Published by De Gruyter

Volume 383 Issue 7-8

Issue of Biological Chemistry

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Contents
Journal Overview

June 1, 2005

The International Proteolysis Society (IPS): A Society for Proteolysis, Proteases and Protease Inhibitors

A. J. Rivett, B. F. Sloane

Page range: 1029-1030

June 1, 2005

Proteases and Their Inhibitors: Traditional Research Subjects in the Munich Area

H. Fritz

Page range: 1031-1034

June 1, 2005

Apoptotic Pathways: Involvement of Lysosomal Proteases

B. Turk, V. Stoka, J. Rozman-Pungercar, T. Cirman, G. Droga-Mazovec, K. Oreic, V. Turk

Page range: 1035-1044

Abstract

Apoptosis or programmed cell death is the major mechanism used by multicellular organisms to remove infected, excessive and potentially dangerous cells. Cysteine proteases from the caspase family play a crucial role in the process. However, there is increasing evidence that lysosomal proteases are also involved in apoptosis. In this review various lysosomal proteases and their potential contribution to propagation of apoptosis are discussed.

June 1, 2005

Human Tissue Kallikreins: A New Enzymatic Cascade Pathway?

G. M. Yousef, E. P. Diamandis

Page range: 1045-1057

Abstract

Serine proteases are proteolytic enzymes with an active serine residue in their catalytic site. Kallikreins are a subgroup of the serine protease family which is known to have diverse physiological functions. The human kallikrein gene family has now been fully characterized and includes 15 members tandemly located on chromosome 19q13.4. Here we discuss the common structural features of kallikreins at the DNA, mRNA and protein levels and summarize their tissue expression and hormonal regulation patterns. Kallikreins are expressed in many tissues including the salivary gland, endocrine tissues such as testis, prostate, breast and endometrium, and in the central nervous system. Most genes appear to be under steroid hormone regulation. The occurrence of several splice variants is common among kallikreins, and some of the splice variants seem to be tissuespecific and might be related to certain pathological conditions. Kallikreins are secreted in an inactive zymogen form which is activated by cleavage of an Nterminal peptide. Some kalikreins can undergo autoactivation while others may be activated by other kallikreins or other proteases. Most kallikreins are predicted to have trypsinlike enzymatic activity except three which are probably chymotrypsinlike. New, but mainly circumstantial evidence, suggests that at least some kallikreins may be part of a novel enzymatic cascade pathway which is turnedon in aggressive forms of ovarian and probably other cancers.

June 1, 2005

Discovery of Chemokine Substrates for Matrix Metalloproteinases by Exosite Scanning: A New Tool for Degradomics

C. M. Overall, G. A. McQuibban, I. Clark-Lewis

Page range: 1059-1066

Abstract

Increasingly it is being recognized that matrix metalloproteinases (MMPs) are important processing enzymes that regulate cellular behaviour and immune cell function by selective proteolysis of cell surface receptors and adhesion molecules, cytokines and growth factors. These functions will likely prove to be as important in vivo as the proposed roles of MMPs in pathological matrix degradation. To screen for new protease substrates we have reported a novel exosite scanning strategy that utilizes protease substrate binding exosite domains as yeast twohybrid baits. We discovered that the chemokine monocyte chemoattractant protein-3 (MCP-3) binds the hemopexin C domain of gelatinase A (MMP-2) leading to its efficient cleavage, converting an agonist to a potent receptor antagonist. We have now found that other MMPs cleave MCP-1, MCP-2, MCP-3, MCP-4, SDF 1α and SDF-1β indicating that the intersection between the chemokine and MMP families is broad with important implications for the control of inflammatory and immune processes. Use of engineered substrates with altered exosite binding affinities further revealed the power of exosites in dictating proteolytic specificity either directing cleavage of nonpreferred sites or in other cases virtually eliminating proteolysis of readily accessible scissile bonds. Hence, bioinformatic searches for protease substrates based on scissile bond preference will only reveal a subset of substrates unless the influence of exosites is considered.

June 1, 2005

Novel Secretory Vesicle Serpins, Endopin 1 and Endopin 2: Endogenous Protease Inhibitors with Distinct Target Protease Specificities

V. Y. H. Hook, S.-R. Hwang

Page range: 1067-1074

Abstract

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 α1-antichymotrypsin (ACT) was detected by Western blots with antiACT. 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 trypsinlike 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 crossclass 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 SDSstable 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 trypsinlike proteases; endopin 2 possesses crossclass inhibition for inhibition of papainlike cysteine proteases and elastaselike serine proteases. It will be of interest in future studies to define the endogenous protease targets of these two novel secretory vesicle serpins.

June 1, 2005

Extracellular Proteases of Staphylococcus spp.

G. Dubin

Page range: 1075-1086

Abstract

Bacterial proteases secreted into an infected host may exhibit a wide range of pathogenic potentials. Staphylococci, in particular Staphylococcus aureus, are known to produce several extracellular proteases, including serine, cysteine and metalloenzymes. Their insensitivity to most human plasma protease inhibitors and, even more, the ability to inactivate some of these make the proteases potentially harmful. Indeed, several recent studies have shown that staphylococcal proteases are able to interact with the host defense mechanisms and tissue components as well as to modify other pathogenderived virulence factors. A tight, cell densitydependent control of proteolytic activity expression, similar to that of the welldefined virulence determinants, further suggests the role of staphylococcal proteases in the infection process. Consistently, alterations in coordinated expression of extracellular proteins markedly diminished the virulence. However, despite these data and the fact that a strain deficient in sspABC operon coding for serine (sspA) and cysteine (sspB) proteases was highly attenuated in virulence in the animal infection model, it was impossible to unambiguously demonstrate the importance of any particular protease as a virulence factor. Therefore, it can be assumed that the orchestrated expression and interaction of a variety of extracellular and cell surface proteins rather than any particular one is responsible for the staphylococcal pathogenicity and that the proteases apparently play an important role in this complex process. Such redundant mechanism is very well suited for promoting the survival of staphylococci under diverse environmental conditions encountered in the infected host.

June 1, 2005

Secreted Aspartic Proteases as Virulence Factors of Candida Species

M. Monod, M. Borg-von Zepelin

Page range: 1087-1093

Abstract

Candida infections have emerged as a significant medical problem during the last few decades. Among the different virulence traits of C. albicans, secreted proteolytic activity has been intensively investigated. Pathogenesis of the various forms of candidiasis was shown to be associated with the differential and temporal regulation of the expression of genes coding for secreted aspartic proteases (Sap). These enzymes act as cytolysins in macrophages after phagocytosis of Candida, are present in tissue penetration and are also involved in adherence to epithelial cells. Since the introduction of new antiretroviral therapeutics such as HIV protease inhibitors, oropharyngeal candidiasis is less often observed in AIDS patients. Different HIV aspartic protease inhibitors were able to inhibit the C. albicans Saps involved in adherence. The lower rates of oropharyngeal candidiasis observed in individuals receiving antiretroviral combination therapy could reflect not only an improvement in the immune system but also direct inhibition of Candida Saps by HIV protease inhibitors. Therefore, the development of specific aspartic protease inhibitors might be of interest for the inhibition of candidiasis.

June 1, 2005

Triple-Helical Peptide Analysis of Collagenolytic Protease Activity

J. L. Lauer-Fields, G. B. Fields

Page range: 1095-1105

Abstract

Matrix metalloproteinase (MMP) family members are involved in the physiological remodeling of tissues and embryonic development as well as pathological destruction of extracellular matrix components. To study the mechanisms of MMP action on collagenous substrates, nonfluorogenic and fluorogenic triplehelical peptide models of MMP-1 cleavage sites in interstitial collagens have been constructed. Triplehelical peptides were assembled by either (a) covalent branching or (b) selfassociation driven by hydrophobic interactions. Fluorogenic triplehelical peptide (fTHP) substrates contained the fluorophore/quencher pair of (7-methoxycoumarin-4-yl)acetyl (Mca) and N-2,4-dinitrophenyl (Dnp) in the P5 and P5 positions, respectively. Investigation of MMP family hydrolysis of THPs showed kcat/Km values in the order of MMP-13 > MMP 1 ~ MMP-1(Δ243-450) ~ MMP-2 >> MMP-3. Studies on the effect of temperature on fTHP and an analogous fluorogenic singlestranded peptide (fSSP) hydrolysis by MMP-1 showed that the activation energies between these two substrates differed by 3.4-fold, similar to the difference in activation energies for MMP-1 hydrolysis of type I collagen and gelatin. The general proteases trypsin and thermolysin were also studied for triplehelical peptidase activity. Both of these enzymes exhibited similar activation energies to MMP-1 for hydrolysis of fTHP versus fSSP. These results suggest that triplehelical peptidase activity can be distinguished from collagenolytic activity, and that mechanistically distinct enzymes convergently evolved to develop collagenolytic activity.

June 1, 2005

Phage Display Substrate: A Blind Method for Determining Protease Specificity

D. Deperthes

Page range: 1107-1112

Abstract

Phage display substrate enables rapid determination of protease specificity by exposing vast numbers of recombinant peptides to a given protease. Peptides released through specific cleavage are amplified in an expression system. Phage display substrate has been widely exploited and developed further. The number of proteases (from various sources) characterized by this approach testifies to its power. To conserve their advantage over chemical methods, however, phage libraries must be constructed accordingly. The current phenomenal progress in genomics steadily increases the number of protease to be studied. Phage display substrate should prove a powerful method to exploit this wealth of new knowledge.

June 1, 2005

Evolution of Placental Proteases

R. W. Mason, D. L. Stabley, G. N. Picerno, J. Frenck, S. Xing, G. P. Bertenshaw, K. Sol-Church

Page range: 1113-1118

Abstract

The placenta is a critical organ in mammals required for the transport of nutrients from the mother to the fetus during gestation. Other critical functions of the placenta include hormone regulation and immune regulation. The origin of the mammals and early placenta is relatively recent in evolutionary terms, and consequently there are few placentaspecific genes. In two separate branches of mammalian evolution, gene duplications have given rise to two large families of protease genes that are expressed only by placental tissues. A family of aspartic protease genes is expressed only in artiodactyls, and a family of cysteine protease genes is expressed only in rodents. These genes have probably evolved to perform specific functions in the placenta that are carried out by broader specificity proteases in mammalian species that do not express these proteases.

June 1, 2005

Factor VII Activating Protease (FSAP): A Novel Protease in Hemostasis

J. Römisch

Page range: 1119-1124

Abstract

Recently a novel serine protease in human plasma was described and was named PHBP, PHBSP or factor seven activating protease (FSAP), respectively, the latter according to the finding that it can support coagulation by factor VII activation. Later on FSAP was identified as a potent activator of single chain plasminogen activators, in particular of prourokinase, as well. The physiological role of FSAP is still speculative, but recent studies suggest a contribution to hemostasis. Due to its affinity to glycosaminoglycans a role in cellassociated or extracellular proteolytic events is also likely. The impact of a very recently uncovered frequent polymorphism impairing the prourokinase activation potential of FSAP needs to be investigated in more detail.

June 1, 2005

Cold-Adapted and Mesophilic Brachyurins

á. Gudmundsdóttir

Page range: 1125-1131

Abstract

Two different types of brachyurins, termed I and II, have been described in the literature. Within type I there are two subtypes, Ia and Ib. The prototype for the type I brachyurins is Fiddler crab collagenase I. Its coldadapted analogue from Antarctic krill, termed euphaulysin, shares many of its characteristics. Both enzymes are distinguished by their broad substrate specificity as well as the ability to cleave collagen. The precursor form of euphaulysin has been expressed in Pichia pastoris and processed to its fully active form using cod trypsin. A molecular model of euphaulysin, based on the known crystal structure of crab collagenase I, indicates that the core structure of these enzymes is almost identical. As a coldadapted enzyme, euphaulysin has a higher catalytic efficiency than crab collagenase I. It is also more sensitive to thermal inactivation and autolysis. Furthermore, euphaulysin has an increased length of several surface loops compared to crab collagenase I. Extended surface loops have been suggested to play a role in the cold activity of some bacterial enzymes. Sensitivity to autolysis is an important factor which contributes to the thermal instability of euphaulysin. Substitution of a highly exposed residue in the autolysis loop of euphaulysin resulted in an increased stability of the enzyme towards thermal inactivation without altering its catalytic efficiency.

June 1, 2005

The Role of Dipeptidyl Peptidase IV (DP IV) Enzymatic Activity in T Cell Activation and Autoimmunity

D. Reinhold, T. Kähne, A. Steinbrecher, S. Wrenger, K. Neubert, S. Ansorge, S. Brocke

Page range: 1133-1138

Abstract

Activated T lymphocytes express high levels of dipeptidyl peptidase IV (DP IV)/CD26. Recent studies support the notion that DP IV may play an important role in the regulation of differentiation and growth of T lymphocytes. This article gives a short overview on DP IV/CD26 expression and effects on immune cells in vitro and in vivo. A major focus of this review are clinical aspects of the function of CD26 on hematopoietic cells and the potential usage of synthetic DP IV inhibitors as therapeutics in inflammatory disorders.

June 1, 2005

Biochemical Features, Molecular Biology and Clinical Relevance of the Human 15-Domain Serine Proteinase Inhibitor LEKTI

M. Walden, P. Kreutzmann, K. Drögemüller, H. John, W.-G. Forssmann, H.-J. Mägert

Page range: 1139-1141

Abstract

Based on the isolation of a 55 amino acid peptide from human hemofiltrate, we cloned the cDNA for a novel human 15-domain serine proteinase inhibitor termed LEKTI. A trypsininhibiting activity was demonstrated for three different domains. High levels of expression of the corresponding gene were detected in oral mucosa, followed by the tonsils, parathyroid glands, thymus, and trachea. Hovnanian and coworkers recently found that certain mutations within the LEKTI gene are linked to the severe congenital disease Netherton syndrome and atopic manifestations (including asthma). Thus, a future therapeutic use of LEKTI is conceivable.

June 1, 2005

Interaction of Plasminogen Activator Inhibitor Type-1 (PAI-1) with Vitronectin (Vn): Mapping the Binding Sites on PAI-1 and Vn

F. Schroeck, N. Arroyo de Prada, S. Sperl, M. Schmitt, V. Magdolen

Page range: 1143-1149

Abstract

The serpin plasminogen activator inhibitor type-1 (PAI-1), as the primary physiological inhibitor of both urokinasetype (uPA) and tissuetype (tPA) plasminogen activator, plays an important role in the regulation of the fibrinolytic system as well as in extracellular remodeling in both physiological and pathophysiological processes. In plasma as well as in the extracellular matrix PAI-1 binds to vitronectin (Vn), an interaction that affects the function of both proteins. As PAI-1/Vn interaction has a significant regulatory function in fibrinolysis, thrombolysis, and cell adhesion in cancer spread, there is a strong interest in defining the binding sites on PAI-1 and Vn as the basis of a rational design of novel drugs that may modulate PAI 1/Vnmediated effects. In this minireview, we give an overview on the approaches to define the Vn binding site of PAI-1 and vice versa. Although in the case of PAI-1 the region around αhelix E and αhelix F of PAI 1 has been demonstrated to be important for its interaction with Vn, the precise location of the Vnbinding region has not completely been resolved. The major highaffinity PAI-1 binding region of Vn is localized within the Nterminal somatomedin B (SMB) domain of Vn. There are indications for at least one other lowaffinity PAI-1 binding site in the Cterminal region of Vn, which seems to be involved in the formation of larger PAI-1/Vn complexes.

June 1, 2005

Structure-Function Relationship of Bromelain Isoinhibitors from Pineapple Stem

K.-i. Hatano, Y. Sawano, M. Tanokura

Page range: 1151-1156

Abstract

Bromelain isoinhibitors from pineapple stem (BIs) are unique doublechain inhibitors and inhibit the cysteine proteinase bromelain competitively. The threedimensional structure was shown to be composed of two distinct domains, each of which is formed by a threestranded antiparallel βsheet. Unexpectedly, BIs were found to share similar folding and disulfidebond connectivities not with the cystatin superfamily, but with BowmanBirk trypsin/chymotrypsin inhibitor (BBI). The structural similarity between them suggests that BIs and BBI have evolved from a common ancestor and differentiated in function during the course of molecular evolution.

June 1, 2005

Structural Basis for the Processive Protein Degradation by Tricorn Protease

H. Brandstetter, J.-S. Kim, M. Groll, P. Göttig, R. Huber

Page range: 1157-1165

Abstract

Cell survival critically depends on the efficient use of available resources. This includes both the clearance and the recycling of those protein components that have become futile or defective. Several proteins sequentially accomplish this complex task. The proteasome serves as an initial protein shredder and generates peptides of 7 12 amino acids in length. In general, these products are useless burden to the cell and need further processing. A few years ago, a proteolytic system was identified in the model organism Thermoplasma acidophilum which indeed performs this processing [Tamura et al., Science 274 (1996), 1385 1389]. The hexameric core protein of this modular system, referred to as tricorn protease, is a 720 kDa protease which is able to assemble further into a giant icosahedral capsid, as determined by electron microscopy. Recently, we determined the crystal structure of the tricorn core particle at 2.0 å resolution [Brandstetter et al., Nature 414 (2001), 466 469]. Here we describe the structural and mechanistic basis for tricorns processive degradation mode, including a novel electrostatic substratetoproduct sink, and suggest how further components might interact with the tricorn protease to complete the cellular waste recycling process.

June 1, 2005

Zinc Ligands in an Astacin Family Metalloprotease Meprin A

B. A. Doll, J. P. Villa, F. T. Ishmael, J. S. Bond

Page range: 1167-1173

Abstract

A conserved tyrosine residue in the astacin family of metalloproteases is one of five ligands proposed to coordinate zinc at the active site. Sitedirected mutagenesis of the conserved Tyr (Y226) of recombinant mouse meprin α was used to test the hypothesis that this residue is essential for zinc binding and enzymatic activity. In addition, another proposed zinc binding ligand, H167, in the conserved (HEXXH) zinc binding motif of the meprin α protease domain was replaced by an alanine residue. Both mutants were expressed and secreted with the same subunit mass as wild type (90 kDa). The Y226F mutant retained the capacity to oligomerize to higher covalently and noncovalentlylinked oligomers as the wild type, whereas H167A was predominantly a monomer. The kcat/Km for Y226F against a fluorgenic bradykinin substrate analog was approximately 15% of the wild type, while the H167A mutant had no detectable activity. Both Y226F and H167A were more susceptible to extensive degradation by trypsin compared with the wildtype protein. The zinc content in the wildtype and Y226F mutant proteins were similar, one molecule of zinc per subunit. The results indicate that Y226 is not essential for zinc binding, but Y226 and H167 are essential for full enzymatic activity and stability of the metalloproteinase.

June 1, 2005

Probing the Active Sites and Mechanisms of Rat Metalloproteases Meprin A and B

G. P. Bertenshaw, J. P. Villa, J. A. Hengst, J. S. Bond

Page range: 1175-1183

Abstract

Meprin A and B are highly regulated, secreted and cellsurface homo and heterooligomeric enzymes. Meprins are abundantly expressed in kidney and intestine. The multidomain α and β subunits have high sequence identity, however they have very different substrate specificities, oligomerization potentials and are differentially regulated. Here we describe that meprin subunit activities are modulated differently by physicochemical factors. Homooligomeric meprin B had an acidic pH optimum. The low pH protonation indicated the existence of at least two ionizable groups. An additional ionizable group generated a shoulder in the basic pH range. Homooligomeric meprin A had a neutral pH optimum and the activity curve revealed that two ionizable groups might be protonated at acidic pH similar to meprin B. Increasing the concentration of salt generally inhibited meprin B activity. Meprin A was inhibited at low salt concentrations but activated as salt was increased. This work has important implications in the elucidation of the catalytic mechanisms of meprins and other metalloproteases. In addition, the activity of meprin oligomers that arise in tissues will be affected by variations in pH and NaCl. This could have profound implications because meprins are exposed to a range of conditions in the extracellular milieu of renal and intestinal tissues and in inflammation and cancer.

June 1, 2005

(R)-3-Amidinophenylalanine-Derived Inhibitors of Factor Xa with a Novel Active-Site Binding Mode

M. M. Mueller, S. Sperl, J. Stürzebecher, W. Bode, L. Moroder

Page range: 1185-1191

Abstract

A putative nonsubstrate like binding mode of (R)-3- amidinophenylalanine derivatives to factor Xa, as derived from modeling experiments based on Xray analysis of their complexes with trypsin, was used to design a new generation of inhibitors. However, the resulting inhibitory potencies were not at all consistent with the working assumption, although with an adamantylureido derivative of (R)-3-amidinophenylalanine phenetyl amide a highly selective nanomolar inhibition of factor Xa was achieved. The Xray analysis of the complex of this ligand with factor Xa revealed an unexpected new binding mode, of which the most important feature is the interaction of the Cterminal aryl moiety with a hydrophobic subregion of the S1 subsite, while the adamantyl group occupies the hydrophobic S3/S4 subsites of the enzyme.

June 1, 2005

Inhibition of Arginine Gingipains (RgpB and HRgpA) with Benzamidine Inhibitors: Zinc Increases Inhibitory Potency

J. A. Krauser, J. Potempa, J. Travis, J. C. Powers

Page range: 1193-1198

Abstract

We assayed several benzamidine derivatives for inhibition potency with HRgpA and RgpB gingipains, enzymes which are involved in the pathogenesis of gingivitis and periodontal disease. The benzamidine derivatives proved to be effective inhibitors of HRgpA and RgpB, with the best inhibitor being a bisbenzamidine with a urea linker (Ki=30 M). The inhibition potency was increased 2 3 fold in the presence of low concentrations of zinc with the benzamidines containing a urea moiety linking the two aromatic rings. We propose an inhibition model involving a tetrahedral zinc atom coordinated with the active site Cys and His of gingipain and the urea linker in the benzamidine inhibitor. In summary, we have discovered a new series of effective inhibitors for the gingipains and found a novel way to increase inhibitor potency with the HRgpA and RgpB gingipains using zinc.

June 1, 2005

Cathepsin B Stability, But Not Activity, Is Affected in Cysteine:Cystine Redox Buffers

C. S. Pillay, C. Dennison

Page range: 1199-1204

Abstract

In order to test the hypothesis that the lysosomal cysteine protease cathepsin B may be redox regulated in vivo, cathepsin B activity and stability were measured in cysteine and/or cystinecontaining buffers. Cathepsin B activity in cysteinecontaining buffers was similar at pH 6.0 and pH 7.0, over all thiol concentrations tested. In contrast, the stability of the enzyme was greater at pH 6.0 than at pH 7.0. This suggests that the enzymes operational pH in vivo may be < pH 7.0. The activity of the enzyme was depressed in glutathionecontaining buffers. When assessed in cysteine:cystine redox buffers (pH 6.0 7.0) cathepsin B was active over a broad redox potential range, suggesting that cathepsin B activity may not be redox regulated. However, at pH 7.0, the stability of cathepsin B decreased with increasing reduction potential and ambient cystine concentration. This suggests that the stability of the enzyme at neutral pH is dependent on redox potential, and on the presence of oxidising agents.

June 1, 2005

Inhibition of Mammalian Legumain by Michael Acceptors and AzaAsn-Halomethylketones

A. J. Niestroj, K. Feußner, U. Heiser, P. M. Dando, A. Barrett, B. Gerhartz, H.-U. Demuth

Page range: 1205-1214

Abstract

Legumain is a lysosomal cysteine peptidase specific for an asparagine residue in the P1-position. It has been classified as a member of clan CD peptidases due to predicted structural similarities to caspases and gingipains. So far, inhibition studies on legumain are limited by the use of endogenous inhibitors such as cystatin C. A series of Michael acceptor inhibitors based on the backbone CbzLAlaLAlaLAsn (Cbz= benzyloxycarbonyl) has been prepared and resulted in an irreversible inhibition of porcine legumain. Variation of the molecular size within the war head revealed the best inhibition for the compound containing the allyl ester (kobs/I=766 M 1s 1). To overcome cyclisation between the amide moiety of the Asn residue and the war head, several asparagine analogues have been synthesised. Integrated in halomethylketone inhibitors, azaasparagine is accepted by legumain in the P1-position. The most potent inhibitor of this series, CbzLAlaLAlaAzaAsnchloromethylketone, displays a kobs/I value of 139 000 M 1s 1. Other cysteine peptidases, such as papain and cathepsin B, are not inhibited by this compound at concentrations up to 100 M. The synthetic inhibitors described here represent useful tools for the investigation of the structural and physiological properties of this unique asparaginespecific peptidase.

June 1, 2005

Role of the Prosegment of Fasciola hepatica Cathepsin L1 in Folding of the Catalytic Domain

M. Cappetta, I. Roth, A. Díaz, J. Tort, L. Roche

Page range: 1215-1221

Abstract

The Nterminal propeptides of cysteine proteinases play regulatory roles in the folding and stability of their catalytic domains, as well as being potent and highly specific inhibitors of their parental mature enzymes. Cysteine proteinases play a major role in the biology of the parasitic trematode Fasciola hepatica; in particular, this organism secretes significant amounts of cathepsin L enzymes. The isolated propeptide of F. hepatica cathepsin L1 functioned as a chaperone for the mature enzyme in renaturation experiments. A double point mutation (N70I/F72I) within the GxNxFxD motif of the propeptide affected its conformation and markedly decreased its affinity for the mature enzyme. When this mutation was introduced into preprocathepsin L1 expressed in yeast, the secretion of active enzyme dropped dramatically. However, significant enzyme activity was recovered from the culture supernatants after denaturation and renaturation in the presence of native propeptide. Thus, the variant prosegment gave rise to an enzyme with altered conformation, which could be refolded to the active form with the assistance of the native propeptide.

June 1, 2005

Roles for Arg- and Lys-Gingipains in the Disruption of Cytokine Responses and Loss of Viability of Human Endothelial Cells by Porphyromonas gingivalis Infection

A. Baba, T. Kadowaki, T. Asao, K. Yamamoto

Page range: 1223-1230

Abstract

Accumulating evidence indicates that periodontal disease is associated with human cardiovascular diseases. The periodontal pathogen Porphyromonas gingivalis was shown to be present in atherosclerotic plaques in addition to periodontal pockets. This bacterium is known to produce two individual cysteine proteinases, Arggingipain (Rgp) and Lysgingipain (Kgp). Here we show that these two enzymes are responsible for either the disruption of cytokine responses in human umbilical vein endothelial cells (HUVEC) to the bacterium infection or the loss of cell viability. The expression of interleukin-8 and monocyte chemoattractant protein-1 mRNA in HUVEC was greatly induced when infected with the wildtype strain, nevertheless, their protein levels in the culture medium were markedly decreased. This decrease was completely abolished in the cells infected with the Rgp/Kgpnull mutant, but not in either the Rgp or Kgpnull mutants. Loss of the adhesion activity and viability of HUVEC were greatly induced by the culture supernatant of the wildtype strain and strongly inhibited by either a combination of the Rgp and the Kgpspecific inhibitors or the deficiency of the Rgp and Kgpencoding genes. These findings indicate that P. gingivalis modulates the cytokine response in the cells and disrupts the adhesion activity and the viability through the cooperative action of Rgp and Kgp and thereby may contribute to pathogenesis of cardiovascular diseases as well as periodontal disease.

June 1, 2005

Aberrant Expression of Serpin Squamous Cell Carcinoma Antigen 2 in Human Tumor Tissues and Cell Lines: Evidence of Protection from Tumor Necrosis Factor-Mediated Apoptosis

A. Takeda, A. Kajiya, A. Iwasawa, Y. Nakamura, T. Hibino

Page range: 1231-1236

Abstract

Squamous cell carcinoma antigens (SCCA) 1 and 2 are highly homologous proteins of the serpin family, although they inhibit different types of proteinases. We investigated the expression of both SCCA mRNAs in tumor tissues, in various cell lines (A431, SAS, Ca9, HeLa, SKGIIIa, HSC-2, HSC-3, HSC-4 and KB) and in HSC cell lines in the presence of tumor necrosis factor α (TNFα). The expression of SCCA2 mRNA could be differentiated from that of SCCA1 in tumor tissues and cell lines by means of reverse transcriptionpolymerase chain reaction (RTPCR). The ratio between SCCA1 and SCCA2 mRNA expression showed selective expression of SCCA2 mRNA in SCC tissues from the uterine cervix compared to SCC tissues from the esophagus or skin. In addition, a significant level of SCCA2 mRNA expression was detected in the HSC-4 cell line, but not in Ca9, HeLa, SKGIIIa, or HSC-3 cells. In contrast, SCCA1 mRNA was detected in all samples tested. These results suggest that the level of expression of SCCA2 mRNA detected by RTPCR can be used to evaluate the status of SCC tumors. Next, we studied the effect of TNFα on SCCA1 and SCCA2 mRNA expression in HSC cell lines. SCCA1 mRNA expression was constantly increased in the three HSC cells examined with increasing time of exposure to TNFα. In contrast, SCCA2 mRNA expression was specific for HSC-4 cells. The survival rate of HSC-4 cells pretreated with TNFα (6.3 ng/ml) for 48 h was found to be 72%, compared with 42% and 9% for HSC-3 and HSC-2 cells, respectively, after apoptotic stimulation by TNFα (10 ng/ml) and cycloheximide (10 g/ml) for 18 h. Furthermore, selective expression of SCCA2 mRNA in HSC-4 pretreated with TNFα protected these cells from TNFαmediated apoptosis. Thus, SCCA2 overexpression in squamous tumor cells contributed to their survival by protecting them against TNFαinduced apoptosis.

June 1, 2005

Endosomal-Lysosomal Proteolysis Mediates Death Signalling by TNFα, Not by Etoposide, in L929 Fibrosarcoma Cells: Evidence for an Active Role of Cathepsin D

M. Démoz, R. Castino, P. Cesaro, F. M. Baccino, G. Bonelli, C. Isidoro

Page range: 1237-1248

Abstract

In several in vitro models of apoptosis, lysosomal proteolysis has been shown to play an active role in mediating the death signal by cytokines or antiblastic drugs. Depending on the experimental cell model and the cytotoxic stimulus applied, an increased expression and the cytosolic translocation of either cathepsin D or B have been reported in apoptotic cells. We have analysed the involvement of these lysosomal proteases in a canonical apoptotic cell model, namely L929 fibroblasts, in which apoptosis was induced by cytotoxic agents acting through different mechanisms: (i) the cytokine TNFα, which triggers the cell suicide via interaction with its membrane receptor, and (ii) the topoisomerase IIinhibitor etoposide (VP16), which directly causes DNA damage. In both cases the activity of cathepsins B and D increased in apoptosing cultures. CA074-Me, a specific inhibitor of cathepsin B, and Leupeptin, a broad inhibitor of serine and cysteine proteases (among which is cathepsin B), did not exert any protection from TNFα. In contrast, preloading the cells with pepstatin A, a specific inhibitor of cathepsin D, protected L929 cells from TNFα cytotoxicity by more than 50%. However, no protection was observed if pepstatin A was added concomitantly with the cytokine. Inhibition of either cathepsin B or D did not impede apoptosis induced by etoposide. Lysosomal integrity was preserved and cathepsin D remained still confined in vesicular structures in apoptotic cells treated with either TNFα or etoposide. It follows that proteolysis by cathepsin D is likely to represent an early event in the death pathway triggered by TNFα and occurs within the endosomal lysosomal compartment.

June 1, 2005

Anti-Inflammatory Effect of Pre-Elafin in Lipopolysaccharide-Induced Acute Lung Inflammation

E. Vachon, Y. Bourbonnais, C.D. Bingle, S. J. Rowe, M. F. Janelle, G. M. Tremblay

Page range: 1249-1256

Abstract

The aim of the present study was to evaluate the antiinflammatory activity of preelafin, an elastasespecific inhibitor, in lipopolysaccharide (LPS)induced acute lung inflammation. C57BL/6 mice were pretreated intranasally with recombinant human preelafin or vehicle only. One hour later, they were instilled intranasally with LPS (2 g/mouse). Animals were sacrificed 6 hours after LPS instillation and bronchoalveolar lavage (BAL) was performed with three 1- ml aliquots of saline. LPS induced a lung inflammation characterised by a 100-fold increase in BAL neutrophils compared to control animals (265.8±54.5 103 and 2.4±1.3 103 neutrophils/ml, respectively). Preelafin dosedependently reduced the neutrophil influx in the lung alveolar spaces by up to 84%. No elastase activity was detectable in all BAL fluids tested. Preelafin also reduced significantly LPSinduced gelatinase activity, as shown by zymography, and BAL macrophage inflammatory protein-2 (MIP-2) and KC levels, two potent neutrophil attractants and activators. Moreover, preelafin also significantly reduced mRNA levels of the three members of the IL-1 ligand family, namely IL-1α, IL-1β and IL-1 receptor antagonist (IL-1Ra), type II IL-1 receptor, and TNFα as assessed in whole lung tissue by RNase protection assay. Thus, preelafin may be considered as a potent antiinflammatory mediator.

June 1, 2005

The 26S Proteasome Rpn10 Gene Encoding Splicing Isoforms: Evolutional Conservation of the Genomic Organization in Vertebrates

Y. Kikukawa, M. Shimada, N. Suzuki, K. Tanaka, H. Yokosawa, H. Kawahara

Page range: 1257-1261

Abstract

Recognition of polyubiquitinated substrates by the 26S proteasome is a key step in the selective degradation of various cellular proteins. The Rpn10 subunit of the 26S proteasome can bind polyubiquitin conjugates in vitro.We have previously reported the unique diversity of Rpn10, which differs from other multiple proteasome subunits, and that the mouse Rpn10 mRNA family is generated from a single gene by developmentally regulated alternative splicing. To determine whether such alternative splicing mechanisms occur in other species, we searched for Rpn10 isoforms in databases and in our original PCR products. Here we report the genomic organization of the Rpn10 gene in lower vertebrates and provide evidence for the competent generation of distinct forms of Rpn10 by alternative splicing through evolution.

June 1, 2005

Reverse Genetic Analysis of the Caenorhabditis elegans 26S Proteasome Subunits by RNA Interference

M. Takahashi, H. Iwasaki, H. Inoue, K. Takahashi

Page range: 1263-1266

Abstract

Reverse genetic analysis was performed on the Caenorhabditis elegans 26S proteasome subunit genes by doublestranded RNAmediated interference (RNAi). Embryonic and postembryonic lethality was caused by interference of all of the eight tested 20S core subunits and all of the 19S regulatory particle subunits except for CeRpn9, CeRpn10, and Ce Rpn12, where RNAi caused no abnormality. However, synthetic suppression of CeRpn10 and CeRpn12 was lethal, whereas neither the combination of Ce Rpn9 with CeRpn10 nor with CeRpn12 resulted in abnormalities in RNAi. These results indicate that the 26S proteasome is indispensable for embryogenesis and postembryonic development, although Ce Rpn9, CeRpn10, and CeRpn12 are not essential, at least under the conditions used. CeRpn10 and Ce Rpn12 are considered to compensate for the suppression of each other.

June 1, 2005

Purification and Characterization of the 20S Proteasome from Ostrich Skeletal Muscle

A. R. Thomas, V. Oosthuizen, R. J. Naudé, K. Muramoto

Page range: 1267-1270

Abstract

The proteasome is a high molecular weight, multisubunit and multicatalytic enzyme. Here we report the purification and characterization of ostrich skeletal muscle 20S proteasome. It was purified to homogeneity with Mr 700 000, pI 6.67 and a ladder of 22.2 33.5 kDa bands on SDSPAGE. The amino acid composition and aminoterminal sequences showed large identities to those of other species. For the three major activities, pH and temperature optima ranged between 8.0 11.0 and 40 70C, and stabilities between 5 12 and up to 40 60C. Substrate specificity and inhibitory effects were also studied. Many similarities to other sources were shown, with a few significant differences.

June 1, 2005

Identification of a Signal Transduction Pathway That Regulates MMP-9 mRNA Expression in Glomerular Injury

I. von Lüttichau, R. Djafarzadeh, A. Henger, C. D. Cohen, A. Mojaat, M. Jochum, C. Ries, P. J. Nelson, M. Kretzler

Page range: 1271-1275

Abstract

Podocytes contribute to the filtration barrier within the kidney. The integrinlinked kinase (ILK) plays an important role in podocyte adhesion to the glomerular basement membrane, signal transduction and phenotype regulation. We demonstrate that ILK activity is also associated with upregulation of matrix metalloproteinase 9 (MMP-9) mRNA levels during podocyte stress. A synthetic ILK inhibitor blocked MMP-9 mRNA upregulation but showed no effect on TIMP-1 or MMP-2 mRNA expression. Interestingly, a corresponding increase in MMP-9 secretion was not observed, suggesting that MMP-9 mRNA production in podocytes is regulated via ILK, whereas additional signaling pathways may mediate the posttranscriptional regulation of MMP-9.

June 1, 2005

Modulation of the Endosomal and Lysosomal Distribution of Cathepsins B, L and S in Human Monocytes/Macrophages

H. Schmid, R. Sauerbrei, G. Schwarz, E. Weber, H. Kalbacher, C. Driessen

Page range: 1277-1283

Abstract

Endosomal and lysosomal fractions of human monocytes/ macrophages prepared from buffy coats were analyzed for activities of cathepsins B, L and S, and expression of cathepsin proteins along with major histocompatibility complex class I and class II molecules under control and immunomodulatory conditions. While the total activity of cathepsins B, L, and S together remained unchanged in lysates of control cells during culture for 72 h, the subcellular distribution of cathepsin activities underwent a shift from a predominantly endosomal localization in freshly isolated cells to a lysosomal pattern after 72 h of culture. Interferonγ treatment for 72 h resulted in an upregulation of both major histocompatibility complex proteins and cathepsins with differential changes in cathepsin B, L and S activities in endosomes versus lysosomes. These changes suggest a remodeling of the endocytic machinery and imply different functions of cathepsins B, L and S during monocyte differentiation.

June 1, 2005

Characterization of Novel Anti-Cathepsin W Antibodies and Cellular Distribution of Cathepsin W in the Gastrointestinal Tract

F. Buhling, U. Kellner, D. Guenther, S. Kahl, D. Brömme, E. Weber, P. Malfertheiner, T. Wex

Page range: 1285-1289

Abstract

Human cathepsin W (lymphopain) is a cysteine protease that is restrictively expressed in cytotoxic cells, in particular NK cells. Several anticathepsin W monoclonal antibodies were tested with respect to their capability to detect cathepsin W by Western blot analysis and immunohistochemistry. Subsequently, the distribution of cathepsin Wexpressing cells was studied in gastrointestinal tissue specimens using the antibody CW-401B1. All cathepsin Wpositive cells had a lymphocyte phenotype. Notably, samples from patients suffering from chronic inflammatory bowel disease (Crohns disease, CD; ulcerative coliltis, UC) or autoimmune gastritis revealed variable amounts of cathepsin Wexpressing cells. The relative portion of cathepsin Wpositive cells among the infiltrating leukocytes (determined by CD45) differed remarkably. In autoimmune gastritis, cathepsin Wexpressing cells made up for 65% of all CD45+ cells, whereas the corresponding values for CD and UC were 11% and 6%, respectively. These differences imply a distinct involvement of cytotoxic cells expressing cathepsin W in the pathogenesis among these diseases. Furthermore, it was tested whether the proinflammatory cytokines TNFα and IFNγ can regulate cathepsin W gene expression in NK-92 cells. Both proinflammatory cytokines had only little effect on the cathepsin W gene expression of these cells.

June 1, 2005

Functional Consequences of Cathepsin L Deficiency in Human Lung Epithelial Cells

A. Wille, A. Heimburg, A. Gerber, A. Reisenauer, T. Welte, F. Bühling

Page range: 1291-1296

Abstract

Cathepsin L is a cysteine protease of the papain family. Lung epithelial cells play an important role in host defence. The aim of the present study was to investigate the functional role of cathepsin L in the human lung carcinoma cell line A549. Cathepsin Ldeficient A549 clones were generated. They showed a significant lower proliferation and secreted 5- to 8-fold more IL-8 than the control cells. The production of IL 6, IL-18, and TGFβ1/2 was not affected significantly. It was shown that the cells upregulate IL-8 transcription and that IL-8 in the culture supernatant is necessary for the containment of cellular proliferation. In conclusion, the data show that suppression of cathepsin L expression in A549 cells leads to a growth inhibition which is partially compensated by an upregulation of IL-8 production.

June 1, 2005

Determination of Cathepsin B Expression May Offer Additional Prognostic Information for Ovarian Cancer Patients

A. Scorilas, S. Fotiou, E. Tsiambas, J. Yotis, F. Kotsiandri, M. Sameni, B. F. Sloane, M. Talieri

Page range: 1297-1303

Abstract

The lysosomal cysteine proteinase cathepsin B has been implicated in the progression of various human tumors including ovarian cancer. Included in this study were 63 patients with epithelial ovarian carcinoma. Followup information (median followup period 7 years) was available for all patients, among whom 42 (66.7%) had relapsed and 32 (50.8%) had died. The immunohistochemistry method was adopted for the detection of cathepsin B using paraffin embedded specimens. Results were compared to clinico pathological data. Statistical analysis showed cathepsin B expression to be significantly associated with the stage of disease, debulking success and interestingly, with progesterone receptors. It was also inversely related to progressionfree survival (PFS) and overall survival (OS). Accordingly, cathepsin B can be regarded as unfavorable and as an independent tumor marker for progressionfree survival and overall survival in ovarian cancer patients with long followup.

June 1, 2005

CA-074, But Not Its Methyl Ester CA-074Me, Is a Selective Inhibitor of Cathepsin B within Living Cells

M. Montaser, G. Lalmanach, L. Mach

Page range: 1305-1308

Abstract

Studies using inhibitors that reportedly discriminate between cathepsin B and related lysosomal cysteine proteinases have implicated the enzyme in a wide range of physiological and pathological processes. The most popular substance to selectively inhibit cathepsin B in vivo is CA-074Me, the methyl ester of the E-64 derivative CA-074. However, we now have found that CA-074Me inactivates both cathepsin B and cathepsin L within murine fibroblasts. In contrast, exposure of these cells to the parental compound CA 074 leads to the selective inhibition of endogenous cathepsin B, while intracellular cathepsin L remains unaffected. These results indicate that CA-074 rather than CA-074Me should be used to specifically inactivate cathepsin B within living cells.

June 1, 2005

Trypsin Mutants for Structure-Based Drug Design: Expression, Refolding and Crystallisation

D. Rauh, S. Reyda, G. Klebe, M.T. Stubbs

Page range: 1309-1314

Abstract

New techniques in drug discovery are essential for the fast and efficient development of novel innovative drugs to deal with the challenges of the future. Structure determinations of various members of serine proteinases have provided a basis for computerbased drug design within this class of enzymes. In many proteins of interest, however, this course is blocked through a lack of suitable crystals. As a strategy for circumventing such problems, we have investigated the use of surrogate proteins for studying protein ligand interactions. To test the feasibility of this approach, we have chosen bovine trypsin as a scaffold to reconstruct the ligand binding site of factor Xa. The simple modular design of trypsin, its readiness to crystallise and straightforward handling lends itself to such drug design by proxy. The expression, folding, purification, crystallographic and kinetic characterisation of bovine trypsin forms with factor Xa phenotype are presented.

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Objective
Biological Chemistry keeps you up-to-date with all new developments in the molecular life sciences. In addition to original research reports, authoritative reviews written by leading researchers in the field keep you informed about the latest advances in the molecular life sciences. Rapid, yet rigorous reviewing ensures fast access to recent research results of exceptional significance in the biological sciences.

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general biochemistry
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