Elastinolytic activity of human cathepsin L.
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ABSTRACT:
SUBMITTER: Mason RW
PROVIDER: S-EPMC1153120 | biostudies-other | 1986 Feb
REPOSITORIES: biostudies-other
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Project description:Galactosialidosis is a human lysosomal storage disease caused by deficiency in the multifunctional lysosomal protease cathepsin A (also known as protective protein/cathepsin A, PPCA, catA, HPP, and CTSA; EC 3.4.16.5). Previous structural work on the inactive precursor human cathepsin A (zymogen) led to a two-stage model for activation, where proteolysis of a 1.6-kDa excision peptide is followed by a conformational change in a blocking peptide occluding the active site. Here we present evidence for an alternate model of activation of human cathepsin A, needing only cleavage of a 3.3-kDa excision peptide to yield full enzymatic activity, with no conformational change required. We present x-ray crystallographic, mass spectrometric, amino acid sequencing, enzymatic, and cellular data to support the cleavage-only activation model. The results clarify a longstanding question about the mechanism of cathepsin A activation and point to new avenues for the design of mechanism-based inhibitors of the enzyme.
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Project description:Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.
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Project description:Cathepsin X is a lysosomal cysteine protease that functions as a carboxypeptidase with broad substrate specificity. Cathepsin X was discovered only recently, and its physiological roles are still not well understood. A number of studies suggest that cathepsin X may be involved in a variety of biological processes, including cancer, aging and degenerative conditions of the brain, inflammation, and cellular communication. Here we present the synthesis and characterization of several activity-based probes (ABPs) that target active cathepsin X. These ABPs were used to label cathepsin X in complex lysates, whole cells, and in vivo. Furthermore, we have developed a method for selectively labeling and visualizing active cathepsin X in vitro and in vivo. Overall, the probes developed in this study are valuable tools for the study of cathepsin X function.
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Project description:Cathepsin H was purified from human liver by a method involving autolysis and acetone fractionation, and chromatography on DEAE-cellulose, Ultrogel AcA 54, hydroxyapatite and concanavalin A-Sepharose. The procedure allowed for the simultaneous isolation of cathepsin B and cathepsin D. Cathepsin H was shown to consist of a single polypeptide chain of 28 000 mol.wt., and affinity for concanavalin A-Sepharose indicated that it was a glycoprotein. The enzyme existed in multiple isoelectric forms, the two major forms having pI values of 6.0 and 6.4; it hydrolysed azocasein (pH optimum 5.5), benzoylarginine 2-naphthylamide (Ba-Arg-NNap), leucyl 2-naphthylamide (Arg-NNap), (pH optimum 6.8). Arg-NNap and Arg-NMec, unlike Bz-Arg-NNap-, were not hydrolysed by human cathepsin B. Cathepsin H was similar to cathepsin B in being irreversibly inactivated by exposure to alkaline pH. Sensitivity to chemical inhibitors by 1 microM-leupeptin, which gave essentially complete inhibition of the other lysosomal cysteine proteinases, cathepsins B and L.
| S-EPMC1162239 | biostudies-other
Project description:Voltage-gated sodium channels (Na(V)) are functionally expressed in highly metastatic cancer cells derived from nonexcitable epithelial tissues (breast, prostate, lung, and cervix). MDA-MB-231 breast cancer cells express functional sodium channel complexes, consisting of Na(V)1.5 and associated auxiliary beta-subunits, that are responsible for a sustained inward sodium current at the membrane potential. Although these channels do not regulate cellular multiplication or migration, their inhibition by the specific blocker tetrodotoxin impairs both the extracellular gelatinolytic activity (monitored with DQ-gelatin) and cell invasiveness leading to the attenuation of colony growth and cell spreading in three-dimensional Matrigel-composed matrices. MDA-MB-231 cells express functional cysteine cathepsins, which we found play a predominant role ( approximately 65%) in cancer invasiveness. Matrigel invasion is significantly decreased in the presence of specific inhibitors of cathepsins B and S (CA-074 and Z-FL-COCHO, respectively), and co-application of tetrodotoxin does not further reduce cell invasion. This suggests that cathepsins B and S are involved in invasiveness and that their proteolytic activity partly depends on Na(V) function. Inhibiting Na(V) has no consequence for cathepsins at the transcription, translation, and secretion levels. However, Na(V) activity leads to an intracellular alkalinization and a perimembrane acidification favorable for the extracellular activity of these acidic proteases. We propose that Na(v) enhance the invasiveness of cancer cells by favoring the pH-dependent activity of cysteine cathepsins. This general mechanism could lead to the identification of new targets allowing the therapeutic prevention of metastases.
| S-EPMC2659227 | biostudies-literature
Project description:Surveillance colonoscopy using random biopsies to detect colitis-associated cancer (CAC) suffers from poor sensitivity. Although chromoendoscopy improves detection, acceptance in the community has been slow. Here, we examine the usefulness of near infrared fluorescence (NIRF) endoscopy to image molecular probes for cathepsin activity in colitis-induced dysplasia.In patient samples, cathepsin activity was correlated with colitis and dysplasia. In mice, cathepsin activity was detected as fluorescent hydrolysis product of substrate-based probes after injection into Il10(-/-) colitic mice. Fluorescence colonoscopy and colonic whole-mount imaging were performed before complete sectioning and pathology review of resected colons.Cathepsin activity was 5-fold and 8-fold higher in dysplasia and CAC, respectively, compared with areas of mild colitis in patient tissue sections. The signal-to-noise ratios for dysplastic lesions seen by endoscopy in Il10(-/-) mice were 5.2 ± 1.3 (P = 0.0001). Lesions with increased NIRF emissions were classified as raised or flat dysplasia, lymphoid tissue, and ulcers. Using images collected by endoscopy, a receiver operating characteristic curve for correctly diagnosing dysplasia was calculated. The area under the curve was 0.927. At a cutoff of 1000 mean fluorescence intensity, the sensitivity and specificity for detecting dysplasia were 100% and 83%, respectively. Analysis revealed that focally enhanced NIRF emissions derived from increased numbers of infiltrating myeloid-derived suppressor cells and macrophages with equivalent cathepsin activity.These studies indicate that cathepsin substrate-based probe imaging correctly identifies dysplastic foci within chronically inflamed colons. Combined white light and NIRF endoscopy presents unique advantages that may increase sensitivity and specificity of surveillance colonoscopy in patients with CAC.
| S-EPMC4306589 | biostudies-literature
Project description:Cathepsin L was purified to apparent homogeneity from human liver obtained post mortem. It was necessary to treat the homogenate at pH 4.2 and 37 degrees C to release active enzyme. The purification procedure involved ion-exchange chromatography on carboxymethyl-Sephadex and the Mono S column of a Pharmacia fast-protein-liquid-chromatography system. The enzyme was found to consist of two polypeptide chains of Mr 25 000 and 5000. The larger chain was shown to contain the active-site cysteine residue. Human cathepsin L proved to be similar to the rat and rabbit enzymes in regard to kinetic constants for the substrate benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide and rates of inactivation by the active-site-directed reagents benzyloxycarbonylphenylalanylphenylalanyldiazomethane and benzyloxycarbonylphenylalanylalanyldiazomethane. Thus clear characteristics of cathepsin L are now emerging, and these should simplify the identification of the enzyme in other tissues and species.
| S-EPMC1144697 | biostudies-other
Project description:Background & aimsAcute pancreatitis is characterized by an activation cascade of digestive enzymes in the pancreas. The first of these, trypsinogen, can be converted to active trypsin by the peptidase cathepsin B (CTSB). We investigated whether cathepsin L (CTSL) can also process trypsinogen to active trypsin and has a role in pancreatitis.MethodsIn CTSL-deficient (Ctsl(-/-)) mice, pancreatitis was induced by injection of cerulein or infusion of taurocholate into the pancreatic duct. Human tissue, pancreatic juice, mouse pancreatitis specimens, and recombinant enzymes were studied by enzyme assay, immunoblot, N-terminal sequencing, immunocytochemistry, and electron microscopy analyses. Isolated acini from Ctsl(-/-) and Ctsb(-/-) mice were studied.ResultsCTSL was expressed in human and mouse pancreas, colocalized with trypsinogen in secretory vesicles and lysosomes, and secreted into pancreatic juice. Severity of pancreatitis was reduced in Ctsl(-/-) mice, whereas apoptosis and intrapancreatic trypsin activity were increased. CTSL-induced cleavage of trypsinogen occurred 3 amino acids toward the C-terminus from the CTSB activation site and resulted in a truncated, inactive form of trypsin and an elongated propeptide (trypsinogen activation peptide [TAP]). This elongated TAP was not detected by enzyme-linked immunosorbent assay (ELISA) but was effectively converted to an immunoreactive form by CTSB. Levels of TAP thus generated by CTSB were not associated with disease severity, although this is what the TAP-ELISA is used to determine in the clinic.ConclusionsCTSL inactivates trypsinogen and counteracts the ability of CTSB to form active trypsin. In mouse models of pancreatitis, absence of CTSL induces apoptosis and reduces disease severity.
| S-EPMC2941736 | biostudies-literature