Publications by authors named "Nobuhiko Katunuma"

59 Publications

An Active 32-kDa Cathepsin L Is Secreted Directly from HT 1080 Fibrosarcoma Cells and Not via Lysosomal Exocytosis.

PLoS One 2015 16;10(12):e0145067. Epub 2015 Dec 16.

Institute for Health Sciences, Tokushima Bunri University, Tokushima, Japan.

Cathepsin L [EC 3.4.22.15] is secreted via lysosomal exocytosis by several types of cancer cells, including prostate and breast cancer cells. We previously reported that human cultured fibrosarcoma (HT 1080) cells secrete cathepsin L into the medium; this secreted cathepsin is 10-times more active than intracellular cathepsin. This increased activity was attributed to the presence of a 32-kDa cathepsin L in the medium. The aim of this study was to examine how this active 32-kDa cathepsin L is secreted into the medium. To this end, we compared the secreted active 32-kDa cathepsin L with lysosomal cathepsin L by using a novel gelatin zymography technique that employs leupeptin. We also examined the glycosylation and phosphorylation status of the proteins by using the enzymes endoglycosidase H [EC 3.2.1.96] and alkaline phosphatase [EC 3.1.3.1]. Strong active bands corresponding to the 32-kDa and 34-kDa cathepsin L forms were detected in the medium and lysosomes, respectively. The cell extract exhibited strong active bands for both forms. Moreover, both forms were adsorbed onto a concanavalin A-agarose column. The core protein domain of both forms had the same molecular mass of 30 kDa. The 32-kDa cathepsin L was phosphorylated, while the 34-kDa lysosomal form was dephosphorylated, perhaps because of the lysosomal marker enzyme, acid phosphatase. These results suggest that the active 32-kDa form does not enter the lysosomes. In conclusion, our results indicate that the active 32-kDa cathepsin L is secreted directly from the HT 1080 cells and not via lysosomal exocytosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0145067PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684495PMC
June 2016

Inhibition of cysteine cathepsin B and L activation in astrocytes contributes to neuroprotection against cerebral ischemia via blocking the tBid-mitochondrial apoptotic signaling pathway.

Glia 2014 Jun 24;62(6):855-80. Epub 2014 Feb 24.

Department of Pharmacology and Laboratory of Cerebrovascular Pharmacology, College of Pharmaceutical Science, Soochow University, Suzhou, 215123, China.

The roles of cathepsins in the ischemic astrocytic injury remain unclear. Here, we test the hypothesis that activation of cathepsin B and L contributes to the ischemic astrocyte injury via the tBid-mitochondrial apoptotic signaling pathways. In the rat models of pMCAO, CA-074Me or Clik148, a selective inhibitor of cathepsin B or cathepsin L, reduced the infarct volume, improved the neurological deficits and increased the MAP2 and GFAP levels. In OGD-induced astrocyte injury, CA-074Me or Clik148 decreased the LDH leakage and increased the GFAP levels. In the ischemic cortex or OGD-induced astrocytes injury, Clik148 or CA-074Me reversed pMCAO or OGD-induced increase in active cathepsin L or cathepsin B at 3 h or 6 h, increase in tBid, reduction in mitochondrial cytochrome-c (Cyt-c) and increase in cytoplastic Cyt-c and active caspase-3 at 12-24 h of the late stage of pMCAO or OGD. CA-074Me or Clik148 also reduced cytosolic and mitochondrial tBid, increased mitochondrial Cyt-c and decreased cytoplastic Cyt-c and active caspase-3 at 6 h of the early stage of Bid activation. CA-074Me or Clik148 blocked the pMCAO-induced release of cathepsin B or L from the lysosomes into the cytoplasm and activation of caspase-3 in ischemic astrocytes at 12 h after ischemia. Concurrent inhibition of cathepsin B and cathepsin L provided better protection on the OGD-induced astrocytic apoptosis than obtained with separate use of each inhibitor. These results suggest that inhibition of the cysteine cathepsin B and cathepsin L activation in ischemic astrocytes contributes to neuroprotection via blocking the tBid-mitochondrial apoptotic signaling pathway.
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http://dx.doi.org/10.1002/glia.22645DOI Listing
June 2014

Autophagy and cathepsin L are involved in the antinociceptive effect of DMBC in a mouse acetic acid-writhing model.

Acta Pharmacol Sin 2013 Aug;34(8):1007-12

Department of Pharmacology and Laboratory of Cerebrovascular Pharmacology, College of Pharmaceutical Science, Soochow University, Suzhou 215123, China.

Aim: 2-(3',5'-Dimethoxybenzylidene) cyclopentanone (DMBC) is a novel synthetic compound with antinociceptive activities. The aim of this study was to investigate the roles of the autophagic-lysosomal pathway in the antinociceptive effect of DMBC in a mouse acetic acid-writhing model.

Methods: Mouse acetic acid-writhing test and hotplate test were used to assess the antinociceptive effects of DMBC, 3-MA (autophagy inhibitor) and Clik148 (cathepsin L inhibitor). The drugs were administered peripherally (ip) or centrally (icv).

Results: Peripheral administration of 3-MA (7.5-30 mg/kg) or Clik148 (10-80 mg/kg) produced potent antinociceptive effect in acetic acid-writhing test. Central administration of 3-MA or Clik148 (12.5-50 nmol/L) produced comparable antinociceptive effect in acetic acid-writhing test. Peripheral administration of DMBC (25-50 mg/kg) produced potent antinociceptive effects in both acetic acid-writhing and hotplate tests. Furthermore, the antinociceptive effect produced by peripheral administration of DMBC (50 mg/kg) in acetic acid-writhing test was antagonized by low doses of 3-MA (3.75 mg/kg) or Clik148 (20 mg/kg) peripherally administered, but was not affected by 3-MA or Clik148 (25 nmol/L) centrally administered.

Conclusion: Activation of central autophagy and cathepsin L is involved in nociception in mice, whereas peripheral autophagy and cathepsin L contributes, at least in part, to the antinociceptive effect of DMBC in mice.
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http://dx.doi.org/10.1038/aps.2013.30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4003021PMC
August 2013

L-tryptophan suppresses rise in blood glucose and preserves insulin secretion in type-2 diabetes mellitus rats.

J Nutr Sci Vitaminol (Tokyo) 2012 ;58(6):415-22

Faculty of Life Science, Tokushima Bunri University, Tokushima, Japan.

Ample evidence indicates that a high-protein/low-carbohydrate diet increases glucose energy expenditure and is beneficial in patients with type-2 diabetes mellitus (T2DM). The present study was designed to investigate the effects of L-tryptophan in T2DM. Blood glucose was measured by the glucose dehydrogenase assay and serum insulin was measured with ELISA in both normal and hereditary T2DM rats after oral glucose administration with or without L-D-tryptophan and tryptamine. The effect of tryptophan on glucose absorption was examined in the small intestine of rats using the everted-sac method. Glucose incorporation in adipocytes was assayed with [(3)H]-2-deoxy-D-glucose using a liquid scintillation counter. Indirect computer-regulated respiratory gas-assay calorimetry was applied to assay energy expenditure in rats. L-Tryptophan suppressed both serum glucose and insulin levels after oral glucose administration and inhibited glucose absorption from the intestine. Tryptamine, but not L-tryptophan, enhanced insulin-stimulated [(3)H]-glucose incorporation into differentiated adipocytes. L-Tryptophan increased glucose-associated energy expenditure in rats in vivo. L-Tryptophan-rich chow consumed from a young age preserved the secretion of insulin and delayed the progression of T2DM in hereditary diabetic rats. The results suggested that L-tryptophan suppresses the elevation of blood glucose and lessens the burden associated with insulin secretion from β-cells.
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http://dx.doi.org/10.3177/jnsv.58.415DOI Listing
August 2013

Processing of human protryptase in mast cells involves cathepsins L, B, and C.

J Immunol 2011 Aug 8;187(4):1912-8. Epub 2011 Jul 8.

Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA.

Human β-tryptase is stored in secretory granules of human mast cells as a heparin-stabilized tetramer. β-Protryptase in solution can be directly processed to the mature enzyme by cathepsin (CTS) L and CTSB, and sequentially processed by autocatalysis at R(-3), followed by CTSC proteolysis. However, it is uncertain which CTS is involved in protryptase processing inside human mast cells, because murine bone marrow-derived mast cells from CTSC-deficient mice convert protryptase (pro-mouse mast cell protease-6) to mature mouse mast cell protease-6. This finding suggests that other proteases are important for processing human β-protryptase. In the current study, reduction of either CTSB or CTSL activity inside HMC-1 cells by short hairpin RNA silencing or CTS-specific pharmacologic inhibitors substantially reduced mature β-tryptase formation. Similar reductions of tryptase levels in primary skin-derived mast cells were observed with these pharmacologic inhibitors. In contrast, protryptase processing was minimally reduced by short hairpin RNA silencing of CTSC. A putative pharmacologic inhibitor of CTSC markedly reduced tryptase levels, suggesting an off-target effect. Skin mast cells contain substantially greater amounts of CTSL and CTSB than do HMC-1 cells, the opposite being found for CTSC. Both CTSL and CTSB colocalize to the secretory granule compartment of skin mast cells. Thus, CTSL and CTSB are central to the processing of protryptase(s) in human mast cells and are potential targets for attenuating production of mature tryptase in vivo.
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http://dx.doi.org/10.4049/jimmunol.1001806DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3150348PMC
August 2011

Promiscuous processing of human alphabeta-protryptases by cathepsins L, B, and C.

J Immunol 2011 Jun 11;186(12):7136-43. Epub 2011 May 11.

Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA.

Human α- and β-protryptase zymogens are abundantly and selectively produced by mast cells, but the mechanism(s) by which they are processed is uncertain. β-Protryptase is sequentially processed in vitro by autocatalysis at R(-3) followed by cathepsin (CTS) C proteolysis to the mature enzyme. However, mast cells from CTSC-deficient mice successfully convert protryptase (pro-murine mast cell protease-6) to mature murine mast cell protease-6. α-Protryptase processing cannot occur by trypsin-like enzymes due to an R(-3)Q substitution. Thus, biological mechanisms for processing these zymogens are uncertain. β-Tryptase processing activity(ies) distinct from CTSC were partially purified from human HMC-1 cells and identified by mass spectroscopy to include CTSB and CTSL. Importantly, CTSB and CTSL also directly process α-protryptase (Q(-3)) and mutated β-protryptase (R(-3)Q) as well as wild-type β-protryptase to maturity, indicating no need for autocatalysis, unlike the CTSC pathway. Heparin promoted tryptase tetramer formation and protected tryptase from degradation by CTSB and CTSL. Thus, CTSL and CTSB are capable of directly processing both α- and β-protryptases from human mast cells to their mature enzymatically active products.
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http://dx.doi.org/10.4049/jimmunol.1001804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3339195PMC
June 2011

CD4-independent human immunodeficiency virus infection involves participation of endocytosis and cathepsin B.

PLoS One 2011 Apr 25;6(4):e19352. Epub 2011 Apr 25.

Department of AIDS Research, Institute of Tropical Medicine, Global Center of Excellence, Nagasaki University, Nagasaki, Japan.

During a comparison of the infectivity of mNDK, a CD4-independent human immunodeficiency virus type 1 (HIV-1) strain, to various cell lines, we found that HeLa cells were much less susceptible than 293T and TE671 cells. Hybridoma cells between HeLa and 293T cells were as susceptible as 293T cells, suggesting that cellular factors enhance the mNDK infection in 293T cells. By screening a cDNA expression library in HeLa cells, cystatin C was isolated as an enhancer of the mNDK infection. Because cathepsin B protease, a natural ligand of cystatin C, was upregulated in HeLa cells, we speculated that the high levels of cathepsin B activities were inhibitory to the CD4-independent infection and that cystatin C enhanced the infection by impairing the excessive cathepsin B activity. Consistent with this idea, pretreatment of HeLa cells with 125 µM of CA-074Me, a cathepsin B inhibitor, resulted in an 8-fold enhancement of the mNDK infectivity. Because cathepsin B is activated by low pH in acidic endosomes, we further examined the potential roles of endosomes in the CD4-independent infection. Suppression of endosome acidification or endocytosis by inhibitors or by an Eps15 dominant negative mutant reduced the infectivity of mNDK in which CD4-dependent infections were not significantly impaired. Taken together, these results suggest that endocytosis, endosomal acidification, and cathepsin B activity are involved in the CD4-independent entry of HIV-1.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019352PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3081840PMC
April 2011

Structure-based development of specific inhibitors for individual cathepsins and their medical applications.

Proc Jpn Acad Ser B Phys Biol Sci 2011 ;87(2):29-39

Institute for Health Sciences, Tokushima Bunri University, Tokushima, Japan.

Specific inhibitors for individual cathepsins have been developed based on their tertiary structures of X-ray crystallography. Cathepsin B-specific inhibitors, CA-074 and CA-030, and cathepsin L specific inhibitors, CLIK-148 and CLIK-195, were designed as the epoxysuccinate derivatives. Cathepsin S inhibitor, CLIK-060, and cathepsin K inhibitor, CLIK-166, were synthesized. These inhibitors can use in vitro and also in vivo, and show no toxicity for experimental animals by the amounts used as the cathepsin inhibitor. Various cathepsins are used in the processing of antigenic proteins. The CLIK-060 treatment to the autoimmune disease, Sjögren model mice, led to strongly suppress the expression of the pathological symptoms. Cathepsins L or K participates to the degradation of bone collagen. The CLIK-148 protects osteoporosis in animals and also protects the bone metastasis of cancer cells. Cathepsin L also enhances insulin-induced glucose uptake into 3T3-L1 adipocytes, suggesting cathepsin L plays the roles in adipogenesis and glucose tolerance in type 2 diabetes.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3043741PMC
http://dx.doi.org/10.2183/pjab.87.29DOI Listing
June 2011

Cathepsin L inhibition prevents murine autoimmune diabetes via suppression of CD8(+) T cell activity.

PLoS One 2010 Sep 22;5(9):e12894. Epub 2010 Sep 22.

Department of Oral Molecular Pathology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.

Background: Type 1 diabetes (T1D) is an autoimmune disease resulting from defects in central and peripheral tolerance and characterized by T cell-mediated destruction of islet β cells. To determine whether specific lysosomal proteases might influence the outcome of a T cell-mediated autoimmune response, we examined the functional significance of cathepsin inhibition on autoimmune T1D-prone non-obese diabetic (NOD) mice.

Methods And Findings: Here it was found that specific inhibition of cathepsin L affords strong protection from cyclophosphamide (CY)-induced insulitis and diabetes of NOD mice at the advanced stage of CD8(+) T cell infiltration via inhibiting granzyme activity. It was discovered that cathepsin L inhibition prevents cytotoxic activity of CD8(+) T cells in the pancreatic islets through controlling dipeptidyl peptidase I activity. Moreover, the gene targeting for cathepsin L with application of in vivo siRNA administration successfully prevented CY-induced diabetes of NOD mice. Finally, cathepsin L mRNA expression of peripheral CD8(+) T cells from NOD mice developing spontaneous T1D was significantly increased compared with that from control mice.

Conclusions: Our results identified a novel function of cathepsin L as an enzyme whose activity is essential for the progression of CD8(+) T cell-mediated autoimmune diabetes, and inhibition of cathepsin L as a powerful therapeutic strategy for autoimmune diabetes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0012894PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943924PMC
September 2010

Quinolinate phosphoribosyl transferase, a key enzyme in de novo NAD(+) synthesis, suppresses spontaneous cell death by inhibiting overproduction of active-caspase-3.

Biochim Biophys Acta 2010 May 3;1803(5):527-33. Epub 2010 Mar 3.

Division of Molecular Biology, Institute for Health Sciences, Tokushima Bunri University, Tokushima, Japan.

Quinolinate phosphoribosyl transferase (QPRT) is a key enzyme in de novo NAD(+) synthesis. QPRT enzyme activity has a restricted tissue distribution, although QPRT mRNA is expressed ubiquitously. This study was designed to elucidate the functions of QPRT protein in addition to NAD(+) synthesis. QPRT was identified as a caspase-3 binding protein using double layer fluorescent zymography, but was not a substrate for caspase-3. Surface plasmon resonance analysis using recombinant proteins showed interaction of QPRT with active-caspase-3 in a dose dependent manner at 55 nM of the dissociation constant. The interaction was also confirmed by immunoprecipitation analysis of actinomycin D-treated QPRT-FLAG expressing cells using anti-FLAG-agarose. QPRT-depleted cells showed increased sensitivity to spontaneous cell death, upregulated caspase-3 activity and strong active-caspase-3 signals. Considered together, the results suggested that QPRT protein acts as an inhibitor of spontaneous cell death by suppressing overproduction of active-caspase-3.
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http://dx.doi.org/10.1016/j.bbamcr.2010.02.007DOI Listing
May 2010

Posttranslational processing and modification of cathepsins and cystatins.

J Signal Transduct 2010 16;2010:375345. Epub 2010 Dec 16.

Institute for Health Sciences, Tokushima Bunri University, 180 Nishihamabouji, Yamashiro-cho, Tokushima City, Tokushima 770-8514, Japan.

Cathepsins are an essential protease family in all living cells. The cathepsins play an essential roles such as protein catabolism and protein synthesis. To targeting to various organella and to regulate their activity, the post translational-processing and modification play an important role Cathepsins are translated in polysome as the pre-pro-mature forms. The pre-peptide is removed cotranslationally and then translocated to Golgi-apparatus and the pro-part is removed and the mature-part is glycosylated, and the mature-part is targeted into the lysosome mediated by mannose-6-phosphate signal and the mature-part is bound with their coenzymes. The degradation of the mature-part is started by the limited proteolysis of the ordered nicked bonds to make hydrophobic peptides. The peptides are incorporated into phagosome or proteasome after ubiquitinated and are degrade into amino-acids. Cystatins are endogenous inhibitors of cathepsins. Cystatin α which is only located in skin is phosphorylated at the near C-terminus by protein kinase-C, and the phosphorylate-cystatin α is incorporated into cornified envelope and conjugated with filaggrin-fiber by transglutaminase to form the linker-fiber of skin. The cystatin α is modified by glutathione or make their dimmer, and they are inactive. Those modifications are regulated by the redox-potential by the glutathione.
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http://dx.doi.org/10.1155/2010/375345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3100116PMC
July 2011

[Structure-based drug development and medical/biological application of cathepsin specific inhibitors].

Seikagaku 2009 Nov;81(11):952-61

Institute for Health Sciences, Tokushima Bumi University, 180 Nishihamabouji, Yamashiro-cho, Tokushima City, Tokushima 770-8514, Japan.

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November 2009

Impaired autophagic flux mediates acinar cell vacuole formation and trypsinogen activation in rodent models of acute pancreatitis.

J Clin Invest 2009 Nov 1;119(11):3340-55. Epub 2009 Oct 1.

Veterans Affairs Greater Los Angeles Healthcare System and University of California at Los Angeles, Los Angeles, California 90073, USA.

The pathogenic mechanisms underlying acute pancreatitis are not clear. Two key pathologic acinar cell responses of this disease are vacuole accumulation and trypsinogen activation. We show here that both result from defective autophagy, by comparing the autophagic responses in rodent models of acute pancreatitis to physiologic autophagy triggered by fasting. Pancreatitis-induced vacuoles in acinar cells were greater in number and much larger than those induced with fasting. Degradation of long-lived proteins, a measure of autophagic efficiency, was markedly inhibited in in vitro pancreatitis, while it was stimulated by acinar cell starvation. Further, processing of the lysosomal proteases cathepsin L (CatL) and CatB into their fully active, mature forms was reduced in pancreatitis, as were their activities in the lysosome-enriched subcellular fraction. These findings indicate that autophagy is retarded in pancreatitis due to deficient lysosomal degradation caused by impaired cathepsin processing. Trypsinogen activation occurred in pancreatitis but not with fasting and was prevented by inhibiting autophagy. A marker of trypsinogen activation partially localized to autophagic vacuoles, and pharmacologic inhibition of CatL increased the amount of active trypsin in acinar cells. The results suggest that retarded autophagy is associated with an imbalance between CatL, which degrades trypsinogen and trypsin, and CatB, which converts trypsinogen into trypsin, resulting in intra-acinar accumulation of active trypsin in pancreatitis. Thus, deficient lysosomal degradation may be a dominant mechanism for increased intra-acinar trypsin in pancreatitis.
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http://dx.doi.org/10.1172/JCI38674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2769194PMC
November 2009

Cathepsin L is required for ecotropic murine leukemia virus infection in NIH3T3 cells.

Virology 2009 Nov 24;394(2):227-34. Epub 2009 Sep 24.

Department of AIDS Research, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan.

Recently it has been reported that a cathepsin B inhibitor, CA-074Me, attenuates ecotropic murine leukemia virus (Eco-MLV) infection in NIH3T3 cells, suggesting that cathepsin B is required for the Eco-MLV infection. However, cathepsin B activity was negative or extremely low in NIH3T3 cells. How did CA-074Me attenuate the Eco-MLV infection? The CA-074Me treatment of NIH3T3 cells inhibited cathepsin L activity, and a cathepsin L specific inhibitor, CLIK148, attenuated the Eco-MLV vector infection. These results indicate that the suppression of cathepsin L activity by CA-074Me induces the inhibition of Eco-MLV infection, suggesting that cathepsin L is required for the Eco-MLV infection in NIH3T3 cells. The CA-074Me treatment inhibited the Eco-MLV infection in human cells expressing the exogenous mouse ecotropic receptor and endogenous cathepsins B and L, but the CLIK148 treatment did not, showing that only the cathepsin L suppression by CLIK148 is not enough to prevent the Eco-MLV infection in cells expressing both of cathepsins B and L, and CA-074Me inhibits the Eco-MLV infection by suppressing both of cathepsins B and L. These results suggest that either cathepsin B or L is sufficient for the Eco-MLV infection.
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http://dx.doi.org/10.1016/j.virol.2009.08.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111982PMC
November 2009

Structural basis for the kexin-like serine protease from Aeromonas sobria as sepsis-causing factor.

J Biol Chem 2009 Oct 4;284(40):27655-63. Epub 2009 Aug 4.

Laboratory of Molecular Microbiological Science, Faculty of Pharmaceutical Sciences, Hiroshima International University, Hiro-Koshingai, Kure, Hiroshima 737-0112, Japan.

The anaerobic bacterium Aeromonas sobria is known to cause potentially lethal septic shock. We recently proposed that A. sobria serine protease (ASP) is a sepsis-related factor that induces vascular leakage, reductions in blood pressure via kinin release, and clotting via activation of prothrombin. ASP preferentially cleaves peptide bonds that follow dibasic amino acid residues, as do Kex2 (Saccharomyces cerevisiae serine protease) and furin, which are representative kexin family proteases. Here, we revealed the crystal structure of ASP at 1.65 A resolution using the multiple isomorphous replacement method with anomalous scattering. Although the overall structure of ASP resembles that of Kex2, it has a unique extra occluding region close to its active site. Moreover, we found that a nicked ASP variant is cleaved within the occluding region. Nicked ASP shows a greater ability to cleave small peptide substrates than the native enzyme. On the other hand, the cleavage pattern for prekallikrein differs from that of ASP, suggesting the occluding region is important for substrate recognition. The extra occluding region of ASP is unique and could serve as a useful target to facilitate development of novel antisepsis drugs.
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http://dx.doi.org/10.1074/jbc.M109.006114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785694PMC
October 2009

Genetic and pharmacologic alteration of cathepsin expression influences reovirus pathogenesis.

J Virol 2009 Oct 29;83(19):9630-40. Epub 2009 Jul 29.

Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

The cathepsin family of endosomal proteases is required for proteolytic processing of several viruses during entry into host cells. Mammalian reoviruses utilize cathepsins B (Ctsb), L (Ctsl), and S (Ctss) for disassembly of the virus outer capsid and activation of the membrane penetration machinery. To determine whether cathepsins contribute to reovirus tropism, spread, and disease outcome, we infected 3-day-old wild-type (wt), Ctsb(-/-), Ctsl(-/-), and Ctss(-/-) mice with the virulent reovirus strain T3SA+. The survival rate of Ctsb(-/-) mice was enhanced in comparison to that of wt mice, whereas the survival rates of Ctsl(-/-) and Ctss(-/-) mice were diminished. Peak titers at sites of secondary replication in all strains of cathepsin-deficient mice were lower than those in wt mice. Clearance of the virus was delayed in Ctsl(-/-) and Ctss(-/-) mice in comparison to the levels for wt and Ctsb(-/-) mice, consistent with a defect in cell-mediated immunity in mice lacking cathepsin L or S. Cathepsin expression was dispensable for establishment of viremia, but cathepsin L was required for maximal reovirus growth in the brain. Treatment of wt mice with an inhibitor of cathepsin L led to amelioration of reovirus infection. Collectively, these data indicate that cathepsins B, L, and S influence reovirus pathogenesis and suggest that pharmacologic modulation of cathepsin activity diminishes reovirus disease severity.
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http://dx.doi.org/10.1128/JVI.01095-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2748054PMC
October 2009

Inhibition of collagenolytic cathepsins by beta-lactoglobulin in milk and its suppressive effect on bone resorption.

J Nutr Sci Vitaminol (Tokyo) 2009 Jun;55(3):264-70

Faculty of Life Science, Tokushima Bunri University, Tokushima, Japan.

It is well known that the collagenolytic cathepsins play an important role in the degradation of bone matrix. Therefore, the purpose of this study was to clarify the prevention effect of bone resorption by milk components. Using double-layer reverse zymography, we found a 20 kDa protein in milk which inhibited cysteine proteases. This inhibitory protein was identified as beta-lactoglobulin B. The inhibitory activity of beta-lactoglobulin B against cathepsin K was stronger than that of beta-lactoglobulin A. Beta-lactoglobulin B specifically inhibited papain type cysteine proteases such as cathepsins K and L, but not serine proteases, aspartic proteases or metallo proteases. Beta-lactoglobulin B inhibited cathepsin K non-competitively and the Ki value was 10(-5) M. The formation of osteoclastic pits in the culture system was effectively inhibited by 10(-4)-10(-5) M beta-lactoglobulin B in vitro. Furthermore, we demonstrated that beta-lactoglobulin B inhibited degradation of type I-collagen by collagenolytic cathepsins. Using the everted-sac method in rat small intestines, it was found that beta-lactoglobulin was more easily absorbed from the intestines of young rats (5 wk-old) than from those of older rats (more than 20 wk-old). The digested products of beta-lactoglobulin B with lysyl-endopeptidase showed a similar inhibitory activity against cathepsin K to the intact beta-lactoglobulin B did. Therefore, peroral intake of beta-lactoglobulin in milk and its digested peptides are expected to help protect osteoclastic bone resorption via inhibition of collagenolytic cathepsins K and L.
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http://dx.doi.org/10.3177/jnsv.55.264DOI Listing
June 2009

Retinal pigment epithelium-derived CTLA-2alpha induces TGFbeta-producing T regulatory cells.

J Immunol 2008 Dec;181(11):7525-36

Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University Graduate School of Medicine, Tokyo, Japan.

T cells that encounter ocular pigment epithelium in vitro are inhibited from undergoing TCR-triggered activation, and instead acquire the capacity to suppress the activation of bystander T cells. Because retinal pigment epithelial (RPE) cells suppress T cell activation by releasing soluble inhibitory factors, we studied whether soluble factors also promote the generation of T regulatory (Treg) cells. We found that RPE converted CD4(+) T cells into Treg cells by producing and secreting CTLA-2alpha, a cathepsin L (CathL) inhibitor. Mouse rCTLA-2alpha converted CD4(+) T cells into Treg cells in vitro, and CTLA-2alpha small interfering RNA-transfected RPE cells failed to induce the Treg generation. RPE CTLA-2alpha induced CD4(+)CD25(+)Foxp3(+) Treg cells that produced TGFbeta in vitro. Moreover, CTLA-2alpha produced by RPE cells inhibited CathL activity in the T cells, and losing CathL activity led to differentiation to Treg cells in some populations of CD4(+) T cells. In addition, T cells in the presence of CathL inhibitor increased the expression of Foxp3. The CTLA-2alpha effect on Treg cell induction occurred through TGFbeta signaling, because CTLA-2alpha promoted activation of TGFbeta in the eye. These results show that immunosuppressive factors derived from RPE cells participate in T cell suppression. The results are compatible with the hypothesis that the eye-derived Treg cells acquire functions that participate in the establishment of immune tolerance in the posterior segment of the eye.
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http://dx.doi.org/10.4049/jimmunol.181.11.7525DOI Listing
December 2008

Structure of an archaeal alanine:glyoxylate aminotransferase.

Acta Crystallogr D Biol Crystallogr 2008 Jun 14;64(Pt 6):696-9. Epub 2008 May 14.

Department of Life System, Institute of Technology and Science, The University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan.

The crystal structure of a novel alanine:glyoxylate aminotransferase from the hyperthermophilic archaeon Thermococcus litoralis was determined at 2.3 A resolution. The asymmetric unit contains four homologous subunits and the functional tetramer is generated by noncrystallographic 222 symmetry. Although the main-chain coordinates of the monomer of the Thermococcus litoralis enzyme showed a high degree of similarity to those of aspartate aminotransferase from Thermus thermophilus HB8, the amino-acid residues involved in substrate binding in the aspartate aminotransferase are only partially conserved in the Thermococcus litoralis enzyme. This may account for the difference in the substrate specificities of the two enzymes.
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http://dx.doi.org/10.1107/S0907444908006732DOI Listing
June 2008

Structural basis of actin recognition and arginine ADP-ribosylation by Clostridium perfringens iota-toxin.

Proc Natl Acad Sci U S A 2008 May 19;105(21):7399-404. Epub 2008 May 19.

Institute for Health Sciences and Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima 770-8514, Japan.

The ADP-ribosylating toxins (ADPRTs) produced by pathogenic bacteria modify intracellular protein and affect eukaryotic cell function. Actin-specific ADPRTs (including Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin) ADP-ribosylate G-actin at Arg-177, leading to disorganization of the cytoskeleton and cell death. Although the structures of many actin-specific ADPRTs are available, the mechanisms underlying actin recognition and selective ADP-ribosylation of Arg-177 remain unknown. Here we report the crystal structure of actin-Ia in complex with the nonhydrolyzable NAD analog betaTAD at 2.8 A resolution. The structure indicates that Ia recognizes actin via five loops around NAD: loop I (Tyr-60-Tyr-62 in the N domain), loop II (active-site loop), loop III, loop IV (PN loop), and loop V (ADP-ribosylating turn-turn loop). We used site-directed mutagenesis to confirm that loop I on the N domain and loop II are essential for the ADP-ribosyltransferase activity. Furthermore, we revealed that Glu-378 on the EXE loop is in close proximity to Arg-177 in actin, and we proposed that the ADP-ribosylation of Arg-177 proceeds by an SN1 reaction via first an oxocarbenium ion intermediate and second a cationic intermediate by alleviating the strained conformation of the first oxocarbenium ion. Our results suggest a common reaction mechanism for ADPRTs. Moreover, the structure might be of use in rational drug design to block toxin-substrate recognition.
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http://dx.doi.org/10.1073/pnas.0801215105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2387182PMC
May 2008

Structure of l-aspartate oxidase from the hyperthermophilic archaeon Sulfolobus tokodaii.

Biochim Biophys Acta 2008 Mar 8;1784(3):563-71. Epub 2008 Jan 8.

Department of Life System, Institute of Technology and Science, The University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan.

The crystal structure of the highly thermostable l-aspartate oxidase (LAO) from the hyperthermophilic archaeon Sulfolobus tokodaii was determined at a 2.09 A resolution. The factors contributing to the thermostability of the enzyme were analyzed by comparing its structure to that of Escherichia coli LAO. Like E. coli LAO, the S. tokodaii enzyme consists of three domains: an FAD-binding domain, an alpha+beta capping domain, and a C-terminal three-helix bundle. However, the situation of the linker between the FAD-binding domain and C-terminal three-helix bundle in S. tokodaii LAO is completely different from that in E. coli LAO, where the linker is situated near the FAD-binding domain and has virtually no interaction with the rest of the protein. In S. tokodaii LAO, this linker is situated near the C-terminal three-helix bundle and contains a beta-strand that runs parallel to the C-terminal strand. This results in the formation of an additional beta-sheet, which appears to reduce the flexibility of the C-terminal region. Furthermore, the displacement of the linker enables formation of a 5-residue ion-pair network between the FAD-binding and C-terminal domains, which strengthens the interdomain interactions. These features might be the main factors contributing to the high thermostability of S. tokodaii LAO.
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http://dx.doi.org/10.1016/j.bbapap.2007.12.012DOI Listing
March 2008

Reciprocal relationship between the apoptosis pathway mediated by executioner caspases and the physiological NAD synthesis pathway.

Adv Enzyme Regul 2008 22;48:19-30. Epub 2007 Nov 22.

Institute for Health Sciences, Tokushima Bunri University, 180 Nishihamabouji, Yamashiro-cho, Tokushima City, Tokushima 770-8514, Japan.

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http://dx.doi.org/10.1016/j.advenzreg.2007.11.007DOI Listing
December 2008

Crystal structure of archaeal highly thermostable L-aspartate dehydrogenase/NAD/citrate ternary complex.

FEBS J 2007 Aug 25;274(16):4315-25. Epub 2007 Jul 25.

Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.

The crystal structure of the highly thermostable L-aspartate dehydrogenase (L-aspDH; EC 1.4.1.21) from the hyperthermophilic archaeon Archaeoglobus fulgidus was determined in the presence of NAD and a substrate analog, citrate. The dimeric structure of A. fulgidus L-aspDH was refined at a resolution of 1.9 A with a crystallographic R-factor of 21.7% (R(free) = 22.6%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. Structural comparison of the A. fulgidus L-aspDH/NAD/citrate ternary complex and the Thermotoga maritima L-aspDH/NAD binary complex showed that A. fulgidus L-aspDH assumes a closed conformation and that a large movement of the two loops takes place during substrate binding. Like T. maritima L-aspDH, the A. fulgidus enzyme is highly thermostable. But whereas a large number of inter- and intrasubunit ion pairs are responsible for the stability of A. fulgidus L-aspDH, a large number of inter- and intrasubunit aromatic pairs stabilize the T. maritima enzyme. Thus stabilization of these two L-aspDHs appears to be achieved in different ways. This is the first detailed description of substrate and coenzyme binding to L-aspDH and of the molecular basis of the high thermostability of a hyperthermophilic L-aspDH.
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http://dx.doi.org/10.1111/j.1742-4658.2007.05961.xDOI Listing
August 2007

Cathepsin L activity controls adipogenesis and glucose tolerance.

Nat Cell Biol 2007 Aug 22;9(8):970-7. Epub 2007 Jul 22.

Cardiovascular Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

Cysteine proteases play an important part in human pathobiology. This report shows the participation of cathepsin L (CatL) in adipogenesis and glucose intolerance. In vitro studies demonstrate the role of CatL in the degradation of the matrix protein fibronectin, insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R), essential molecules for adipogenesis and glucose metabolism. CatL inhibition leads to the reduction of human and murine pre-adipocyte adipogenesis or lipid accumulation, protection of fibronectin from degradation, accumulation of IR and IGF-1R beta-subunits, and an increase in glucose uptake. CatL-deficient mice are lean and have reduced levels of serum glucose and insulin but increased levels of muscle IR beta-subunits, fibronectin and glucose transporter (Glut)-4, although food/water intake and energy expenditure of these mice are no less than their wild-type littermates. Importantly, the pharmacological inhibition of CatL also demonstrates reduced body weight gain and serum insulin levels, and increased glucose tolerance, probably due to increased levels of muscle IR beta-subunits, fibronectin and Glut-4 in both diet-induced obese mice and ob/ob mice. Increased levels of CatL in obese and diabetic patients suggest that this protease is a novel target for these metabolic disorders.
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http://dx.doi.org/10.1038/ncb1623DOI Listing
August 2007

Significance of 32-kDa cathepsin L secreted from cancer cells.

Cancer Biother Radiopharm 2006 Jun;21(3):217-24

Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, Chikusa-ku, Nagoya, Japan.

It is recognized that many cancer cells secrete cathepsin L to degrade the components of extracellular matrices and basement membranes, thus promoting tumor invasion and metastasis. However, very little information is available concerning the secreted forms of cathepsin L and their possible role in human cancer. We initially demonstrated that approximately 10-fold higher mature cathepsin L activity was secreted in a medium of human fibrosarcoma (HT 1080) cells, compared with their intracellular activity. A 32-kDa major-activity band, together with a 41-kDa faint-activity band, was detected in the medium by our newly developed gelatin zymography. The two forms were further confirmed to be cathepsin L by immunoblot analysis. Both were apparently secreted directly from the cells, as neither was affected when the cells were cultured in the presence of various kinds of proteinase inhibitors. Human tumor necrosis factor-alpha (TNF-alpha) stimulated not only the production of the 32-kDa cathepsin L, but also its secretion. Moreover, the 32-kDa cathepsin L activities in 3 colon and 2 lung cancer tissues were significantly higher than in normal tissues. Based on the foregoing, there are good reasons to speculate that the 32-kDa cathepsin L found in HT 1080 cell medium is involved in cancer invasion and metastasis.
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http://dx.doi.org/10.1089/cbr.2006.21.217DOI Listing
June 2006

A novel apoptosis cascade mediated by lysosomal lactoferrin and its participation in hepatocyte apoptosis induced by D-galactosamine.

FEBS Lett 2006 Jun 5;580(15):3699-705. Epub 2006 Jun 5.

Institute for Health Sciences, Tokushima Bunri University, 180 Nishihamabouji, Yamashiro-cho, Tokushima 770-8514, Japan.

A new apoptosis cascade mediated by lysosomal lactoferrin was found in apoptotic liver induced by d-galactosamine. Caspase-3 and lactoferrin were increased in the apoptotic liver cytoplasm and serum transaminases were elevated. Recombinant lactoferrin stimulated procaspase-3 processing at 10(-6)-10(-7)M to an extent similar to that by granzyme B in vitro. Lactoferrin changed procaspase-3 structure susceptible to the processing. Synthetic peptide Y(679)-K(695) in lactoferrin molecule inhibited the processing of procaspase-3 by lactoferrin. Lactoferrin in lysosomes was decreased and lactoferrin released into cytoplasm was increased quantitatively in d-galactosamine induced apoptotic liver, and procaspase-3 in cytoplasm was processed to caspase-3.
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http://dx.doi.org/10.1016/j.febslet.2006.05.057DOI Listing
June 2006

Structural basis of the sphingomyelin phosphodiesterase activity in neutral sphingomyelinase from Bacillus cereus.

J Biol Chem 2006 Jun 4;281(23):16157-67. Epub 2006 Apr 4.

Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.

Sphingomyelinase (SMase) from Bacillus cereus (Bc-SMase) hydrolyzes sphingomyelin to phosphocholine and ceramide in a divalent metal ion-dependent manner. Bc-SMase is a homologue of mammalian neutral SMase (nSMase) and mimics the actions of the endogenous mammalian nSMase in causing differentiation, development, aging, and apoptosis. Thus Bc-SMase may be a good model for the poorly characterized mammalian nSMase. The metal ion activation of sphingomyelinase activity of Bc-SMase was in the order Co2+ > or = Mn2+ > or = Mg2+ >> Ca2+ > or = Sr2+. The first crystal structures of Bc-SMase bound to Co2+, Mg2+, or Ca2+ were determined. The water-bridged double divalent metal ions at the center of the cleft in both the Co2+- and Mg2+-bound forms were concluded to be the catalytic architecture required for sphingomyelinase activity. In contrast, the architecture of Ca2+ binding at the site showed only one binding site. A further single metal-binding site exists at one side edge of the cleft. Based on the highly conserved nature of the residues of the binding sites, the crystal structure of Bc-SMase with bound Mg2+ or Co2+ may provide a common structural framework applicable to phosphohydrolases belonging to the DNase I-like folding superfamily. In addition, the structural features and site-directed mutagenesis suggest that the specific beta-hairpin with the aromatic amino acid residues participates in binding to the membrane-bound sphingomyelin substrate.
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http://dx.doi.org/10.1074/jbc.M601089200DOI Listing
June 2006

Processing of human cathepsin D is independent of its catalytic function and auto-activation: involvement of cathepsins L and B.

J Biochem 2006 Mar;139(3):363-71

Inserm, U540, Montpellier, F-34000 France.

The current mechanism proposed for the processing and activation of the 52 kDa lysosomal aspartic protease cathepsin D (cath-D) is a combination of partial auto-activation generating a 51 kDa pseudo-cath-D, followed by enzyme-assisted maturation involving cysteine and/or aspartic proteases and yielding successively a 48 kDa intermediate and then 34 + 14 kDa cath-D mature species. Here we have investigated the in vivo processing of human cath-D in a cath-D-deficient fibroblast cell line in order to determine whether its maturation occurs through already active cath-D and/or other proteases. We demonstrate that cellular cath-D is processed in a manner independent of its catalytic function and that auto-activation is not a required step. Moreover, the cysteine protease inhibitor E-64 partially blocks processing, leading to accumulation of 52-48 kDa cath-D intermediates. Furthermore, two inhibitors, CLICK148 and CA-074Met, specific for the lysosomal cath-L and cath-B cysteine proteases induce accumulation of 48 kDa intermediate cath-D. Finally, maturation of endocytosed pro-cath-D is also independent of its catalytic function and requires cysteine proteases. We therefore conclude that the mechanism of cath-D maturation involves a fully-assisted processing similar to that of pro-renin.
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http://dx.doi.org/10.1093/jb/mvj037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2376303PMC
March 2006

The first crystal structure of an archaeal helical repeat protein.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2005 Jul 30;61(Pt 7):636-9. Epub 2005 Jun 30.

Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, Tokushima 770-8506, Japan.

The crystal structure of ST1625p, a protein encoded by a hypothetical open reading frame ST1625 in the genome of the hyperthermophilic archaeon Sulfolobus tokodaii, was determined at 2.2 A resolution. The only sequence similarity exhibited by the amino-acid sequence of ST1625p was a 33% identity with the sequence of SSO0983p from S. solfataricus. The 19 kDa monomeric protein was observed to consist of a right-handed superhelix assembled from a tandem repeat of ten alpha-helices. A structural homology search using the DALI and MATRAS algorithms indicates that this protein can be classified as a helical repeat protein.
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http://dx.doi.org/10.1107/S1744309105019263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1952456PMC
July 2005

Catechin derivatives: specific inhibitor for caspases-3, 7 and 2, and the prevention of apoptosis at the cell and animal levels.

FEBS Lett 2006 Feb 6;580(3):741-6. Epub 2006 Jan 6.

Tokushima Bunri University, Institute for Health Sciences, 180 Nishihamabouji, Tokushima 770-8514, Japan.

Tea-catechin derivatives are shown to inhibit activities of caspases-3, 2 and 7 in vitro, and prevented experimental apoptosis at the cell and animal levels. Epigallo-catechin-gallate showed the strongest inhibition at 1 x 10(-7)M to these caspases, but cysteine cathepsins and caspase-8 were not inhibited. Caspase-3 inhibition showed a 2nd-order allosteric-type, but the inhibition of caspases-2 and 7 showed a non-competitive-type. The apoptosis-test using cultured HeLa cells was inhibited by these catechins. In rat hepatocytes, apoptosis was induced by d-galactosamine in vivo. In this case, caspase-3 activity in the cytoplasm, the serum aminotransferases and dUTP nick formation detected by TUNNEL-staining were effects, and these elevations were suppressed by administration of catechin.
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http://dx.doi.org/10.1016/j.febslet.2005.12.087DOI Listing
February 2006
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