus of the hypothalamus (PVN), pro-TRH is synthesized to control adenohypophysial thyrotropin and prolactin release. Biochemical and immunological approaches have shown that in the hypothalamus, pro-TRH is extensively cleaved at pairs of basic amino acids. We quantified, by two different approaches, in situ hybridization (ISH) on consecutive cryostat sections or double label ISH, the proportion of PVN TRH neurons containing either PC1 or PC2 mRNAs. Both techniques gave similar results: PC2 mRNA was present in 60-70% of TRH neurons, and PC1 mRNA in 37-46%. Values were similar in the anterior and medial parts of the parvocellular PVN. TRH neurons containing either PC1 or PC2 mRNA were found throughout the areas containing TRH cells without any evidence of anatomical segregation. These results suggest a biochemical heterogeneity in PVN TRH biosynthetic machinery.

A 32 amino acid peptide called histatin-3 (H3; 22% His) and its N-terminal 24 amino acid fragment histatin-5 (H5, 33% His), are found in human saliva and possess powerful antimicrobial properties. These His-rich peptides have been synthesized by Fmoc-based solid-phase chemistry. Their sequences are: DSHAKRHHGYKRKFHEKHHSHRGYRSNYLYDN (H3) and DSHAKRHHGYKRKFHEKHHSHRGY (H5). In addition, we also prepared two H5 and one H3 mutants. The H5 mutants were: DH5 (all amino acids in D configuration) and H5F (where all His are replaced by Phe at positions 3, 7, 8, 15, 18, 19, 21). The 9-24 segment of H3 with all the His at positions 15,18,19,21 replaced by Tyr was also prepared (delta 1-8 H3Y). The behavior of these five peptides was examined with three proprotein convertases (PC's) which possess cleavage specificity directed towards single and pairs of basic residues. These were: human (h)PC1, an endocrine and neural convertase, hfurin and rat (r)PC7, two widely expressed enzymes. All are serine endoproteases belonging to the kexin/subtilisin family. Our in vitro study revealed that H3 behaves as a substrate for PC1, being cleaved by this endoprotease primarily at a site carboxy terminal to the single Arg25 residue (HRGYR decrease SN). On prolonged incubation some minor cleavage was also observed C-terminal to the first LysArg6 pairs of basic amino acids namely at: HAKR decrease HH, which contains a P4 as well as P'1 and P'2 His residues. The second potential site YKRK12-FH which does not have a P4 basic residues is not cleaved, even upon incubation with excess protease. PC1 only poorly cleaves H5 at the same site mentioned above for H3, i.e., at HAKR decrease HH. As expected, neither the D-amino acid analogue (DH5), nor the Phe and Tyr mutant analogues of the long and short histatins, respectively, are cleaved at all. In contrast to the above findings for hPC1, the convertase hfurin did not cleave any of the five synthetic peptides. Instead, H3 and H5 were found to be moderately potent inhibitors of the furin-mediated cleavage of the pentapeptide pGlu-Arg-Thr-Lys-Arg-MCA fluorogenic substrate. This inhibition was reversible and competitive, with an estimated inhibition constant Ki of 1.98 microM for H3 and 2.98 microM for H5. The other analogs exhibited only a moderate to weak inhibition of furin, suggesting that substitution of all His by aromatic residues (Phe or Tyr) drastically reduces their inhibitory potency. When tested against rPC7, H3 exhibited almost identical inhibition profile with a measured Ki of 2.4 microM. The partial sequence identity of H3 to the inhibitory pro-peptide of furin and PC7 provides a rationale for our observation.

Nerve growth factor (NGF) in mouse submandibular glands (SGs) is generated from a 35-kD precursor by proteolytic enzymes that have yet to be identified. Prohormone convertases (PCs) cleave the NGF precursor in vitro, and in this study we questioned whether PCs could process salivary NGF in vivo. mRNA coding for PC2 (but not PC1) was detected on Northern blots of SG mRNA and also by in situ hybridization within parasympathetic neurons of intralobular ganglia. Northern blot and in situ hybridization analyses also detect mRNA coding for furin. In SGs of male mice, furin mRNA levels are high at birth and remain high throughout development. In glands from female mice, levels decline during postnatal development and are lower in adults than in newborns. Immunocytochemistry detects furin immunoreactivity in pro-acinar and ductal cells of glands from newborn and pubescent mice. In glands of adults, furin immunoreactivity is detectable in acinar cells but highest levels are present in NGF-containing granular convoluted tubule cells. These data, taken together with those from previous studies, suggest that furin is a candidate processing enzyme for NGF in mouse submandibular glands.