Mutations of the PC2 substrate binding pocket alter enzyme specificity

By taking advantage of the recently published furin structure, whose catalytic domain shares high homology with other proprotein convertases, we designed mutations in the catalytic domain of PC2, altering residues Ser206, Thr271, Asp278, ArgGlu282, AlaSer323, Leu341, Asn365, and Ser380, which are both conserved and specific to this convertase, and substituting residues specific to PC1 and/or furin. In order to investigate the determinants of PC2 specificity, we have tested the mutated enzymes against a set of proenkephalin-derived substrates, as well as substrates representing Arg, Ala, Leu, Phe, and Glu positional scanning variants of a peptide B-derived substrate. We found that the exchange of the Ser206 residue with Arg or Lys led to a total loss of activity. Increased positive charge of the substrate generally resulted in an increased specificity constant. Most intriguingly, the RE281GR mutation, corresponding to a residue placed distantly in the S6 pocket, evoked the largest changes in the specificity pattern. The D278E and N356S mutations resulted in distinct alterations in PC2 substrate preferences. However, when other residues that distinguish PC2 from other convertases were substituted with PC1-like or furin-like equivalents, there was no significant alteration of the PC2 specificity pattern, suggesting that the overall structure of the substrate binding cleft rather than individual residues specifies substrate binding.     

Propeptides Are Sufficient to Regulate Organelle-Specific pH-Dependent Activation of Furin and Proprotein Convertase 1/3

The proprotein convertases (PCs) furin and proprotein convertase 1/3 (PC1) cleave substrates at dibasic residues along the eukaryotic secretory/endocytic pathway. PCs are evolutionarily related to bacterial subtilisin and are synthesized as zymogens. They contain N-terminal propeptides (PRO) that function as dedicated catalysts that facilitate folding and regulate activation of cognate proteases through multiple-ordered cleavages. Previous studies identified a histidine residue (His69) that functions as a pH sensor in the propeptide of furin (PRO(FUR)), which regulates furin activation at pH~6.5 within the trans-Golgi network. Although this residue is conserved in the PC1 propeptide (PRO(PC1)), PC1 nonetheless activates at pH~5.5 within the dense core secretory granules. Here, we analyze the mechanism by which PRO(FUR) regulates furin activation and examine why PRO(FUR) and PRO(PC1) differ in their pH-dependent activation. Sequence analyses establish that while both PRO(FUR) and PRO(PC1) are enriched in histidines when compared with cognate catalytic domains and prokaryotic orthologs, histidine content in PRO(FUR) is ~2-fold greater than that in PRO(PC1), which may augment its pH sensitivity. Spectroscopy and molecular dynamics establish that histidine protonation significantly unfolds PRO(FUR) when compared to PRO(PC1) to enhance autoproteolysis. We further demonstrate that PRO(FUR) and PRO(PC1) are sufficient to confer organelle sensing on folding and activation of their cognate proteases. Swapping propeptides between furin and PC1 transfers pH-dependent protease activation in a propeptide-dictated manner in vitro and in cells. Since prokaryotes lack organelles and eukaryotic PCs evolved from propeptide-dependent, not propeptide-independent prokaryotic subtilases, our results suggest that histidine enrichment may have enabled propeptides to evolve to exploit pH gradients to activate within specific organelles.     

Cleavage specificity of the serine protease of Aeromonas sobria, a member of the kexin family of subtilases

Subtilisin-like proteases have been grouped into six families based on a sequence of the catalytic domain. One of the six is the kexin family, of which furin is a representative protease. All members of the kexin family, except one, are from eukaryotes. The one prokaryotic protease is a serine protease of Aeromonas sorbria (ASP). Here, we examined the substrate specificity of ASP based on the cleavage of short peptides. The results showed that ASP preferentially cleaves the peptide bond following two basic residues, one of which is Lys, but not the bond following a single basic residue. This indicates that the tertiary structure around the catalytic domain of ASP resembles, but is not identical to that of furin. Prekallikrein was cleaved into four fragments by ASP, indicating that the protein must be cleaved at specific sequences.     

Selective expression of the proprotein convertases furin, pc5, and pc7 in proliferating vascular smooth muscle cells of the rat aorta in vitro.

The aim of this study was to investigate whether transformation of quiescent vascular smooth muscle cells (VSMCs) into proliferating secretory cells is accompanied by an expression of processing enzymes that activate de novo-synthesized growth factors. Three enzymes belonging to the family of the kexin/subtilisin-like mammalian proprotein convertases (PCs), furin, PC5, and PC7, were found to be upregulated after balloon denudation in vivo. To determine their importance in these cell processes, we investigated their gene regulation using a short-term organ culture system. After incubation of rat aorta for 4 and 24 hr in serum-free medium, we demonstrated a significant induction of VSMC proliferation. The affected subset of VSMCs, positive for alpha-smooth muscle actin, also expressed proliferating cell nuclear antigen (PCNA). Our results revealed a parallel upregulation of furin, PC5, and PC7 in PCNA-immunolabeled cells. As a substrate model for comparison with PCs we used nerve growth factor (NGF). NGF is known to be activated by PCs. As shown by Northern blotting analysis, NGF mRNA concentration was significantly increased in cultured explants. NGF was released into the culture medium. In conclusion, both PCs and NGF are coordinately modulated on induction of VSMC proliferation.     

Prohormone convertases 1/3, 2, furin and protein 7B2 (Secretogranin V) in endocrine cells of the human pancreas

Prohormone convertases (PCs) are proteinases that cleave inactive prohormones to biologically active peptides. Seven PCs have been identified; two of them, PC1/3 and PC2, have only been localized in neuroendocrine (NE) tissues; a third, furin, in both endocrine and exocrine tissues. We have studied the immunoreactivity of PC1/3, PC2 and furin in the four major NE cell types of the human pancreas by using double immunofluorescence techniques. The study also included the expression of NE secretory protein 7B2 (secretogranin V), a member of the granin family, which influences the function of PC2. The results showed that the three PCs and 7B2 were expressed only in endocrine pancreas, furin also in exocrine cells. Insulin (B) cells harboured PC1/3 and PC2, but not furin. Glucagon (A) cells were immunoreactive to all three PCs; all glucagon cells expressed PC2, but one subpopulation showed PC1/3 immunoreactivity and another furin. Only a few somatostatin (D) cells contained PC2, but no other proconvertase. Pancreatic polypeptide (PP) cells were non-reactive to all three PCs. 7B2 occurred only in insulin and glucagon cells. A varying co-localization pattern was observed between PCs and between PCs and 7B2, with the exception of PC1/3 and furin which were not co-localized. In conclusion, our study shows that PCs are localized in insulin and glucagon cells and do seem to be important in these cell types for processing of hormone and other protein precursors, especially chromogranins, but for the two other major cell types probably other enzymes are of importance.     

The prosegments of furin and PC7 as potent inhibitors of proprotein convertases. In vitro and ex vivo assessment of their efficacy and selectivity

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.     

Furin: the prototype mammalian subtilisin-like proprotein-processing enzyme. Endoproteolytic cleavage at paired basic residues of proproteins of the eukaryotic secretory pathway.

Furin, the translational product of the recently discovered fur gene, appears to be the first known mammalian member of the subtilisin family of serine proteases and the first known mammalian proprotein-processing enzyme with cleavage selectivity for paired basic amino acid residues. Structurally and functionally, it resembles the prohormone-processing enzyme, kexin (EC 3.4.21.61), which is encoded by the KEX2 gene of yeast Saccharomyces cerevisiae. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Here, we review the discovery of the fur gene and describe the isolation of cDNA clones corresponding to human and mouse fur and to two fur-like genes of Drosophila melanogaster, Dfur1 and Dfur2. We also compare the structural organization of the various deduced furin proteins to that of yeast kexin, and of other members of the subtilisin family of serine proteases. Furthermore, the biosynthesis of biologically active human and mouse furin is evaluated. Finally, the cleavage specificity for paired basic amino acid residues of human and mouse furin is demonstrated by the correct processing of the precursor for von Willebrand factor.     

Identification of the fourth member of the mammalian endoprotease family homologous to the yeast Kex2 protease. Its testis-specific expression.

We used the polymerase chain reaction to identify a mouse testis cDNA that represented another member of a growing class of mammalian endoproteases involved in the processing of precursor proteins. This cDNA encoded a 655-residue protein, designated PC4, containing a bacterial subtilisin-like catalytic domain closely related to those of the recently characterized precursor-processing endoproteases, furin, PC1/PC3, PC2, and Kex2. Within this domain, the amino acid sequence of PC4 was 70, 58, 55, and 45% identical with those of mouse furin, mouse PC1/PC3, mouse PC2, and yeast Kex2, respectively. Northern blot analysis indicated that the PC4 mRNA was detectable only in the testes after the 20th day of postnatal development. Moreover, this message was mainly expressed in the round spermatids. These data suggest that PC4 represents a prime candidate for a precursor-processing endoprotease in the testicular germ cells and that its gene expression is regulated during spermatogenesis.     

The Multifaceted Proprotein Convertases: Their Unique,Redundant, Complementary,and Opposite Functions

The secretory proprotein convertase (PC) family comprises nine members: PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, SKI-1/S1P, and PCSK9. The first seven PCs cleave their substrates at single or paired basic residues, and SKI-1/S1P cleaves its substrates at non-basic residues in the Golgi. PCSK9 cleaves itself once, and the secreted inactive protease escorts specific receptors for lysosomal degradation. It regulates the levels of circulating LDL cholesterol and is considered a major therapeutic target in phase III clinical trials. In vivo, PCs exhibit unique and often essential functions during development and/or in adulthood, but certain convertases also exhibit complementary, redundant, or opposite functions.     

Comparative study of the binding pockets of mammalian proprotein convertases and its implications for the design of specific small molecule inhibitors

Proprotein convertases are enzymes that proteolytically cleave protein precursors in the secretory pathway to yield functional proteins. Seven mammalian subtilisin/Kex2p-like proprotein convertases have been identified: furin, PC1, PC2, PC4, PACE4, PC5 and PC7. The binding pockets of all seven proprotein convertases are evolutionarily conserved and highly similar. Among the seven proprotein convertases, the furin cleavage site motif has recently been characterized as a 20-residue motif that includes one core region P6-P2´ inside the furin binding pocket. This study extended this information by examining the 3D structural environment of the furin binding pocket surrounding the core region P6-P2´ of furin substrates. The physical properties of mutations in the binding pockets of the other six mammalian proprotein convertases were compared. The results suggest that: 1) mutations at two positions, Glu230 and Glu257, change the overall density of the negative charge of the binding pockets, and govern the substrate specificities of mammalian proprotein convertases; 2) two proprotein convertases (PC1 and PC2) may have reduced sensitivity for positively charged residues at substrate position P5 or P6, whereas the substrate specificities of three proprotein convertases (furin, PACE4, and PC5) are similar to each other. This finding led to a novel design of a short peptide pattern for small molecule inhibitors: [K/R]-X-V-X-K-R. Compared with the widely used small molecule dec-RVKR-cmk that inhibits all seven proprotein convertases, a finely-tuned derivative of the short peptide pattern [K/R]-X-V-X-K-R may have the potential to more effectively inhibit five of the proprotein convertases (furin, PC4, PACE4, PC5 and PC7) compared to the remaining two (PC1 and PC2). The results not only provide insights into the molecular evolution of enzyme function in the proprotein convertase family, but will also aid the study of the functional redundancy of proprotein convertases and the development of therapeutic applications.     

Mammalian neural and endocrine pro-protein and pro-hormone convertases belonging to the subtilisin family of serine proteinases.

Conversion of pro-hormones and precursor proteins into biologically active peptides and proteins involves the concerted action of a number of convertases and post-translation modification enzymes. The identification of the yeast convertase kexin as a prototype processing enzyme led to the discovery of the mammalian convertase designated furin, PC1 and PC2. Whereas furin is ubiquitously expressed, PC1 and PC2 are found only in endocrine and neural tissues and cell lines. In man and mouse, the genes coding for furin, PC1 and PC2 reside on three different chromosomes. The analysis of the intracellular processing of PC1 and PC2 and the removal of their pro-segment is presented, together with a summary of the cleavage specificity of these enzymes for precursors such as pro-opiomelanocortin (POMC) and human pro-renin. The distinct tissue distribution of PC1 and PC2 and their coregulation with POMC in the pituitary neurointermediate lobe adds credence to their physiological role as convertases involved in the tissue-specific processing of precursor proteins.     

Barley serine proteinase inhibitor 2-derived cyclic peptides as potent and selective inhibitors of convertases PC1/3 and furin

Proprotein convertases (PCs) are serine proteases containing a subtilisin-like catalytic domain that are involved in the conversion of hormone precursors into their active form. This study aims at designing small cyclic peptides that would specifically inhibit two members of this family of enzymes, namely, the neuroendocrine PC1/3 and the ubiquitously expressed furin. We studied peptide sequences related to the 18-residue loop identified as the active site of the 83 amino acid barley serine protease inhibitor 2 (BSPI-2). Peptides incorporating mutations at various positions in the sequence were synthesized on solid phase and purified by HPLC. Cyclization was achieved by the introduction of a disulfide bridge between the two Cys residues located at both the N- and C-terminal extremities. Peptides VIIA and VIIB incorporating P4Arg, P2Lys, P1Arg, and P2'Lys were the most potent inhibitors with K(i) around 4 microM for furin and around 0.5 microM for PC1/3. Whereas peptide VIIB behaved as a competitive inhibitor of furin, peptide VIIA acted as a noncompetitive one. However, all peptides were eventually cleaved after variable incubation times by PC1/3 or furin. To avoid this problem, we incorporated at the identified cleavage site a nonscissile aminomethylene bond (psi[CH(2)-NH]). Those pseudopeptides, in particular peptide VIID, were shown not to be cleaved and to inhibit potently furin. Conversely, they were not able to inhibit PC1/3 at all. Those results show the validity of this approach in designing new effective PC inhibitors showing a certain level of discrimination between PC1/3 and furin.     

Solution structure of the pro-hormone convertase 1 pro-domain from Mus musculus

The solution structure of the mouse pro-hormone convertase (PC) 1 pro-domain was determined using heteronuclear NMR spectroscopy and is the first structure to be obtained for any of the domains in the convertase family. The ensemble of NMR-derived structures shows a well-ordered core consisting of a four-stranded antiparallel beta-sheet with two alpha-helices packed against one side of this sheet. Sequence homology suggests that the other eukaryotic PC pro-domains will have the same overall fold and most of the residues forming the hydrophobic core of PC1 are highly conserved within the PC family. However, some of the core residues are predicted by homology to be replaced by polar amino acid residues in other PC pro-domains and this may help to explain their marginal stability. Interestingly, the folding topology observed here is also seen for the pro-domain of bacterial subtilisin despite little or no sequence homology. Both the prokaryotic and eukaryotic structures have hydrophobic residues clustered on the solvent-accessible surface of their beta-sheets although the individual residue types differ. In the bacterial case this region is buried at the binding interface with the catalytic domain and, in the eukaryotic PC family, these surface residues are conserved. We therefore propose that the hydrophobic patch in the PC1 pro-domain is involved in the binding interface with its cognate catalytic domain in a similar manner to that seen for the bacterial system. The PC1 pro-domain structure also reveals potential mechanisms for the acid-induced dissociation of the complex between pro- and catalytic domains.     

The crystal structure of the proprotein processing proteinase furin explains its stringent specificity

In eukaryotes, many essential secreted proteins and peptide hormones are excised from larger precursors by members of a class of calcium-dependent endoproteinases, the prohormone-proprotein convertases (PCs). Furin, the best-characterized member of the mammalian PC family, has essential functions in embryogenesis and homeostasis but is also implicated in various pathologies such as tumor metastasis, neurodegeneration and various bacterial and viral diseases caused by such pathogens as anthrax and pathogenic Ebola virus strains. Furin cleaves protein precursors with narrow specificity following basic Arg-Xaa-Lys/Arg-Arg-like motifs. The 2.6 A crystal structure of the decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk)-inhibited mouse furin ectodomain, the first PC structure, reveals an eight-stranded jelly-roll P domain associated with the catalytic domain. Contoured surface loops shape the active site by cleft, thus explaining furin's stringent requirement for arginine at P1 and P4, and lysine at P2 sites by highly charge-complementary pockets. The structure also explains furin's preference for basic residues at P3, P5 and P6 sites. This structure will aid in the rational design of antiviral and antibacterial drugs.     

Quantitative characterization of furin specificity. Energetics of substrate discrimination using an internally consistent set of hexapeptidyl methylcoumarinamides.

Furin, an essential mammalian proprotein processing enzyme of the kexin/furin family of subtilisin-related eukaryotic processing proteases, is implicated in maturation of substrates involved in development, signaling, coagulation, and pathogenesis. We examined the energetics of furin specificity using a series of peptidyl methylcoumarinamide substrates. In contrast to previous reports, we found that furin can cleave such substrates with kinetics comparable to those observed with extended peptides and physiological substrates. With the best of these hexapeptidyl methylcoumarinamides, furin displayed k(cat)/K(m) values greater than 10(6) M(-1) s(-1). Furin exhibited striking substrate inhibition with hexapeptide but not tetrapeptide substrates, an observation of significance to the evaluation of peptide-based furin inhibitors. Quantitative comparison of furin and Kex2 recognition at P(1), P(2), and P(4) demonstrates that whereas interactions at P(1) make comparable contributions to catalysis by the two enzymes, furin exhibited a approximately 10-fold lesser dependence on P(2) recognition but a 10-100-fold greater dependence on P(4) recognition. Furin has recently been shown to exhibit P(6) recognition and we found that this interaction contributes approximately 1.4 kcal/mol toward catalysis independent of the nature of the P(4) residue. We have also shown that favorable residues at P(2) and P(6) will compensate for less than optimal residues at either P(1) or P(4). The quantitative analysis of furin and Kex2 specificity sharply distinguish the nature of substrate recognition by the processing and degradative members of subtilisin-related proteases.     

The crystal structure of an oxidatively stable subtilisin-like alkaline serine protease, KP-43, with a C-terminal beta-barrel domain

The crystal structure of an oxidatively stable subtilisin-like alkaline serine protease, KP-43 from Bacillus sp. KSM-KP43, with a C-terminal extension domain, was determined by the multiple isomorphous replacements method with anomalous scattering. The native form was refined to a crystallographic R factor of 0.134 (Rfree of 0.169) at 1.30-A resolution. KP-43 consists of two domains, a subtilisin-like alpha/beta domain and a C-terminal jelly roll beta-barrel domain. The topological architecture of the molecule is similar to that of kexin and furin, which belong to the subtilisin-like proprotein convertases, whereas the amino acid sequence and the binding orientation of the C-terminal beta-barrel domain both differ in each case. Since the C-terminal domains of subtilisin-like proprotein convertases are essential for folding themselves, the domain of KP-43 is also thought to play such a role. KP-43 is known to be an oxidation-resistant protease among the general subtilisin-like proteases. To investigate how KP-43 resists oxidizing reagents, the structure of oxidized KP-43 was also determined and refined to a crystallographic R factor of 0.142 (Rfree of 0.212) at 1.73-A resolution. The structure analysis revealed that Met-256, adjacent to catalytic Ser-255, was oxidized similarly to an equivalent residue in subtilisin BPN'. Although KP-43, as well as proteinase K and subtilisin Carlsberg, lose their hydrolyzing activity against synthetic peptides after oxidation treatment, all of them retain 70-80% activity against proteinaceous substrates. These results, as well as the beta-casein digestion pattern analysis, have indicated that the oxidation of the methionine adjacent to the catalytic serine is not a dominant modification but might alter the substrate specificities.     

The kindest cuts of all: crystal structures of Kex2 and furin reveal secrets of precursor processing

Pro-hormone or pro-protein convertases are a conserved family of eukaryotic serine proteases found in the secretory pathway. These endoproteases mature precursors for peptides and proteins that perform a wide range of physiologically important and clinically relevant functions. The first member of this family to be identified was Kex2 in the yeast Saccharomyces cerevisiae. One mammalian member of this family - furin - is responsible for processing substrates that include insulin pro-receptor, human immunodeficiency virus gp160 glycoprotein, Ebola virus glycoprotein, and anthrax protective antigen. Recent determination of the crystal structures for the catalytic core domains of both Kex2 and furin - the first for any members of this family - provide remarkable insights and a new level of understanding of substrate specificity and catalysis by the pro-protein convertases.     

Furin is the major processing enzyme of the cardiac-specific growth factor bone morphogenetic protein 10

Bone morphogenetic protein 10 (BMP10) is a member of the TGF-β superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (～60 kDa) is processed into active BMP10 (～14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR(316)↓ site (human), suggesting the involvement of proprotein convertase(s) (PCs). In vitro digestion of a 12-mer peptide encompassing the predicted cleavage site with furin, PACE4, PC5/6, and PC7, showed that furin cleaves the best, whereas PC7 is inactive on this peptide. Ex vivo studies in COS-1 cells, a cell line lacking PC5/6, revealed efficient processing of pro-BMP10 by endogenous PCs other than PC5/6. The lack of processing of overexpressed pro-BMP10 in the furin- and PACE4-deficient cell line, CHO-FD11, and in furin-deficient LoVo cells, was restored by stable (CHO-FD11/Fur cells) or transient (LoVo cells) expression of furin. Use of cell-permeable and cell surface inhibitors suggested that endogenous PCs process pro-BMP10 mostly intracellularly, but also at the cell surface. Ex vivo experiments in mouse primary hepatocytes (wild type, PC5/6 knock-out, and furin knock-out) corroborated the above findings that pro-BMP10 is a substrate for endogenous furin. Western blot analyses of heart right atria extracts from wild type and PACE4 knock-out adult mice showed no significant difference in the processing of pro-BMP10, implying no in vivo role of PACE4. Overall, our in vitro, ex vivo, and in vivo data suggest that furin is the major convertase responsible for the generation of BMP10.