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.     

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.     

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.     

Plasticity of extended subsites facilitates divergent substrate recognition by Kex2 and furin

Yeast Kex2 and human furin are subtilisin-related proprotein convertases that function in the late secretory pathway and exhibit similar though distinguishable patterns of substrate recognition. Although both enzymes prefer Arg at P(1) and basic residues at P(2), the two differ in recognition of P(4) and P(6) residues. To probe P(4) and P(6) recognition by Kex2p, furin-like substitutions were made in the putative S(4) and S(6) subsites of Kex2. T252D and Q283E mutations were introduced to increase the preference for Arg at P(4) and P(6), respectively. Glu(255) was replaced with Ile to limit recognition of P(4) Arg. The effects of putative S(4) and S(6) mutations were determined by examining the cleavage by purified mutant enzymes of a series of fluorogenic substrates with systematic changes in P(4) and/or P(6). Whereas wild Kex2 exhibited little preference type for Arg at P(6), the T252D mutant and T252D/Q283E double mutant exhibited clear interactions with P(6) Arg. Moreover, the T252D and T252D/Q283E substitutions altered the influence of the P(6) residue on P(4) recognition. We infer that cross-talk between S(4) and S(6), not seen in furin, allows wild type and mutant forms of Kex2 to adapt their subsites for altered modes of recognition. This apparent plasticity may allow the subsites to rearrange their local environment to interact with different substrates in a productive manner. E255I-Kex2 exhibited significantly decreased recognition of P(4) Arg in a tetrapeptide substrate with Lys at P(1), although the general pattern of selectivity for aliphatic residues at P(4) remained unchanged.     

Proteolytic cleavage of wild type and mutants of the F protein of human parainfluenza virus type 3 by two subtilisin-like endoproteases, furin and Kex2.

The fusion (F) protein of human parainfluenza virus type 3 contains the tribasic cleavage site R-T-K-R, which was altered by site-directed mutagenesis. Wild-type F protein and various mutants were expressed by recombinant vaccinia viruses. The endogenous endoprotease present in CV-1 cells cleaves F variants containing the furin recognition motif R-X-K/R-R but not variants containing the dibasic site K-R or a single R at the cleavage site. A similar cleavage pattern was obtained when the subtilisin-like endoproteases Kex2 and furin were coexpressed with the wild type and mutants of the F protein. Peptidylchloromethylketone inhibitors mimicking basic cleavage sites prevent cleavage of the precursor Fo by the endogenous protease only when the furin-specific motif is present in the peptidyl portion. The data support the concept that furin is a cellular protease responsible for the activation of the F protein of human parainfluenza virus type 3.     

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.     

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.     

Human fur gene encodes a yeast KEX2-like endoprotease that cleaves pro- beta-NGF in vivo

Extracts from BSC-40 cells infected with vaccinia recombinants expressing either the yeast KEX2 prohormone endoprotease or a human structural homologue (fur gene product) contained an elevated level of a membrane-associated endoproteolytic activity that could cleave at pairs of basic amino acids (-LysArg- and -ArgArg-). The fur-directed activity (furin) shared many properties with Kex2p including activity at pH 7.3 and a requirement for calcium. By using antifurin antibodies, immunoblot analysis detected two furin translation products (90 and 96 kD), while immunofluorescence indicated localization to the Golgi apparatus. Coexpression of either Kex2p or furin with the mouse beta- nerve growth factor precursor (pro-beta-NGF) resulted in greatly enhanced conversion of the precursor to mature nerve growth factor. Thus, the sequence homology shared by furin and the yeast KEX2 prohormone processing enzyme is reflected by significant functional homology both in vitro and in vivo.     

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].     

Furin inhibition by compounds of copper and zinc

Furin, a human subtilisin-related proprotein convertase (SPC), is emerging as an important pharmaceutical target because it processes vital proteins of many aggressive pathogens. Furin inhibitors reported as yet are peptide derivatives and proteins, with the exception of andrographolides, which are natural compounds. Here we report that the small and highly stable compounds M(chelate)Cl(2) (M is copper or zinc) inhibit furin and Kex2, with Cu(TTP)Cl(2) and Zn(TTP)Cl(2) as the most efficient inhibitors. (TTP is 4'-[p-tolyl]-2,2 ':6',2"-terpyridine.) Inhibition is irreversible, competitive with substrate, and affected by substituents on the chelate. The free chelates are not inhibitors. Solvated Zn(2+) is less potent than its complexes. This is true also for copper and Kex2. However, solvated Cu(2+) (k(on) of 25,000 +/- 2,500 s(-1)) is more potent than Cu(TTP)Cl(2) (k(on) = 140 +/- 13 s(-1) and allows recovery of furin activity prior to a second inhibition phase. A mechanism that involves coordination to the catalytic histidine is proposed for all inhibitors. Target specificity is indicated by the fact that these metal chelate inhibitors are much less potent toward Kex2, the yeast homologue of furin. For example, k(on) with Zn(TTP)Cl(2) is 120 +/- 20 s(-1) for furin, but only 1.2 +/- 0.1 s(-1) for Kex2.     

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.     

Purification and characterization of furin, a Kex2-like processing endoprotease, produced in Chinese hamster ovary cells.

Furin, a mammalian homolog of the yeast Kex2 protease, is associated with Golgi membranes and is involved in cleavage of precursor proteins at sites marked by the Arg-X-Lys/Arg-Arg (RXK/RR) motif. We have recently shown that a furin mutant lacking the transmembrane domain can be secreted from cDNA-transfected cells with proteolytic activity for the fluorogenic peptide t-butoxycarbonyl-Arg-Val-Arg-Arg-4-methylcoumarin-7- amide. In this study, we purified and characterized the recombinant furin from the conditioned medium of these cells. Furin was purified as a mixture of 83- and 81-kDa forms and a 96-kDa form. The differences in molecular mass were not due to differences in molecular mass were not due to differences in glycosylation. Moreover, all forms had the same NH2-terminal sequence beginning at the residue after the Arg-Ala-Lys-Arg sequence. These data suggest that the three different forms may be produced by differential COOH-terminal processing of a furin molecule and that mature furin may be autocatalytically produced. Both enzyme preparations showed a pH optimum at 7.0, required Ca2+ for the activity, and showed essentially the same inhibitor profile. These properties resembled those of the Kex2 protease. Both preparations efficiently cleaved fluorogenic peptides with an RXK/RR sequence and moderately cleaved a peptide with an RXXR sequence, but did not cleave dibasic peptides. The sequence requirements determined in vitro were compatible with those determined by expression studies in cultured cells. These data unequivocally demonstrate that furin is an endogenous cellular protease responsible for cleavage of precursor proteins mainly at RXK/RR sites.     

cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases

Based on the concept of sequence conservation around the active sites of serine proteinases, polymerase chain reaction applied to mRNA amplification allowed us to obtain a 260-bp probe which was used to screen a mouse pituitary cDNA library. The primers used derived from the cDNA sequence of active sites Ser* and Asn* of human furin. Two cDNA sequences were obtained from a number of positive clones. These code for two similar but distinct structures (mPC1 and mPC2), each being homologous to yeast Kex2 and human furin. In situ hybridization (mPC1) and Northern blots (mPC1 = 3.0 kb and mPC2 = 2.8 and 4.8 kb) demonstrated tissue and cellular specificity of expression, only within endocrine and neuroendocrine cells. These data suggest that mPC1 and mPC2 represent prime candidates for tissue-specific pro-hormone converting proteinases.     

Specific modulation of Kex2/furin family proteases by potassium

Kex2 protease is the prototype for a family of proteases responsible for endoproteolytic cleavage at multi-basic motifs in the eukaryotic secretory pathway. Here we demonstrate that potassium ion can act as a modulator of Kex2 activity with an apparent affinity of approximately 20 mm. Other monovalent cations (Li(+), Na(+), etc.) display similar effects, but affinities are all over 20-fold lower. Potassium ion binding stimulates turnover at physiologically relevant Lys-Arg cleavage sites but reduces turnover with at least one incorrect sequence. Furthermore, the mammalian Kex2 homolog furin displays similar effects. In contrast, the neuroendocrine homolog PC2 is inhibited by potassium ion with all substrates examined. The pre-steady-state behavior of Kex2 is also altered upon binding of potassium ion, with opposite effects on acylation and deacylation rates. These biochemical data indicate that potassium ion concentration may function as a regulator of processing protease specificity and activity in the eukaryotic secretory pathway, with such enzymes potentially encountering compartments high in potassium ion caused by the action of antiporters such as yeast NHX1 (VPS44) or the mammalian NHE7.     

Cloning and functional expression of a novel endoprotease involved in prohormone processing at dibasic sites.

We cloned and sequenced a cDNA from a library of mouse pituitary AtT-20 cells which are known to cleave an endogenous and various foreign prohormones at dibasic sites. This cDNA encodes a novel 753-residue protein, named PC3, which is structurally related to the yeast Kex2 protease involved in precursor cleavage at dibasic sites and to recently identified mammalian Kex2-like proteins, furin and PC2. Among examined cell lines and tissues, PC3 mRNA was only detected in AtT-20 cells. The substrate specificity of PC3 expressed in mammalian cells was similar to that observed in AtT-20 cells. We conclude that PC3 is a resident prohormone processing endoprotease in AtT-20 cells.     

Intracellular activation of human adamalysin 19/disintegrin and metalloproteinase 19 by furin occurs via one of the two consecutive recognition sites

Adamalysin 19 (a disintegrin and metalloproteinase 19, ADAM19, or meltrin beta) is a plasma membrane metalloproteinase. Human ADAM19 zymogen contains two potential furin recognition sites (RX(K/R)R), (196)KRPR(200)R and (199)RRMK(203)R, between its pro- and catalytic domains. Protein N-terminal sequencing revealed that the cellular mature forms of hADAM19 started at (204)EDLNSMK, demonstrating that the preferred furin cleavage site was the (200)RMK(203)R downward arrow(204)EDLN. Those mature forms were catalytically active. Both Pittsburgh mutant of alpha(1)-proteinase inhibitor and dec-Arg-Val-Lys-Arg-chloromethyl ketone, two specific furin inhibitors, blocked the activation of hADAM19. Activation of hADAM19 was also blocked by brefeldin A, which inhibits protein trafficking from the endoplasmic reticulum to the Golgi, or, a calcium ionophore known to inhibit the autoactivation of furin. When (202)KR were mutated to AA, the proenzyme was also activated, suggesting that (197)RPRR is an alternative activation site. Furthermore, only pro-forms of hADAM19 were detected in the (199)RR to AA mutant, which abolished both furin recognition sites. Moreover, the zymogens were not converted into their active forms in two furin-deficient mammalian cell lines; co-expression of hADAM19 and furin in these two cell lines restored zymogen activation. Finally, co-localization between furin and hADAM19 was identified in the endoplasmic reticulum-Golgi complex and/or the trans-Golgi network. This report is the first thorough investigation of the intracellular activation of adamalysin 19, demonstrating that furin activated pro-hADAM19 in the secretory pathway via one of the two consecutive furin recognition sites.     

Structure and expression of Furin mRNA in the ovary of the medaka, Oryzias latipes

A cDNA for furin was cloned from the ovary of the medaka, Oryzias latipes, by a combination of cDNA library screening, 5'-rapid amplification of cDNA ends (RACE), and 3'- RACE. The cDNA sequence codes for a protein of 814 amino acid residues highly homologous to other vertebrate furins, Ca(2+)-dependent serine proteases belonging to the subtilysin-like proprotein convertase family. The medaka preprofurin consists of a leader sequence, a propeptide with autoactivation sites, a Kex2-like catalytic domain, a P domain, a cysteine-rich domain, a putative transmembrane domain, and a cytoplasmic domain. The catalytic triad residues (Asp-164, His-205, and Ser-379) were all conserved. Furin mRNA was expressed in many tissues of this, including the ovary. In the ovary, the greatest expression of furin mRNA occurred in oocytes of small growing follicles, as demonstrated by Northern blotting, RT-PCR, and in situ hybridization analysis. Temporary and spatial expression patterns of the medaka fish furin were similar to those of stromelysin-3 and MT5-MMP during oocyte growth and postnatal development.     

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.     

Differential utilization of enzyme-substrate interactions for acylation but not deacylation during the catalytic cycle of Kex2 protease

Kex2 protease from Saccharomyces cerevisiae is the prototype for a family of eukaryotic proprotein processing proteases belonging to the subtilase superfamily of serine proteases. Kex2 can be distinguished from degradative subtilisins on the basis of stringent substrate specificity and distinct pre-steady-state behavior. To better understand these mechanistic differences, we have examined the effects of substrate residues at P(1) and P(4) on individual steps in the Kex2 catalytic cycle with a systematic series of isosteric peptidyl amide and ester substrates. The results demonstrate that substrates based on known, physiological cleavage sites exhibit high acylation rates (> or =550 s(-1)) with Kex2. Substitution of Lys for the physiologically correct Arg at P(1) resulted in a > or =200-fold drop in acylation rate with almost no apparent effect on binding or deacylation. In contrast, substitution of the physiologically incorrect Ala for Nle at P(4) resulted in a much smaller defect in acylation and a modest but significant effect on binding with Lys at P(1). This substitution also had no effect on deacylation. These results demonstrate that Kex2 utilizes enzyme-substrate interactions in different ways at different steps in the catalytic cycle, with the S(1)-P(1) contact providing a key specificity determinant at the acylation step.