Biomedical subjects

J S Mort

Publications and source records attributed to J S Mort.

At least 37 records · Page 2Linked to original sources

PubMed1999

Identification of UDP-N-acetylglucosamine-phosphotransferase-binding sites on the lysosomal proteases, cathepsins A, B, and D.

A key step in the targeting of soluble lysosomal enzymes is their recognition and phosphorylation by a 540 kDa multisubunit enzyme, UDP-N-acetylglucosamine-phosphotransferase (phosphotransferase). The molecular mechanism of recognition is still unknown, but previous experiments suggested that the phosphotransferase-binding sites on lysosomal proteins are represented by structurally conserved surface patches of amino acids. We identified four such regions on nonhomologous lysosomal enzymes, cathepsins A, B, and D, which were superimposed by rotating their structures around the Calpha atom of the glycosylated Asn residue. We proposed that these regions represent putative phosphotransferase-binding sites and tested synthetic peptides, derived from these regions on the basis of surface accessibility, for their ability to inhibit in vitro phosphorylation of purified cathepsins A, B, and D. Our results indicate that cathepsin A and cathepsin D have one closely related phosphotransferase recognition site represented by a structurally and topologically conserved beta-hairpin loop, similar to that previously identified in lysosomal beta-glucuronidase. The most potent inhibition of phosphorylation was demonstrated by homologous peptides derived from the regions located on cathepsin molecules opposite the oligosaccharide chains which are phosphorylated by the phosphotransferase. We propose that recognition and catalytic sites of the phosphotransferase are located on different subunits, therefore, providing an effective mechanism for binding and phosphorylation of lysosomal proteins of different molecular size.

Animals↗

PubMed1999

The use of cleavage site specific antibodies to delineate protein processing and breakdown pathways.

The hydrolysis of peptide bonds is an integral part of most physiological and pathological processes, yet knowledge is often lacking as to which peptide bonds are cleaved, in which protein substrates, in which order, and by which proteolytic enzymes. An increase in our understanding of these processes will enhance understanding of the pathogenesis underlying many diseases and might aid in the recognition of new targets for therapeutic intervention. This article reviews the development, design, and use of antibodies for the detection of specific peptide bond cleavage events, and describes how the application of such antibodies can increase our understanding of the roles played by proteolytic enzymes in physiology and pathology.

Antibodies↗

PubMed1998

Cathepsin B: an alternative protease for the generation of an aggrecan 'metalloproteinase' cleavage neoepitope.

Previously, only matrix metalloproteinases were believed capable of cleaving the cartilage proteoglycan, aggrecan, between Asn341 and Phe342, to yield a small G1 fragment terminating in the residues VDIPEN. We show that the combined endo- and exopeptidase activities of the cysteine protease, cathepsin B, also generate this epitope, suggesting that it should no longer be considered as an exclusive marker of metalloproteinase activity.

Aggrecans↗

PubMed1998

Lysine-based structure responsible for selective mannose phosphorylation of cathepsin D and cathepsin L defines a common structural motif for lysosomal enzyme targeting.

Previous studies have shown that lysine residues on the surface of cathepsins and other lysosomal proteins are a shared component of the recognition structure involved in mannose phosphorylation. In this study, the involvement of specific lysine residues in mannose phosphorylation of cathepsin D was explored by site-directed mutagenesis. Mutation of two lysine residues in the mature portion of the protein, Lys-203 and Lys-293, cooperated to inhibit mannose phosphorylation by 70%. Other positively charged residues could not substitute for lysine at these positions, and comparison of thermal denaturation curves for the wild type and mutant proteins indicated that the inhibition could not be explained by alterations in protein folding. Structural comparisons of the two lysine residues with those required for phosphorylation of cathepsin L, using models generated from recently acquired crystal structures, revealed several relevant similarities. On both molecules, the lysine residues were positioned approximately 34 A apart (34.06 A for cathepsin D and 33.80 A for cathepsin L). When the lysine pairs were superimposed, N-linked glycosylation sites on the two proteins were found to be oriented so that oligosaccharides extending out from the sites could share a common region of space. Further similarities in the local environments of the critical lysines were also observed. These results provide details for a common lysosomal targeting structure based on a specific arrangement of lysine residues with respect to each other and to glycosylation sites on the surface of lysosomal proteins.

Animals↗

PubMed1998

An NMR-based identification of peptide fragments mimicking the interactions of the cathepsin B propeptide.

Selected fragments of the 62-residue proregion (or residues 1p-62p) of the cysteine protease cathepsin B were synthesized and their interactions with cathepsin B studied by use of proton NMR spectroscopy. Peptide fragments 16p-51p and 26p-51p exhibited differential perturbations of their proton resonances in the presence of cathepsin B. These resonance perturbations were lost for the further truncated 36p-51p fragment, but remained in the 26p-43p and 28p-43p peptide fragments. Residues 23p-26p or TWQ25A in the N-terminal 1p-29p fragment did not show cathepsin B-induced resonance perturbations although the same residues had strongly perturbed proton resonances within the 16p-51p peptide. Both the 1p-29p and 36p-51p fragments lack a common set of hydrophobic residues 30p-35p or F30YNVDI35 from the proregion. The presence of residues F30YNVDI35 appears to confer a conformational preference in peptide fragments 16p-51p, 26p-51p, 28p-43p and 26p-43p, but the same residues induce the aggregation of peptides 16p-36p and 1p-36p. The peptide fragment 26p-43p binds to the active site, as indicated by its inhibition of the catalytic activity of cathepsin B. The cathepsin B prosegment can therefore be reduced into smaller, but functional subunits 28p-43p or 26p-43p that retain specific binding interactions with cathepsin B. These results also suggest that residues F30YNVDI35 may constitute an essential element for the selective inhibition of cathepsin B by the full-length cathepsin B proregion.

Cathepsin B↗

PubMed1998

Autocatalytic processing of recombinant human procathepsin L. Contribution of both intermolecular and unimolecular events in the processing of procathepsin L in vitro.

The autocatalytic processing of procathepsin L was investigated in vitro using purified recombinant proenzyme expressed in Pichia pastoris. Pure intermolecular processing was studied by incubating the mutant procathepsin L (C25S), which cannot autoactivate with a small amount of mature active cathepsin L. The results clearly establish that, contrary to recent reports, intermolecular processing of procathepsin L is possible. The main cleavage sites are located at or near the N terminus of the mature enzyme, in an accessible portion of the proregion, which contains sequences corresponding to the known substrate specificity of cathepsin L. Contrary to procathepsins B, K, and S, autocatalytic processing of procathepsin L can generate the natural mature form of the enzyme. A continuous assay using the substrate benzyloxycarbonyl-Phe-Arg 4-methylcoumarinyl-7-amide hydrochloride has also been used to obtain information on the nature of the steps involved in the autocatalytic processing of wild-type procathepsin L. Processing is initiated by decreasing the pH from 8.0 to 5.3. The influence of proenzyme concentration on the rate of processing indicates the existence of both unimolecular and bimolecular steps in the mechanism of processing. The nature of the unimolecular event that triggers processing remains elusive. Circular dichroism and fluorescence measurements indicate the absence of large scale conformational change in the structure of procathepsin L on reduction of pH. However, the bimolecular reaction can be attributed to intermolecular processing of the zymogen.

Amino Acid Sequence↗

PubMed1998

Two-step mechanism of inhibition of cathepsin B by cystatin C due to displacement of the proteinase occluding loop.

Stopped-flow kinetics showed that the inhibition of the lysosomal cysteine proteinase, cathepsin B, by its endogenous inhibitor, cystatin C, occurs by a two-step mechanism, in which an initial, weak interaction is followed by a conformational change. The initial interaction most likely involves binding of the N-terminal region of the inhibitor to the proteinase. Considerable evidence indicates that the subsequent conformational change is due to the inhibitor displacing the occluding loop of the proteinase that partially obscures the active site. The presence of this loop, which allows the enzyme to function as an exopeptidase, thus complicates the inhibition mechanism, rendering cathepsin B much less susceptible than other cysteine proteinases to inhibition by cystatins.

Binding Sites↗

PubMed1998

Immunolocalization of the cleavage of the aggrecan core protein at the Asn341-Phe342 bond, as an indicator of the location of the metalloproteinases active in the lysis of the rat growth plate.

In view of the extensive lysis of hyaline cartilage known to take place during endochondral bone formation, the current study was designed to test the hypothesis that metalloproteinases are the agents that mediate this lysis. Since these enzymes have been shown in vitro to cleave the core protein of the major proteoglycan of cartilage, aggrecan, at the Asn341-Phe342 bond, an immunohistochemical method has been developed to find out whether or not there are sites in the growth plate of the rat tibia where cleavage of this bond takes place. The cleavage of aggrecan by metalloproteinases is followed by the retention of the fragment known as G1, for it includes the G1 domain. Since the G1 fragment terminates in the amino acid residues ...FVDIPEN, we prepared an antiserum against FVDIPEN, confirmed its specificity, then applied it to the growth plate of 21-day-old rat tibia in the hope of localizing the G1 fragments. The antiserum specificity was shown by its recognition of the ...FVDIPEN sequence at the C-terminus of peptides and of G1 fragments produced by aggrecan cleavage. When the antiserum was applied to Western blots of guanidinium chloride extracts prepared from epiphyseal growth plate, it recognized two species (56 and 52 kDa), which differed only in the degree of glycosylation. These fragments were comparable in size to the G1 fragments generated by the action of recombinant metalloproteinase in vitro, thus confirming antiserum specificity for these fragments. Applying the antiserum to cryosections of 21-day-old rat tibiae revealed immunostaining at two intensities within the growth plate matrix: a strong staining was observed in a 1-5 microm-wide layer designated "peripheral" matrix, which borders the epiphyseal and metaphyseal marrow spaces as well as the perichondrium, while a weak staining was found in the rest of the plate, designated "central" matrix. The abundance of G1 fragments terminating in ...FVDIPEN in the peripheral matrix indicates that this is where the growth plate is lysed to achieve longitudinal and latitudinal bone growth. The site where metalloproteinases exert their main lytic activity is a thin layer of matrix separating central from peripheral matrix.

Aggrecans↗

PubMed1997

Aggrecan degradation in human intervertebral disc and articular cartilage.

Aggrecan degradation in human intervertebral disc and articular cartilage has been studied by using anti-neoepitope antibodies specific for the N-terminal degradation products generated by cleavage within the interglobular domain at the metalloproteinase and aggrecanase sites. Immunoblot analysis of extracts of annulus fibrosus, nucleus pulposus and articular cartilage demonstrated age-related patterns in the abundance of both degradation products. In all three tissues the metalloproteinase-generated fragment was present at very low levels in young individuals but increased in abundance with age. In the disc tissues, the abundance of this degradation product levelled off in the juvenile; for cartilage this occurred in early adulthood. Despite these temporal differences, the levels attained in adults were comparable for the three tissues. In contrast, the aggrecanase-generated degradation product exhibited tissue-specific differences in the variation of its abundance with age. Whereas this degradation product increased with age in annulus fibrosus and articular cartilage and had levelled off by adulthood, in nucleus pulposus it was present in greatest abundance in young individuals and decreased to very low levels with age. Examination of discs exhibiting various degrees of degeneration did not reveal any differences in the levels of the metalloproteinase and aggrecanase-generated cleavage products that could not be accounted for by differences in age. In adults the product of aggrecanase action was much more abundant in articular cartilage than in either of the disc tissues, despite the age-related increase also observed for annulus fibrosus. Analysis of tissue extracts with an antibody recognizing the G1 domain of aggrecan identified two major degradation products whose abundance and size were correlated with the fragments detected by the anti-neoepitope antibodies. Taken together, these results indicate that cleavage at the metalloproteinase and aggrecanase sites are quantitatively important events in aggrecan catabolism in both articular cartilage and intervertebral disc in vivo. Moreover the two enzyme systems act independently and exhibit differences in the degree to which they contribute to aggrecan degradation in these tissues.

Adolescent↗

PubMed1997

Role of the occluding loop in cathepsin B activity.

Within the lysosomal cysteine protease family, cathepsin B is unique due to its ability to act both as an endopeptidase and a peptidyldipeptidase. This latter capacity to remove C-terminal dipeptides has been attributed to the presence of a 20-residue insertion, termed the occluding loop, that blocks the primed terminus of the active site cleft. Variants of human procathepsin B, where all or part of this element was deleted, were expressed in the yeast Pichia pastoris. A mutant, where the 12 central residues of the occluding loop were deleted, autoprocessed, albeit more slowly than the wild type proenzyme, to yield a mature form of the enzyme with endopeptidase activity comparable with the wild-type cathepsin B, but totally lacking exopeptidase activity. This deletion mutant showed a 40-fold higher affinity for the inhibitor cystatin C, suggesting that the occluding loop normally restricts access of this inhibitor to the active site. In addition, the binding affinity of the cathepsin B propeptide, which is a potent inhibitor of this enzyme, was 50-fold increased, consistent with the finding that the loop reorients on activation of the proenzyme. These results suggest that the endopeptidase activity of cathepsin B is an evolutionary remnant since, as a consequence of its membership in the papain family, the propeptide must be able to bind unobstructed through the full length of the active site cleft.

Amino Acid Sequence↗

PubMed1997

Macrophage cathepsin L, a factor in the erosion of subchondral bone in rheumatoid arthritis.

OBJECTIVE: To test the hypothesis that the proteinase cathepsin L is involved in the subchondral bone lesions found in chronic rheumatoid arthritis (RA). METHODS: The medial tibial plateaus from 4 control cases and 30 patients diagnosed as having end-stage RA were examined immunochemically for cathepsin L. RESULTS: RA lesions include large groups of mononuclear cells, many of which are rich in cathepsin L. Since these mononuclear cells contained the CD68 glycoprotein and, in the electron microscope, displayed an irregular cell surface, cytoplasmic vacuoles, lysosomes, and phagosomes, they were identified as belonging to the macrophage family. The lesions were classified into 2 main patterns, both displaying these cathepsin L-rich cells, which, in at least 1 of the 2, were closely associated with bone degradation. CONCLUSION: The cathepsin L-rich macrophages are sufficiently numerous to be considered a major factor in producing the erosion of subchondral bone found in chronic RA lesions.

Adult↗

PubMed1997

Proregion structure of members of the papain superfamily. Mode of inhibition of enzymatic activity.

Papain-like cysteine proteases have been divided into two subfamilies represented by mammalian enzymes cathepsin L and cathepsin B, respectively. The recent determination of the three-dimensional structures of four cysteine protease proenzymes showed that the mechanism of inhibition of the activity by the proregions is the same in both subfamilies despite significant differences in the proregion lengths. Here we describe the structures of the proregions, their binding to cognate enzymes and analyze similarities and differences between them.

Amino Acid Sequence↗

PubMed1997

Cathepsin B.

Cathepsin B is a lysosomal cysteine protease of the papain family. It functions in intracellular protein catabolism and in certain situations may also be involved in other physiological processes, such as processing of antigens in the immune response, hormone activation and bone turnover. There is also evidence that cathepsin B is implicated in the pathology of chronic inflammatory diseases of airways and joints, and in cancer and pancreatitis. In this short review we outline the major structural features of the enzyme, and describe how these relate to its synthesis, trafficking, processing and function.

Animals↗

PubMed1997

Inhibition of carcinoma cell invasion and liver metastases formation by the cysteine proteinase inhibitor E-64.

Cysteine proteinases, in particular cathepsins B and L, have been implicated in tumor invasion and are thought to be important mediators of metastasis. Using two clonal sublines of the Lewis lung carcinoma with distinct patterns of metastasis, we previously reported that H-59 carcinoma cells, which are highly invasive and preferentially metastatic to the liver, express high levels of cathepsin L and lower levels of cathepsin B whereas M-27 cells which are less invasive and only moderately metastatic to the lung express cathepsin B only. In the present study, the role of these enzymes in invasion and metastasis, in particular the involvement of cysteine proteinases in liver metastasis of H-59 cells was further investigated. Using a reconstituted basement membrane (Matrigel) invasion assay we found that the cysteine proteinase inhibitor, E-64, blocked the invasion of H-59 cells under conditions which did not affect cell viability. A more minor but significant inhibitory effect (up to 32%) was also seen with the propeptide of cathepsin B, implicating this enzyme in the invasion process. Furthermore, treatment of H-59 cells with E-64 inhibited experimental liver metastases formation by up to 90%. On the other hand, invasion of M-27 cells could not be blocked by cysteine proteinase inhibitors even under conditions which resulted in complete abrogation of intracellular enzymatic activity, as assessed using synthetic substrates. Together, these results confirm our previous conclusion that the two carcinoma sublines utilize distinct proteolytic mechanisms for invasion and identify the cysteine proteinases as key mediators of H-59 carcinoma invasion and metastasis.

Animals↗

PubMed1996

Structure of human procathepsin L reveals the molecular basis of inhibition by the prosegment.

Cathepsin L is a member of the papain superfamily of cysteine proteases and, like many other proteases, it is synthesized as an inactive proenzyme. Its prosegment shows little homology to that of procathepsin B, whose structure, the first for a cysteine protease proenzyme, has been determined recently. We report here the 3-D structure of a mutant of human procathepsin L determined at 2.2 A resolution, describe the mode of binding employed by the prosegment and discuss the molecular basis for other possible roles of the prosegment. The N-terminal part of the prosegment is globular and contains three alpha-helices with a small hydrophobic core built around aromatic side chains. This domain packs against a loop on the enzyme's surface, with the aromatic side chain from the prosegment being located in the center of this loop and providing a large contact area. The C-terminal portion of the prosegment assumes an extended conformation and follows along the substrate binding cleft toward the N-terminus of the mature enzyme. The direction of the prosegment in the substrate binding cleft is opposite to that of substrates. The previously described role of the prosegment in the interactions with membranes is supported by the structure of its N-terminal domain. The fold of the prosegment and the mechanism by which it inhibits the enzymatic activity of procathepsin L is similar to that observed in procathepsin B despite differences in length and sequence, suggesting that this mode of inhibition is common to all enzymes from the papain superfamily.

Amino Acid Sequence↗

PubMed1996

Deacylation and reacylation for a series of acyl cysteine proteases, including acyl groups derived from novel chromophoric substrates.

In order to investigate structure-reactivity relationships within a series of acyl cysteine proteases [Doran, J. D., & Carey, P. R. (1996) Biochemistry 35, 12495-12502], deacylation kinetics have been measured for a number of acyl intermediates involving members of the papain superfamily. Derivatives of the "simple" chromophoric ligand (5-methylthienyl)acrylate (5MTA) and those based on two chromophorically labeled derivatives of peptidyl substrates, viz., 2-[(N-acetyl-L-phenylalanyl)amino]-3-(5-methylthienyl)acrylate (Phe5MTA) and 2-[(N-acetyl-L-alanyl)amino]-3-(5-methylthienyl)acrylate (Ala5MTA), were used to create acyl enzyme adducts with papain, cathepsin B, and cathepsin L. The chromophoric specific substrates were designed to utilize hydrogen-bonding and hydrophobic interactions which are known to be important in promoting catalysis by papain. For cathepsins B and L, removing one of the hydrogen-bonding donors making up the putative oxyanion hole retards deacylation by 3-25-fold, demonstrating that the oxyanion hole has a modest effect on catalysis for these substrates. With the above substrates and the wild-type and oxyanion hole mutants, the values of the deacylation rate constants stretch over a 214-fold range, from 0.07 to 15 x 10(-3) s-1. The pKa for deacylation of [(5-methylthienyl)-acryloyl]papain is 4.9, close to that reported for similar papain intermediates, while that for Ala5MTA-papain is at 3.5, which in the latter likely represents the effect of the P1-S1 and P2-S2 interactions on the environment of histidine-159. For the Phe5MTA-papain the extent of deacylation was found to depend on the pH and the starting acyl enzyme concentration. A simple model has been derived which accounts quantitatively for this behavior, using the assumptions that the protonated form of the acyl product reacylates the enzyme and that in the pH range 5.0-7.5 the ionization of active-site groups has no effect on reacylation. The validity of the first assumption was demonstrated by following the deacylation of Phe5MTA-papain in the presence of the potent inhibitor E-64 [1-(L-trans-epoxysuccinyl-L-leucylamino)-4-guanidinobutane], whereupon complete deacylation occurred at all pHs with a pKa identical to that for Ala5MTA-papain, viz., 3.5.

Acrylates↗

PubMed1996

Presence of pro-forms of decorin and biglycan in human articular cartilage.

The proteoglycans decorin and biglycan in extracts of human articular cartilage were analysed by SDS/PAGE and immunoblotting, using antisera raised to peptide sequences present in the pro-regions and the mature core proteins. In adult cartilage, both pro-forms and mature processed forms of the proteoglycan core protein were observed for both decorin and biglycan. In the case of biglycan, it was also shown that additional proteolytic processing takes place after removal of the propeptide and that this accounts for the presence of non-glycanated forms of the molecule. For both decorin and biglycan, the relative abundance of the pro-forms was much less in the juvenile than the adult. Different adult connective tissues, including meniscus, tendon and intervertebral disc were also examined for the presence of pro-forms of the proteoglycans. While the mature form of decorin was present at a similar level in extracts of all tissues examined, the pro-form was only detected in the articular cartilage. In the case of biglycan, the abundance of the mature form was more varied, with high levels in articular cartilage, intermediate levels in meniscus and the annulus fibrosus of the intervertebral disc, low levels in the nucleus pulposus of the intervertebral disc, and non-detectable levels in the patellar tendon. The pro-form of biglycan was detected in the disc tissue extracts, albeit at a lower level than in articular cartilage, but was not detected in the meniscus or tendon. The proportion of the pro-form relative to the mature form of biglycan was, however, higher in the nucleus pulposus of the intervertebral disc than in articular cartilage. Thus, the persistence of pro-forms of both decorin and biglycan is a feature of the extracellular matrix of some connective tissues, although their abundance is both tissue- and age-dependent, with adult articular cartilage being a particularly rich source.

Adult↗

PubMed1996

Crystallization of rat procathepsin B.

Rat procathepsin B has been expressed in the yeast Pichia pastoris. To facilitate crystallization of the proform two mutations were introduced: Cys29Ser to avoid self-processing and Ser115Ala to eliminate an N-glycosylation site. The recombinant protein was purified and crystallized by vapor diffusion against mother liquor containing 100 mM KSCN, 100 mM phosphate buffer, pH 6.5 and polyethylene glycol (PEG) 3350 as a precipitating agent. Crystal size was increased by multiple macroseeding. At a 16% PEG concentration trigonal crystals were obtained, with the space group P3(1)21 and a = 99.6, c = 141.4 A, gamma = 120 degrees. They diffract to 2.8 A resolution using a rotating-anode source. At a concentration of 11% PEG, rod-shaped crystals were grown. They are monoclinic, space group P2(1), a = 62.8, b = 67.9, c = 100.4 A, beta = 98.2 degrees and diffract to approximately 3.5 A.

Journal Article↗