2.4 A resolution crystal structure of the prototypical hormone-processing protease Kex2 in complex with an Ala-Lys-Arg boronic acid inhibitor

This paper reports the first structure of a member of the Kex2/furin family of eukaryotic pro-protein processing proteases, which cleave sites consisting of pairs or clusters of basic residues. Reported is the 2.4 A resolution crystal structure of the two-domain protein ssKex2 in complex with an Ac-Ala-Lys-boroArg inhibitor (R = 20.9%, R(free) = 24.5%). The Kex2 proteolytic domain is similar in its global fold to the subtilisin-like superfamily of degradative proteases. Analysis of the complex provides a structural basis for the extreme selectivity of this enzyme family that has evolved from a nonspecific subtilisin-like ancestor. The P-domain of ssKex2 has a novel jelly roll like fold consisting of nine beta strands and may potentially be involved, along with the buried Ca(2+) ion, in creating the highly determined binding site for P(1) arginine.     

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.     

Direct measurement of acylenzyme hydrolysis demonstrates rate-limiting deacylation in cleavage of physiological sequences by the processing protease Kex2

Saccharomyces cerevisiae Kex2 protease is the prototype for the family of eukaryotic proprotein convertases that includes furin, PC1/3, and PC2. These enzymes belong to the subtilase superfamily of serine proteases and are distinguished from degradative subtilisins by structural features and by their much more stringent substrate specificity. Pre-steady-state studies have shown that both Kex2 and furin exhibit an initial burst of 7-amino-4-methylcoumarin release in cleavage of peptidyl methylcoumarinamide substrates that are based on physiological cleavage sites. Thus, in cleavage of such substrates, formation of the acylenzyme intermediate is fast relative to some later step (deacylation or N-terminal product release). This behavior is significant, because Kex2 also exhibits burst kinetics in cleavage of peptide bonds. k(cat) for cleavage of a tetrapeptidyl methylcoumarinamide substrate based on the physiological yeast substrate pro-alpha-factor exhibits a weak solvent isotope effect, but neither this isotope effect nor temperature dependence studies with this substrate conclusively identify the rate-limiting step for Kex2 cleavage of this substrate. We therefore developed an assay to measure deacylation directly by pulse-chase incorporation of H(2)(18)O in a rapid-quenched-flow mixer followed by mass spectrometric quantitation. The results given by this assay rule out rate-limiting product release for cleavage of this substrate by Kex2. These experiments demonstrate that cleavage of the acylenzyme ester bond, as opposed to either the initial attack on the amide bond or product release, is rate-limiting for the action of Kex2 at physiological sequences. This work demonstrates a fundamental difference in the catalytic strategy of proprotein processing enzymes and degradative subtilisins.     

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.     

Interaction of EGLIN C variants with the extended subsites of the precursor processing proteases

Potent inhibitors of the Kex2/furin family precursor processing proteases were developed by randomizing adventitious contact sites and screening for optimized affinity using inhibition assays in 96-well format [1]. In this review, the binding interactions of the developed inhibitors will be examined in light of the three dimensional structures of Kex2 and furin [2-4].     

Secretion of mouse ZP3, the sperm receptor, requires cleavage of its polypeptide at a consensus furin cleavage-site

The mouse egg extracellular coat, or zona pellucida, consists of three glycoproteins, called mZP1-3. Each glycoprotein possesses a consensus sequence recognized by the furin family of proprotein convertases. Previously, it was reported that mZP2 and mZP3 are cleaved at their consensus furin cleavage-sites located near the C-terminus of the polypeptides [Litscher, E. S., Qi, H., and Wassarman, P. M. (1999) Biochemistry 38, 12280-12287]. Here, use of site-directed mutagenesis of the mZP3 gene and a specific inhibitor of furin-like enzymes revealed that secretion of nascent mZP3 from transfected cells is dependent on cleavage of mZP3 at its consensus furin cleavage-site. The dependence of secretion on cleavage represents a novel function for furin family enzymes.     

Molecular and enzymatic properties of furin, a Kex2-like endoprotease involved in precursor cleavage at Arg-X-Lys/Arg-Arg sites.

We have recently shown that furin, a mammalian homologue of the yeast precursor-processing endoprotease Kex2, is involved in precursor cleavage at sites marked by the Arg-X-Lys/Arg-Arg motif within the constitutive secretory pathway. In this study, we analyzed molecular and enzymatic properties of furin expressed in Chinese hamster ovary cells using gene transfer techniques. COOH-terminal truncation analyses indicate that the polypeptide region significantly conserved among the Kex2 family members is required for the endoprotease activity of furin, while the COOH-terminal unconserved region containing the Cys-rich domain and the transmembrane domain is dispensable. A mutant of furin truncated up to the transmembrane domain from the COOH-terminus was secreted into the culture medium as an active form. The sequence requirements for precursor cleavage of this truncated furin determined in vitro were similar to those of wild-type furin determined by expression studies in cultured cells. It had a strong resemblance to the Kex2 protease in the inhibitor profile and pH dependency. These observations support the notion that furin is the endogenous endoprotease involved in precursor cleavage at Arg-X-Lys/Arg-Arg sites.     

Ectodomain shedding of furin: kinetics and role of the cysteine-rich region

Furin, a member of the subtilisin-like pro-protein convertase family, is a type I membrane protein that undergoes ectodomain shedding. Metabolic labeling of cells stably expressing furin demonstrated that the shed form of furin is detected after 30 min. Moreover, sequence analysis revealed that specific residues of the cysteine-rich region of furin aligned with those of tumor necrosis factor receptor, which is also shed. Introduction within furin's cysteine-rich region of mutations that impair TNFR1 shedding also abolished furin shedding. Our results show that shedding of furin occurs rapidly and further suggest that specific cysteine residues may impart a conformation to the enzyme, thereby affecting its susceptibility to proteolysis.     

Maturation of HIV envelope glycoprotein precursors by cellular endoproteases

The entry of enveloped viruses into its host cells is a crucial step for the propagation of viral infection. The envelope glycoprotein complex controls viral tropism and promotes the membrane fusion process. The surface glycoproteins of enveloped viruses are synthesized as inactive precursors and sorted through the constitutive secretory pathway of the infected cells. To be infectious, most of the viruses require viral envelope glycoprotein maturation by host cell endoproteases. In spite of the strong variability of primary sequences observed within different viral envelope glycoproteins, the endoproteolytical cleavage occurs mainly in a highly conserved domain at the carboxy terminus of the basic consensus sequence (Arg-X-Lys/Arg-Arg downward arrow). The same consensus sequence is recognized by the kexin/subtilisin-like serine proteinases (so called convertases) in many cellular substrates such as prohormones, proprotein of receptors, plasma proteins, growth factors and bacterial toxins. Therefore, several groups of investigators have evaluated the implication of convertases in viral envelope glycoprotein cleavage. Using the vaccinia virus overexpression system, furin was first shown to mediate the proteolytic maturation of both human immunodeficiency virus (HIV-1) and influenza virus envelope glycoproteins. In vitro studies demonstrated that purified convertases directly and specifically cleave viral envelope glycoproteins. Although these studies suggested the participation of several enzymes belonging to the convertases family, recent data suggest that other protease families may also participate in the HIV envelope glycoprotein processing. Their role in the physiological maturation process is still hypothetical and the molecular mechanism of the cleavage is not well documented. Crystallization of the hemagglutinin precursor (HA0) of influenza virus allowed further understanding of the molecular interaction between viral precursors and the cellular endoproteases. Furthermore, relationships between differential pathogenicity of influenza strains and their susceptibility to cleavage are molecularly funded. Here we review the most recent data and recent insights demonstrating the crucial role played by this activation step in virus infectivity. We discuss the cellular endoproteases that are implicated in HIV gp160 endoproteolytical maturation into gp120 and gp41.     

Engineered eglin c variants inhibit yeast and human proprotein processing proteases, Kex2 and furin

We engineered eglin c, a potent subtilisin inhibitor, to create inhibitors for enzymes of the Kex2/furin family of proprotein processing proteases. A structural gene was synthesized that encoded "R(1)-eglin", having Arg at P(1) in the reactive site loop in place of Leu(45). Ten additional variants were created by cassette mutagenesis of R(1)-eglin. These polypeptides were expressed in Escherichia coli, purified to homogeneity, and their interactions with secreted, soluble Kex2 and furin were examined. R(1)-eglin itself was a modest inhibitor of Kex2, with a K(a) of approximately 10(7) M(-)(1). Substituting Arg (in R(4)R(1)-eglin) or Met (in M(4)R(1)-eglin) for Pro(42) at P(4) created potent Kex2 inhibitors exhibiting K(a) values of approximately 10(9) M(-)(1). R(4)R(1)-eglin inhibited furin with a K(a) of 4.0 x 10(8) M(-)(1). Introduction of Lys at P(1), in place of Arg in R(4)R(1)-eglin reduced affinity only approximately 3-fold for Kex2 but 15-fold for furin. The stabilities of enzyme-inhibitor complexes were characterized by association and dissociation rate constants and visualized by polyacrylamide gel electrophoresis. R(4)R(1)-eglin formed stable 1:1 complexes with both Kex2 and furin. However, substitution of Lys at P(2) in place of Thr(44) resulted in eglin variants that inhibited both Kex2 and furin but which were eventually cleaved (temporary inhibition). Surprisingly, R(6)R(4)R(1)-eglin, in which Arg was substituted for Gly(40) in R(4)R(1)-eglin, exhibited stable, high-affinity complex formation with Kex2 (K(a) of 3.5 x 10(9) M(-)(1)) but temporary inhibition of furin. This suggests that enzyme-specific interactions can alter the conformation of the reactive site loop, converting a permanent inhibitor into a substrate. Eglin variants offer possible avenues for affinity purification, crystallization, and regulation of proprotein processing proteases.     

Structural basis for differences in substrate selectivity in Kex2 and furin protein convertases

Kex2 is the yeast prototype of a large family of serine proteases that are highly specific for cleavage of their peptide substrates C-terminal to paired basic sites. This paper reports the 2.2 A resolution crystal structure of ssKex2 in complex with an Ac-Arg-Glu-Lys-Arg peptidyl boronic acid inhibitor (R = 19.7, R(free) = 23.4). By comparison of this structure with the structure of the mammalian homologue furin [Henrich, S., et al. (2003) Nat. Struct. Biol. 10, 520-526], we suggest a structural basis for the differences in substrate recognition at the P(2) and P(4) positions between Kex2 and furin and provide a structural rationale for the lack of P(6) recognition in Kex2. In addition, several monovalent cation binding sites are identified, and a mechanism of activation of Kex2 by potassium ion is proposed.     

Furin overexpression suppresses tumor growth and predicts a better postoperative disease-free survival in hepatocellular carcinoma

Furin is a member of the pro-protein convertase family. It processes several growth regulatory proteins into their active forms, which are critical to tumor progression, metastasis, and angiogenesis. Furin over-expression could occur in liver cancer and a previous study showed that over-expression of furin promoted HepG2 cell invasion in tail vein xenograft models. However, the clinical relevance of furin expression in hepatocellular carcinoma (HCC) remained unknown. Surprisingly, in a postoperative survival analysis for HCC patients, it was found that the tumor/non-tumor (T/N) ratio of furin expression ≥ 3.5 in HCC tissues predicted a better postoperative disease-free survival (DFS) (P = 0.010; log-rank test). Furthermore, subcutaneous xenograft experiments demonstrated a significant suppression effect of tumor growth in the furin-overexpressed xenografts (Huh7-Furin) compared to the mock control. Administration of a synthetic furin inhibitor for inhibition of the pro-protein convertase activity, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (decRVKR-CMK), to the Huh7-Furin xenograft bearing mice restored the repression effect of tumor growth. In contrast, administration of decRVKR-CMK to the mock Huh7 xenograft bearing mice showed no change in growth rate. In conclusion, furin overexpression inhibited HCC tumor growth in a subcutaneous xenograft model and predicted a better postoperative DFS in clinical analysis.     

Proteolytic cleavages of proalbumin and complement Pro-C3 in vitro by a truncated soluble form of furin, a mammalian homologue of the yeast Kex2 protease.

We have recently purified and characterized a truncated soluble form of furin from which the predicted transmembrane domain and cytoplasmic tail were deleted (Hatsuzawa, K., Nagahama, M., Takahashi, S., Takada, K., Murakami, K., and Nakayama, K. (1992) J. Biol. Chem. 267, 16094-16099). Our results showed that furin resembles the yeast Kex2 protease with respect to both its enzymic properties and substrate specificity. Here we demonstrate that the soluble form of furin is capable of converting the precursors of albumin and the third component of complement (proalbumin and pro-C3, respectively) in vitro to mature proteins. Thus furin mimics the Ca(2+)-dependent proalbumin and pro-C3 convertases found in the Golgi membranes (Brennan, S. O., and Peach, R. J. (1988) FEBS Lett. 229, 167-170; Oda, K. (1992) J. Biol. Chem. 267, 17465-17471). Furthermore we show that the variant alpha 1-antitrypsin Pittsburgh, which is a specific inhibitor of the Golgi proalbumin convertase, inhibits not only the Golgi pro-C3 convertase, but also the soluble furin. These results suggest a role for furin in the cleavage of proproteins transported via the constitutive pathway.     

Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing protease Kex2

We have identified a human insulinoma cDNA (PC2) that encodes a protein homologous to the precursor processing Kex2 endoprotease of yeast by using a polymerase chain reaction to detect and amplify conserved sequences within the catalytic site. The 638-residue amino acid sequence of PC2 begins with a cleavable signal peptide, indicating that it enters the secretory pathway, and contains a 282-residue domain that is homologous to the catalytic modules of both Kex2 and the related bacterial subtilisins. Within this region 49 and 27% of the amino acids are identical to those in the aligned Kex2 and subtilisin BPN' sequences, respectively, and the catalytically essential Asp, His, and Ser residues are all conserved. Northern blot analysis revealed the presence of 2.8- and 5.0-kilobase hybridizing bands in mRNA from the insulinoma. The PC2 protein also shows great similarity to the incomplete NH2-terminal sequence of the human furin gene product, a putative membrane-inserted receptor-like molecule. We propose that PC2 is a member of a family of mammalian Kex2/subtilisin-like proteases that includes members involved in a number of specific proteolytic events within cells, including the processing of prohormones.     

Role of host cellular proteases in the pathogenesis of influenza and influenza-induced multiple organ failure

Influenza A virus (IAV) is one of the most common infectious pathogens in humans. Since the IVA genome does not have the processing protease for the viral hemagglutinin (HA) envelope glycoprotein precursors, entry of this virus into cells and infectious organ tropism of IAV are primarily determined by host cellular trypsin-type HA processing proteases. Several secretion-type HA processing proteases for seasonal IAV in the airway, and ubiquitously expressed furin and pro-protein convertases for highly pathogenic avian influenza (HPAI) virus, have been reported. Recently, other HA-processing proteases for seasonal IAV and HPAI have been identified in the membrane fraction. These proteases proteolytically activate viral multiplication at the time of viral entry and budding. In addition to the role of host cellular proteases in IAV pathogenicity, IAV infection results in marked upregulation of cellular trypsins and matrix metalloproteinase-9 in various organs and cells, particularly endothelial cells, through induced pro-inflammatory cytokines. These host cellular factors interact with each other as the influenza virus-cytokine-protease cycle, which is the major mechanism that induces vascular hyperpermeability and multiorgan failure in severe influenza. This mini-review discusses the roles of cellular proteases in the pathogenesis of IAV and highlights the molecular mechanisms of upregulation of trypsins as effective targets for the control of IAV infection. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

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.     

Proteolytic cleavage of the ectodomain of the L1 CAM family member Tractin

Tractin is a member of the L1 family of cell adhesion molecules in leech. Immunoblot analysis suggests that Tractin is constitutively cleaved in vivo at a proteolytic site with the sequence RKRRSR. This sequence conforms to the consensus sequence for cleavage by members of the furin family of convertases, and this proteolytic site is shared by a majority of other L1 family members. We provide evidence with furin-specific inhibitor experiments, by site-specific mutagenesis of Tractin constructs expressed in S2 cells, as well as by Tractin expression in furin-deficient LoVo cells that a furin convertase is the likely protease mediating this processing. Cross-immunoprecipitations with Tractin domain-specific antibodies suggest that the resulting NH(2)- and COOH-terminal cleavage fragments interact with each other and that this interaction provides a means for the NH(2)-terminal fragment to be tethered to the membrane. Furthermore, in S2 cell aggregation assays we show that the NH(2)-terminal fragment is necessary for homophilic adhesion and that cells expressing only the transmembrane COOH-terminal fragment are non-adhesive. However, tethering of exogeneously provided Tractin NH(2)-terminal fragment to S2 cells expressing only the COOH-terminal fragment can functionally restore the adhesive properties of Tractin.     

Beta-amyloid protein converting enzyme 1 and brain-specific type II membrane protein BRI3: binding partners processed by furin

Using a yeast two-hybrid system, we screened a human brain cDNA library for possible interacting proteins with the C-terminal cytosolic tail of the beta-secretase beta-amyloid protein converting enzyme (BACE)1. This identified seven potential candidates, including the brain-specific type II membrane protein BRI3. Co-localization and co-immunoprecipitation experiments confirmed that BACE1 and BRI3 co-localize and interact with each other via the cytosolic tail of BACE1. Furthermore, pulse and pulse-chase analyses revealed that the pro-protein convertases furin, and to a lesser extent PC7 and PC5A, process BRI3 into a C-terminal secreted approximately 4-kDa product. Thus, furin efficiently processes both pro-BACE1 and its novel interacting protein pro-BRI3.