Mapping surface sequences of the tubulin dimer and taxol-induced microtubules with limited proteolysis

Native tubulin alpha beta dimers and microtubules have been subjected to limited proteolysis with trypsin, chymotrypsin, elastase, clostripain, proteinase lysine-C, thermolysin, protease V8, papain, subtilisin, proteinase K, proteinase aspartic-N, and bromelain. Eighty nicking points have been mapped onto the alpha- and beta-tubulin sequences with the aid of site-directed antibodies, of which 18 sites have been exactly determined by N-terminal sequencing, and the probable position of 6 others deduced from protease specificities. Proteolytic sites cluster into five characteristic zones, including the C termini of both chains. Residues accessible to proteases in the tubulin dimer include alpha-tubulin Lys40-Thr41-Ile42, Glu168-Phe169-Ser170, Ser178-Thr179-Ala180-Val181, Lys280-Ala281, Glu290-Ile291, Ala294-Cys295, Arg339-Ser340 (plus probably Lys60-His61 and Glu183-Pro184) and beta-tubulin Gly93-Gln94, Lys174-Val175, Gly277-Ser278, Tyr281-Arg282-Ala283, Cys354-Asp355 (plus probably Arg121-Lys122, Phe167-Ser168, Tyr183-Asn184, and Glu426-Asp427 or Ala430-Asp431). While the majority of these sites remain accessible at the outer surface of taxol-induced microtubules, alpha-tubulin Lys280-Ala281, Arg339-Ser340 and beta-tubulin Tyr281-Arg282-Ala283 (and probably Arg121-Lys122) become protected from limited proteolysis, suggesting that they are close to or at intermolecular contacts in the assembled structure. The protease nicking points constitute sets of surface constraints for any three-dimensional model structures of tubulin and microtubules. The dimer tryptic site at alpha-tubulin 339-340 jumps approximately 12-22 residues upstream (probably to Lys326-Asp327 or Lys311-Tyr312) in taxol microtubules, suggesting a tertiary structural change. The cleavage of the approximately 10 C-terminal residues of alpha-tubulin by protease V8, papain, and subtilisin is inhibited in taxol microtubules compared to tubulin dimers, while the approximately 20 C-terminal residues of beta-tubulin are similarly accessible to protease V8, subtilisin, proteinase K, proteinase AspN, and bromelain and show enhanced papain cleavage. This is consistent with models in which the alpha-tubulin C-terminal zone is near the interdimer contact zone along the protofilaments, whereas the C terminus of beta is near the interface between both subunits.     

Cross-class inhibition of the cysteine proteinases cathepsins K, L, and S by the serpin squamous cell carcinoma antigen 1: a kinetic analysis

The human squamous cell carcinoma antigens (SCCA) 1 and 2 are tandemly arrayed genes that encode two high-molecular-weight serine proteinase inhibitors (serpins). Although these proteins are 92% identical, differences in their reactive site loops suggest that they inhibit different types of proteinases. Our previous studies show that SCCA2 inhibits chymotrypsin-like serine proteinases [Schick et al. (1997) J. Biol. Chem. 272, 1849-1855]. We now show that, unlike SCCA2, SCCA1 lacks inhibitory activity against any of the more common types of serine proteinases but is a potent cross-class inhibitor of the archetypal lysosomal cysteine proteinases cathepsins K, L, and S. Kinetic analysis revealed that SCCA1 interacted with cathepsins K, L, and S at 1:1 stoichiometry and with second-order rate constants >/= 1 x 10(5) M-1 s-1. These rate constants were comparable to those obtained with the prototypical physiological cysteine proteinase inhibitor, cystatin C. Also relative to cystatin C, SCCA1 was a more potent inhibitor of cathepsin K-mediated elastolytic activity by forming longer lived inhibitor-proteinase complexes. The t1/2 of SCCA1-cathepsin S complexes was >1155 min, whereas that of cystatin C-cathepsin complexes was 55 min. Cleavage between the Gly and Ser residues of the reactive site loop and detection of a stable SCCA1-cathepsin S complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the serpin interacted with the cysteine proteinase in a manner similar to that observed for typical serpin-serine proteinase interactions. These data suggest that, contingent upon their reactive site loop sequences, mammalian serpins, in general, utilize their dynamic tertiary structure to trap proteinases from more than one mechanistic class and that SCCA1, in particular, may be involved in a novel inhibitory pathway aimed at regulating a powerful array of lysosomal cysteine proteinases.     

Azapeptides as inhibitors and active site titrants for cysteine proteinases

Ester and amide derivatives of alpha-azaglycine (carbazic acid, H2NNHCOOH), alpha-azaalanine, and alpha-azaphenylalanine (i.e., Ac-l-Phe-NHN(R)CO-X, where X = H, CH3, or CH2Ph, respectively) were synthesized and evaluated as inhibitors of the cysteine proteinases papain and cathepsin B. The ester derivatives inactivated papain and cathepsin B at rates which increased dramatically with leaving group hydrophobicity and electronegativity. For example, with 8 (R = H, X = OPh) the apparent second-order rate constant for papain inactivation was 67 600 M-1 s-1. Amide and P1-thioamide derivatives do not inactivate papain, nor are they substrates; instead they are weak competitive inhibitors (0.2 mM < Ki < 4 mM). Inactivation of papain involves carbamoylation of the enzyme, as demonstrated by electrospray mass spectrometry. Active site titration indicated a 1:1 stoichiometry for the inactivation of papain with 8, and both inactivated papain and cathepsin B are highly resistant to reactivation by dialysis (t1/2 > 24 h at 4 degrees C). Azaalanine derivatives Ac-L-Phe-NHN(CH3)CO-X inactivate papain ca. 400- 900-fold more slowly than their azaglycine analogues, consistent with the planar configuration at Nalpha of the P1 residue and the very substantial stereoselectivity of papain for L- vs D- residues at the P1 position of its substrates. Azaglycine derivative 9 (R = H, X = OC6H4NO2-p) inactivates papain extremely rapidly (>70 000 M-1 s-1), but it also decomposes rapidly in buffer with release of nitrophenol (kobs = 0.13 min-1); under the same conditions 8 shows <7% hydrolysis over 24 h. This nitrophenol release probably involves cyclization to an oxadiazolone since 17 (R = CH3, X = OC6H4NO2-p), which cannot form an isocyanate, releases nitrophenol almost as rapidly (kobs = 0.028 min-1). Cathepsin C, another cysteine proteinase with a rather different substrate specificity (i.e., aminopeptidase), was not inactivated by 8, indicating that the inactivation of papain and cathepsin B by azapeptide esters is a specific process. Their ease of synthesis coupled with good solution stability suggests that azapeptide esters may be useful as active site titrants of cysteine proteinases and probes of their biological function in vivo.     

Difference between human and guinea pig Hageman factors in activation by bacterial proteinases: cleavage site shift due to local amino acid substitutions may determine the activation efficiency of serine proteinase zymogens

Human and guinea pig Hageman factors have been subjected to the action of pseudomonal elastase and serratial E15 proteinase. The pseudomonal elastase cleaved 22-24% of the human molecule at Arg353-Val354, and the remainder at Gly357-Leu358 resulting in the generation of about 20% of potential activity as activated Hageman factor, compared with trypsin activation, while it hydrolyzed Arg340-Ile341 bond in guinea pig molecule and generated about 75% of activity as activated Hageman factor. The serratial proteinase did not hydrolyze the essential cleavage site (Arg353-Val354) of the human zymogen but Gly356-Gly357 (30%) and Gly357-Leu358 (70%) bonds. Both products showed no activity. The guinea pig zymogen, in contrast, was cleaved mostly at Arg340-Ile341 (70%) and less abundantly at Gly344-Leu345 (30%), generating about 85% of the whole potential activity as activated Hageman factor. From the high correspondence between the proportions of activation and of hydrolysis at the essential cleavage site in activation, it was concluded that hydrolysis of the bonds different from the essential bond did not cause activation, even when the spatial separation was only 3 or 4 residues. Considering the amino acid differences between human and guinea pig Hageman factors, -Met351-Thr-Arg-Val-Val-Gly-Gly-Leu-Val-Ala360- and -Leu338-Ser-Arg-Ile-Val-Gly-Gly-Leu-Val-Ala347-, respectively, it was realized that even the minor amino acid substitutions caused the cleavage site shift which resulted in significant differences in activation efficiency of the proteinase zymogens.     

Solution structure of bromelain inhibitor IV from pineapple stem: structural similarity with Bowman-Birk trypsin/chymotrypsin inhibitor from soybean

Bromelain inhibitor VI from pineapple stem (BI-VI) is a unique double-chain inhibitor with an 11-residue light chain and a 41-residue heavy chain by disulfide bonds and inhibits the cysteine proteinase bromelain competitively. The structure of BI-VI in aqueous solution was determined using nuclear magnetic resonance spectroscopy and simulated annealing-based calculations. Its three-dimensional structure was shown to be composed of two distinct domains, each of which is formed by a three-stranded antiparallel beta-sheet. Unexpectedly, BI-VI was found to share a similar folding and disulfide bond connectivities not with cystatin superfamily inhibitors which inhibit the same cysteine proteinases but with the Bowman-Birk trypsin/chymotrypsin inhibitor from soybean (BBI-I). BBI-I is a 71-residue inhibitor which has two independent inhibitory sites toward the serine proteinases trypsin and chymotrypsin. These structural similarities with BBI-I suggest that they have evolved from a common ancestor and differentiated in function during a course of molecular evolution.     

The kinetic mechanism of serpin-proteinase complex formation. An intermediate between the michaelis complex and the inhibited complex

Serine proteinase inhibitors (serpins) form enzymatically inactive, 1:1 complexes (denoted E*I*) with their target proteinases that release free enzyme and cleaved inhibitor only very slowly. The mechanism of E*I* formation is incompletely understood and continues to be a source of controversy. Kinetic evidence exists that formation of E*I* proceeds via a Michaelis complex (E.I) and so involves at least two steps. In this paper, we determine the rate of E*I* formation from alpha-chymotrypsin and alpha1-antichymotrypsin using two approaches: first, by stopped-flow spectrofluorometric monitoring of the fluorescent change resulting from reaction of alpha-chymotrypsin with a fluorescent derivative of alpha1-antichymotrypsin (derivatized at position P7 of the reactive center loop); and second, by a rapid mixing/quench approach and SDS-polyacrylamide gel electrophoresis analysis. In some cases, serpins are both substrates and inhibitors of the same enzyme. Our results indicate the presence of an intermediate between E.I and E*I* and suggest that the partitioning step between inhibitor and substrate pathways precedes P1-P1' cleavage.     

Influence of cardiopulmonary bypass surgery on cancer-specific survival rate of patients with colorectal cancer

We investigated whether cystatins and cystatin-derived peptides, encompassing sequences of secondary structures of cystatin S and papain binding domains of cystatin C, display antimicrobial properties. Of the different microorganisms tested, only the growth of P. gingivalis was inhibited by chicken cystatin and cystatin C. Cystatin S, cystatin S:1-14, cystatin S:61-73 and cystatin S:108-121 also inhibited its growth, whereas cystatin S:21-38, cystatin S:39-55, cystatin S:81-95, cystatin S:94-109, and cystatin C: 9-12/55-60/106-107 did not. No inhibition of the cysteine proteinase activity of P. gingivalis was observed for all cystatin-derived peptides. On the other hand, leupeptin and antipain inhibited P. gingivalis proteinase activity, but had no effect on the growth. These data suggest that cystatins contain antibacterial sequences active against P. gingivalis and that the growth inhibition does not depend on the inhibition of P. gingivalis cysteine proteinases.     

Characterization of proteolytic activities of rumen bacterial isolates from forage-fed cattle

The proteolytic activities of eight strains of ruminal bacteria isolated from New Zealand cattle were characterized with respect to their cellular location, response to proteinase inhibitors and hydrolysis of artificial proteinase substrates. The Streptococcus bovis strains had predominantly cell-bound activity, which included a mixture of serine and cysteine-type proteinases which had high activity against leucine p-nitroanilide (LPNA). The Eubacterium strains had a mainly cell-associated activity with serine and metallo-type proteinases which showed high activity against the chymotrypsin substrate, N-succinyl alanine alanine phenylalanine proline p-nitroanilide (NSAAPPPNA) and some LPNA activity. A Butyrivibrio strain, C211, had a cell-bound mixture of cysteine and metallo-proteinase activities and strongly hydrolysed NSAAPPPNA and LPNA while the high activity Butyrivibrio-like strain, B316, had a cell-bound, mainly serine proteinase activity which strongly hydrolysed NSAAPPPNA. A Prevotella-like strain, C21a, had a mixture of cysteine, serine and metallo-proteinase activities which were cell-bound and hydrolysed LPNA. The activities of these strains did not match those of the bacterial fraction of rumen fluid, which contained activities mainly of the cysteine type with specificity towards the substrate N-succinyl phenylalanine p-nitroanilide. The contribution of these strains to proteolysis in the rumen is discussed.     

The reactive site loop of the serpin SCCA1 is essential for cysteine proteinase inhibition

The high-molecular-weight serine proteinase inhibitors (serpins) are restricted, generally, to inhibiting proteinases of the serine mechanistic class. However, the viral serpin, cytokine response modifier A, and the human serpins, antichymotrypsin and squamous cell carcinoma antigen 1 (SCCA1), inhibit different members of the cysteine proteinase class. Although serpins employ a mobile reactive site loop (RSL) to bait and trap their target serine proteinases, the mechanism by which they inactivate cysteine proteinases is unknown. Our previous studies suggest that SCCA1 inhibits papain-like cysteine proteinases in a manner similar to that observed for serpin-serine proteinase interactions. However, we could not preclude the possibility of an inhibitory mechanism that did not require the serpin RSL. To test this possibility, we employed site-directed mutagenesis to alter the different residues within the RSL. Mutations to either the hinge or the variable region of the RSL abolished inhibitory activity. Moreover, RSL swaps between SCCA1 and the nearly identical serpin, SCCA2 (an inhibitor of chymotrypsin-like serine proteinases), reversed their target specificities. Thus, there were no unique motifs within the framework of SCCA1 that independently accounted for cysteine proteinase inhibitory activity. Collectively, these data suggested that the sequence and mobility of the RSL of SCCA1 are essential for cysteine proteinase inhibition and that serpins are likely to utilize a common RSL-dependent mechanism to inhibit both serine and cysteine proteinases.     

An ester bond linking a fragment of a serine proteinase to its serpin inhibitor

Most known members of the serpin superfamily are serine proteinase inhibitors. Serpins are therefore important regulators of blood coagulation, complement activation, fibrinolysis, and turnover of extracellular matrix. Serpins form SDS-resistant complexes of 1:1 stoichiometry with their target proteinases by reaction of their P1-P1' peptide bond with the active site of the proteinases. The nature of the interactions responsible for the high stability of the complexes is a controversial issue. We subjected the complex between the serine proteinase urokinase-type plasminogen activator (uPA) and the serpin plasminogen activator inhibitor-1 (PAI-1) to proteolytic digestion under nondenaturing conditions. The complex could be degraded to a fragment containing two disulfide-linked peptides from uPA, one of which included the active site Ser, while PAI-1 was left undegraded. By further proteolytic digestion after denaturation and reduction, it was also possible to degrade the PAI-1 moiety, and we isolated a fragment containing 10 amino acids from uPA, encompassing the active site Ser, and 6 amino acids from PAI-1, including the P1 Arg. Characterization of the fragment gave results fully in agreement with the hypothesis that it contained an ester bond between the hydroxyl group of the active site Ser and the carboxyl group of the P1 Arg. These data for the first time provide direct evidence that serine proteinases are entrapped at an acyl intermediate stage in serine proteinase-serpin complexes.     

Lysine- and arginine-specific proteinases from Porphyromonas gingivalis. Isolation, characterization, and evidence for the existence of complexes with hemagglutinins

Porphyromonas gingivalis contains many virulence factors that have been implicated as participants in the progression of periodontal disease. It has been shown to produce proteinases of "trypsin-like" specificity in a number of molecular forms, but previous work in our laboratory resulted in the purification of a major arginine-specific cysteine proteinase, gingipain, which contradicted this supposed specificity. In this study, separate proteinases with arginine and lysine specificity were isolated from a high molecular mass fraction of the P. gingivalis culture fluid. The arginine-specific enzyme was found, by amino acid sequencing studies, to be a high molecular mass form of gingipain, formed by the 50-kDa gingipain noncovalently complexed with 44-kDa binding proteins, subsequently identified as hemagglutinins. The 60-kDa lysine-specific proteinase, referred to as Lys-gingipain, was also found to have one of these hemagglutinins complexed with it in the same manner. Lys-gingipain was found to be a cysteine proteinase with optimal activity and stability at pH 8.0-8.5 and was extensively characterized in terms of its specificity and activation characteristics. The proteinase-hemagglutinin complexes may be important in the uptake of hemin, a vital metabolite for P. gingivalis, via hemagglutination and subsequent hemolysis of erythrocytes.     

Cathepsin L2, a novel human cysteine proteinase produced by breast and colorectal carcinomas

We have identified and cloned a new member of the papain family of cysteine proteinases from a human brain cDNA library. The isolated cDNA codes for a polypeptide of 334 amino acids that exhibits all of the structural features characteristic of cysteine proteinases, including the active site cysteine residue essential for peptide hydrolysis. Pairwise comparisons of this amino acid sequence with the remaining human cysteine proteinases identified to date showed a high percentage of identity (78%) with cathepsin L; the percentage of identity with all other members of the family was much lower (<40%). On the basis of these structural characteristics, we have tentatively called this novel protein cathepsin L2. The cDNA encoding the mature cathepsin L2 was expressed in Escherichia coli, and after purification, the recombinant protein was able to degrade the synthetic peptide benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin, which is commonly used as a substrate for cysteine proteinases. Cathepsin L2 proteolytic activity on this substrate was abolished by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cysteine proteinases, thus providing additional evidence that the isolated cDNA encodes a functional cysteine proteinase. Northern blot analysis of polyadenylated RNAs isolated from a variety of human tissues demonstrated that cathepsin L2 is predominantly expressed in the thymus and testis. This finding is in marked contrast with the wide tissue distribution of most cysteine proteinases characterized to date, including cathepsin L, and suggests that cathepsin L2 may play a specialized role in the thymus and testis. Expression analysis of cathepsin L2 in human tumors revealed a widespread expression in colorectal and breast carcinomas but not in normal colon or mammary gland or in peritumoral tissues. Cathepsin L2 was also expressed by colorectal and breast cancer cell lines as well as by some tumors of diverse origin, including ovarian and renal carcinomas. These results open the possibility that this novel enzyme may be involved in tumor processes, as already reported for other cysteine proteinases, including cathepsin L.     

Expression and characterization of recombinant Manduca sexta serpin-1B and site-directed mutants that change its inhibitory selectivity

Hemolymph of Manduca sexta contains a number of serine proteinase inhibitors from the serpin superfamily. During formation of a stable complex between a serpin and a serine proteinase, the enzyme cleaves a specific peptide bond in an exposed loop (the reactive-site region) at the surface of the serpin. The amino acid residue on the amino-terminal side of this scissile bond, the P1 residue, is important in defining the selectivity of a serpin for inhibiting different types of serine proteinases. M. sexta serpin-1B, with alanine at the position predicted from sequence alignments to be the P1 residue, was previously named alaserpin. This alanyl residue was changed by site-directed mutagenesis to lysine (A343K) and phenylalanine (A343F). The serpin-1B cDNA and its mutants were inserted into an expression vector, H6pQE-60, and the serpin proteins were expressed in Escherichia coli. Affinity-purified recombinant serpins selectively inhibited mammalian serine proteinases: serpin-1B inhibited elastase; serpin-1B(A343K) inhibited trypsin, plasmin, and thrombin; serpin-1B(A343F) inhibited chymotrypsin as well as trypsin. All three serpins inhibited human cathepsin G. This insect serpin and its site-directed mutants associated with mammalian serine proteinases at rates similar to those reported for mammalian serpins. Serpin-1B and its mutants formed SDS-stable complexes with the enzymes they inhibited. The scissile bond was determined to be between residues 343 and 344 in wild-type serpin-1B and in serpin-1B with mutations at residue 343. These results demonstrate that the P1 alanine residue defines the primary selectivity of serpin-1B for elastase-like enzymes, and that this selectivity can be altered by mutations at this position.     

Antichymotrypsin interaction with chymotrypsin. Reactions following encounter complex formation

Serpins, serine proteinase inhibitors, form enzymatically inactive, 1:1 complexes (denoted E*I*) with their target proteinases, that only slowly release I*, in which the P1-P1' linkage is cleaved. Recently we presented evidence that the serpin antichymotrypsin (ACT, I) reacts with the serine proteinase chymotrypsin (Chtr, E) to form an E*I* complex via a three-step mechanism, E + I <==> E .I <==> EI' <==> E*I* in which EI', which retains the P1-P1' linkage, is formed in a partly or largely rate-determining step, depending on temperature (O'Malley, K. H, Nair, S. A., Rubin, H., and Cooperman, B. S. (1997) J. Biol. Chem. 272, 5354-5359). Here we extend these studies through the introduction of a new assay for the formation of the postcomplex fragment, corresponding to ACT residues 359 (the P1' residue) to 398 (the C terminus), coupled with rapid quench flow kinetic analysis. We show that the E.I encounter complex of wild type-rACT and Chtr forms both E*I* and postcomplex fragment with the same rate constant, so that both species arise from EI' conversion to E*I*. These results support our earlier conclusion that the P1-P1' linkage is preserved in EI' and imply that E*I* corresponds to a covalent adduct of E and I, either acyl enzyme or the tetrahedral intermediate formed by water attack on acyl enzyme. Furthermore, we show that the A347R (P12) variant of rACT, which is a substrate rather than an inhibitor of Chtr, has a rate constant for postcomplex fragment formation from the E.I complex very similar to that observed for WT-rACT, implying that EI' is the common intermediate from which partitioning to inhibitor and substrate pathways occurs. These results are used to elaborate a proposed scheme for ACT interaction with Chtr that is considered in the light of relevant results from studies of other serpin-serine proteinase pairs.     

Histatin 5 is a substrate and not an inhibitor of the Arg- and Lys-specific proteinases of Porphyromonas gingivalis

The salivary peptide histatin 5 has been reported to be an inhibitor of the Arg- and Lys-specific proteinases of Porphyromonas gingivalis, an oral pathogen associated with periodontitis. In this study a purified P. gingivalis proteinase preparation consisting of a complex of the Arg- and Lys-specific proteinases and adhesins was assayed using chromogenic substrates in the presence of histatin 5. Histatin 5 produced a concentration-dependent decrease in the initial rate of hydrolysis of the chromogenic substrates by both proteinases. However, pre-incubation of histatin 5 with the purified proteinase preparation or a P. gingivalis cell sonicate for 10 min prior to assay with the chromogenic substrates showed that under these conditions the salivary peptide did not decrease the initial rate of chromogen release. Mass spectrometric analysis revealed rapid degradation of histatin 5 at all four lysyl and all three arginyl residues by the P. gingivalis proteinases. This study demonstrates that histatin 5 is a substrate for the P. gingivalis extracellular Arg- and Lys-specific cysteine proteinases and not an inhibitor.     

Tumor-associated cysteine proteinase activities in human melanoma cells and fibroblasts of different origin

Specific catalytic activities of cysteine proteinases including cathepsins B (EC 3.4.22.1) and L (EC 3.4.22.15) in human melanoma cell lines SK-MEL-28, SK-MEL-30, MEL-HO and in fibroblasts of different origin are reported. Cell line-specific pH profiles of these cysteine proteinases were determined fluorometrically with benzyloxycarbonyl-phenylalanyl-arginine-amidomethylcoumarine (Z-Phe-Arg-AMC) under saturated conditions. Single activities of cathepsins B and L were inactivated by urea and by benzyloxycarbonyl-phenylalanyl-phenylalanine-diazomethylketone (Z-Phe-Phe-CHN2) in order to describe the activities of these enzymes separately. The melanoma cell line MEL-HO, which originated from a primary lesion, showed highest activity of an unknown cysteine proteinase. This enzyme is not inactivated by urea and Z-Phe-Phe-CHN2 and has a Michaelis constant (K(M) value) of approximately 1 mM. The specific characteristics suggest that it is a tumor-associated cathepsin B. In addition, high invasive subpopulations of SK-MEL-28 and SK-MEL-30 cell lines isolated by an invasion assay showed higher proteinase activities than the low invasive subpopulations. Furthermore, in fibroblasts originating from melanoma tissue cysteine proteinase activities were increased compared to normal skin fibroblasts. In conclusion, these results indicate that these cysteine proteinases shown here are tumor-associated proteinases, possibly facilitating invasion and dissemination of melanoma cells.     

Reactive oxygen metabolites and arterial thrombosis

A new cysteine proteinase, salmon miltpain, was isolated and purified from the milt of chum salmon (Oncorhynchus keta). Native molecular mass was estimated as 67,000 by gel filtration column chromatography (Shodex WS2003) and 22,300 by SDS-polyacrylamide gel electrophoresis. Isoelectoric point was determined to be 3.9 by isoelectric focusing. The first 15 amino acid residues in the N-terminal region were LPSFLY-AEMVGYNIL. The cysteine proteinase, which had a pH optimum of 6.0 for Z-Arg-Arg-MCA hydrolysis, required a thiol-reducing reagent for activation and was inhibited by E-64, iodacetamide, CA-074 Me, TLCK, TPCK and ZPCK. The cysteine proteinase exhibited unique substrate specificity toward paired basic residues such as Lys-Arg, Arg-Arg at the subsites of P2-P1 and had a K(m) of 16.3 microM and kcat of 20.3 s-1 with Z-Arg-Arg-MCA as substrate and a K(m) of 52.9 microM and kcat of 1.79 s-1 with Z-Phe-Arg-MCA. This proteinase was found to considerably hydrolyze basic proteins such as histone, salmine and clupaine but not milk casein.     

Acid-activatable cysteine proteinases in the cellular slime mold Dictyostelium discoideum

Studies of the cysteine proteinases of the cellular slime mold Dictyostelium discoideum have been aided by a simple acid treatment step that was incorporated into the standard one-dimensional gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay procedure. The step involved immersing the separating gel in 10% (v/v) glacial acetic acid for 30-60 s immediately after electrophoresis. This modified approach revealed the presence of acid-activatable forms of some enzymes with noticeable increases in their ability to hydrolyze gelatin, a substrate present in the sodium dodecyl sulfate-polyacrylamide gels, and peptidyl amidomethylcoumarins. The activation has been analyzed using extracts of dormant spores from which cysteine proteinase activity had previously appeared low or virtually absent. The major acid-activatable proteinase had an apparent molecular mass of 48 kDa. Its activation was not due to autocatalysis as it was not prevented by mercuric chloride, an inhibitor of the enzyme, and was not accompanied by a significant change in electrophoretic mobility. It was most likely due to a conformational change and/or the removal of a low molecular weight inhibitor. The acid treatment has also revealed the presence of acid-activatable cysteine proteinases in vegetative cells, in which cysteine proteinase activity is present at high levels, as well as among enzymes from the developmental cells which have much lower cysteine proteinase activity. Indeed novel developmental forms were detected at some stages. These results provide additional insight concerning cysteine proteinase expression at various stages during development in the slime molds. A developmental model is presented which suggests that the crypticity of the cysteine proteinases in dormant spores may be governed by proton pumps and endogenous lysosomotropic agents.     

Class specific inhibition of house dust mite proteinases which cleave cell adhesion, induce cell death and which increase the permeability of lung epithelium

1. House dust mite (HDM) allergens with cysteine and serine proteinase activity are risk factors for allergic sensitization and asthma. A simple method to fractionate proteinase activity from HDM faecal pellets into cysteine and serine class activity is described. 2. Both proteinase fractions increased the permeability of epithelial cell monolayers. The effects of the serine proteinase fraction were inhibited by 4-(2-aminoethyl)-benzenesulphonyl fluoride hydrochloride (AEBSF) and soybean trypsin inhibitor (SBTI). The effects of the cysteine proteinase fraction could be inhibited by E-64. No reciprocity of action was found. 3. Treatment of epithelial monolayers with either proteinase fraction caused breakdown of tight junctions (TJs). AEBSF inhibited TJ breakdown caused by the serine proteinase fraction, whereas E-64 inhibited the cysteine proteinase fraction. 4. Agarose gel electrophoresis revealed that the proteinases induced DNA cleavage which was inhibited by the matrix metalloproteinase inhibitor BB-250. Compound E-64 inhibited DNA fragmentation caused by the cysteine proteinase fraction, but was without effect on the serine proteinase fraction. Staining of proteinase-treated cells with annexin V (AV) and propidium iodide (PI) revealed a diversity of cellular responses. Some cells stained only with AV indicating early apoptosis, whilst others were dead and stained with both AV and PI. 5. HDM proteinases exert profound effects on epithelial cells which will promote allergic sensitization; namely disruption of intercellular adhesion, increased paracellular permeability and initiation of cell death. Attenuation of these actions by proteinase inhibitors leads to the conclusion that compounds designed to be selective for the HDM enzymes may represent a novel therapy for asthma.     

The differential specificity of chymotrypsin A and B is determined by amino acid 226

The A and B isoforms of the pancreatic serine proteinase, chymotrypsin are known to cleave substrates selectively at peptide bonds formed by some hydrophobic residues, like tryptophan, phenylalanine and tyrosine. We found, however, that the B forms of native bovine and recombinant rat chymotrypsins are two orders of magnitude less active on the tryptophanyl than on the phenylalanyl or tyrosyl substrates, while bovine chymotrypsin A cleaves all these substrates with comparable catalytic efficiency. Analysing the structure of substrate binding pocket of chymotrypsin A prompted us to perform an Ala226Gly substitution in rat chymotrypsin B. The specificity profile of the Ala226Gly rat chymotrypsin B became similar to that of bovine chymotrypsin A suggesting that only the amino acid at sequence position 226 is responsible for the differential specificities of chymotrypsin A and B isoenzymes.