Biomedical subjects

E Kominami

Publications and source records attributed to E Kominami.

At least 55 records · Page 3Linked to original sources

PubMed1999

Characterization of new fluorogenic substrates for the rapid and sensitive assay of cathepsin E and cathepsin D.

Cathepsin E and cathepsin D are two major intracellular aspartic proteinases implicated in the physiological and pathological degradation of intra- and extracellular proteins. In this study, we designed and constructed highly sensitive synthetic decapeptide substrates for assays of cathepsins E and D based on the known sequence specificities of their cleavage sites. These substrates contain a highly fluorescent (7-methoxycoumarin-4-yl)acetyl (MOCAc) moiety and a quenching 2,4-dinitrophenyl (Dnp) group. When the Phe-Phe bond is cleaved, the fluorescence at an excitation wavelength of 328 nm and emission wavelength of 393 increases due to diminished quenching resulting from the separation of the fluorescent and quenching moieties. The first substrate, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Le u-Lys(Dnp)gamma-NH2, in which the Lys-Pro combination at positions P5 and P4 was designed for specific interaction with cathepsin E, is hydrolyzed equally well by cathepsins E and D (kcat/Km = 10.9 microM(-1) x s(-1) for cathepsin E and 15.6 microM(-1) x s(-1) for cathepsin D). A very acidic pH optimum o was obtained for both enzymes. The second substrate, MOCAc-Gly-Lys-Pro-Ile-Ile-Phe-Phe-Arg-Le u-Lys(Dnp)gamma-NH2, in which the isoleucine residue at position P2 was meant to increase the specificity for cathepsin E, is also hydrolyzed equally by both enzymes (kcat/Km = 12.2 microM(-1) x s(-1) for cathepsin E and 16.3 microM(-1) x s(-1) for cathepsin D). The kcat/Km values for both substrates are greater than those for the best substrates for cathepsins E and D described so far. Unfortunately, each substrate shows little discrimination between cathepsin E and cathepsin D, suggesting that amino acids at positions far from the cleavage site are important for discrimination between the two enzymes. However, in combination with aspartic proteinase inhibitors, such as pepstatin A and Ascaris pepsin inhibitor, these substrates enable a rapid and sensitive determination of the precise levels of cathepsins E and D in crude cell extracts of various tissues and cells. Thus these substrates represent a potentially valuable tool for routine assays and for mechanistic studies on cathepsins E and D.

Amino Acid Sequence↗

PubMed1999

Function of the propeptide region in recombinant expression of active procathepsin L in Escherichia coli.

In order to determine the functional role of the procathepsin L propeptide region for the preparation of active recombinant rat cathepsin L (CL), cDNAs encoding two short-length propeptides (C-terminal 2 and 27 residues) and the full-length (96 residues) one plus the entire CL were expressed as two soluble fusion proteins with a fragment of maltose-binding protein and an insoluble fusion protein with glutathione-S-transferase in Escherichia coli, respectively. After refolding of the insoluble fusion protein, each gene product was purified to homogeneity by amylose or glutathione-Sepharose-4B affinity column, and digestion with factor Xa and alpha-thrombin under alkaline conditions (pH approximately 8.0) led to the elution of two pure short-length procathepsin Ls (PCLs) and a full-length one, respectively. The enzymatic activity, estimated by hydrolytic assaying of benzoxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide under acidic conditions (pH 5.5), indicated that the two short-length PCLs exhibited in a great loss of the activity, as compared with the full-length PCL. The CD spectra of the short-length PCLs were different from that of the full-length one. The present results clearly show that the full-length propeptide is essential for construction of the active tertiary structure of CL at the stage of recombinant protein expression, although the expression of CL itself in E. coli does not require the propeptide. Based on the tertiary structure of PCL, the propeptide region necessary for the construction of the CL active structure has been discussed.

Animals↗

PubMed1999

The induction of autophagic vacuoles and the unique endocytic compartments, C-shaped multivesicular bodies, in GH4C1 cells after treatment with 17beta-estradiol, insulin and EGF.

The mechanisms for the formation of autophagic vacuoles were investigated using GH4C1 cells, a rat pituitary tumor cell line, whose induction increases intracellular levels of lysosomal proteinases and their mRNA by treatment with a combination of hormones (17beta-estradiol, insulin and EGF). By ordinary electron microscopy, autophagic vacuoles containing various undigested structures with or without limiting membranes were abundant in the hormone-induced cells. These vacuoles, also containing numerous small vesicles, appeared to be derived from multivesicular bodies. In fact, there were also numerous C-shaped multivesicular bodies which enclosed cytoplasmic portions, suggesting that these unique structures are involved in the production of the autophagic vacuoles. Moreover, the cytoplasmic portions enlapped by the C-shaped multivesicular bodies were high in electron density and contained filamentous structures. By the cryothin-section immunogold method, the C-shaped multivesicular bodies in some cases contained lysosomal marker proteins such as cathepsins B and H, and Igp 120. Using an anti-actin monoclonal antibody, immunogold particles clearly labeled the cytoplasmic portions enclosed by the C-shaped multivesicular bodies. Pulse-chase experiments with horse radish peroxidase, a fluid-phase endocytic marker, revealed that the incidence of the C-shaped multivesicular bodies labeled with horse radish peroxidase peaked at 30 min after the beginning of chase incubation, whereas no C-shaped multivesicular body with horse radish peroxidase was detected in the cells by cytochalasin D treatment. These results suggest that the C-shaped multivesicular bodies occur in a transitional process from endosomes to lysosomes by the action of actin filaments, and that this morphological change may be essential for the production of autophagic vacuoles in the hormone-induced GH4C1 cells.

Actins↗

PubMed1999

Efficient mitochondrial import of newly synthesized ornithine transcarbamylase (OTC) and correction of secondary metabolic alterations in spf(ash) mice following gene therapy of OTC deficiency.

BACKGROUND: The mouse strain sparse fur with abnormal skin and hair (spf(ash)) is a model for the human ornithine transcarbamylase (OTC) deficiency, an X-linked inherited urea cycle disorder. The spf(ash) mouse carries a single base-pair mutation in the OTC gene that leads to the production of OTC enzyme at 10% of the normal level. MATERIALS AND METHODS: Recombinant adenoviruses carrying either mouse (Ad.mOTC) or human (Ad.hOTC) OTC cDNA were injected intravenously into the spf(ash) mice. Expression of OTC enzyme precursor and its translocation to mitochondria in the vector-transduced hepatocytes were analyzed on an ultrastructural level. Liver OTC activity and mitochondrial OTC concentration were significantly increased (300% of normal) in mice treated with Ad.mOTC and were moderately increased in mice receiving Ad.hOTC (34% of normal). The concentration and subcellular location of OTC and associated enzymes were studied by electron microscope immunolocalization and quantitative morphometry. RESULTS: Cytosolic OTC concentration remained unchanged in Ad.mOTC-injected mice but was significantly increased in mice receiving Ad.hOTC, suggesting a block of mitochondria translocation for the human OTC precursor. Mitochondrial ATPase subunit c [ATPase(c)] was significantly reduced and mitochondrial carbamy delta phosphate synthetase I (CPSI) was significantly elevated in spf(ash) mice relative to C3H. In Ad.mOTC-treated mice, the hepatic mitochondrial concentration of ATPase(c) was completely normalized and the CPSI concentration was partially corrected. CONCLUSIONS: Taken together, we conclude that newly synthesized mouse OTC enzyme was efficiently imported into mitochondria following vector-mediated gene delivery in spf(ash) mice, correcting secondary metabolic alterations.

Adenoviridae↗

PubMed1998

Interaction of Doc2 with tctex-1, a light chain of cytoplasmic dynein. Implication in dynein-dependent vesicle transport.

Doc2 has one Munc13-interacting domain at the N-terminal region and two C2-like domains interacting with Ca2+ and phospholipid at the C-terminal region. Doc2 consists of two isoforms, Doc2alpha and -beta. Doc2alpha is specifically expressed in neuronal cells and implicated in Ca2+-dependent neurotransmitter release, whereas Doc2beta is ubiquitously expressed and its function is unknown. We show here that both Doc2alpha and -beta interact with rat tctex-1, a light chain of cytoplasmic dynein, in both cell-free and intact cell systems. Overexpression of the N-terminal fragment of Doc2 containing the tctex-1-interacting domain induces changes in the intracellular localization of cation-independent mannose 6-phosphate receptor and its ligand, cathepsin D, which are transported from trans-Golgi network to late endosomes. Overexpression of the C-terminal fragment containing two C2-like domains shows the similar effect, but to a lesser extent, whereas overexpression of full-length Doc2 or the C-terminal fragment of rabphilin3 containing two C2-like domains does not show this effect. Because dynein is a minus-end-directed microtubule-based motor protein, these results suggest that Doc2, especially Doc2beta, plays a role in dynein-dependent intracellular vesicle transport.

Amino Acid Sequence↗

PubMed1998

Multiple processing of procathepsin L to cathepsin L in vivo.

Three amino-terminal-specific peptidic antibodies against cathepsin L were generated. These antibodies recognize in vitro processing products of procathepsin L in time-course-dependent fashion. Immunoblot analyses with these antibodies followed by immunoprecipitation with anti-cathepsin L antibody showed that the amino terminal amino acid sequences of intracellular cathepsin L are heterogeneous: the single chain form of cathepsin L starts with either EPLML, LKIPK or IPKSV, and the heavy chain of the two chain form with IPKSV. Percoll density gradient and fluorescence immunohistochemistry suggested that these three species of cathepsin L localize in the lysosomes where procathepsin L processing occurs.

3T3 Cells↗

PubMed1998

Overexpression of cation-dependent mannose 6-phosphate receptor prevents cell death induced by serum deprivation in PC12 cells.

PC12 cells express well cation-independent mannose 6-phosphate receptors (CI-MPR), but not cation-dependent (CD)-MPR as much. To examine CD-MPR dependency of transport of cathepsins B and D to lysosomes in PC12 cells, we prepared the cells overexpressing CD-MPR. Immunoreactivity for cathepsin B became more distinct and larger in size in the transfected cells than in wild-type cells. No difference in the distribution of cathepsin D was seen between these two cells. The viability of the cells following serum deprivation was significantly higher in the transfected cells than in wild-type cells. This increased viability of the transfected cells was blocked by CA074, a specific inhibitor of cathepsin B, while pepstatin A suppressed the action of CA074. The results suggest that CD-MPR preferentially transport cathepsin B in PC12 cells, and cathepsins B and D participate in the regulation of PC12 cell apoptosis.

Amino Acid Sequence↗

PubMed1998

Participation of cathepsins B and D in apoptosis of PC12 cells following serum deprivation.

Cathepsin D, a lysosomal aspartic proteinase, has been shown to induce apoptosis of HeLa cells when overexpressed. To further understand regulatory mechanisms of cathepsin D-induced cell death, we examined whether lysosomal cysteine and aspartic proteinases are involved in apoptosis of PC12 cells following serum deprivation. In serum deprived culture, PC12 cells overexpressing cathepsin D died more rapidly than wild-type cells. When the active forms of cathepsins B and D were examined during the apoptotic process of wild-type cells, the amount of cathepsin B was drastically reduced 24 hr after the onset of culture, whereas that of cathepsin D considerably increased. The viability of PC12 cells overexpressing cathepsin B was significantly higher in serum-deprived culture than wild-type cells. In this situation, the amount of the cathepsin B protein did not decrease. The results suggest that there exists an apoptotic pathway regulated by lysosomal cathepsins B and D.

Animals↗

PubMed1998

The antigen presentation pathway in medullary thymic epithelial cells, but not that in cortical thymic epithelial cells, conforms to the endocytic pathway.

Murine medullary thymic epithelial cells (mTEC), but not cortical thymic epithelial cells (cTEC), are able to present a soluble antigen, ovalbumin, to helper T cells (Mizuochi, T. et al., J. Exp. Med. 1992. 175: 1601-1605). This functional difference between the mTEC and the cTEC is particularly important when we consider the thymic selection of the T cell repertoire. In the previous report, we proposed that mTEC and cTEC utilize two distinct antigen processing/presenting pathways (Kasai, M. et al., Eur. J. Immunol. 1996. 26: 2101-2107). In this report, we further confirmed this difference by analyzing (a) localization of MHC class II, H2-DM, and invariant chain (li) molecules, (b) the biochemical nature of MHC class II molecules, (c) the sensitivity of MHC class II alphabeta heterodimer formation to concanamycin A, a vacuolar H+-ATPase inhibitor, and (d) the subcellular distribution of MHC class II, H2-DM, and li molecules, in both TEC. Our results demonstrated that, in the mTEC, MHC class II, H2-DM and li molecules gain access to the endocytic pathway, where the luminal condition is acidic and thus li molecules are efficiently degraded and H2-DM molecules function well. In the cTEC, however, such molecules seemed to gain access to an alternative transport pathway, e.g. a secretory pathway, where the luminal condition is not fully acidic. These two distinct antigen processing pathways may account for the functional difference between mTEC and cTEC.

Amino Acid Sequence↗

PubMed1998

Inhibition of ischaemic hippocampal neuronal death in primates with cathepsin B inhibitor CA-074: a novel strategy for neuroprotection based on 'calpain-cathepsin hypothesis'.

Although Cornu Ammonis (CA) 1 neurons of the hippocampus are known to be vulnerable to transient ischaemia, the mechanism of ischaemic neuronal death is still unknown, and there are very few strategies to prevent neuronal death at present. In a previous report we demonstrated micro-calpain activation at the disrupted lysosomal membrane of postischaemic CA1 neurons in the monkey undergoing a complete 20 min whole brain ischaemia. Using the same experimental paradigm, we observed that the enzyme activity of the lysosomal protease cathepsin B increased throughout the hippocampus on days 3-5 after the transient ischaemia. Furthermore, by immunocytochemistry cathepsin B showed presence of extralysosomal immunoreactivity with specific localization to the cytoplasm of CA1 neurons and the neuropil of the vulnerable CA1 sector. When a specific inhibitor of cathepsin B, the epoxysuccinyl peptide CA-074 (C18H29N3O6) was intravenously administered immediately after the ischaemic insult, approximately 67% of CA1 neurons were saved from delayed neuronal death on day 5 in eight monkeys undergoing 20 min brain ischaemia: the extent of inhibition was excellent in three of eight and good in five of eight monkeys. The surviving neurons rescued by blockade of lysosomal activity, showed mild central chromatolysis and were associated with the decreased immunoreactivity for cathepsin B. These observations indicate that calpain-induced cathepsin B release is crucial for the development of the ischaemic neuronal death, and that a specific inhibitor of cathepsin B is of potential therapeutic utility in ischaemic injuries to the human CNS.

Animals↗

PubMed1998

Suppression of lysosomal proteolysis at three different steps in regenerating rat liver.

Decreased lysosomal proteolysis in regenerating liver after 70% hepatectomy was analyzed. The activities of cathepsins B and L increased transiently 4 h after hepatectomy, began to decrease gradually reaching about 30% of the control level at 24 h, then returned to near control level after 7 days. Immunoblot and RNA blot analyses confirmed that the changes in cathepsin activities coincided with changes in protein levels and mRNA levels. In parallel with the changes in cathepsins, we found that the amounts of LGP120, LGP110, and LGP85, three integral lysosomal membrane proteins, declined significantly after hepatectomy, suggesting that the lysosomal levels are also diminished in regenerating liver. We isolated dextran-loaded lysosomes and found that the protein content and marker enzyme activities of dextran-loaded lysosomes from partially hepatectomized livers are lower by 50 and 40%, respectively, compared with control livers. This indicates that there is a significant reduction in the cellular lysosomal level in regenerating liver. In addition, we used a sensitive biochemical assay to quantify leupeptin-induced autolysosomes and found that the autophagic activity is markedly suppressed in regenerating liver as compared with normal liver. Thus, the suppression of lysosomal proteolysis in regenerating liver is attained through three steps, i. e., decreased biosynthesis of cathepsins, decreased lysosomal biogenesis, and decreased cellular autophagy.

Animals↗

PubMed1998

Cystatin A expression reduces bile salt-induced apoptosis in a rat hepatoma cell line.

We have previously demonstrated abrogation of bile salt-induced apoptosis by cathepsin B inhibitors. However, caspases have been strongly implicated in apoptosis, and the mechanistic interface between caspase and cathepsin B activation is unclear. Thus our aims were to determine the mechanistic relationship between caspases and cathepsin B in bile salt-induced apoptosis in a rat hepatoma cell line. Expression of cystatin A was used to inhibit cathepsin B, whereas Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) was used to inhibit caspases. Cystatin A expression prevented cathepsin B activation and apoptosis during treatment with glycochenodeoxycholate (GCDC), a toxic bile salt. Caspase N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC) hydrolytic activity increased in both wild-type and cystatin A-transfected cells treated with GCDC, demonstrating caspase activation despite inhibition of cathepsin B. In contrast, Z-VAD-FMK blocked both DEVD-AMC hydrolytic activity and cathepsin B activity during GCDC treatment. Our data demonstrate that 1) bile salt-induced apoptosis can be inhibited by the cystatin A transgene and 2) caspase and cathepsin B activation are linked mechanistically with cathepsin B downstream of caspases.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Deimination of 70-kD nuclear protein during epidermal apoptotic events in vitro.

Peptidylarginine deiminase (PAD) is the enzyme responsible for converting protein-bound arginine residues to citrulline. It has recently been shown that a number of epidermal proteins, including filaggrin, trichohyalin, and keratins, are deiminated by the action of PAD, suggesting a possible role for protein deimination during the final stages of epidermal differentiation. We report here a novel PAD substrate found during the course of identifying deiminated proteins in cultured rat epidermal keratinocytes. We found that a 70-kD protein localized to the periphery of the nucleus was preferentially deiminated after ionomycin treatment in the presence of 2 mM calcium and was associated with apoptotic events in these cells. Furthermore, we discovered that the deimination of nuclear protein could be induced by transfection of a PAD cDNA into rat epidermal keratinocytes. These data suggest that PAD may act on the 70-kD nuclear protein to induce disassembly of the nuclear lamina and promote apoptosis during terminal epidermal differentiation.

Animals↗

PubMed1998

Gene regulation and extracellular functions of procathepsin L.

Cathepsin L, a lysosomal cysteine proteinase, belongs to the papain family. This proteinase is different from the other members of the mammalian papain family cysteine proteinase in the following ways: (i) The cathepsin L gene is activated by a variety of growth factors and activated oncogenes. (ii) Procathepsin L, a precursor form of cathepsin L is secreted from various cells. (iii) The mRNA level of cathepsin L is related to the in vivo metastatic potential of the transformed cells. Thus, the regulation of the cathepsin L gene and the extracellular functions of secreted procathepsin L are tightly coupled. In this review, we describe these two points, which have recently been addressed in our laboratory.

Animals↗

PubMed1998

Molecular weight-dependent effects of hyaluronate on the arthritic synovium.

Intra-articular injection of hyaluronate (HA) is widely used in the treatment of arthropathies. However, the mechanism of the effects of HA preparations on the arthritic synovium and the relationship between their effects and molecular weights (MW) remains unknown. The objectives of this study were to compare the effects of two hyaluronate preparations, HA84 (MW: 84 X 10(4)) and HA230 (MW: 230 X 10(4)), on the synovium of an arthritis model and to examine the mechanism of the effects of HA. The HA preparations were intra-articularly injected in a model of canine arthritis induced by anterior cruciate ligament transection for a total trial of 5 weeks. To define the accessibility of HA preparations to the synovial lining layers, fluorescein-labeled HA84 or HA230 was injected at the last administration. Pathological changes analyzed included increases in volumes and prostaglandin E2 concentrations in synovial fluids, thickening of the synovial lining layers, vacuolar alterations in the lining cells, and stainability of HA in the synovium. Expression levels of Heat shock protein 72 (Hsp72) were immunohistochemically detected in the tissues to investigate the ability of the cells to survive the degeneration. The pathological changes described above were more significantly suppressed in the HA84-treated than in the HA230-treated groups. In most cases of the HA84-treated group (five cases out of six), fluorescein particles were intensely distributed in the synovial lining layers, but only two cases in the HA230-treated group showed a weak distribution of fluorescein particles in the layers, indicating a certain difference in the accessibility of HA preparations to the lining cells between the two HA molecules. Moreover, the immunoreactivity for Hsp72 in the lining cells as more intense in the HA84-treated than in the HA230-treated groups. The difference in the accessibility of HA molecules corresponded well with that in the inducibility of Hsp72 in the lining cells. These results suggest that the up-regulation of Hsp72 may offer a new concept concerning mechanism of the effects of HA preparations on the arthritic synovium.

Animals↗

PubMed1998

An ultrastructural and immunohistochemical study of PC12 cells during apoptosis induced by serum deprivation with special reference to autophagy and lysosomal cathepsins.

In addition to the caspase family of proteinases, cathepsin D, a lysosomal aspartic proteinase, has been suggested to act as a proapoptotic mediator in mammalian cells. To further understand the roles of cathepsins B and D in apoptosis of the cells, we examined the precise alteration processes of ultrastructures and immunoreactivity for these enzymes in PC12 cells cultured under serum deprivation. Laser scanning microscopy showed immunoreactivity for cathepsins B and D to be finely distributed in the cytoplasm of PC12 cells at the onset of culture under serum deprivation. At 3 h after the onset of culture, the immunoreactivity for cathepsin B slightly decreased in the cells, while immunodeposits for cathepsin D in the cells became more intense and larger in size than those at 0 h. Positive staining for TUNEL in nuclei of the cells appeared at 6 h, though fewer in number. Corresponding to the increase in the number of TUNEL-positive cells at 12 h and 24 h, the immunoreactivity for cathepsin B was drastically diminished in the cells, whereas that for cathepsin D was significantly augmented, especially in TUNEL-positive cells. Electron microscopically, autophagic vacuoles/autolysosomes appeared in the cytoplasm of the cells 3 h after the onset of culture. A distinct nuclear change showing relatively condensed chromatin first appeared in the peripheral part of the nuclei at 6 h. The number of PC12 cells having nuclei with chromatin condensation increased especially at 24 h, while these cells showed shrinkage of both their cytoplasm and nuclei. Dense bodies and autophagic vacuoles with limiting membranes were seen in these cells. These results showing the occurrence of autophagy and imbalance of protein amounts between cathepsins B and D during apoptosis may argue for our hypothesis that these enzymes are, in part, involved in the cell death cascade for PC12 cells following serum deprivation.

Animals↗

PubMed1998

[Batten disease].

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Biomarkers↗

PubMed1997

Egr family member proteins are involved in the activation of the cathepsin L gene in v-src-transformed cells.

Both the protein and mRNA levels of cathepsin L in SR-3Y1-2, v-src-transformed 3Y1 cell lines were higher than in 3Y1 cells. Results of CAT assays suggested that the v-src responsible region in the cathepsin L gene localizes to 300 bp in the 5'-upstream region and 60 bp in the first exon. DNaseI footprinting analyses showed that transcription factors bind to the region from 29bp to 55bp from the transcription start site. This region contains a CAAT-box and a 5'-GGCGGGGGCGG-3' sequence containing two repeat copies of consensus Sp-1 binding sites, overlapping a consensus Egr family binding site. DNA band shift assays showed that Sp-1 and NF-1 binding proteins bind to this region in 3Y1 cells while Egr family protein binds in SR-3Y1-2 cells. These results suggest that Egr family proteins are involved in the activation of the cathepsin L gene in SR-3Y1-2 cells.

Animals↗