Inhibitory effect of cathepsin B inhibitor CA 074 on mouse anti-rat mixed lymphocyte reaction: novel immunosuppressive strategy for indirect xenoantigen recognition.
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Animals↗
Biomedical subjects
N Katunuma
Publications and source records attributed to N Katunuma.
A novel procaspase-3 activating cascade in liver lysosomes, and lack of the cascade in hepatoma cells.
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Carcinoma, Hepatocellular↗
Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain inovalbumin-immunized mice.
We previously reported that CA074, a specific inhibitor of cathepsin B, modulates specific immune responses from the T helper 2 (Th2) type to Th1 type in BALB/c mice infected with Leishmania major. In the present study, we found that a similar type of immune deviation was also induced in mice immunized with ovalbumin (OVA). However, treatment of mice with pepstatin A, a specific cathepsin D inhibitor, suppressed the OVA-specific proliferation of lymphocytes and blocked the development of both Th1 and Th2 cellular responses. These inhibitors did not appear to have any direct influence in vitro on functions of naive lymphocytes. OVA antigen (47 000 MW) was digested mainly into 40 000 MW protein in vitro by lysosomal proteases from naive BALB/c mice, and its digestion was markedly inhibited by the addition of CA074, but not by addition of pepstatin A, during incubation. However, pepstatin A strongly suppressed the degradation of the major histocompatibility complex class II-associated invariant chain (Ii) molecule in vivo and in vitro. Thus, cathepsin B appears to process antigens directed to preferential activation of Th2 cells, while cathepsin D may be responsible for the degradation of Ii, the processing of which is essential in initiating the antigen-specific activation of Th1 and Th2 CD4+ T cells. These lysosomal proteases may have different functions in regulating immune responses.
Animals↗
[Development of new specific inhibitors for cathepsins and its application for medical field].
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Animals↗
Inhibition mechanism of cathepsin L-specific inhibitors based on the crystal structure of papain-CLIK148 complex.
Papain was used as an experimental model structure to understand the inhibition mechanism of newly developed specific inhibitors of cathepsin L, the papain superfamily. Recently, we developed a series of cathepsin L-specific inhibitors which are called the CLIK series [(1999) FEBS Lett. 458, 6-10]. Here, we report the complex structure of papain with CLIK148, which is a representative inhibitor from the CLIK series. The inhibitor complex structure was solved at 1.7 A resolution with conventional R 0.177. Unlike other epoxisuccinate inhibitors (E64, CA030, and CA074), CLIK148 uses both prime and nonprime sites, which are important for the specific inhibitory effect on cathepsin L. Also, the specificity for cathepsin L could be explained by the existence of Phe in the P2 site and hydrophobic interaction of N-terminal pyridine ring.
Cathepsin L↗
Modulation of invasive properties of murine squamous carcinoma cells by heterologous expression of cathepsin B and cystatin C.
Murine SCC-VII squamous carcinoma cells have the capacity to penetrate reconstituted basement membranes (Matrigel) in vitro. The invasion of Matrigel layers by SCC-VII cells was significantly reduced by E-64, a specific inhibitor of lysosomal cysteine proteinases. The cathepsin-B-selective E-64 derivative, CA-074, inhibited penetration of Matrigel by SCC-VII cells to the same extent, indicating a major role for this particular lysosomal enzyme in extracellular-matrix degradation during squamous-carcinoma-cell invasion. SCC-VII cells were stably transfected with a cDNA encoding human procathepsin B, in an attempt to modulate the invasive properties of the cell line. The transfected cells expressed the heterologous gene, secreted increased amounts of procathepsin B and displayed enhanced invasive potential. In vivo, the activity of cathepsin B is strictly regulated by endogenous inhibitors. SCC-VII cells were therefore also stably transfected with a cDNA encoding human cystatin C, the most potent cysteine-proteinase inhibitor in mammalian tissues. The expression of this transgene resulted in the production of active recombinant cystatin C and a pronounced reduction in Matrigel invasion. These studies demonstrate that the invasive properties of squamous-cell carcinomas can be changed by modulation of the balance between cathepsin B and its endogenous inhibitors, and provide further evidence for the involvement of this lysosomal cysteine proteinase in tumour invasion and metastasis.
Animals↗
Structure based development of novel specific inhibitors for cathepsin L and cathepsin S in vitro and in vivo.
Specific inhibitors for cathepsin L and cathepsin S have been developed with the help of computer-graphic modeling based on the stereo-structure. The common fragment, N-(L-trans-carbamoyloxyrane-2-carbonyl)-phenylalanine-dimethyla mide, is required for specific inhibition of cathepsin L. Seven novel inhibitors of the cathepsin L inhibitor Katunuma (CLIK) specifically inhibited cathepsin L at a concentration of 10(-7) M in vitro, while almost no inhibition of cathepsins B, C, S and K was observed. Four of the CLIKs are stable, and showed highly selective inhibition for hepatic cathepsin L in vivo. One of the CLIK inhibitors contains an aldehyde group, and specifically inhibits cathepsin S at 10(-7) M in vitro.
Animals↗
Crystallization and preliminary X-ray studies of the Ia component of Clostridium perfringens iota toxin complexed with NADPH.
A recombinant Ia component of Clostridium perfringens iota toxin, which ADP-ribosylates actin, was crystallized by the hanging drop vapor diffusion method using PEG4000 as a precipitating agent. The crystals were obtained in the presence of NADPH, which is similar to a real substrate, NADH, and belongs to the space group P1 (a = 47.9 A, b = 54.5 A, c = 103.1 A, alpha = 99.0 degrees, beta = 93.3 degrees, and gamma = 107.2 degrees ). The Matthews coefficient of native crystal was 2.7, assuming 2 mol/asymmetric unit. Native data were collected at 2.4-A resolution. The results from a heavy-atom search showed that lanthanide ions (samarium, holmium) altered the molecular packing, judging from the unit-cell difference. The crystals also belonged to the space group P1 (a = 47.7 A, b = 53.9 A, c = 54.6 A, alpha = 68.9 degrees, beta = 78.3 degrees, and gamma = 73.7 degrees ), which is consistent with only one molecule per asymmetric unit.
ADP Ribose Transferases↗
Cleavage of RXRalpha by a lysosomal enzyme, cathepsin L-type protease.
In this study, we characterized a protease responsible for the cleavage of RXRalpha in two human derived cell lines, HepG2 and JEG-3 cells. Electrophoretic mobility shift assay (EMSA) combined with antibody supershift analysis suggested that contamination of cytoplasmic components during nuclear extract preparation could result in complete cleavage of RXRalpha at its N-terminus in JEG-3 cells, while such proteolytic activity was much less evident in HepG2. When the nuclei were purified in the presence of leupeptin, only full-length RXRalpha was found in the extracts prepared from both JEG-3 and HepG2 cells, suggesting a member of cysteine protease family is responsible for the cleavage. The presence of the protease in the cytoplasm, but not in the nucleus, was confirmed by incubating full-length 35S-labeled RXRalpha with each fraction. The cytoplasmic fraction from JEG-3 and HepG2 cells cleaved RXRalpha into smaller sizes with molecular mass of 45, 43, and 31 kD. Immunoprecipitation with antibodies recognizing distinct epitopes indicated that the cleaved RXRalpha with the size of 45 and 43 kD were truncated at N-terminus in which most of the A/B domain was absent. Using a series of protease inhibitors, the enzyme cleaving RXRalpha was characterized as cathepsin L-type protease. The enzyme activity in JEG-3 cells was much higher than that in HepG2 cells. This is the first demonstration that RXRalpha is cleaved by a lysosomal enzyme, cathepsin L-type protease.
Cathepsin L↗
A comparative study of chemonucleolysis with recombinant human cathepsin L and chymopapain. A radiologic, histologic, and immunohistochemical assessment.
STUDY DESIGN: Investigation of the effects of recombinant human cathepsin L on intervertebral discs and comparison with the effects of chymopapain. OBJECTIVE: To evaluate the effects of cathepsin L on intervertebral discs as an agent for chemonucleolysis. SUMMARY BACKGROUND DATA: Cathepsin L is a typical cysteine proteinase that belongs to the papain superfamily. It plays a major role in intracellular proteolysis and is not believed to induce anaphylactic reactions. METHODS: In vivo: Rabbit intervertebral discs were injected with recombinant human cathepsin L, its buffer solution, and chymopapain. After 1, 4, and 16 weeks the animals were killed, and radiologic and histologic examinations were performed. In vitro: The enzymatic actions of recombinant human cathepsin L and chymopapain on human intervertebral disc proteoglycans were examined immunohistochemically using antiproteoglycan antibodies. RESULTS: In rabbit models, roentgenography showed that disc spaces treated with cathepsin L and chymopapain had become narrower 1 week after injection. Histologically, loss of safranin-O staining was observed in the anulus fibrosus of discs treated with cathepsin L. After 16 weeks, nucleus pulposus had regenerated with chondrocyte-like cells, and the safranin-O staining characteristics of the matrix also had recovered. In an immunohistochemical study, all components of the proteoglycan stained weakly after chymopapain digestion. After cathepsin L digestion, unsulfated chondroitin and core protein staining was weaker, but the chondroitin 6-sulfate staining was unaffected. CONCLUSIONS: Cathepsin L seems to be an effective agent for chemonucleolysis. Its enzymatic action on proteoglycan appears to be different from that of chymopapain.
Adult↗
Inhibition of cysteine protease and growth of Staphylococcus aureus V8 and poliovirus by phosphorylated cystatin alpha conjugate of skin.
The inhibitory properties of phosphorylated cystatin alpha (P-cystatin alpha) and a conjugated protein of the P-cystatin alpha with filaggrin linker segment peptide (FLSP) against the growth of Staphylococcus bacteria and poliovirus were investigated. Both the P-cystatin alpha and the conjugated protein (P-cystatin alpha-FLSP conjugate) as a model for the cornified envelope of skin inhibited the cysteine protease activity of Staphylococcus aureus V8. The protease activity was inhibited by normal cornified envelope of newborn rat skin, which contains P-cystatin alpha, and P-cystatin alpha in cornified envelope of newborn rat skin also suppressed the growth of S. aureus V8. When P-cystatin alpha or P-cystatin alpha-FLSP conjugate was added to cultured HeLa cells infected with poliovirus, 50-70% of the cell-death due to poliovirus infection was prevented. The poliovirus 3C protease activity in the infected HeLa cells was inhibited by P-cystatin alpha or P-cystatin alpha-FLSP conjugate. As a result, the processing of viral capsid peptides was suppressed. These findings suggest that P-cystatin alpha and P-cystatin alpha-FLSP conjugate could play the role of the barrier against microorganism infections due to inhibition of their cysteine protease activities.
Amino Acid Sequence↗
Study of the functional share of lysosomal cathepsins by the development of specific inhibitors.
To analyze the functional share of individual cathepsins, we developed powerful and specific inhibitors for individual cathepsins using computer graphics of substrate binding pockets based on X-ray crystallography. These new inhibitors were named CLIK group. Epoxy succinate peptide derivatives, CLIK-066, 088, 112, 121, 148, 181, 185 and 187, are typical specific inhibitors for cathepsin L. Aldehyde derivatives CLIK-060 and CLIK-164 showed specific inhibition against cathepsin S and cathepsin K, respectively. We found that pyridoxal phosphate (PLP), a coenzyme form of vitamin B6, inhibits all cathepsins and also new artificially synthesized pyridoxal derivatives, CLIK-071 and -072, in which the phosphate esters of PLP were replaced by propionic acid, exhibited strong inhibition for cathepsins. Furthermore, CLIK-071 was easy to incorporate into cells and showed powerful inhibition for intracellular cathepsins. Using these selective inhibitors, the allotment of individual cathepsin functions in cells has been studied as follows. Cathepsin L and/or K participate in bone resorption based on bone type-1 collagen degradation and the L-type protease inhibitors suppressed the bone resorption. Cathepsins B and S participate in antigen presentations based on antigen processing and invariant chain degradation, respectively. Also cathepsin L participates in cell apoptosis mediated by caspase III activation.
Animals↗
Participation of a cathepsin L-type protease in the activation of caspase-3.
A previous paper from this laboratory reported the activation of a caspase-3-like protease by a digitonin-treated lysosomal fraction [FEBS Lett. 435, 233-236, 1998]. In this study, we examined the effects of specific inhibitors of lysosomal cysteine proteases, such as cathepsins B, S, and L, on the activation of caspase-3 to find out which cathepsin is responsible for the activation. Pro-caspase-3 in the cytosol was cleaved by a lysosomal protease(s) contained in the supernatant of a digitonin-treated crude mitochondrial fraction containing lysosomes (ML) and the cleaved product was detected by Western blotting using anti-caspase-3 antibody. The activation of caspase-3 by the lysosomal protease(s) was pH dependent and the optimum pH for activation was pH 6.6-6.8. This activation was not inhibited by CA-074, a specific inhibitor of cathepsin B, but was strongly inhibited by CLIK-066 and CLIK-181, specific inhibitors of cathepsin L. The inhibitory effect of CLIK-060, a specific inhibitor of cathepsin S, was very weak. Furthermore, the activation of caspase-3 was enhanced by addition of purified cathepsin L only in the presence of the supernatant of the digitonin-treated ML. These results suggested that a cathepsin L-type protease activity might participate in the activation mechanism of caspase-3 in the presence of the supernatnat from the ML.
Adenosine Triphosphate↗
[New function of vitamin B6 derivatives: inhibition of cathepsins].
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Bone and Bones↗
Intracellular proteolytic cleavage of 9-cis-retinoic acid receptor alpha by cathepsin L-type protease is a potential mechanism for modulating thyroid hormone action.
We previously reported that the responsiveness of hepatocytes to thyroid hormone is markedly attenuated when they were cultured as monolayers rather than spheroids. To elucidate the mechanisms underlying the altered responsiveness, thyroid hormone receptor auxiliary proteins in the hepatocytes were analyzed by electrophoretic mobility shift assay. The major thyroid hormone receptor auxiliary protein was identified as 9-cis-retinoic acid receptor alpha (RXRalpha) in the hepatocytes regardless of the culture conditions. The cytoplasmic fraction was shown to contain a protease(s) that cleaves RXRalpha at its amino terminus. The presence of the protease in the cytosol, but not in the nucleus, was ascertained by incubating full-length 35S-labeled RXRalpha with each fraction. Using various protease inhibitors, it was shown that cathepsin L-type protease could participate in the cleavage of the RXRalpha. The enzyme activity was much higher in the monolayers than the spheroids. Inhibition of this enzyme activity in the monolayer hepatocyte resulted in the increase of nuclear RXRalpha protein and the augmentation of T3-dependent induction of spot 14 mRNA. These results suggest that the changes in cathepsin L-type protease activity in the cytosol may alter the turnover of RXRalpha in the nucleus and modify the function of steroid receptor superfamilies that heterodimerize with RXRalpha.
Animals↗
Switch of CD4+ T cell differentiation from Th2 to Th1 by treatment with cathepsin B inhibitor in experimental leishmaniasis.
When activated, CD4+ T helper cells differentiate functionally into one of two subsets, Th1 or Th2. Before the Th differentiation, Ags must be processed into peptide epitopes and presented to CD4+ T cells in association with MHC class II molecules. However, the proteases responsible for this Ag processing have not been well defined. When BALB/c mice susceptible to infection with Leishmania major were treated with a specific inhibitor (CA074) of cathepsin B, a lysosomal cysteine protease that digests exogenous antigenic proteins, those mice acquired resistance against infection with L. major and showed the shift of immune responses from Th2 to Th1; that is, they produced specific IgG2a Ab and generated IFN-gamma in contrast to untreated and infected mice that produced IgG1 and IgE and generated IL-4. CA074 interfered with the digestion of L. major Ags with lysosomal enzymes in vivo as well as in vitro. However, this inhibitor did not show any direct influence on the growth of L. major and the functions of T cells stimulated with anti-CD3 Ab. These findings indicate that cathepsin B inhibitor could switch CD4+ T cell differentiation from Th2 to Th1, suggesting that the alteration in Ag processing modulates the polarity of Th differentiation.
Animals↗
Novel physiological functions of cathepsins B and L on antigen processing and osteoclastic bone resorption.
Lysosomal cathepsin B plays an essential role in the processing of ovalbumin as an exogenous antigen to produce the complex between antigenic-peptide and major histocompatibility-complex class II. Administration of cathepsin B inhibitors, E-64, CA-074 and vitamin B6, caused the strong suppression of the Th-2 type immune responses. We found that pyridoxal phosphate (PAP), a coenzyme form of vitamin B6, inhibits the activities of cathepsin B and L in vitro and vitamin B6 administration induces the inhibition of the lysosomal cathepsin activities in vivo. The production of an antigenic epitope (I323-R339) of ovalbumin by antigen presenting cells was suppressed by cathepsin B specific inhibitors. The ovalbumin dependent production of immunoglobulins (IgE and IgG1) and of the corresponding interleukin (IL-4) was suppressed by cathepsin B inhibitors, while the production of IgG2a and interferon (INF-gamma) was increased. The switch of helper T lymphocyte functions from the type-2 to the type-1 may be induced by the cathepsin B inhibition. The experimental bone pit formation, i.e., osteoclastic bone collagen degradation test, induced by parathyroid hormone was markedly suppressed by the administration of pyridoxal, because of the inhibition of cathepsin L type cysteine proteases in bone.
Animals↗
Electrophoretic analysis of the "cross-class" interaction between novel inhibitory serpin, squamous cell carcinoma antigen-1 and cysteine proteinases.
We investigated the "cross-class" interaction between cysteine proteinases and a novel inhibitory serpin, recombinant squamous cell carcinoma (rSCC) antigen-1, which inhibits a serine proteinase, chymotrypsin. rSCC antigen-1 inhibited the cysteine proteinases, papain, papaya proteinase IV and cathepsin L. Interestingly, although rSCC antigen-1 formed sodium dodecyl sulfate (SDS)- and heat-stable complexes with chymotrypsin, rSCC antigen-1 gave the 40 kDa fragment and small molecular mass peptide by incubation with papain without forming an SDS- and heat-stable complex. The cleavage was observed between the Gly353-Ser354 bond, indicating that rSCC antigen-1 interacts with cysteine proteinases not at the predicted reactive site P1-P1' portion (Ser354-Ser355), but at the Gly353-Ser354 of the P2-P1 portion. These findings promote understanding of the "suicide inhibition" mechanism of SCC antigen-1 against cysteine proteinases.
Animals↗