Specific Assay Method for the Activities of Cathepsin L-Type Cysteine Proteinases

Inubushi, Tomoko; Kakegawa, Hisao; Kawaguchi, Yasuo; Katunuma, Nobuhiko

doi:10.1093/oxfordjournals.jbchem.a124520

Cited by 45 publications

(37 citation statements)

References 13 publications

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“…These findings indicate that nippocystatin can inhibit cysteine proteases, like other members of the cystatin family. In order to investigate the function of nippocystatin in a more physiological condition, a crude mitochondrial lysosomal (ML) fraction obtained from mouse spleens was incubated with a substrate in the presence of rNbCys, and the activities of the cysteine proteases in the ML fraction were determined by a specific assay method (10). The activities of the cysteine proteases cathepsins L, B, and J/C in the ML fraction were specifically inhibited (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The amount of product was monitored fluorometrically with excitation at 370 nm and emission at 460 nm by using a fluorescence spectrometer (Hitachi, Ibaraki, Japan). The activities of the cysteine proteases in the mitochondrion-lysosome (ML) fraction of mouse spleens were measured by a specific assay method using E64 and CA074, as reported previously (10). The activity of cathepsin D was measured by a Folin-Lowry reaction method, as reported previously (25).…”

Section: Animalsmentioning

confidence: 99%

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Nippocystatin, a Cysteine Protease Inhibitor fromNippostrongylus brasiliensis, Inhibits Antigen Processing and Modulates Antigen-Specific Immune Response

et al. 2001

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During infection, parasites evade the host immune system by modulating or exploiting the immune system; e.g., they suppress expression of major histocompatibility complex class II molecules or secrete cytokine-like molecules. However, it is not clear whether helminths disturb the immune responses of their hosts by controlling the antigen-processing pathways of the hosts. In this study, we identified a new cysteine protease inhibitor, nippocystatin, derived from excretory-secretory (ES) products of an intestinal nematode, Nippostrongylus brasiliensis. Nippocystatin, which belongs to cystatin family 2, consists of 144 amino acids and is secreted as a 14-kDa mature form. In vivo treatment of ovalbumin (OVA)-immunized mice with recombinant nippocystatin (rNbCys) profoundly suppressed OVA-specific proliferation of splenocytes but not non-antigenspecific proliferation of splenocytes. OVA-specific cytokine production was also greatly suppressed in rNbCystreated mice. Although the serum levels of both OVA-specific immunoglobulin G1 (IgG1) and IgG2a were not affected by rNbCys treatment, OVA-specific IgE was preferentially downregulated in rNbCys-treated mice. In vitro rNbCys inhibited processing of OVA by lysosomal cysteine proteases from the spleens of mice. Mice with anti-nippocystatin antibodies became partially resistant to infection with N. brasiliensis. Based on these findings, N. brasiliensis appears to skillfully evade host immune systems by secreting nippocystatin, which modulates antigen processing in antigen-presenting cells of hosts.

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Section: Resultsmentioning

confidence: 99%

Section: Animalsmentioning

confidence: 99%

Nippocystatin, a Cysteine Protease Inhibitor fromNippostrongylus brasiliensis, Inhibits Antigen Processing and Modulates Antigen-Specific Immune Response

et al. 2001

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“…The soluble fraction was removed and co-incubated with 20 M N-CBZ-Phe-Arg-7-amido-4-methylcoumarin (Sigma), a substrate for cathepsins B and L, at 37°C for 10 min. To determine the specific contributions of the two proteases, other tubes were supplemented with 0.75 M CA-074 (Peptides International, Louisville, KY), a specific inhibitor of cathepsin B (Kakegawa et al, 1993;Inubushi et al, 1994). The activity of cathepsin L, determined as the fraction insensitive to CA-074, was completely suppressed when 0.25 M chymostatin (Sigma) was added to the assay.…”

Section: Methodsmentioning

confidence: 99%

Suppression of Cathepsins B and L Causes a Proliferation of Lysosomes and the Formation of Meganeurites in Hippocampus

Bednarski¹,

Ribak

Lynch³

1997

J. Neurosci.

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Cultured hippocampal slices exhibited prominent ultrastructural features of brain aging after exposure to an inhibitor of cathepsins B and L. Six days of treatment with N-CBZ-Lphenylalanyl-L-alanine-diazomethylketone (ZPAD) resulted in a dramatic increase in the number of lysosomes in the perikarya of neurons and glial cells throughout the slices. Furthermore, lysosomes in CA1 and CA3 pyramidal cells were not restricted to the soma but instead were located throughout dendritic processes. Clusters of lysosomes were commonly found within bulging segments of proximal dendrites that were notable for an absence of microtubules and neurofilaments. Although pyknotic nuclei were sometimes encountered, most of the cells in slices exposed to ZPAD for 6 d appeared relatively normal. Slices given 7 d of recovery contained several unique features, compared with those processed immediately after incubation with the inhibitor. Cell bodies of CA1 neurons were largely cleared of the excess lysosomes but had gained fusiform, somatic extensions that were filled with fused lysosomes and related complex, dense bodies. These appendages, similar in form and content to structures previously referred to as "meganeurites," were not observed in CA3 neurons or granule cells. Because meganeurites were often interposed between cell body and axon, they have the potential to interfere with processes requiring axonal transport. It is suggested that inactivation of cathepsins B and L results in a proliferation of lysosomes and that meganeurite generation provides a means of storing residual catabolic organelles. The accumulated material could be eliminated by pinching off the meganeurite but, at least in some cases, this action would result in axotomy. Reduced cathepsin L activity, increased numbers of lysosomes, and the formation of meganeurites are all reported to occur during brain aging; thus, it is possible that the infusion of ZPAD into cultured slices sets in motion a greatly accelerated gerontological sequence.

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“…Cathepsin L activities were measured with Z-Phe-Arg-MCA as substrate at pH 5.5 [13]. The enzyme fraction was preincubated with 10 7 M of CA-074 for 5 min at 37°C in 0.1 M sodium acetate buffer, pH 79 5.5, containing 8 mM cysteine and 1 mM EDTA.…”

Section: Enzyme Assaymentioning

confidence: 99%

Secretion and processing mechanisms of procathepsin L in bone resorption

Kakegawa¹,

Tagami²,

Ohba³

et al. 1995

FEBS Letters

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Secretion of procathepsin L into the culture medium from a bone cell mixture was markedly enhanced by addition of parathyroid hormone (PTH), 1 a,25-(OH)2D 3 or tumor necrosis factor a (TNFa). These stimulators of secretion of procathepsin L enhanced bone pit formation, which was inhibited by E-64, but not by CA-074, a specific inhibitor of cathepsin B. Procathepsin L may thus participate in the process of bone collagenolysis during bone resorption. Procathepsin L partially purified from rat long bones under cold conditions was rapidly converted to the mature form under acidic conditions at room temperature. This conversion was inhibited by E-64, suggesting that the procathepsin L secreted into lacunae is catalytically converted to the mature enzyme by cysteine proteinase(s).In the present study, designed to clarify the mechanism and regulation of both the secretion of procathepsin L and its processing in lacunae, we examined the secretion of procathepsin L induced by 10~,25-(OH)2D 3, TNFc~ or by PTH, and also the effects of the cysteine proteinase inhibitors, E-64 and CA-074, on the pit formation induced by these effectors. Furthermore, to clarify the participation of cysteine proteinase(s) in processing from the precursor to the active form, the inhibitory effect of E-64 on the processing of procathepsin L partially purified from rat long bones was also examined. Experimental

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Specific Assay Method for the Activities of Cathepsin L-Type Cysteine Proteinases

Cited by 45 publications

References 13 publications

Nippocystatin, a Cysteine Protease Inhibitor fromNippostrongylus brasiliensis, Inhibits Antigen Processing and Modulates Antigen-Specific Immune Response

Nippocystatin, a Cysteine Protease Inhibitor fromNippostrongylus brasiliensis, Inhibits Antigen Processing and Modulates Antigen-Specific Immune Response

Suppression of Cathepsins B and L Causes a Proliferation of Lysosomes and the Formation of Meganeurites in Hippocampus

Secretion and processing mechanisms of procathepsin L in bone resorption

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