Crystal structure of an actinidin-E-64 complex.

E-64, 1-(L-trans-epoxysuccinylleucylamino)-4-guanidinobutane, is a potent and highly selective irreversible inhibitor of cysteine proteases. The crystal structure of a complex of actinidin and E-64 has been determined at 1.86-A resolution by using the difference Fourier method and refined to an R-factor of 14.5%. The electron density map clearly shows that the C2 atom of the E-64 epoxide ring is covalently bonded to the S atom of the active-site cysteine 25. The charged carboxyl group of E-64 forms four H-bonds with the protein and thus may play an important role in favorably positioning the inhibitor molecule for nucleophilic attack by the active-site thiolate anion. The interaction features between E-64 and actinidin are very similar to those seen in the papain-E-64 complex; however, the amino-4-guanidinobutane group orients differently. The crystals of the actinidin-E-64 complex diffracted much better than the papain-E-64 complex, and consequently the present study provides more precise geometrical information on the binding of the inhibitor. Moreover, this study provides yet another confirmation that the binding of E-64 is at the S subsites and not at the S' subsites as has been previously proposed. The original actinidin structure has been revised using the new cDNA sequence information.     

Refined x-ray structure of papain.E-64-c complex at 2.1-A resolution.

E-64-c, a synthetic cysteine protease inhibitor designed from E-64, binds to papain through a thioether covalent bond. The x-ray diffraction data for 2.1-A resolution were used to determine the three-dimensional structure of this complex and refined it to R = 0.159. 0.159. In the complex structure, the configurational conversion from S to R took place on the epoxy carbon of E-64-c, implying that the nucleophilic attack of the Cys-25 thiol group occurs at the opposite side of the epoxy oxygen atom. The leucyl and isoamylamide groups of E-64-c were strongly fixed to papain S subsites by specific interactions, including hydrogen bonding to the Gly-66 residue. The carboxyl-terminal anion of E-64-c formed an electrostatic interaction with the protonated His-159 imidazole ring (O-...HN+ = 3.76 A) and consequently prevented the participation of this residue in the hydrolytic charge-relay system. No significant distortion caused by the binding of E-64-c was shown in the secondary structure of papain. It is important to note that inhibitor and substrate have opposite binding modes for the peptide groups. The possible relationship between the binding mode and inhibitory activity is discussed on the basis of the crystal structure of this complex.     

Structural basis for development of cathepsin B-specific noncovalent-type inhibitor: crystal structure of cathepsin B-E64c complex

In order to elucidate the substrate specificity of the Sn subsites (n=1-3) of cathepsin B, its crystal structure inhibited by E64c [(+)-(2S,3S)-3-(1-[N-(3-methylbutyl)amino]-leucylcarbonyl)oxirane-2-carboxylic acid] was analyzed by the X-ray diffraction method. Iterative manual rebuilding and convenient conjugate refinement of structure decreased R- and free R-factors to 19.7% and to 23.9%, respectively, where 130 water molecules were included for the refinement using 14,759 independent reflections from 10 to 2.3 A resolution. The epoxy carbonyl carbon of E64c was covalently bonded to the Cys(29) S(gamma) atom and the remaining parts were located at Sn subsites (n=1-3). The substrate specificity of these subsites was characterized based on their interactions with the inhibitor. Base on these structural data, we developed a novel cathepsin B-specific noncovalent-type inhibitor, which may bind to S2'-S3. The molecular design of possessing structural elements of both CA074 and E64c, assisted by energy minimization and molecular dynamics (MD) simulation, may lead to a new lead noncovalent-type inhibitor.     

Impact of cysteine proteinase inhibition in midgut fluid and oral secretion on fecundity and pollen consumption of western corn rootworm (Diabrotica virgifera virgifera)

Cysteine proteinases predominate in the midgut fluid (MF) and oral secretion (OS) of adult western corn rootworm (WCR) based on their mild acidic pH optima (pH 6.0), enhanced activities after treatment with thiol reducing agents, and inhibition by selective cysteine proteinase inhibitors (PIs). Four cysteine PIs including E-64, calpeptin, calpain inhibitor II, and leupeptin (also a serine PI) strongly inhibited azocaseinolytic activity in a dose-dependent manner in both the MF and OS. The most significant effect on adult female WCR of cysteine PI consumption with corn pollen was the reduction in fecundity, but female survival was not apparently affected. Mean fresh weights for all PI-fed females were also lower than control groups. All PI-fed groups [E-64, calpain inhibitor I (Cal I) and leupeptin] had a significantly lower daily egg production than respective corn pollen-fed controls. E-64 was more potent than leupeptin and Cal I on inhibiting fecundity, which correlates with their relative anti-proteinase potency in vitro. E-64, Cal I, and leupeptin at 1.5-2 nmol/beetle/day reduced fecundity down to 25-45% of control values. Reduced egg production by PI-fed beetles results from a combination of the direct inhibition of protein digestion and a post-ingestive negative feedback mechanism, which reduces food intake. The supplement of ten essential amino acids into the E-64-treated pollen enhanced up to 3.7-fold the number of eggs laid compared to the E-64-fed group without these amino acids, suggesting that egg production is dependent on the supply of essential amino acids from corn pollen proteolysis.     

The cysteine proteinase inhibitor, E-64, reduces proteinuria in an experimental model of glomerulonephritis

Proteinuria is a major manifestation of glomerular disease (glomerulonephritis, GN). We examined the effect of trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a specific and irreversible cysteine proteinase inhibitor, on urinary protein excretion in a complement- and neutrophil-independent model of antiglomerular basement membrane (GBM) antibody disease. A single injection of rabbit antirat-GBM IgG produced a marked increase in urinary protein excretion 24hr after injection. In two separate studies using different pools of antiGBM IgG, administration of E-64 (5mg every 6h starting 2hr prior to induction of GN) reduced proteinuria (-45 +/- 7%, and -41 +/- 14%, Mean +/- SEM, n = 6; P less than 0.001) in the 24 hour period following induction of the disease. This reduction in urinary protein excretion was accompanied by a marked decrease in the specific activity of the cysteine proteinases cathepsins B and L in glomeruli (B: -97%; L: -84%) and renal cortex (B: -87%; L: -75%) isolated from the same E-64-treated rats compared to same saline-treated controls. These data, combined with the specificity of E-64 for cysteine proteinases, suggest a potential role for cysteine proteinases in the increased GBM permeability and proteinuria in this experimental model of glomerular disease.     

A high-affinity competitive inhibitor of type A botulinum neurotoxin protease activity

The peptide N-acetyl-CRATKML-amide is an effective inhibitor of type A botulinum neurotoxin (BoNT A) protease activity [Schmidt et al., FEBS Lett. 435 (1998) 61-64]. To improve inhibitor binding, the peptide was modified by replacing cysteine with other sulfhydryl-containing compounds. Ten peptides were synthesized. One peptide adapted the structure of captopril to the binding requirements of BoNT A, but it was a weak inhibitor, suggesting that angiotensin-converting enzyme is not a good model for BoNT A inhibitor development. However, replacing cysteine with 2-mercapto-3-phenylpropionyl yielded a peptide with K(i) of 330 nM, the best inhibitor of BoNT A protease activity reported to date. Additional modifications of the inhibitor revealed structural elements important for binding and supported our earlier findings that, with the exception of P1' arginine, subsites on BoNT A are not highly specific for particular amino acid side chains.     

Invasion of ras-transformed breast epithelial cells depends on the proteolytic activity of cysteine and aspartic proteinases

It has been suggested that the lysosomal proteinases cathepsin B, L and D participate in tumour invasion and metastasis. Whereas for cathepsins B and L the role of active enzyme in invasion processes has been confirmed, cathepsin D was suggested to support tumour progression via its pro-peptide, rather than by its proteolytic activity. In this study we have compared the presence of active cathepsins B, L and D in ras-transformed human breast epithelial cells (MCF-10A neoT) with their ability to invade matrigel. In this cell line high expression of all three cathepsins was detected by immunofluorescence microscopy. The effect of proteolytic activity on cell invasion was studied by adding various natural and synthetic cysteine and aspartic proteinase inhibitors. The most effective compound was chicken cystatin, a general natural inhibitor of cysteine proteinases, (82.8+/-1.6% inhibition of cell invasion), followed by the synthetic inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64). CLIK-148, a specific inhibitor of cathepsin L, showed a lower effect than chicken cystatin and E-64. Pepstatin A weakly inhibited invasion, whereas the same molar concentrations of squash aspartic proteinase (SQAPI)-like inhibitor, isolated from squash Cucurbita pepo, showed significant inhibition (65.7+/-1.8%). We conclude that both cysteine and aspartic proteinase activities are needed for invasion by MCF-10A neoT cells in vitro.     

Involvement of cysteine proteinases in excystment of Paragonimus ohirai metacercariae induced by sodium cholate and A23187

The involvement of intrinsic proteinases in the excystment of Paragonimus ohirai metacercariae was studied in in vitro excystment induced by sodium (Na) cholate, a bile salt and A23187, a Ca2+ ionophore. The effects of various proteinase inhibitors on the in vitro excystment were examined and similar inhibitory profiles were obtained. Benzyloxycarbonyl-L-leucyl-L-leucinal (Z-Leu-Leu-H), a cysteine proteinase inhibitor and 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc SC), a serine proteinase inhibitor completely inhibited excystment, while L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido (4-guanidino)-butane (E-64), a cysteine proteinase inhibitor and leupeptin, a cysteine/serine proteinase inhibitor permitted partial excystment at a lower rate, but inhibited it from proceeding from the partial excystment stage. In secretions released from metacercariae during excystment, proteinase activities detected towards various fluorogenic peptidyl substrates were almost completely inhibited by Z-Leu-Leu-H and E-64, but not by Pefabloc SC. Sodium cholate induced a higher secretion of cysteine proteinases and a higher rate of excystment than A23187. Profiles of cysteine proteinase activities towards five peptidyl substrates detected were markedly different among the two secretions and the lysate of newly excysted juveniles. Newly excysted juveniles released cysteine proteinases with similar activity profiles and levels to metacercariae induced by Na cholate-incubation, whereas the release of cysteine proteinases was reduced compared with metacercariae induced by A23187-incubation. These results provide valuable information about the involvement of intrinsic proteinases in metacercarial excystment.     

L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L.

1. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) at a concentration of 0.5 mM had no effect on the serine proteinases plasma kallikrein and leucocyte elastase or the metalloproteinases thermolysin and clostridial collagenase. In contrast, 10 muM-E-64 rapidly inactivated the cysteine proteinases cathepsins B, H and L and papain (t0.5 = 0.1-17.3s). The streptococcal cysteine proteinase reacted much more slowly, and there was no irreversible inactivation of clostripain. The cysteine-dependent exopeptidase dipeptidyl peptidase I was very slowly inactivated by E-64. 2. the active-site-directed nature of the interaction of cathepsin B and papain with E-64 was established by protection of the enzyme in the presence of the reversible competitive inhibitor leupeptin and by the stereospecificity for inhibition by the L as opposed to the D compound. 3. It was shown that the rapid stoichiometric reaction of the cysteine proteinases related to papain can be used to determine the operational molarity of solutions of the enzymes and thus to calibrate rate assays. 4. The apparent second-order rate constants for the inactivation of human cathepsins B and H and rat cathepsin L by a series of structural analogues of E-64 are reported, and compared with those for some other active-site-directed inhibitors of cysteine proteinases. 5. L-trans-Epoxysuccinyl-leucylamido(3-methyl)butane (Ep-475) was found to inhibit cathepsins B and L more rapidly than E-64. 6. Fumaryl-leucylamido(3-methyl)butane (Dc-11) was 100-fold less reactive than the corresponding epoxide, but was nevertheless about as effective as iodoacetate.     

Characterization of novel cysteine proteases from germinating cotyledons of soybean [Glycine max (L.) Merrill].

The enzymatic properties of novel cysteine proteases D3-alpha and beta which were purified from germinating soybean cotyledons were investigated. The enzyme activities were exhibited in the presence of a thiol reagent, such as 2-mercaptoethanol, and apparently inhibited by E-64, a cysteine protease inhibitor. Hydrolytic activities toward carbobenzoxy-Phe-Arg-MCA were detected at a pH above 4.0. The optimum temperature for activities was about 40 degrees C. The isoelectric point of D3-alpha and beta was 4.4 and 4. 7, respectively. The molecular mass of D3-alpha and beta, measured by MALDI/TOF mass spectrometry, was 26,178 and 26,429 Da, respectively. The substrate specificities of the enzymes were examined using peptide-MCAs and peptides, and cathepsin L-like broad specificity was observed at pH 4.0. These results demonstrated that these enzymes are cysteine endopeptidases [EC 3.4.22.-] like papain [EC 3.4.22.2].     

Effects of proteasome inhibitor (lactacystin) and cysteine protease inhibitor (E-64-d) on processes of mitosis in Xenopus embryonic cells

At least two different protease pathways have been implicated in the degradation that is required to control the eukaryotic cell cycle; these two pathways center on the activities of ubiquitin/proteasome and cysteine protease. The proteasome inhibitors, lactacystin and AcLLnL and the cysteine protease inhibitor E-64-d were tested for their ability to inhibit the cell cycles of Xenopus embryos. Lactacystin, AcLLnL and E-64-d all caused the complete arrest of the cell cycle. To define the specific cell cycle processes that were affected by the two inhibitors, we performed a cytological analysis. Inhibition of the cell cycle by lactacystin and E-64-d occurred during prophase and metaphase. The number of cells that arrested in prophase was 1.4-times higher in the E-64-d-treated group than in the control group and the number of arrested cells in the lactacystin-treated group was 1.4-times higher than in the E-64-d-treated group. The number of cells that arrested in metaphase was 3-to-4-times higher in the E-64-d and lactacystin groups than in the control group. These results indicate that both cysteine protease(s) and proteasomes are involved in the prophase and metaphase stages of cell division.     

Purification of active cysteine proteases by affinity chromatography with attached E-64 inhibitor

Cysteine proteases are implicated in many regulatory and degradative processes in animal and plant cells. Many of the proteases are strongly inhibited by an irreversible inhibitor, trans-(epoxysuccinyl)-l-leucylamino-4-guanidinobutane (E-64) from Aspergillus japonicus. Here we report a method for purification of cysteine proteases by affinity chromatography on E-64. Attachment of the inhibitor to thiopropyl Sepharose through its epoxy group resulted in the loss of its irreversible activity but did not affect the specificity of interaction or its capability to bind cysteine proteases. Papain that served as a model cysteine protease was fully active after elution. We also provide evidence for purification of active proteases from a mixture of extracellular fluid of Botrytis cinerea- and Trichoderma harzianum-inoculated bean plants. Since the proteases are eluted with urea after the column is washed with 1 M NaCl, this procedure may provide highly efficient purification.     

A comparison between the binding modes of a substrate and inhibitor to papain as observed in complex crystal structures

On the basis of the crystal structures of papain complexed with the substrate analogue benzyloxycarbonyl-L-phenylalanyl-L-alanine chloromethyl-ketone (Drenth, J., Kalk, K.H., and Swen, H.M. (1976) Biochemistry 15, 3731-3738) and with the inhibitor E-64-c, the binding modes were compared at the atomic level to clarify the functional difference between the substrate and inhibitor. Irrespective of the reverse chemical bonding in the peptide bonds, both the molecules are located at the S subsites of papain with similar interactions. However, the inhibitory activity of E-64-c is characterized by the stereochemical function of a carboxyoxirane ring and the tight binding of the isopentylaminoleucyl side chain to the S subsites.     

Effective pretreatment by cysteine proteinase inhibitor for improved analysis of protein components of trematodes on SDS-PAGE

Since Fasciola sp. contained proteolytic enzyme(s), it was confirmed that degradation took place in protein components in extracts of the liver flukes, which resulted in lack of clarity of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Degradation was shown to occur mostly during a heating process of the extract samples. The proteolytic activity in the extracts was completely blocked and electrophoretic patterns were improved only by the use of cysteine proteinase inhibitor N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). Great improvement was also noted in electrophoretic patterns of the extracts of other trematodes, such as Paragonimus westermani, P. miyazakii and Clonorchis sinesis, when their extracts were treated with E-64.     

Activation of caspase-3 by lysosomal cysteine proteases and its role in 2,2'-azobis-(2-amidinopropane)dihydrochloride (AAPH)-induced apoptosis in HL-60 cells

We previously reported that in addition to mitochondrial cytochrome c dependent activation, lysosomal cysteine proteases were also involved in the activation of caspase-3. In this study, we have separately obtained the lysosomal and mitochondrial caspase-3 activating factors in a crude mitochondrial fraction and characterized their ability to activate pro-caspase-3 in the in vitro assay system. When a rat liver crude mitochondrial fraction containing lysosomes (ML) was treated with a low concentration of digitonin, lysosomal factors were selectively released without the release of a mitochondrial factor (cytochrome c, Cyt.c). Treatment of ML with Ca(2+) in the presence of inorganic phosphate (P(i)), in contrast, released mitochondrial Cyt.c without the release of lysosomal factors. The obtained lysosomal and mitochondrial factors activated caspase-3 in different manners; caspase-3 activation by lysosomal and mitochondrial factors was specifically suppressed by E-64, a cysteine protease inhibitor, and caspase-9 inhibitor, respectively. Thus, the activation of caspase-3 by lysosomal factors was found to be distinct from the activation by mitochondrial Cyt.c dependent formation of the Apaf-1/caspase-9 complex. To further determine whether or not the activation of caspase-3 by lysosomal cysteine proteases is involved in cellular apoptosis, the effect of E-64-d, a cell-permeable inhibitor of cysteine protease, on 2,2'-azobis-(2-amidinopropane)dihydrochloride (AAPH)-induced apoptosis in HL-60 cells was investigated. As a result, DNA fragmentation induced by AAPH was found to be remarkably (up to 50%) reduced by pretreatment with E-64-d, indicating the participation of lysosomal cysteine proteases in AAPH-induced apoptosis in HL-60 cells.     

Effects of lysosomal proteinase inhibition on the development of the rat embryo in vitro

Altered lysosomal function in the visceral yolk sac can result in abnormal development. As proteolysis is an important function of the rodent visceral yolk sac during early and mid-gestation, we characterized the lysosomal proteolytic enzyme activity of this extraembryonic membrane and determined the effects of inhibitors of protein degradation on embryonic development. Constituent activities of cysteine and aspartic acid proteinases were measured in rat visceral yolk sac on gestation day 12, and the effects of the cysteine proteinase inhibitors leupeptin, E-64 [trans-epoxysuccinyl-l-leucylamido(4-guanido)butane] and N-ethylmaleimide and the aspartic acid proteinase inhibitor pepstatin were determined in Sprague-Dawley rat embryos cultured in vitro from gestation days 10-12. It was determined that only cysteine proteinases, primarily cathepsins B and L, are active in the mid-gestation visceral yolk sac. The cysteine proteinase inhibitors leupeptin and E-64 both produced a concentration-related decrease in embryonic growth, as measured by crown-rump length, somite number, and embryonic protein content, and a concentration-related increase in incidence of abnormalities. A characteristic pattern of abnormalities was produced which involved a decrease in neural tube volume and the formation of a subectodermal blister opposite the point of attachment of the vitelline vessels. At high concentrations, anophthalmia was also observed. The decreased neural tube volume was associated with increased osmolality of the exocoelomic fluid, the major extraembryonic fluid compartment. It is possible that the osmotic change decreased neural tube volume by causing water to move to the compartment with a higher solute concentration, out of the embryo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of arginine aminopeptidase by bestatin and arphamenine analogues. Evidence for a new mode of binding to aminopeptidases

The synthesis and inhibition kinetics of a new, potent inhibitor of arginine aminopeptidase (aminopeptidase B; EC 3.4.11.6) are reported. The inhibitor is a reduced isostere of bestatin in which the amide carbonyl is replaced by the methylene (-CH2-) moiety. Analysis of the inhibition of arginine aminopeptidase by this inhibitor according to the method of Lineweaver and Burk yields an unusual noncompetitive double-reciprocal plot. The replot of the slopes versus [inhibitor] is linear (Kis = 66 nM), but the replot of the y intercepts (1/V) versus [inhibitor] is hyperbolic (Kii = 10 nM, Kid = 17 nM). These results provide evidence for a kinetic mechanism in which the inhibitor binds to the S1' and S2' subsites on the enzyme, not the S1 and S1' subsites occupied by dipeptide substrates. Furthermore, structure-activity data for a series of ketomethylene dipeptide isosteres in which the amide (-CONH-) of a dipeptide is replaced with the ketomethylene (-COCH2-) moiety show that the S1 and S1' subsites preferentially bind basic and aromatic side chains, respectively. These results are in agreement with the known substrate specificity of arginine aminopeptidase. The structure-activity data for several bestatin analogues, however, show that these compounds do not bind to the S1 and S1' sites of arginine aminopeptidase. A comparison of the data provides evidence that bestatin inhibits arginine aminopeptidase and possibly other aminopeptidases by binding to the S1' and S2' sites of the enzyme.     

Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum

The protein from chicken egg white that inhibits cysteine proteinases, and has been named 'cystatin', was purified by ovomucin precipitation, affinity chromatography on carboxymethylpapain-Sepharose and chromatofocusing. The final purification step separated two major forms of the protein (pI 6.5 and 5.6), with a total recovery of about 20% from egg white. By use of affinity chromatography and immunodiffusion it was shown that the inhibitor is also present at low concentrations in the serum of male and female chickens. Tryptic peptide maps of the separated forms 1 and 2 of egg-white cystatin were closely similar, and each form had the N-terminal sequence Ser-Glx-Asx. The two forms showed complete immunological identity, and neither contained carbohydrate. Ki values for the inhibition of cysteine proteinases were as follows: papain (less than 1 X 10(-11)M), cathepsin B (8 X 10(-10)M), cathepsin H (about 2 X 10(-8)M) and cathepsin L (about 3 X 10(-12)M). Some other cysteine proteinases, and several non-cysteine proteinases, were found not to be significantly inhibited by cystatin. The inhibition of the exopeptidase dipeptidyl peptidase I by cystatin was confirmed and the Ki found to be 2 X 10(-10)M. Inhibitor complexes with active cysteine proteinases and the inactive derivatives formed by treatment with iodoacetate, E-64 [L-trans-epoxysuccinylleucylamido(4-guanidino)butane] and benzyloxycarbonylphenylalanylalanyldiazomethane were demonstrated by isoelectric focusing and cation-exchange chromatography. The complexes dissociated in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with or without reduction) with no sign of fragmentation of the inhibitor. Cystatin was found not to contain a free thiol group, and there was no indication that disulphide exchange plays any part in the mechanism of inhibition.     

Inhibition of proteasome activity strongly affects kiwifruit pollen germination. Involvement of the ubiquitin/proteasome pathway as a major regulator.

The 26S proteasome is a multicatalytic complex that acts as primary protease of the ubiquitin-mediated proteolytic pathway in eukaryotes. We provide here the first evidence that the proteasome plays a key role in regulating pollen tube growth. Immunoblotting experiments revealed the presence of high levels of free ubiquitin and ubiquitin conjugates in rehydrated and germinating pollen of kiwifruit [Actinidia deliciosa var. deliciosa (A. Chev) C. F. Liang et A. R. Ferguson]. Proteasome activity, assayed fluorometrically, accompanied the progression of germination. Specific inhibitors of proteasome function such as benzyloxycarbonyl-leucinyl-leucinyl-leucinal (MG-132), clasto-lactacystin beta-lactone, and epoxomicin significantly decreased tube growth or altered tube morphology. High-molecular mass, ubiquitinated proteins accumulated in MG-132- and beta-lactone-treated pollen, indicating that proteasome function was effectively impaired. The inhibitors were also able to decrease in vitro proteasome activity in pollen extracts. Because MG-132 can inhibit calpains, as well as the proteasome, trans-epoxy succinyl-L-leucylamido-(4-guanidino) butane (E-64), an inhibitor of cysteine proteases, was investigated. Some reduction in tube growth rate was observed, but only at 80 microM E-64, and no abnormal tubes were produced. Furthermore, no inhibition of tube growth was observed when another inhibitor of cysteine proteases, leupeptin, or inhibitors of serine and aspartic proteases (phenylmethylsulfonyl fluoride and pepstatin) were used. Our results indicate that protein turnover during tube organization and elongation in kiwifruit pollen is important, and our results also implicate the ubiquitin/26S proteasome as the major proteolytic pathway involved.     

Specificity determinants of human cathepsin s revealed by crystal structures of complexes

Cathepsin S, a lysosomal cysteine protease of the papain superfamily, has been implicated in the preparation of MHC class II alphabeta-heterodimers for antigen presentation to CD4+ T lymphocytes and is considered a potential target for autoimmune-disease therapy. Selective inhibition of this enzyme may be therapeutically useful for attenuating the hyperimmune responses in a number of disorders. We determined the three-dimensional crystal structures of human cathepsin S in complex with potent covalent inhibitors, the aldehyde inhibitor 4-morpholinecarbonyl-Phe-(S-benzyl)Cys-Psi(CH=O), and the vinyl sulfone irreversible inhibitor 4-morpholinecarbonyl-Leu-Hph-Psi(CH=CH-SO(2)-phenyl) at resolutions of 1.8 and 2.0 A, respectively. In the structure of the cathepsin S-aldehyde complex, the tetrahedral thiohemiacetal adduct favors the S-configuration, in which the oxygen atom interacts with the imidazole group of the active site His164 rather than with the oxyanion hole. The present structures provide a detailed map of noncovalent intermolecular interactions established in the substrate-binding subsites S3 to S1' of cathepsin S. In the S2 pocket, which is the binding affinity hot spot of cathepsin S, the Phe211 side chain can assume two stable conformations that accommodate either the P2-Leu or a bulkier P2-Phe side chain. This structural plasticity of the S2 pocket in cathepsin S explains the selective inhibition of cathepsin S over cathepsin K afforded by inhibitors with the P2-Phe side chain. Comparison with the structures of cathepsins K, V, and L allows delineation of local intermolecular contacts that are unique to cathepsin S.