Search PubMed⌕ Search

Biomedical subjects

J S Mort

Publications and source records attributed to J S Mort.

At least 55 records · Page 3Linked to original sources
PubMed1996

Potency and selectivity of the cathepsin L propeptide as an inhibitor of cysteine proteases.

The cathepsin L propeptide (phcl-2) was expressed in Saccharomyces cerevisiae using a human procathepsin L/alpha-factor fusion construct containing a stop codon at position -1 (the C-terminal amino acid of the proregion). Since the yield after purification was very low, the cathepsin L propeptide was also obtained by an alternate procedure through controlled processing of an inactive mutant of procathepsin L (Cys25Ser/Thrl10Ala) expressed in Pichia pastoris, by small amounts of cathepsin L. The peptide resulting from the cleavage of the proenzyme (phcl-1) was then purified by HPLC. The purified propeptides were characterized by N-terminal sequencing and mass spectrometry and correspond to incomplete forms of the proregion (87 and 81 aa for phcl-1 and phcl-2 respectively, compared to 96 aa for the complete cathepsin L propeptide). The two peptides were found to be potent and selective inhibitors of cathepsin L at pH 5.5, with Ki values of 0.088 nM for phcl-1 and 0.66 nM for phcl-2. The Ki for inhibition of cathepsin S was much higher (44.6 nM with phcl-1), and no inhibition of cathepsin B or papain could be detected at up to 1 microM of the propeptide. The inhibitory activity was also found to be strongly pH-dependent. Two synthetic peptides of 75 and 44 aa corresponding to N-terminal truncated versions of the propeptide were also prepared by solid phase synthesis and displayed Ki values of 11 nM and 2900 nM, respectively, against cathepsin L. The data obtained for the 4 propeptide derivatives of various lengths indicate that the first 20 residues in the N-terminal region of the propeptide are more important for inhibition than the C-terminal region which contributes little to the overall inhibitory activity.

Amino Acid Sequence↗
PubMed1996

Structure of rat procathepsin B: model for inhibition of cysteine protease activity by the proregion.

BACKGROUND: Cysteine proteases of the papain superfamily are synthesized as inactive precursors with a 60-110 residue N-terminal prosegment. The propeptides are potent inhibitors of their parent proteases. Although the proregion binding mode has been elucidated for all other protease classes, that of the cysteine proteases remained elusive. RESULTS: We report the three-dimensional structure of rat procathepsin B, determined at 2.8 A resolution. The 62-residue proregion does not form a globular structure on its own, but folds along the surface of mature cathepsin B. The N-terminal part of the proregion packs against a surface loop, with Trp24p (p indicating the proregion) playing a pivotal role in these interactions. Inhibition occurs by blocking access to the active site: part of the proregion enters the substrate-binding cleft in a similar manner to a natural substrate, but in a reverse orientation. CONCLUSIONS: The structure of procathepsin B provides the first insight into the mode of interaction between a mature cysteine protease from the papain superfamily and its prosegment. Maturation results in only one loop of cathepsin B changing conformation significantly, replacing contacts lost by removal of the prosegment. Contrary to many other proproteases, no rearrangement of the N terminus occurs following activation. Binding of the prosegment involves interaction with regions of the enzyme remote from the substrate-binding cleft and suggests a novel strategy for inhibitor design. The region of the prosegment where the activating cleavage occurs makes little contact with the enzyme, leading to speculation on the activation mechanism.

Animals↗
PubMed1996

Crystallization and preliminary X-ray diffraction studies of human procathepsin L.

Human procathepsin L has been expressed in the yeast Pichia pastoris and its inactive (Cys25Ser) and unglycosylated (Thr110Ala) mutant purified, concentrated to 4 mg/ml, and crystallized by vapor diffusion against solution containing 1.4 M (Na,K)PO4 buffer, pH 7.8. Crystal size was increased by multiple macroseeding. The crystals are orthorhombic, of space group P2 1 2 1 2 1, with cell dimensions of a = 40.2 A, b = 88.4 A, and c = 94.9 A. A 2.2 A native data set was collected using synchrotron radiation. Although molecular replacement solution for the mature portion of the enzyme was easily found, the resulting maps could not be interpreted in the proregion. Heavy-atom derivative search is in progress.

Cathepsin L↗
PubMed1996

Modulation of the catabolic effects of interleukin-1 beta on human articular chondrocytes by transforming growth factor-beta.

The effects of IL-1 beta and TGF-beta on the biosynthesis of extracellular matrix structural components relative to the metalloproteinases and their inhibitor TIMP1 in human articular chondrocytes were investigated. It has been proposed that TGF-beta, acting as a positive regulator of matrix accretion, can counteract the increased loss of cartilage matrix induced by IL-1 beta. To allow a comparison of their effects on mRNA levels for these different components, quantitation by competitive RT/PCR was employed. This method was found to give reproducible estimates of mRNA levels and the observed effects of IL-1 beta and TGF-beta on individual components of this system agree with qualitative data obtained by northern blotting. IL-1 beta had a more pronounced effect on aggrecan mRNA levels than on those for type II collagen. Similar quantitative differences were observed between collagenase and stromelysin mRNA levels. TGF-beta generally counteracted the effects of IL-1 beta, and new steady state levels were attained within 24 h. However, the reversal of IL-1 beta induced suppression of matrix protein mRNA levels appeared more effective than its suppression of the increase in stromelysin and collagenase mRNA levels. Similarly TGF-beta did not reduce the extent of IL-1 beta induced secretion of stromelysin at the protein level. TIMP1 mRNA levels were only slightly reduced by IL-1 beta; however this cytokine effectively suppressed its induction by TGF-beta. The higher concentrations of TGF-beta and longer exposure times required to overcome the suppressive effects of IL-1 beta suggest that the interaction between IL-1 beta and TGF-beta in the regulation of TIMP1 expression follows a different mechanism to that operating for the metalloproteinases and matrix proteins. Thus the overall potential of TGF-beta to inhibit proteolysis is attenuated by its much slower effect on TIMP1 mRNA levels.

Aggrecans↗
PubMed1996

Enhanced denaturation of the alpha (II) chains of type-II collagen in normal adult human intervertebral discs compared with femoral articular cartilage.

The mechanical strength of connective tissues is dependent on the integrity of their fibrillar collagen frameworks. The objective of the present study was to assess type-II collagen damage (denaturation) in the adult human intervertebral disc compared with articular cartilage, in order to determine whether damage to this molecule may vary in different anatomical sites in the same person. A new immunochemical assay was used to measure the amounts of denatured and total type-II collagen in the annulus fibrosus and nucleus pulposus of the L5-S1 disc and in cartilage from the femoral condyles of the same individuals (n = 7). Denaturation of type-II collagen was significantly higher in both the annulus fibrosus and the nucleus pulposus than in articular cartilage. Such increased damage to type-II collagen in the adult disc may have relevance to the more pronounced degenerative changes observed in this tissue compared with articular cartilage.

Adult↗
PubMed1996

Changes with age in the structure of fibromodulin in human articular cartilage.

An anti-peptide antibody was raised in a rabbit against the carboxy terminal region of the human fibromodulin core protein. The antibody was purified from other components of the resulting antiserum by affinity chromatography using the immobilized peptide, and was used to study the structural heterogeneity of fibromodulin extracted from human articular cartilage of different ages by the use of immunoblotting following sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the extracted macromolecules. In the fetus and neonate, fibromodulin was visualized as a diffuse component with a relative molecular weight of 70-110 kDa, whereas in the mature adult a more discrete component of smaller size was apparent with a relative molecular weight of 67 kDa. The size of the fibromodulin from mature adult cartilage could not be altered by pretreatment of the samples with keratanase II or endo-beta-galactosidase before analysis. In contrast, the size of the fibromodulin from younger cartilage could be decreased with both glycosidases, with the endo-beta-galactosidase yielding a smaller product than the keratanase. The size of the product resulting from endo-beta-galactosidase treatment of the fibromodulin from young cartilage was the same as that of the intact fibromodulin from mature adult cartilage. Thus, fibromodulin is present in human articular cartilage at all ages, but the extracted molecules only appear to exist in a proteoglycan form possessing keratan sulfate chains in the juvenile and young adult, and the size of these chains decreases with age. In the mature adult the fibromodulin does not possess either keratan sulfate or non-sulfated polylactosamine chains, though it appears to possess the same number of N-linked oligosaccharides as its counterparts from the younger tissue, but they are not modified further. The majority of the fibromodulin extracted from arthritic cartilage is of the same size as that found in the normal mature adult, although there is evidence for proteolytic processing. The degree of such processing is greater for the fibromodulin obtained from the cartilage of rheumatoid arthritic joints than osteoarthritic joints.

Acetylglucosaminidase↗
PubMed1995

Proteolysis of glucagon within hepatic endosomes by membrane-associated cathepsins B and D.

The acidic glucagon-degrading activity of hepatic endosomes has been attributed to membrane-bound forms of cathepsins B and D. Endosomal lysates processed full-length nonradiolabeled glucagon to 32 different peptides that were identified by amino acid analysis and full-length sequencing. These indicated C-terminal carboxypeptidase, endopeptidase as well as N-terminal tripeptidyl-aminopeptidase activities in endosomes. Glucagon proteolysis was inhibited 95% by E-64 and pepstatin A, inhibitors of cathepsins B and D, respectively. This was confirmed by the pH 6-dependent chemical cross-linking of [125I]iodoglucagon to a polypeptide of 30 kDa, which was immunodepleted by polyclonal anti-cathepsin B antibody, and the removal of greater than 80% of glucagon-degrading activity by polyclonal antibodies to cathepsins B and D. By similar criteria, insulin-degrading enzyme was ruled out as a candidate enzyme for endosomal proteolysis of glucagon. Lysosomal contamination was unlikely since all forms of cathepsin B in endosomes, i.e. the major 45-kDa inactive precursor as well as the lesser amounts of the 32- and 28-kDa active forms, were tightly bound to endosomal membranes. Furthermore the mature 29-kDa single-chain and 22-kDa heavy-chain forms of cathepsin L were undetectable in endosomes, although high levels of the 37-kDa proform were observed. Membrane association of the cathepsins B and D was not to the mannose 6-phosphate receptor since association was unaffected by mannose 6-phosphate and/or EDTA, thereby indicating a distinct endosomal receptor. Hence, a pool of active cathepsins B and D as well as a poorly defined tripeptidyl aminopeptidase is maintained in endosomes by selective membrane retention. These hydrolases degrade glucagon internalized into liver parenchyma early in endocytosis.

Amino Acid Sequence↗
PubMed1995

Crystal structures of recombinant rat cathepsin B and a cathepsin B-inhibitor complex. Implications for structure-based inhibitor design.

The lysosomal cysteine proteinase cathepsin B (EC 3.4.22.1) plays an important role in protein catabolism and has also been implicated in various disease states. The crystal structures of two forms of native recombinant rat cathepsin B have been determined. The overall folding of rat cathepsin B was shown to be very similar to that of the human liver enzyme. The structure of the native enzyme containing an underivatized active site cysteine (Cys29) showed the active enzyme conformation to be similar to that determined previously for the oxidized form. In a second structure Cys29 was derivatized with the reversible blocking reagent pyridyl disulfide. In this structure large side chain conformational changes were observed for the two key catalytic residues Cys29 and His199, demonstrating the potential flexibility of these side chains. In addition the structure of the complex between rat cathepsin B and the inhibitor benzyloxycarbonyl-Arg-Ser(O-Bzl) chloromethylketone was determined. The complex structure showed that very little conformational change occurs in the enzyme upon inhibitor binding. It also allowed visualization of the interaction between the enzyme and inhibitor. In particular the interaction between Glu245 and the P2 Arg residue was clearly demonstrated, and it was found that the benzyl group of the P1 substrate residue occupies a large hydrophobic pocket thought to represent the S'1 subsite. This may have important implications for structure-based design of cathepsin B inhibitors.

Amino Acid Chloromethyl Ketones↗
PubMed1995

Monoclonal antibodies that specifically recognize neoepitope sequences generated by 'aggrecanase' and matrix metalloproteinase cleavage of aggrecan: application to catabolism in situ and in vitro.

Monoclonal antibodies have been prepared that react specifically with the neoepitopes present on proteoglycan degradation products generated from the proteolytic cleavage of aggrecan in the interglobular domain. Antibody BC-3 recognizes the new N-terminus (ARGSV...) on aggrecan degradation products produced by the action of the as yet uncharacterized proteolytic activity, 'aggrecanase', and antibody BC-4 recognizes the new C-terminus (...DIPEN) generated by the proteolytic action of matrix metalloproteinases. Specificity for these neoepitope sequences was determined in competitive e.l.i.s.a. using synthetic peptide antigens as inhibitors. Antibody BC-3 was used in the detection of aggrecan degradation products in the culture medium obtained from two different in vitro culture systems: bovine cartilage explants treated with either retinoic acid or interleukin-1, and secondly, rat chondrosarcoma cells treated with retinoic acid. Both interleukin-1 and retinoic acid treatment caused an increase in aggrecan catabolism resulting in an increased release to the medium of specific aggrecan degradation products containing the BC-3 neoepitope generated by the action of 'aggrecanase'. However, several additional aggrecan catabolites were present that were not immunoreactive with antibody BC-3. In addition, under control conditions, in the bovine cartilage cultures the BC-3 epitope was found on some of these aggrecan catabolites. In contrast, no immune-reactive material was found in the aggrecan degradation products present in control media of rat chondrosarcoma cells cultured in the absence of retinoic acid. Collectively, these results demonstrate that 'aggrecanase' activity is not a constitutive event in all cartilage culture systems and also suggest that proteolytic agents other than 'aggrecanase' are involved in aggrecan catabolism in normal turnover compared with pathological conditions. Antibody BC-4 was used to demonstrate the identity of the G1 domain of aggrecan following proteolytic cleavage of a purified G1-G2 preparation with collagenase, gelatinase A or stromelysin. The G2 product of this cleavage did not react with antibody BC-3, indicating that, under the experimental conditions used, none of these enzymes exhibited 'aggrecanase' activity. It is expected that both of these antibodies will play a pivotal role in detailed studies elucidating molecular mechanisms of aggrecan degradation and they will be particularly useful for the sensitive monitoring of aggrecan degradation products in tissue extracts and body fluids.

Aggrecans↗
PubMed1995

Modification of S1 subsite specificity in the cysteine protease cathepsin B.

Cysteine proteases of the papain family generally exhibit broad P1 specificity. A notable exception is papaya proteinase IV (PPIV), which only accepts Gly at this position. In all other cysteine proteases the S1 subsite residues 23 and 65 (papain numbering) are absolutely conserved as Gly, while in PPIV they are replaced by Glu and Arg, respectively. These differences appear to underlie both PPIV specificity and its resistance to inhibition by cystatins. To test this hypothesis, the equivalent residues (Gly27 and Gly73) in the mammalian cysteine protease cathepsin B were changed to Glu and Arg, respectively. Relative to the wild-type enzyme, the Gly27Glu and Gly73Arg mutants showed a drastic reduction in activity with substrates containing a P1 Arg. In contrast, substrates having a Gly residue in P1 were hydrolyzed effectively. The double mutant (Gly27Glu:Gly73Arg) exhibited no detectable activity against any substrate studied. Inhibition of the Gly73Arg mutant by E-64 [1-(L-trans-epoxysuccinyl-L-leucylamino)-4-guanidinobutane] was found to be similar to that of the wild-type enzyme. In contrast, inhibition by cystatin C exhibited a 20,000-fold reduction. These results demonstrate the dramatic influence of side chains at sequence locations 27 and 73 on the S1 subsite specificity of cysteine proteases.

Animals↗
PubMed1995

The septoclast, a cathepsin B-rich cell involved in the resorption of growth plate cartilage.

At the transition between growth plate cartilage and the endochondral bone region, transverse septa are being eroded to allow the advance of invasive capillaries. To find out whether resorption is due to proteinase activity, tissue sections prepared from the growth plate/metaphyseal interface of young rats were immunostained with antibodies to the cysteine proteinase cathepsin B. Intense staining was found in a cell that is associated with the growing portion of the invasive capillaries and extends between them and the transverse septum. This cell has a single nucleus, actively synthesizes protein, and shows two other characteristic features: the cytoplasm is packed with multivesicular and dense bodies rich in cathepsin B, and the cell apex ends in a ruffled border extending into the transverse septum and signs of dissociated extracellular matrix. Even though the ruffled border resembles that of the osteoclast, the cell was not immunostained by a monoclonal antibody that recognizes a 97 KD protein known as ED1 which characterizes rat osteoclasts, monocytes, and macrophages. Therefore, this distinctive cell produces the proteinase cathepsin B and appears to be involved in the resorption of the transverse septum. The cell has been named the "septoclast."

Animals↗
PubMed1994

Maturation of human procathepsin B. Proenzyme activation and proteolytic processing of the precursor to the mature proteinase, in vitro, are primarily unimolecular processes.

Recombinant latent human procathepsin B produced in yeast was purified to near homogeneity. The purified recombinant proenzyme is activated in vitro under acidic conditions resulting in rapid conversion into the mature form of the proteinase. Activation as well as proteolytic maturation of the recombinant cathepsin B precursor were shown to be primarily concentration-independent processes indicating a unimolecular (i.e. intramolecular) mechanism. Only one cleavage site was identified, yielding a mature polypeptide with the same amino-terminal sequence as that found in recombinant active human cathepsin B obtained from yeast culture media. The same peptide bond is cleaved during processing of a nonactivatable mutant of procathepsin B by the purified mature enzyme (i.e. intermolecular processing). Thus, the complete proregion is liberated during procathepsin B processing. This peptide may then act as a reversible inhibitor and stabilizer of the mature proteinase, and it appears likely that cathepsin B-propeptide complexes occur transiently during proteolytic maturation.

Amino Acid Sequence↗
PubMed1994

Noncovalent complexes between the lysosomal proteinase cathepsin B and its propeptide account for stable, extracellular, high molecular mass forms of the enzyme.

Although the lysosomal cysteine proteinase cathepsin B is alkaline pH-labile, active, stable high molecular mass forms have been reported previously from the culture medium of human and murine mammary tumor explants and the sputum of patients with purulent bronchiectasis. A similar, catalytically active, high molecular mass form of recombinant human cathepsin B produced in yeast has now been found to represent a noncovalent complex between the 30-kDa single chain enzyme and its 6-kDa propeptide formed during autocatalytic maturation of the proenzyme (see accompanying article; Mach, L., Mort, J. S., and Glössl, J. (1994) J. Biol. Chem. 269, 13030-13035). Incubation of the complex under acidic conditions resulted in dissociation and degradation of the inhibitory propeptide leading to increased enzymatic activity, as also observed for partially purified cathepsin B isoenzymes from purulent sputum and mammary tumor explant media. The stabilization of the processed proteinase as a noncovalent complex with its proregion provides an important mechanism whereby extracellular cathepsin B can lie dormant until regional acidification mediates its activity.

Amino Acid Sequence↗
PubMed1994

Evidence for the interaction of valine-10 in cystatin C with the S2 subsite of cathepsin B.

The interactions between wild-type or mutant recombinant forms of human cystatin C and rat cathepsin B were characterized by measuring progress curves for substrate hydrolysis in the presence of inhibitor. The investigation was guided by the use of computer modeling and explores the possibility that amino acid residues in the N-terminal region of cystatin C interact with substrate-binding regions in the target enzyme. With cystatin C that has Val-10 replaced by an Arg residue (Val10Arg cystatin C), the inhibition constant, K(i), increased 31-fold if the isosteric substitution Glu-245 to Gln was made in cathepsin B. When the wild-type form of the inhibitor was used, the corresponding effect on K(i) was less than 2-fold. In a similar study, using cathepsin B in which the substitution to Gln is instead at Glu-171, no such difference in how K(i) is affected was observed. Both Glu-245 and Glu-171 are located in the S2 subsite of cathepsin B. The observed effects on K(i) indicate that the additional positive charge introduced in Val10Arg cystatin C is interacting with the negative charge on Glu-245 in cathepsin B when these two proteins form a complex; the cystatin variant is thus binding in a substratelike manner with this region of the enzyme. Indirectly, these results suggest that when native cystatin C and cathepsin B form a complex, Val-10 in the inhibitor interacts with the S2 subsite of the enzyme. A K(i) value of 0.13 nM was obtained for the interaction of Val10Arg cystatin C with papain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Differential changes in the association and dissociation rate constants for binding of cystatins to target proteinases occurring on N-terminal truncation of the inhibitors indicate that the interaction mechanism varies with different enzymes.

The importance of the N-terminal region of human cystatin C or chicken cystatin for the kinetics of interactions of the inhibitors with four cysteine proteinases was characterized. The association rate constants for the binding of recombinant human cystatin C to papain, ficin, actinidin and recombinant rat cathepsin B were 1.1 x 10(7), 7.0 x 10(6), 2.4 x 10(6) and 1.4 x 10(6) M-1.s-1, whereas the corresponding dissociation rate constants were 1.3 x 10(-7), 9.2 x 10(-6), 4.6 x 10(-2) and 3.5 x 10(-4) s-1. N-Terminal truncation of the first ten residues of the inhibitor negligibly affected the association rate constant with papain or ficin, but increased the dissociation rate constant approx. 3 x 10(4)- to 2 x 10(6)-fold. In contrast, such truncation decreased the association rate constant with cathepsin B approx. 60-fold, while minimally affecting the dissociation rate constant. With actinidin, the truncated cystatin C had both an approx. 15-fold lower association rate constant and an approx. 15-fold higher dissociation rate constant than the intact inhibitor. Similar results were obtained for intact and N-terminally truncated chicken cystatin. The decreased affinity of human cystatin C or chicken cystatin for cysteine proteinases after removal of the N-terminal region is thus due to either a decreased association rate constant or an increased dissociation rate constant, or both, depending on the enzyme. This behaviour indicates that the contribution of the N-terminal segment of the two inhibitors to the interaction mechanism varies with the target proteinase as a result of structural differences in the active-site region of the enzyme.

Animals↗
PubMed1993

Non-proteoglycan forms of biglycan increase with age in human articular cartilage.

Polyclonal anti-peptide antibodies were raised to the C-terminal regions of human biglycan and decorin. These antibodies were used in immunoblotting to study structural variations with age in the proteoglycan core proteins present in extracts of human articular cartilage and intervertebral disc. Three forms of the biglycan core protein were identified. The largest form was detected only after chondroitinase treatment and represents the proteoglycan form of the molecule from which the glycosaminoglycan chains have been removed. However, chondroitinase treatment did not alter the electrophoretic mobility of the two smaller proteins, which appear to represent non-proteoglycan forms of the molecule, resulting either from a failure to substitute the intact proteoglycan core protein with glycosaminoglycan chains during its synthesis or from proteolytic processing of the intact proteoglycan causing removal of the N-terminal region bearing the glycosaminoglycan chains. The non-proteoglycan forms constituted a minor proportion of biglycan in the newborn, but were the major components in the adult. A similar trend was seen in both articular cartilage and intervertebral disc. In comparison, decorin appears to exist predominantly as a proteoglycan at all ages, with two core protein sizes being present after chondroitinase treatment. Non-proteoglycan forms were detected in the adult, but they were always a minor constituent.

Adolescent↗
PubMed1993

Matrix metalloproteinases cleave at two distinct sites on human cartilage link protein.

The actions of human recombinant stromelysins-1 and -2, collagenase, gelatinases A and B and matrilysin on neonatal human proteoglycan aggregates were examined. With the exception of gelatinase B, aggrecan was degraded extensively by most metalloproteinases studied, whereas link protein showed only limited proteolysis. Sequencing studies of modified link protein components revealed that stromelysins-1 and -2, gelatinases A and B and collagenase cleaved specifically between His16 and Ile17, and matrilysin, stromelysin-2 and gelatinase A cleaved between Leu25 and Leu26. Cleavage at the former bond generated a link protein component with the same N-terminus as that isolated from newborn human cartilage. Based on previously determined in situ cleavage sites it is evident that matrix metalloproteinases are not solely responsible for the accumulation of link protein degradation products in adult human cartilage, indicating that additional proteolytic agents are involved in the normal catabolism of human cartilage matrix.

Aggrecans↗