Amino-acid sequence and glycan structures of cysteine proteases with proline specificity from ginger rhizome Zingiber officinale.

The ginger proteases (GP-I and GP-II), isolated from the ginger rhizome Zingiber officinale, have an unusual substrate specificity preference for cleaving peptides with a proline residue at the P2 position. The complete amino-acid sequence of GP-II, a glycoprotein containing 221 amino acids, and about 98% that of GP-I have been determined. Both proteases, which are 82% similar, have cysteine residues at positions 27 and histidines at position 161, corresponding to the essential cysteine-histidine diads found in the papain family of cysteine proteases, and six corresponding cysteine residues that form the three invariant disulfide linkages seen in this family of proteins. The sequence homology with other members (papain, bromelain, actinidin, protease omega, etc.) of this family is approximately 50%. GP-II has two predicted glycosylation sites at Asn99 and Asn156. Analyisis by electrospray and collision-induced dissociation MS showed that both sites were occupied by the glycans (Man)3(Xyl)1(Fuc)1(GlcNAc)2 and (Man)3(Xyl)1(Fuc)1(GlcNAc)3, in a ratio of approximately 7 : 1. Both glycans are xylose containing biantennary complex types that share the common core structural unit, Man1-->6(Man1-->3) (Xyl1-->2)Man1-->4GlcNAc1-->4(Fuc1-->3)GlcNAc for the major form, with an additional N-acetylglucosamine residue being linked, in the minor form, to one of the terminal mannose units of the core structure.     

Binding modes of a new epoxysuccinyl-peptide inhibitor of cysteine proteases. Where and how do cysteine proteases express their selectivity?

Papain from Carica papaya, an easily available cysteine protease, is the best-studied representative of this family of enzymes. The three dimensional structure of papain is very similar to that of other cysteine proteases of either plant (actinidin, caricain, papaya protease IV) or animal (cathepsins B, K, L, H) origin. As abnormalities in the activities of mammalian cysteine proteases accompany a variety of diseases, there has been a long-lasting interest in the development of potent and selective inhibitors for these enzymes. A covalent inhibitor of cysteine proteases, designed as a combination of epoxysuccinyl and peptide moieties, has been modeled in the catalytic pocket of papain. A number of its configurations have been generated and relaxed by constrained simulated annealing-molecular dynamics in water. A clear conformational variability of this inhibitor is discussed in the context of a conspicuous conformational diversity observed earlier in several solid-state structures of other complexes between cysteine proteases and covalent inhibitors. The catalytic pockets S2 and even more so S3, as defined by the pioneering studies on the papain-ZPACK, papain-E64c and papain-leupeptin complexes, appear elusive in view of the evident flexibility of the present inhibitor and in confrontation with the obvious conformational scatter seen in other examples. This predicts limited chances for the development of selective structure-based inhibitors of thiol proteases, designed to exploit the minute differences in the catalytic pockets of various members of this family. A simultaneous comparison of the three published proenzyme structures suggests the enzyme's prosegment binding loop-prosegment interface as a new potential target for selective inhibitors of papain-related thiol proteases.     

The 2.1 A structure of a cysteine protease with proline specificity from ginger rhizome, Zingiber officinale.

A cysteine protease from ginger rhizome (GP-II) cleaves peptides and proteins with proline at the P(2) position. The unusual specificity for proline makes GP-II an attractive tool for protein sequencing and identification of stably folded domains in proteins. The enzyme is a 221 amino acid glycoprotein possessing two N-linked oligosaccharide chains (8% glycosylated by weight) at Asn99 and Asn156. The availability of the sequence of these glycosyl chains afforded the opportunity to observe their structure and impact on protein conformation. The three-dimensional structure of GP-II has been determined by X-ray crystallography to a resolution of 2.1 A (overall R-factor = 0.214, free R = 0.248). The overall structure of GP-II is similar to that of the homologous cysteine proteases papain, actinidin, and glycyl endopeptidase, folding into two distinct domains of roughly equal size which are divided by a cleft. The observed N-linked glycosyl chains (half the total carbohydrate sequence) participate in both crystallographic and noncrystallographic contacts, tethering the proteins together via hydrogen bonds to the carbohydrate residues without intervening ordered water molecules. The putative S(2) binding pocket (the proline recognition site) was identified by superposition of the GP-II structure with structures of four previously determined papain-inhibitor complexes. The particular enzymic amino acids forming the S(2) pocket of GP-II (Trp, Met, and Ala) are similar to those found in the proline binding pockets of the unrelated enzymes alpha-lytic protease and cyclophilin. However, there is no conserved three-dimensional arrangement of these residues between the three enzymes (i.e., no proline binding motif). Thus, the particular amino acids found at S(2) are consistent with a binding pocket for a moiety with the steric characteristics and charge distribution of proline. Size exclusion is also a mechanism for selectivity compared to the S(2) binding pocket of papain. The S(2) binding pocket of GP-II greatly restricts the size of the side chain which could be bound because of the occurrence of a tryptophan in place of the corresponding tyrosine in papain. In light of the nature of the binding pocket, the specificity of GP-II for proline over other small nonpolar amino acids may be attributed to a direct effect of proline on the substrate peptide backbone conformation.     

Theoretical studies of binding modes of two covalent inhibitors of cysteine proteases

Physiological and pathological roles of cysteine proteases make them important targets for inhibitor development. Although highly potent inhibitors of this group of enzymes are known, their major drawback is a lack of sufficient specificity. Two cysteine protease covalent inhibitors, viz. (i) Z-RL-deoxo-V-peptide-epoxysuccinyl hybrid, and (ii) Z-RLVG-methyl-, have been developed and modeled in the catalytic pocket of papain, an archetypal thiol protease. A number of configurations have been generated and relaxed for each system using the AMBER force field. The catalytic pockets S3 and S4 appear rather elusive in view of the observed inhibitors' flexibility. This suggest rather limited chances for the development of selective structure-based inhibitors of thiol proteases, designed to exploit differences in the structure of catalytic pockets of various members of this family.     

Inhibition of cysteine proteases by a natural biflavone: behavioral evaluation of fukugetin as papain and cruzain inhibitor

Cruzain is the major cysteine protease of Trypanosoma cruzi, the infectious agent responsible for Chagas disease, and cruzain inhibitors display considerable antitrypanosomal activity. In the present work we elucidated crystallographic data of fukugetin, a biflavone isolated from Garcinia brasiliensis, and investigated the role of this molecule as cysteine protease inhibitor. The kinetic analyses demonstrated that fukugetin inhibited cruzain and papain by a slow reversible type inhibition with K_I of 1.1 and 13.4 µM, respectively. However, cruzain inhibition was about 12 times faster than papain inhibition. Lineweaver–Burk plots demonstrated partial competitive inhibition for cruzain and hyperbolic mixed-type inhibition for papain. Furthermore, the docking results showed that the biflavone binds to ring C′ in the S2 pocket and to ring C in the S3 pocket through hydrophobic interactions and hydrogen bonds. Finally, fukugetin also presented inhibitory activity on proteases of the T. cruzi extract, with IC₅₀ of 7 µM.     

Peptide ketobenzoxazole inhibitors bound to cathepsin K

Potent inhibitors of human cysteine proteases of the papain family have been made and assayed versus a number of relevant family members. We describe the synthesis of peptide alpha-ketoheterocyclic inhibitors that occupy binding subsites S1'-S3 of the cysteine protease substrate recognition cleft and that form a reversible covalent bond with the Cys 25 nucleophile. X-ray crystal structures of cathepsin K both unbound and complexed with inhibitors provide detailed information on protease/inhibitor interactions and suggestions for the design of tight-binding, selective molecules.     

Processing of the papain precursor. Purification of the zymogen and characterization of its mechanism of processing

The precursor of the cysteine protease papain has been expressed and secreted as propapain from insect cells infected with a recombinant baculovirus expressing a synthetic gene coding for prepropapain. This 39-kDa secreted propapain zymogen molecule is glycosylated and can be processed in vitro into an enzymatically active authentic papain molecule of 24.5 kDa (Vernet, T., Tessier, D.C., Richardson, C., Laliberté, F., Khouri, H. E., Bell, A. W., Storer, A. C., and Thomas, D. Y. (1990) J. Biol. Chem. 265, 16661-16666). Recombinant propapain was stabilized with Hg2+ and purified to homogeneity using affinity chromatography, gel filtration, and ion-exchange chromatographic procedures. The maximum rate of processing in vitro was achieved at approximately pH 4.0, at a temperature of 65 degrees C and under reducing conditions. Precursor processing is inhibited by a variety of reversible and irreversible cysteine protease inhibitors but not by specific inhibitors of serine, metallo or acid proteases. Replacement by site-directed mutagenesis of the active site cysteine with a serine at position 25 also prevents processing. The inhibitor 125I-N-(2S,3S)-3-trans-hydroxycarbonyloxiran-2-carbonyl-L-tyrosine benzyl ester covalently labeled the wild type papain precursor, but not the C25S mutant, indicating that the active site is accessible to the inhibitor and is in a native conformation within the precursor. Based on biochemical and kinetic analyses of the activation and processing of propapain we have shown that the papain precursor is capable of autoproteolytic cleavage (intramolecular). Once free papain is released processing can then occur in trans (intermolecular).     

Crystal structure of human cathepsin V

Cathepsin V is a lysosomal cysteine protease that is expressed in the thymus, testis and corneal epithelium. We have determined the 1.6 A resolution crystal structure of human cathepsin V associated with an irreversible vinyl sulfone inhibitor. The fold of this enzyme is similar to the fold adopted by other members of the papain superfamily of cysteine proteases. This study provides a framework for understanding the structural basis for cathepsin V's activity and will aid in the design of inhibitors of this enzyme. A comparison of cathepsin V's active site with the active sites of related proteases revealed a number of differences, especially in the S2 and S3 subsites, that could be exploited in identifying specific cathepsin V inhibitors or in identifying inhibitors of other cysteine proteases that would be selective against cathepsin V.     

A target within the target: probing cruzain's P1' site to define structural determinants for the Chagas' disease protease

BACKGROUND: Cysteine proteases of the papain superfamily are present in nearly all groups of eukaryotes and play vital roles in a wide range of biological processes and diseases, including antigen and hormone processing, bacterial infection, arthritis, osteoporosis, Alzheimer's disease and cancer-cell invasion. Because they are critical to the life-cycle progression of many pathogenic protozoa, they represent potential targets for selective inhibitors. Chagas' disease, the leading cause of death due to heart disease in Latin American countries, is transmitted by Trypanosoma cruzi. Cruzain is the major cysteine protease of T cruzi and has been the target of extensive structure-based drug design. RESULTS: High-resolution crystal structures of cruzain bound to a series of potent phenyl-containing vinyl-sulfone, sulfonate and sulfonamide inhibitors have been determined. The structures show a consistent mode of interaction for this family of inhibitors based on a covalent Michael addition formed at the enzyme's active-site cysteine, hydrophobic interactions in the S2 substrate-binding pocket and a strong constellation of hydrogen bonding in the S1' region. CONCLUSIONS: The series of vinyl-sulfone-based inhibitors examined in complex with cruzain was designed to probe recognition and binding potential of an aromatic-rich region of the enzyme. Analysis of the interactions formed shows that aromatic interactions play a less significant role, whereas the strength and importance of hydrogen bonding in the conformation adopted by the inhibitor upon binding to the enzyme was highlighted. A derivative of one inhibitor examined is currently under development as a therapeutic agent against Chagas' disease.     

Crystal structures of two engineered thiol trypsins

We have determined the three-dimensional structures of engineered rat trypsins which mimic the active sites of two classes of cysteine proteases. The catalytic serine was replaced with cysteine (S195C) to test the ability of sulfur to function as a nucleophile in a serine protease environment. This variant mimics the cysteine trypsin class of thiol proteases. An additional mutation of the active site aspartate to an asparagine (D102N) created the catalytic triad of the papain-type cysteine proteases. Rat trypsins S195C and D102N,S195C were solved to 2.5 and 2.0 A, respectively. The refined structures were analyzed to determine the structural basis for the 10(6)-fold loss of activity of trypsin S195C and the 10(8)-fold loss of activity of trypsin D102N,S195C, relative to rat trypsin. The active site thiols were found in a reduced state in contrast to the oxidized thiols found in previous thiol protease structures. These are the first reported structures of serine proteases with the catalytic centers of sulfhydryl proteases. Structure analysis revealed only subtle global changes in enzyme conformation. The substrate binding pocket is unaltered, and active site amino acid 102 forms hydrogen bonds to H57 and S214 as well as to the backbone amides of A56 and H57. In trypsin S195C, D102 is a hydrogen-bond acceptor for H57 which allows the other imidazole nitrogen to function as a base during catalysis. In trypsin D102N,S195C, the asparagine at position 102 is a hydrogen-bond donor to H57 which places a proton on the imidazole nitrogen proximal to the nucleophile. This tautomer of H57 is unable to act as a base in catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of human cathepsin S.

We have determined the 2.5 A structure (Rcryst = 20.5%, Rfree = 28.5%) of a complex between human cathepsin S and the potent, irreversible inhibitor 4-morpholinecarbonyl-Phe-hPhe-vinyl sulfone-phenyl. Noncrystallographic symmetry averaging and other density modification techniques were used to improve electron density maps which were nonoptimal due to systematically incomplete data. Methods that reduce the number of parameters were implemented for refinement. The refined structure shows cathepsin S to be similar to related cysteine proteases such as papain and cathepsins K and L. As expected, the covalently-bound inhibitor is attached to the enzyme at Cys 25, and enzyme binding subsites S3-S1' are occupied by the respective inhibitor substituents. A somewhat larger S2 pocket than what is found in similar enzymes is consistent with the broader specificity of cathepsin S at this site, while Lys 61 in the S3 site may offer opportunities for selective inhibition of this enzyme. The presence of Arg 137 in the S1' pocket, and proximal to Cys 25 may have implications not only for substrate specificity C-terminal to the scissile bond, but also for catalysis.     

Human rhinovirus 3C protease: a cysteine protease showing the trypsin(ogen)-like fold

Viral-encoded proteases cleave precursor polyprotein(s) leading to maturation of infectious virions. Strikingly, human rhinovirus 3C protease shows the trypsin(ogen)-like serine protease fold based on two topologically equivalent six-stranded β-barrels, but displays residue Cys147 as the active site nucleophile. By contrast, papain, which is representative of most cysteine proteases, does not display the trypsin(ogen)-like fold. Remarkably, in human rhinovirus 3C cysteine protease, the catalytic residues Cys147, His40 and Glu71 are positioned as Ser195, His57 and Asp102, respectively, building up the catalytic triad of serine proteases in the chymotrypsin–trypsin–elastase family. However, as compared to trypsin-like serine proteases and their zymogens, residue His40 and the oxyanion hole of human rhinovirus 3C cysteine protease, both key structural components of the active site, are located closer to the protein core. Human rhinovirus 3C cysteine protease cleaves preferentially GlnGly peptide bonds or, less commonly, the GlnSer, GlnAla, GluSer or GluGly pairs. Finally, human rhinovirus 3C cysteine protease and the 3CD cysteine protease–polymerase covalent complex bind the 5′ non-coding region of rhinovirus genomic RNA, an essential function for replication of the viral genome.     

Subsite specificity of trypanosomal cathepsin L-like cysteine proteases. Probing the S2 pocket with phenylalanine-derived amino acids.

The S2 subsite of mammalian cysteine proteinases of the papain family is essential for specificity. Among natural amino acids, all these enzymes prefer bulky hydrophobic residues such as phenylalanine at P2. This holds true for their trypanosomal counterparts: cruzain from Trypanosoma cruzi and congopain from T. congolense. A detailed analysis of the S2 specificity of parasitic proteases was performed to gain information that might be of interest for the design of more selective pseudopeptidyl inhibitors. Nonproteogenic phenylalanyl analogs (Xaa) have been introduced into position P2 of fluorogenic substrates dansyl-Xaa-Arg-Ala-Pro-Trp, and their kinetic constants (Km, kcat/Km) have been determined with congopain and cruzain, and related host cathepsins B and L. Trypanosomal cysteine proteases are poorly stereoselective towards D/L-Phe, the inversion of chirality modifying the efficiency of the reaction but not the Km. Congopain binds cyclohexylalanine better than aromatic Phe derivatives. Another characteristic feature of congopain compared to cruzain and cathepsins B and L was that it could accomodate a phenylglycyl residue (kcat/Km = 1300 mM-1.s-1), while lengthening of the side chain by a methylene group only slightly impaired the specificity constant towards trypanosomal cysteine proteases. Mono- and di-halogenation or nitration of Phe did not affect Km for cathepsin L-like enzymes, but the presence of constrained Phe derivatives prevented a correct fitting into the S2 subsite. A model of congopain has been built to study the fit of Phe analogs within the S2 pocket. Phe analogs adopted a positioning within the S2 pocket similar to that of the Tyr of the cruzain/Z-Tyr-Ala-fluoromethylketone complex. However, cyclohexylalanine has an energetically favorable chair-like conformation and can penetrate deeper into the subsite. Fitting of modeled Phe analogs were in good agreement with kinetic parameters. Furthermore, a linear relationship could be established with logP, supporting the suggestion that fitting into the S2 pocket of trypanosomal cysteine proteases depends on the hydrophobicity of Phe analogs.     

Modelling family 2 cystatins and their interaction with papain

Cystatins are extensively studied cysteine protease inhibitors, found in wide range of organisms with highly conserved structural folds. S-type of cystatins is well known for their abundance in saliva, high selectivity and poorer activity towards host cysteine proteases in comparison to their immediate ancestor cystatin C. Despite more than 90% sequence similarity, the members of this group show highly dissimilar binding affinity towards papain. Cystatin M/E is a potent inhibitor of legumain and papain like cysteine proteases and recognized for its involvement in skin barrier formation and potential role as a tumor suppressor gene. However, the structures of these proteins and their complexes with papain or legumain are still unknown. In the present study, we have employed computational methods to get insight into the interactions between papain and cystatins. Three-dimensional structures of the cystatins are generated by homology modelling, refined with molecular dynamics simulation, validated through numerous web servers and finally complexed with papain using ZDOCK algorithm in Discovery Studio. A high degree of shape complementarity is observed within the complexes, stabilized by numerous hydrogen bonds (HB) and hydrophobic interactions. Using interaction energy, HB and solvent accessible surface area analyses, we have identified a series of key residues that may be involved in papain–cystatin interaction. Differential approaches of cystatins towards papain are also noticed which are possibly responsible for diverse inhibitory activity within the group. These findings will improve our understanding of fundamental inhibitory mechanisms of cystatin and provide clues for further research.     

Quantifying cathepsin S activity in antigen presenting cells using a novel specific substrate

Cathepsin S (CatS) is a lysosomal cysteine protease belonging to the papain superfamily. Because of the relatively broad substrate specificity of this family, a specific substrate for CatS is not yet known. Based on a detailed study of the CatS endopeptidase specificity, using six series of internally quenched fluorescent peptides, we were able to design a specific substrate for CatS. The peptide series was based on the sequence GRWHTVGLRWE-Lys(Dnp)-DArg-NH2, which shows only one single cleavage site between Gly and Leu and where every substrate position between P-3 and P-3' was substituted with up to 15 different amino acids. The endopeptidase specificity of CatS was mainly determined by the P-2, P-1', and the P-3' substrate positions. Based on this result, systematically modified substrates were synthesized. Two of these modified substrates, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2 and Mca-GRWHPMGAPWE-Lys(Dnp)-DArg-NH2, did not react with the purified cysteine proteases cathepsin B (CatB) and cathepsin L (CatL). Using a specific CatS inhibitor, we could further show that these two peptides were not cleaved by endosomal fractions of antigen presenting cells (APCs), when CatS was inhibited and related cysteine proteases cathepsin B, H, L and X were still active. Although aspartic proteases like cathepsin E and cathepsin D were also present, our substrates were suitable to quantify cathepsin S activity specifically in APCs, including B cells, macrophages, and dendritic cells without the use of any protease inhibitor. We find that CatS activity differs significantly not only between the three types of professional APCs but also between endosomal and lysosomal compartments.     

Versatile Loops in Mycocypins Inhibit Three Protease Families

Mycocypins, clitocypins and macrocypins, are cysteine protease inhibitors isolated from the mushrooms Clitocybe nebularis and Macrolepiota procera. Lack of sequence homology to other families of protease inhibitors suggested that mycocypins inhibit their target cysteine protease by a unique mechanism and that a novel fold may be found. The crystal structures of the complex of clitocypin with the papain-like cysteine protease cathepsin V and of macrocypin and clitocypin alone have revealed yet another motif of binding to papain like-cysteine proteases, which in a yet unrevealed way occludes the catalytic residue. The binding is associated with a peptide-bond flip of glycine that occurs before or concurrently with the inhibitor docking. Mycocypins possess a β-trefoil fold, the hallmark of Kunitz-type inhibitors. It is a tree-like structure with two loops in the root region, a stem comprising a six-stranded β-barrel, and two layers of loops (6 + 3) in the crown region. The two loops that bind to cysteine cathepsins belong to the lower layer of the crown loops, whereas a single loop from the crown region can inhibit trypsin or asparaginyl endopeptidase, as demonstrated by site-directed mutagenesis. These loops present a versatile surface with the potential to bind to additional classes of proteases. When appropriately engineered, they could provide the basis for possible exploitation in crop protection.     

Novel secretory vesicle serpins,endopin 1 and endopin 2: endogenous protease inhibitors with distinct target protease specificities

Secretory vesicles of neuroendocrine cells possess multiple proteases for proteolytic processing of proteins into biologically active peptide components, such as peptide hormones and neurotransmitters. The importance of proteases within secretory vesicles predicts the presence of endogenous protease inhibitors in this subcellular compartment. Notably, serpins represent a diverse class of endogenous protease inhibitors that possess selective target protease specificities, defined by the reactive site loop domains (RSL). In the search for endogenous serpins in model secretory vesicles of neuroendocrine chromaffin cells, the presence of serpins related to alpha1-antichymotrypsin (ACT) was detected by Western blots with anti-ACT. Molecular cloning revealed the primary structures of two unique serpins, endopin 1 and endopin 2, that possess homology to ACT. Of particular interest was the observation that distinct RSL domains of these new serpins predicted that endopin 1 would inhibit trypsin-like serine proteases cleaving at basic residues, and endopin 2 would inhibit both elastase and papain that represent serine and cysteine proteases, respectively. Endopin 1 showed selective inhibition of trypsin, but did not inhibit chymotrypsin, elastase, or subtilisin. Endopin 2 demonstrated cross-class inhibition of the cysteine protease papain and the serine protease elastase. Endopin 2 did not inhibit chymotrypsin, trypsin, plasmin, thrombin, furin, or cathepsin B. Endopin 1 and endopin 2 each formed SDS-stable complexes with target proteases, a characteristic property of serpins. In neuroendocrine chromaffin cells from adrenal medulla, endopin 1 and endopin 2 were both localized to secretory vesicles. Moreover, the inhibitory activity of endopin 2 was optimized under reducing conditions, which required reduced Cys-374; this property is consistent with the presence of endogenous reducing agents in secretory vesicles in vivo. These new findings demonstrate the presence of unique secretory vesicle serpins, endopin 1 and endopin 2, which possess distinct target protease selectivities. Endopin 1 inhibits trypsin-like proteases; endopin 2 possesses cross-class inhibition for inhibition of papain-like cysteine proteases and elastase-like serine proteases. It will be of interest in future studies to define the endogenous protease targets of these two novel secretory vesicle serpins.     

Aza-peptide epoxides: potent and selective inhibitors of Schistosoma mansoni and pig kidney legumains (asparaginyl endopeptidases)

Aza-peptide epoxides are a new class of irreversible cysteine protease inhibitors. Derivatives containing a P1 aza-asparagine residue are specific for Schistosoma mansoni and pig kidney legumains, which are clan CD cysteine proteases. The inhibitors have second-order rate constants of up to 10(4) M(-1) s(-1) with pig kidney legumain and IC50 values as low as 45 nM with S. mansoni legumain. The most potent epoxides contain an ester moiety with S,S stereochemistry attached to the epoxide. Interestingly, amide and amino acid derivatives of the epoxysuccinate moiety were not inhibitors of legumain, while disubstituted amide derivatives are quite potent. The inhibitors have little or no inhibitory activity with other proteases such as caspases, chymotrypsin, papain, cathepsin B, granzyme B, and various aspartyl proteases.     

Evidence that serpin architecture intrinsically supports papain-like cysteine protease inhibition: engineering alpha(1)-antitrypsin to inhibit cathepsin proteases

The closely related serpins squamous cell carcinoma antigen-1 and -2 (SCCA-1 and -2, respectively) are capable of inhibiting cysteine proteases of the papain superfamily. To ascertain whether the ability to inhibit cysteine proteases is an intrinsic property of serpins in general, the reactive center loop (RCL) of the archetypal serine protease inhibitor alpha(1)-antitrypsin was replaced with that of SCCA-1. It was found that this simple substitution could convert alpha(1)-antitrypsin into a cysteine protease inhibitor, albeit an inefficient one. The RCL of SCCA-1 is three residues longer than that of alpha(1)-antitrypsin, and therefore, the effect of loop length on the cysteine protease inhibitory activity was investigated. Mutants in which the RCL was shortened by one, two, or three residues were effective inhibitors with second-order rate constants of 10(5)-10(7) M(-)(1) s(-)(1). In addition to loop length, the identity of the cysteine protease was of considerable importance, since the chimeric molecules inhibited cathepsins L, V, and K efficiently, but not papain or cathepsin B. By testing complexes between an RCL-mimicking peptide and the mutants, it was found that the formation of a stable serpin-cysteine protease complex and the inhibition of a cysteine protease were both critically dependent on RCL insertion. The results strongly indicate that the serpin body is intrinsically capable of supporting cysteine protease inhibition, and that the complex with a papain-like cysteine protease would be expected to be analogous to that seen with serine proteases.     

Specific reaction of alpha,beta-unsaturated carbonyl compounds such as 6-shogaol with sulfhydryl groups in tubulin leading to microtubule damage

6-Shogaol and 6-gingerol are ginger components with similar chemical structures. However, while 6-shogaol damages microtubules, 6-gingerol does not. We have investigated the molecular mechanism of 6-shogaol-induced microtubule damage and found that the action of 6-shogaol results from the structure of alpha,beta-unsaturated carbonyl compounds. alpha,beta-Unsaturated carbonyl compounds such as 6-shogaol react with sulfhydryl groups of cysteine residues in tubulin, and impair tubulin polymerization. The reaction with sulfhydryl groups depends on the chain length of alpha,beta-unsaturated carbonyl compounds. In addition, alpha,beta-unsaturated carbonyl compounds are more reactive with sulfhydryl groups in tubulin than in 2-mercaptoethanol, dithiothreitol, glutathione and papain, a cysteine protease.