Faculty Profiles - OKAMOTO Kuniaki

Papers - OKAMOTO Kuniaki

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Cathepsin E: An Aspartic Protease with Diverse Functions and Biomedical Implications Reviewed

K. Yamamoto, K. Okamoto, T. Tsukuba

Encyclopedia of Cell Biology 1 681 - 690 2015.1

Language：English Publishing type：Part of collection (book) Publisher：Elsevier Inc.

Aspartic peptidases are widely distributed among vertebrates, yeast, plants, fungi, bacteria, and viruses. Of these, the A1 family members, including cathepsin E and ß-secretase, are involved in specific and/or nonspecific degradation of proteins and peptides in intra- and/or extracellular spaces. In the last decade, cathepsin E and ß-secretase have received enormous interest because of their involvement in important biological processes and the close association of their abnormal expression and uncontrolled activity with human diseases. This review summarizes the current knowledge on biochemical properties and functions of cathepsin E and highlights the pathophysiological conditions caused by its deficiency.

DOI： 10.1016/B978-0-12-394447-4.10078-1

Defective adipose tissue development associated with hepatomegaly in cathepsin E-deficient mice fed a high-fat diet Reviewed

Tomoko Kadowaki, Mizuho A. Kido, Junko Hatakeyama, Kuniaki Okamoto, Takayuki Tsukuba, Kenji Yamamoto

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 446 ( 1 ) 212 - 217 2014.3

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Cathepsin E is an intracellular aspartic proteinase, which is predominantly distributed in immune-related and epithelial cells. However, the role of the enzyme in adipose tissues remains unknown. In this study, we investigated the characteristics of cathepsin E-deficient (CatE(-/-)) mice fed a high-fat diet (HFD), as a mouse model of obesity. HFD-fed CatE(-/-) mice displayed reduced body weight gain and defective development of white adipose tissue (WAT) and brown adipose tissue (BAT), compared with HFD-fed wild-type mice. Moreover, fat-induced CatE(-/-) mice showed abnormal lipid accumulation in non-adipose tissues characterized by hepatomegaly, which is probably due to defective adipose tissue development. Detailed pathological and biochemical analyses showed that hepatomegaly was accompanied by hepatic steatosis and hypercholesterolemia in HFD-induced CatE(-/-) mice. In fat-induced CatE(-/-) mice, the number of macrophages infiltrating into WAT was significantly lower than in fat-induced wild-type mice. Thus, the impaired adipose tissue development in HFD-induced CatE(-/-) mice was probably due to reduced infiltration of macrophages and may lead to hepatomegaly accompanied by hepatic steatosis and hypercholesterolemia. (C) 2014 Elsevier Inc. All rights reserved.

File： 47. Kadowaki (BBRC).pdf

DOI： 10.1016/j.bbrc.2014.02.089

Repression of cathepsin E expression increases the risk of mammary carcinogenesis and links to poor prognosis in breast cancer Reviewed

Tomoyo Kawakubo, Atsushi Yasukochi, Tatsuya Toyama, Satoru Takahashi, Kuniaki Okamoto, Takayuki Tsukuba, Seiji Nakamura, Yasuhiko Ozaki, Koichi Nishigaki, Hiroko Yamashita, Kenji Yamamoto

CARCINOGENESIS 35 ( 3 ) 714 - 726 2014.3

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Despite advances in detection and treatment for breast cancer (BC), recurrence and death rates remain unacceptably high. Therefore, more convenient diagnostic and prognostic methods still required to optimize treatments among the patients. Here, we report the clinical significance of the serum cathepsin E (CatE) activity as a novel prognostic marker for BC. Correlation analysis between the serum levels of CatE expression and clinicopathological parameters revealed that the activity levels, but not the protein levels, were negatively associated with the stages and progression of BC. Univariate and multivariate analyses demonstrated that the serum CatE activity was significantly correlated with favorable prognostic outcomes of the patients. The functional link of CatE expression to BC progression was further corroborated by in vivo and in vitro studies with mice exhibiting different levels of CatE expression. Multiparous CatE(/) mice spontaneously developed mammary tumors concomitant with morphological transformation and altered growth characteristics of the mammary glands. These alterations were associated in part with the induction of epithelialmesenchymal transition and the activation of -catenin-dependent pathway in mammary cells. Loss of CatE strongly induced the translocation and accumulation of Wnt5a in the nuclei, thereby leading to the aberrant trafficking, maturation and secretion of Wnt5a and the impaired signaling. The interaction of CatE and Wnt5a was verified by proximity ligation assay and by knockdown or restoration of CatE expression in the mammary cells. Consequently, our data demonstrate that CatE contributes to normal growth and development of mammary glands through proper trafficking and secretion of Wnt5a.

File： 47. Kawakubo (Carcinogenesis).pdf

DOI： 10.1093/carcin/bgt373

Inhibitory effects of tert-butylhydroquinone on osteoclast differentiation via up-regulation of heme oxygenase-1 and down-regulation of HMGB1 release and NFATc1 expression Reviewed

Yu Yamaguchi, Eiko Sakai, Hiroshi Sakamoto, Reiko Fumimoto, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

JOURNAL OF APPLIED TOXICOLOGY 34 ( 1 ) 49 - 56 2014.1

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

Osteoclasts (OCLs) are multinucleated bone-resorbing cells that are differentiated by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Our recent studies have shown that heme-oxygenase-1 (HO-1), a stress-induced cytoprotective enzyme, plays an important role in OCL differentiation, although the pharmacological significance of this effect remains unknown. In this study, we investigated the effects of tert-butylhydroquinone (tBHQ), a pharmacological HO-1 inducer, on in vitro differentiation of bone marrow-derived macrophages (BMMs) or murine monocytic cell line RAW-D into OCLs. tBHQ inhibited the formation and the bone-resorbing activity of OCLs. Moreover, tBHQ treatment decreased the expression of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), a master regulator of OCL differentiation, and of OCL markers transcriptionally regulated by NFATc1, such as Src and cathepsin K. In addition, tBHQ impaired phosphorylation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase, Akt, and inhibitor of nuclear factor kappa B alpha (I kappa B alpha). Finally, we show that tBHQ inhibited the release of high mobility group box 1 (HMGB1), a recently identified activator of OCL differentiation. Thus, tBHQ inhibits OCL differentiation through the HO-1/HMGB1 pathways. Copyright (c) 2012 John Wiley & Sons, Ltd.

File： 48. Yamaguchi (J Appl Toxicol).pdf

DOI： 10.1002/jat.2827

Cobalt protoporphyrin prevents osteoclastogenesis via blocking of I kappa B phosphorylation Reviewed

Yuka Yashima, Kuniaki Okamoto, Eiko Sakai, Kazuhisa Nishishita, Takayuki Tsukuba

JOURNAL OF PHARMACOLOGICAL SCIENCES 124 232P - 232P 2014

Cathepsin E Deficiency Impairs Autophagic Proteolysis in Macrophages Reviewed

Takayuki Tsukuba, Michiyo Yanagawa, Tomoko Kadowaki, Ryosuke Takii, Yoshiko Okamoto, Eiko Sakai, Kuniaki Okamoto, Kenji Yamamoto

PLOS ONE 8 ( 12 ) 2013.12

Language：English Publishing type：Research paper (scientific journal) Publisher：PUBLIC LIBRARY SCIENCE

Cathepsin E is an endosomal aspartic proteinase that is predominantly expressed in immune-related cells. Recently, we showed that macrophages derived from cathepsin E-deficient (CatE(-/-)) mice display accumulation of lysosomal membrane proteins and abnormal membrane trafficking. In this study, we demonstrated that CatE(-/-) macrophages exhibit abnormalities in autophagy, a bulk degradation system for aggregated proteins and damaged organelles. CatE(-/-) macrophages showed increased accumulation of autophagy marker proteins such as LC3 and p62, and polyubiquitinated proteins. Cathepsin E deficiency also altered autophagy-related signaling pathways such as those mediated by the mammalian target of rapamycin (mTOR), Akt, and extracellular signal-related kinase (ERK). Furthermore, immunofluorescence microscopy analyses showed that LC3-positive vesicles were merged with acidic compartments in wild-type macrophages, but not in CatE(-/-) macrophages, indicating inhibition of fusion of autophagosome with lysosomes in CatE(-/-) cells. Delayed degradation of LC3 protein was also observed under starvation-induced conditions. Since the autophagy system is involved in the degradation of damaged mitochondria, we examined the accumulation of damaged mitochondria in CatE(-/-) macrophages. Several mitochondrial abnormalities such as decreased intracellular ATP levels, depolarized mitochondrial membrane potential, and decreased mitochondrial oxygen consumption were observed. Such mitochondrial dysfunction likely led to the accompanying oxidative stress. In fact, CatE(-/-) macrophages showed increased reactive oxygen species (ROS) production and up-regulation of oxidized peroxiredoxin-6, but decreased antioxidant glutathione. These results indicate that cathepsin E deficiency causes autophagy impairment concomitantly with increased aberrant mitochondria as well as increased oxidative stress.

File： 46. Tsukuba (Plosone).pdf

DOI： 10.1371/journal.pone.0082415

Structural and phylogenetic comparison of napsin genes: The duplication, loss of function and human-specific pseudogenization of napsin B Reviewed

Kazuhisa Nishishita, Eiko Sakai, Kuniaki Okamoto, Takayuki Tsukuba

GENE 517 ( 2 ) 147 - 157 2013.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Aspartic proteinases form a widely distributed protein superfamily, including cathepsin D, cathepsin E, pepsins, renin, BACE and napsin. Human napsin genes are located on human chromosome 19q13, which comprises napsin A and napsin B. Napsin B has been annotated as a pseudogene because it lacks an in-frame stop codon; its nascent chains are cotranslationally degraded. Until recently, there have been no studies concerning the molecular evolution of the napsin protein family in the human genome. In the present study, we investigated the evolution and gene organization of the napsin protein family. Napsin B orthologs are primarily distributed in primates, while napsin A orthologs are the only napsin genes in other species. The corresponding regions of napsin B in the available sequences from primate species contain an in-frame stop codon at a position equivalent to that of human napsin A. In addition, a rare single-nucleotide polymorphism (SNP) that creates a proper stop codon in human napsin B was identified using HapMap populations. Recombinant protein expression and three-dimensional comparative modeling revealed that napsin B exhibits residual activity toward synthetic aspartic protease substrates compared with napsin A, presumably through a napsin B-specific Arg287 residue. Thus, napsin B was duplicated from napsin A during the early stages of primate evolution, and the subsequent loss of napsin B function during primate evolution reflected ongoing human-specific napsin B pseudogenization. (C) 2013 Elsevier B.V. All rights reserved.

File： 44. NIshishita (Gene).pdf

DOI： 10.1016/j.gene.2013.01.013

Fisetin inhibits osteoclastogenesis through prevention of RANKLInduced ROS production by Nrf2-mediated Up-regulation of Phase II antioxidant enzymes Reviewed

Eiko Sakai, Megumi Shimada-Sugawara, Yu Yamaguchi, Hiroshi Sakamoto, Reiko Fumimoto, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

Journal of Pharmacological Sciences 121 ( 4 ) 288 - 298 2013

Language：English Publishing type：Research paper (scientific journal)

Osteoclasts (OCLs) are multinucleated bone-resorbing cells that are differentiated by stimulation with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor. We recently demonstrated that regulation of heme-oxygenase 1 (HO-1), a stress-induced cytoprotective enzyme, also functions in OCL differentiation. In this study, we investigated effects of fisetin, a natural bioactive flavonoid that has been reported to induce HO-1 expression, on the differentiation of macrophages into OCLs. Fisetin inhibited the formation of OCLs in a dose-dependent manner and suppressed the bone-resorbing activity of OCLs. Moreover, fisetin-treated OCLs showed markedly decreased phosphorylation of extracellular signal-regulated kinase, Akt, and Jun N-terminal kinase, but fisetin did not inhibit p38 phosphorylation. Fisetin up-regulated mRNA expression of phase II antioxidant enzymes including HO-1 and interfered with RANKL-mediated reactive oxygen species (ROS) production. Studies with RNA interference showed that suppression of NF-E2-related factor 2 (Nrf2), a key transcription factor for phase II antioxidant enzymes, rescued fisetin-mediated inhibition of OCL differentiation. Furthermore, fisetin significantly decreased RANKL-induced nuclear translocation of cFos and nuclear factor of activated T cells cytoplasmic-1 (NFATc1), which is a transcription factor critical for osteoclastogenic gene regulation. Therefore, fisetin inhibits OCL differentiation through blocking RANKLmediated ROS production by Nrf2-mediated up-regulation of phase II antioxidant enzymes. © The Japanese Pharmacological Society.

File： 45. Sakai (J Pharmacol Sci).pdf

DOI： 10.1254/jphs.12243FP

Deltamethrin inhibits osteoclast differentiation via regulation of heme oxygenase-1 and NFATc1 Reviewed

Hiroshi Sakamoto, Eiko Sakai, Reiko Fumimoto, Yu Yamaguchi, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

TOXICOLOGY IN VITRO 26 ( 6 ) 817 - 822 2012.9

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

Deltamethrin is a widely used pyrethroid pesticide. Although the cytotoxicity of deltamethrin has been reported, especially in neuronal cells, there is no information concerning the effects of deltamethrin on osteoclasts (OCLs). In this study, we showed that deltamethrin inhibited OCL differentiation in vitro. The effects of deltamethrin on OCL differentiation by receptor activator of nuclear factor kappa-B ligand (RANKL) were investigated in bone marrow-derived macrophages (BMMs) or the murine monocytic cell line RAW-D. Treatment with deltamethrin inhibited OCL formation and bone resorption and up-regulated expression of heme oxygenase-1 (HO-1), an anti-oxidative stress enzyme. Deltamethrin also decreased the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), which is a master regulator for OCL differentiation, and concomitantly reduced the expression levels of Src and cathepsin K, which are transcriptionally regulated by NFATc1. The effects of deltamethrin on intracellular signaling during the OCL differentiation of BMMs indicated that deltamethrin-treated OCLs displayed impaired phosphorylation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, Jun N-terminal kinase, and Akt, and slightly delayed phosphorylation of inhibitor of nuclear factor kappa B alpha (I kappa beta alpha) compared with untreated OCLs. Thus, deltamethrin possibly affects bone metabolism by inhibiting OCL differentiation. (c) 2012 Elsevier Ltd. All rights reserved.

File： 43. Sakamoto (Toxicol In Vitro).pdf

DOI： 10.1016/j.tiv.2012.05.005

The Coffee Diterpene Kahweol Prevents Osteoclastogenesis via Impairment of NFATc1 Expression and Blocking of Erk Phosphorylation Reviewed

Reiko Fumimoto, Eiko Sakai, Yu Yamaguchi, Hiroshi Sakamoto, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

JOURNAL OF PHARMACOLOGICAL SCIENCES 118 ( 4 ) 479 - 486 2012.4

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE PHARMACOLOGICAL SOC

Osteoclasts (OCLs) are multinucleated bone resorbing cells whose differentiation is regulated by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). It is known that inflammatory cytokines and oxidative stress stimulate differentiation of OCLs. Here we evaluated the effects of kahweol, a coffee-specific diterpene, which has been reported to possess anti-oxidant and anti-inflammatory properties, on the differentiation of bone marrow derived macrophages (BMMs) or murine monocytic cell line RAW-D cells into OCLs. Kahweol dose-dependently inhibited the formation of tartrate-resistant acid phosphatase staining positive OCLs from both BMMs and RAW-D cells. In addition, kahweol prevented the bone resorbing activity of OCLs. Kahweol completely abolished RANKL-stimulated phosphorylation of extracellular signal-regulated kinase and impaired phosphorylation of Akt. Moreover, the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), a master regulator for OCL differentiation; and OCL markers transcriptionally regulated by NFATc1 such as Src and cathepsin K were down-regulated by kahweol treatment. As one of the molecular mechanisms for the inhibitory effects of kahweol, we also showed that kahweol up-regulated heme oxygenase-1 and inhibited high mobility group box I release. Thus, kahweol in coffee is a useful constituent for inhibition of OCL differentiation.

File： 42. Fumimoto (J Pharmacol Sci).pdf

DOI： 10.1254/jphs.11212FP

Genetic backgrounds and redox conditions influence morphological characteristics and cell differentiation of osteoclasts in mice Reviewed

Shun Narahara, Haruna Matsushima, Eiko Sakai, Yutaka Fukuma, Kazuhisa Nishishita, Kuniaki Okamoto, Takayuki Tsukuba

CELL AND TISSUE RESEARCH 348 ( 1 ) 81 - 94 2012.4

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

Osteoclasts (OCLs) are multinucleated giant cells and are formed by the fusion of mononuclear progenitors of monocyte/macrophage lineage. It is known that macrophages derived from different genetic backgrounds exhibit quite distinct characteristics of immune responses. However, it is unknown whether OCLs from different genetic backgrounds show distinct characteristics. In this study, we showed that bone-marrow macrophages (BMMs) derived from C57BL/6, BALB/c and ddY mice exhibited considerably distinct morphological characteristics and cell differentiation into OCLs. The differentiation of BMMs into OCLs was comparatively quicker in the C57BL/6 and ddY mice, while that of BALB/c mice was rather slow. Morphologically, ddY OCLs showed a giant cell with a round shape, C57BL/6 OCLs were of a moderate size with many protrusions and BALB/c OCLs had the smallest size with fewer nuclei. The intracellular signaling of differentiation and expression levels of marker proteins of OCLs were different in the respective strains. Treatment of BMMs from the three different strains with the reducing agent N-acetylcysteine (NAC) or with the oxidation agent hydrogen peroxide (H2O2) induced changes in the shape and sizes of the cells and caused distinct patterns of cell differentiation and survival. Thus, genetic backgrounds and redox conditions regulate the morphological characteristics and cell differentiation of OCLs.

File： 41. Narahara (Cell Tissue Res).pdf

DOI： 10.1007/s00441-012-1325-8

Role of the transcription factor Sp1 in regulating the expression of the murine cathepsin E gene Reviewed

Kuniaki Okamoto, Yoshiko Okamoto, Tomoyo Kawakubo, Jun-ichi Iwata, Yoshiyuki Yasuda, Takayuki Tsukuba, Kenji Yamamoto

JOURNAL OF BIOCHEMISTRY 151 ( 3 ) 263 - 272 2012.3

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Cathepsin E (CE) is an intracellular aspartic proteinase that is exclusively expressed in cells of the gastrointestinal tracts, lymphoid tissues, urinary organs and red blood cells. However, the molecular mechanism by which CE is predominantly expressed in these cells remains unknown. Here, we report the identification of several transcription start sites of the CE gene and their regulatory factors in gastric adenosarcoma cells. We first identified several unique transcription start sites in mouse CE genes by an oligo cap method. Their analysis also revealed the existence of a non-coding region similar to 24-kb upstream of exon 1 in the CE gene and also the existence of two transcripts for CE. Luciferase analyses in upstream of exon 1 revealed that this site contained putative binding regions for the transcription factors Sp1, AP-1 and cEts-1 essential for the expression of CE gene. Moreover, electrophoretic mobility shift assays revealed that the protein-oligonucleotides complex of the Sp1 site were supershifted by an anti-Sp1 antibody. The chromatin immunoprecipitation assay showed that Sp1 bound to the CE promoter region. In addition, overexpression of the Sp1 protein increased the expression of the CE protein. Altogether, these results suggest that Sp1 binding plays a particularly important role in the regulation of CE gene expression.

File： 40. Okamoto (j Biochem).pdf

DOI： 10.1093/jb/mvr135

Suppression of RANKL-dependent heme oxygenase-1 is required for high mobility group box 1 release and osteoclastogenesis Reviewed

Eiko Sakai, Megumi Shimada-Sugawara, Kazuhisa Nishishita, Yutaka Fukuma, Mariko Naito, Kuniaki Okamoto, Koji Nakayama, Takayuki Tsukuba

JOURNAL OF CELLULAR BIOCHEMISTRY 113 ( 2 ) 486 - 498 2012.2

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

The differentiation of osteoclasts is regulated by several essential cytokines, such as receptor activator of nuclear factor ?B ligand (RANKL) and macrophage colony-stimulating factor. Recently, high mobility group box 1 (HMGB1), a chromatin protein, also has been identified as one of these osteoclast differentiation cytokines. However, the molecular mechanisms that control HMGB1 release from osteoclast precursor cells are not known. Here, we report that RANKL-induced suppression of heme oxygenase-1 (HO-1), a heme-degrading enzyme, promotes HMGB1 release during osteoclastogenesis. In contrast, induction of HO-1 with hemin or curcumin in bone marrow-derived macrophages or RAW-D murine osteoclast precursor cells inhibited osteoclastogenesis and suppressed HMGB1 release. Since an inhibitor for p38 mitogen-activated protein kinase (MAPK) prevented the RANKL-mediated HO-1 suppression and extracellular release of HMGB1, these effects were p38 MAPK-dependent. Moreover, suppression of HO-1 in RAW-D cells by RNA interference promoted the activation of caspase-3 and HMGB1 release, whereas overexpression of HO-1 inhibited caspase-3 activation as well as HMGB1 release. Furthermore, these effects were regulated by redox conditions since antioxidant N-acetylcysteine abolished the HO-1/HMGB1/caspase-3 axis. These results suggest that RANKL-dependent HO-1 suppression leads to caspase-3 activation and HMGB1 release during osteoclastogenesis. J. Cell. Biochem. 113: 486498, 2012. (C) 2011 Wiley Periodicals, Inc.

DOI： 10.1002/jcb.23372

Suppression of RANKL-dependent heme oxygenase-1 is required for high mobility group box 1 release and osteoclastogenesis Reviewed

Eiko Sakai, Megumi Sugawara, Kazuhisa Nishishita, Yutaka Fukuma, Kuniaki Okamoto, Takayuki Tsukuba

JOURNAL OF PHARMACOLOGICAL SCIENCES 118 ( 2 ) 208P - 208P 2012

Cathepsin E is critical for proper trafficking of cell surface proteins Reviewed

Takayuki Tsukuba, Kuniaki Okamoto, Kenji Yamamoto

Journal of Oral Biosciences 54 ( 1 ) 48 - 53 2012

Language：English Publishing type：Research paper (international conference proceedings) Publisher：Japanese Association for Oral Biology

Cathepsin E is an intracellular aspartic proteinase of the pepsin superfamily, which is predominantly expressed in certain cell types, such as the immune-related cells and rapidly regenerating cells. Recent studies have demonstrated that loss of cathepsin E in mice impairs their normal immune responses. In antigen-presenting cells (APC) such as macrophages, dendritic cells, and microglia, cathepsin E is localized mainly in the endosomal component and regulates the nature and functions of these cells. Deficiency of cathepsin E in macrophages induces a novel form of lysosomal storage disorder manifesting the accumulation of major lysosomal membrane glycoproteins such as LAMP-1 and LAMP-2 and elevated lysosomal pH. Such alterations in these cells are linked to abnormal intracellular trafficking of secretory and cell surface proteins. In fact, cathepsin E deficiency leads to increased secretion of a variety of soluble lysosomal enzymes including cathepsins and glycosidases, into the culture medium. By contrast, the expression and localization of cell surface proteins including Toll-like receptors, chemotactic receptors, and cell-adhesion receptors, as well as LAMPs, is significantly decreased by cathepsin E deficiency. While these alterations are not observed with cathepsin E-deficient (CatE-/-) dendritic cells, the cell surface expression and localization of the costimulatory molecules CD86, CD80, and CD40 were significantly increased in these cells, indicating that cathepsin E differentially regulates the nature and function of these two APC. This review focuses on the emerging roles of cathepsin E in proper intracellular trafficking of both secretory and cell surface proteins in APC. © 2012 Japanese Association for Oral Biology.

DOI： 10.1016/j.job.2011.10.001

The role of cathepsin E in terminal differentiation of keratinocytes. Reviewed

Kawakubo T, Yasukochi A, Okamoto K, Okamoto Y, Nakamura S, Yamamoto K

Biological chemistry 392 ( 6 ) 571 - 585 2011.4

Cathepsin E-deficient mice show increased susceptibility to bacterial infection associated with the decreased expression of multiple cell surface Toll-like receptors. Reviewed

Tsukuba T, Yamamoto S, Yanagawa M, Okamoto K, Okamoto Y, Nakayama KI, Kadowaki T, Yamamoto K

Journal of biochemistry 140 ( 1 ) 57 - 66 2006.7

Mutational analysis of residues in two consensus motifs in the active sites of cathepsin E1 Reviewed

Jian Liu, Takayuki Tsukuba, Kuniaki Okamoto, Masamichi Ohishi, Kenji Yamamoto

Journal of Biochemistry 132 ( 3 ) 493 - 499 2002

Language：English Publishing type：Research paper (scientific journal)

Cathepsin E, an intracellular aspartic proteinase of the pepsin family, is composed of two homologous domains, each containing the catalytic Asp residue in a consensus DTG motif. Here we examine the significance of residues in the motifs of rat cathepsin E by substitution of Asp98, Asp283, and Thr284 with other residues using site-directed mutagenesis. Each of the mutant proenzymes, as well as the wild-type protein, was found in culture media and cell extracts when heterologously expressed in human embryonic kidney 293T cells. The single mutants D98A, D283A, and D283E, and the double mutants D98A/D283A and D98E/D283E showed neither autocatalytic processing nor enzymatic activities under acidic conditions. However, the D98E and T284S mutants retained the ability to transform into the mature forms, although they exhibited only about 13 and 40% of the activity of the wild-type enzyme, respectively. The Km values of these two mutants were similar to those of the wild-type enzyme, but their kcat values were greatly decreased. The Ki values for pepstatin and the Ascaris pepsin inhibitor of the mutants and the wild-type enzyme were almost the same. The circular dichroism spectra of the two mutants were essentially the same as those of the wild-type enzyme at various pH values. These results indicate that (i) Asp98, Asp283, and Thr284 are indeed critical for catalysis, and (ii) the decrease in the catalytic activity of D98E and T284S mutants is brought about by an effect on the kinetic process from the enzyme-substrate complex to the release of the product. © 2002 Japanese Biochemical Society.

DOI： 10.1093/oxfordjournals.jbchem.a003247

Characterization of phagosomal subpopulations along endocytic routes in osteoclasts and macrophages. Reviewed

Sakai E, Miyamoto H, Okamoto K, Kato Y, Yamamoto K, Sakai H

J Biochem. 130 ( 6 ) 823 - 831 2001

Design and synthesis of sensitive fluorogenic substrates specific for Lys-gingipain Reviewed

Naoko Abe, Atsuyo Baba, Tomoko Kadowaki, Kuniaki Okamoto, Shinji Okazaki, Tetsuji Asao, Kenji Yamamoto

Journal of Biochemistry 128 ( 5 ) 877 - 881 2000

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press

Lys-gingipain (Kgp) is a major cysteine proteinase produced by the oral anaerobic bacterium Porphyromonas gingivalis, and has been implicated as a major pathogen in the development and progression of advanced adult periodontitis. This enzyme is believed to act as a major virulence factor of the disease, yet there exist no convenient and sensitive substrates for analyzing its biological activity. For a better understanding of the importance of this enzyme in the organism, there is an urgent need for specific substrates. Here we designed and synthesized two peptide 4-methyl-coumaryl-7-amides (MCA), carbobenzoxy (Z)-His-Glu-Lys-MCA, and Z-Glu-Lys-MCA, and tested their possible use as sensitive substrates for Kgp with limited specificity. Both substrates exhibited greater k(cat)/K(m) values than the best known Kgp substrates described so far. Both substrates were resistant to Arg-gingipain, another pathogenic cysteine proteinase from P. gingivalis, as well as trypsin and cathepsins B, L, and H. The levels of Kgp in various microorganisms and human cells were determined with Z-His-Glu-Lys-MCA. Little or no Kgp-like activity was detected in either other microorganisms or human cells tested. These results indicate that the present substrates are a valuable and fast tool for routine assays and for mechanistic studies on Kgp.

DOI： 10.1093/oxfordjournals.jbchem.a022826

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