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Biomedical subjects

K Ishidoh

Publications and source records attributed to K Ishidoh.

At least 37 records · Page 2Linked to original sources
PubMed1995

Autocatalytic inactivation of lysosomal cathepsins is associated with inhibition of protein breakdown by insulin-like growth factor-1 (IGF-1) in myotubes.

Protein breakdown was monitored in C2C12 myotubes as the rate of release of radioactivity after prelabeling cell protein with [3H] tyrosine. IGF-1 (13 nM) and insulin (100 nM) prolonged the half-life of long-lived proteins. Enzymatic activities of cathepsins B and B+L were inhibited by the addition of IGF-1 or insulin. Immunoblotting of cathepsins B and L revealed extensive degradation of heavy chain forms by IGF-1. However, neither expression of cathepsins B and L genes nor expression of cystatin beta, an intrinsic inhibitor for cathepsins, was influenced. The addition of E-64, L-3-carboxy-trans-2,3-epoxypropionyl-leucylamide-(4-guanidin o) butane, a inhibitor of cathepsins B and L, increased protein contents of heavy chains of cathepsins B and L in the IGF-1 treated cells. Inhibition of protein breakdown by IGF-1 is mediated by autocatalytic inactivation of lysosomal cathepsins B and L.

Analysis of Variance↗
PubMed1995

Specific delay of degradation of mitochondrial ATP synthase subunit c in late infantile neuronal ceroid lipofuscinosis (Batten disease).

Subunit c is normally present as an inner mitochondrial membrane component of the F0 section of the ATP synthase complex, but in the late infantile form of neuronal ceroid lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. To explore the mechanism of storage of subunit c, the rates of degradation and synthesis of subunit c were measured in fibroblast cell types from controls and patients with the late infantile form of NCL. The radiolabel from subunit c decreased with time in control cells, whereas no apparent loss of radioactivity of subunit c was found in patients' cells. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. A combination of pulse-chase and subcellular fractionation analysis showed that a delay of intramitochondrial loss from prelabeled subunit c was seen in all diseased cells tested. Lysosomal appearance of labeled subunit c could be detected after chase for more than 1 week and its radioactivities were variable among diseased cell types. The biosynthetic rate of subunit c was almost the same in both control and patient cells. Northern blotting analyses showed that mRNAs for P1 and P2 genes had no significant difference in lengths and amounts between control and patient cells. Results suggest a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes. This is the first direct evidence to show a delay of subunit c degradation in the cells from the late infantile form of NCL.

Fibroblasts↗
PubMed1995

Changes of proteasomes and cathepsins activities and their expression during differentiation of C2C12 myoblasts.

C2C12 myoblasts fuse to form multinucleated myotubes and express muscle specific proteins during differentiation. To elucidate developmental regulation of intracellular proteolytic systems, enzymatic activities, protein and mRNA levels of proteasomes (20S and 26S) and lysosomal cathepsins (B, L, and H) were examined. Myoblasts were differentiated fully to myotubes 6 days after starting differentiation. In this developmental process, the 26S proteasome activity decreased, while the 20S proteasome activity increased. Expression of proteasome subunits of 20S (RC2, RC8) and regulatory components of 26S (S4, S7) was down-regulated, though total protein levels of proteasomes showed no remarkable changes. On the other hand, enzymatic activities of cathepsins B and B + L increased in association with an increase of their transcriptional and translational levels. Expression of their specific endogenous inhibitor, cystatin beta, also increased. Maturation of the lysosomal proteolytic system was tightly linked to the differentiation process. These results suggested that signals for differentiation of myoblasts mediate a change of intracellular proteolytic systems, involving up-regulation of lysosomal cathepsins and down-regulation of proteasomes.

Animals↗
PubMed1995

Cysteine proteinases in GH4C1 cells, a rat pituitary tumor cell line, are secreted by the constitutive and regulated secretory pathways.

Secretory granules of GH4C1 cells, a rat pituitary tumor cell line, are known to be induced by the treatment of estradiol (E2), insulin, and epidermal growth factor (EGF). We examined changes in the localization of cathepsins B, H, and L, lysosomal cysteine proteinases, in GH4C1 cells before and after hormonal treatment. Northern blotting and immunofluorescence microscopy showed that both mRNAs and intracellular protein concentrations of these enzymes were increased in the hormone-induced cells. By immunoelectron microscopy, immunogold particles indicating cathepsins B, H, and L were localized not only in lysosomes but also in some secretory granules. To further examine the molecular forms of these proteinases in secretory granules, radiolabeling and immunoprecipitation methods were applied to the media of the cells incubated with or without secretagogues (100 nM 12-O-tetradecanoylphorbol-13-acetate and 50 microM forskolin); the proforms of cathepsins B, H, and L were secreted from the cells by the constitutive pathway, whereas the mature forms of cathepsins B and H, and the proform and mature form of cathepsin L were secreted by the regulated pathway. These results suggest that in hormone-induced GH4C1 cells, cathepsins B, H, and L are sorted from the Golgi complex not only into lysosomes but also into secretory granules, in which proforms of cathepsins B and H, and a part of procathepsin L are processed into mature forms.

Animals↗
PubMed1994

Multi-step processing of procathepsin L in vitro.

The proteolytic processes involved in the conversion of procathepsin L to cathepsin L on a negatively charged surface, dextran sulfate, were studied. Upon incubation for 30 min at 37 degrees C, pH 5.5 with dextran-sulfate and dithiothreitol, purified procathepsin L showed maximal activation and, correspondingly, the complete conversion to the 30 kDa, single chain mature form of enzyme was observed. In contrast, incubation under the same conditions on ice rather than at 37 degrees C for 30 or 60 min resulted in partial proteolysis to produce a 31 kDa form without a significant increase in activity. Amino terminal amino acid sequence analyses showed that the 30 kDa form obtained by incubation at 37 degrees C corresponds to the purified form of mature cathepsin L with a 2 amino acid extension at the amino terminal, and that the 31 kDa form generated by incubation on ice possesses a 6 amino acid amino terminal extension, suggesting that the activation and processing of procathepsin L are different processes, and that 4 amino acid residues (Glu-Pro-Leu-Met) at the carboxyterminal in the propeptide function to prevent the activation of processed cathepsin L.

3T3 Cells↗
PubMed1993

Procathepsin L-specific antibodies that recognize procathepsin L but not cathepsin L.

Procathepsin L was purified to apparent homogeneity from the culture medium of v-Ha-ras transformed NIH3T3 (Ras-NIH) cells in three steps; anion-exchange chromatography, gel filtration, and re-gel filtration. SDS-PAGE analyses revealed that the purified samples contained only the precursor form, procathepsin L, but not the mature enzyme, cathepsin L. Antibodies against purified procathepsin L were raised. These recognized both rat cathepsin L and the purified procathepsin L. To isolate procathepsin L-specific antibodies that did not recognize cathepsin L, sequential affinity chromatography procedures were carried out. Immunoblot analyses showed that the procathepsin L-specific antibodies recognized only procathepsin L, but not cathepsin L.

3T3 Cells↗
PubMed1993

Processing and transport of the precursor of cathepsin C during its transfer into lysosomes.

The biosynthesis and processing of a lysosomal cysteine proteinase, cathepsin C (dipeptidylaminopeptidase I), was investigated by pulse-chase experiments in cultured rat macrophages. Cathepsin C is first synthesized as procathepsin C with a molecular mass of 55 kDa. Procathepsin C is then cleaved and modified within 1 h into mature cathepsin C with two chains of 25 and 7.8 kDa. A combination of pulse-chase experiments and the subcellular fractionation analysis showed that procathepsin C and cathepsin C are located in low-buoyant-density organelles and lysosomes, respectively. The reactivity of endoglycosidase H and N-glycanase and analysis of phosphorylation indicated that both precursor and mature cathepsin C are phosphorylated and N-glycosylated to give a high-mannose-type. The addition of 300-kDa mannose 6-phosphate receptor antiserum to the chase medium caused extensive release of procathepsin C into the medium, whereas the addition of control serum did not. The membrane association of procathepsin C was tested by successive extraction of cells pulse labeled for 75 min with hypotonic buffer, alkaline solution, and Triton X-100. Procathepsin C was totally extracted by hypotonic solution, whereas procathepsin D was a membrane-associated form requiring Triton X-100 for its extraction. Gel-filtration chromatography analysis of the pulse-labeled products revealed that the precursor product exists as an oligomeric form. It is suggested that the oligomerization of cathepsin C occurs before its entry into lysosomes.

Amino Acid Sequence↗
PubMed1993

Cathepsin L activity is increased in alveolar macrophages and bronchoalveolar lavage fluid of smokers.

Elastinolytic enzymes derived from alveolar macrophages (AM) are considered to play an important role in the development of emphysema associated with cigarette smoking. In this study, the enzyme activity and mRNA expression of cathepsin L were quantitated in AM and bronchoalveolar lavage (BAL) fluid obtained from current smokers and compared with those from nonsmokers. Activity was measured with the synthetic substrate Z-Phe-Arg-MCA combined with a novel cathepsin B inhibitor, CA-074. We found that the specific activity of cathepsin L was significantly elevated in BAL cells from smokers (7.1 +/- 0.7 mumol/mg protein/h, mean +/- SEM) compared with cells from nonsmokers (2.9 +/- 0.3) (p < 0.01). The expression of cathepsin L mRNA in BAL cells as determined by dot-blot analysis was also higher in BAL cells from smokers, which was comparable to the increase in the enzyme activity. About 5 to 6% of the specific activity of cathepsin L in BAL cell lysates was detected in unconcentrated BAL fluid; specific activity was also significantly higher in samples from smokers (0.38 +/- 0.04 mumol/mg protein/h) than from nonsmokers (0.14 +/- 0.02). In addition, procathepsin L (42 kD) and the mature form of cathepsin L (33 kD) were demonstrated in BAL fluid by immunoblot analyses. These data suggest that cigarette smoking induces mRNA expression and the synthesis of cathepsin L in AM and the release of procathepsin from AM into extracellular milieu. Furthermore, increased activity levels of cathepsin L in extracellular compartments may contribute to the proteolysis of elastin in the process of lung destruction associated with cigarette smoking.

Adult↗
PubMed1992

Structural organization of the gene encoding rat cystatin beta.

A genomic DNA clone encompassing the gene (cy beta) encoding rat cystatin beta (Cy beta) was isolated by screening with a rat cy beta cDNA as a probe. The gene spans about 2.6 kb and comprises three exons. The first intron is located between Lys22 and Val23 and the second between Lys56 and Val57 in the deduced amino acid sequence of Cy beta. The second exon contains the highly conserved QVVAG sequence which, unlike the sequence of other cystatin family members, is not split by an intron. In the 5'-upstream region, three SP-1-binding sites exist, but no typical TATA-box or CAAT-box sequences are found. The difference in the organization of the rat cy beta gene, encoding a family-1 cystatin, from that encoding members of the other cystatin families, suggests that cy beta diverged from a common ancestral gene earlier than the separation of genes encoding family-2 and family-3 cystatins.

Amino Acid Sequence↗
PubMed1991

Molecular cloning of cDNA for rat cathepsin C. Cathepsin C, a cysteine proteinase with an extremely long propeptide.

A cDNA for rat cathepsin C (dipeptidylaminopeptidase I) was isolated. The deduced amino acid sequence of cathepsin C comprises 462 amino acid residues: 28 NH2-terminal residues corresponding to the signal peptide, 201 residues corresponding to the propeptide, and 233 COOH-terminal residues corresponding to the mature enzyme region. Four potential glycosylation sites were found, three located in the propeptide region, and one in the mature enzyme region. The amino acid sequence of mature cathepsin C has 39.5% identity to that of cathepsin H, 35.1% to that of cathepsin L, 30.1% to that of cathepsin B, and 33.3% to that of papain. Cathepsin C, therefore, is a member of the papain family, although its propeptide region is much longer than those of other cysteine proteinases and shows no significant amino acid sequence similarity to any other cysteine proteinase.

Amino Acid Sequence↗
PubMed1991

Gene structures of rat cathepsins H and L.

The gene structures of rat cathepsins H and L have been determined. Cathepsin H gene spans more than 21.5 Kbp and comprises more than 12 exons. On the other hand, cathepsin L gene spans 8.5 Kbp and comprises 8 exons. In both genes, two intron insertion positions are conserved at the amino acid level. Both enzymes, therefore, diverged from a common ancestral gene. In the 5'-upstream region of the cathepsin L gene, no TATA box, one CAAT box and some SP-1 binding sites exist, common with other lysosomal enzyme genes. Furthermore, two AP-2 binding sites exist: a promoter under the control by the tumor promoter (TPA) and cAMP, and a cAMP response element (CRE). These results suggest that the cathepsin L gene expression is controlled by these factors.

Amino Acid Sequence↗
PubMed1989

Gene structure and 5'-upstream sequence of rat cathepsin L.

The structure of rat cathepsin L gene has been determined. The gene spans 8.5 kilobase pairs comprising 8 exons, and has an intron located near the active site cysteine residue. The gene structure does not correspond well to the functional units of the proteinase. These characteristics are found to be in common with the cysteine proteinase gene family. In the 5'-upstream region, one CAAT-box and four SP-1 binding sites, together with two AP-2 binding sites and CRE, but no typical TATA-box are found. Further, SP-1 and AP-2 binding sites and an octamer motif are also found in the 1st intron, suggesting a complex regulatory mechanism for the expression of the cathepsin L gene.

Amino Acid Sequence↗
PubMed1989

Gene structure of rat cathepsin H.

The gene structure of rat cathepsin H was determined. It comprises at least 12 exons of various lengths (32-433 bp) spanning in total more than 17.5 kbp. The gene structure does not correspond well to the functional unit of the proteinase. The region around the active site Cys residue, the most conserved region among cysteine proteinases, is split by an intron. This is a common characteristic among the gene structures of cysteine proteinases.

Amino Acid Sequence↗
PubMed1989

Diacylglycerols enhance the anti-tumor effect of glucocorticoid on L5178Y lymphoblasts in vivo.

1,2-Dioleoyl-rac-glycerol, a potent activator of protein kinase C, was found to enhance the growth-inhibitory effect of triamcinolone acetonide on L5178Y lymphoblasts in adrenalectomized, male DBA/2 mice. On the other hand, in mice without adrenalectomy, it markedly inhibited tumor growth without increasing the plasma level of corticosterone or adrenocorticotropic hormone or reducing the body weight. These results suggest that diacylglycerol enhances the action of endogenous glucocorticoid to a sufficient level to inhibit the growth of lymphoblasts. Of various diacylglycerols with different carbon chain lengths tested, 1,2-dioleoyl-rac-glycerol was the most potent growth inhibitor and was maximally effective at a dose of above 30 micrograms/100 g body weight. This finding suggests that diacylglycerols may be useful for enhancing the antitumor effect of a low dose of glucocorticoid or endogenous glucocorticoid on lymphoblasts without any significant side effect.

Adrenalectomy↗
PubMed1987

Molecular cloning and sequencing of cDNA for rat cathepsin H. Homology in pro-peptide regions of cysteine proteinases.

A cDNA for rat cathepsin H was isolated and sequenced. The deduced protein comprising 333 amino acid residues is composed of a typical signal sequence (21 residues), a pro-peptide region (92 residues) and a mature enzyme region (220 residues). The amino acid sequence in the pro-peptide region, in particular, residues Phe-(-41) to Ser-(-29) of cathepsin H, is highly homologous to the pro-peptide regions of other cysteine proteinases. This homologous region may play a role in the processing of cysteine proteinases.

Amino Acid Sequence↗
PubMed1987

Molecular cloning and sequencing of cDNA for rat cathepsin L.

A near full-length cDNA for rat cathepsin L was isolated. The deduced protein comprises 334 amino acid residues (Mr 37,685) containing a typical signal sequence (N-terminal 17 residues), pro-peptide (96 residues), and the sequence for mature cathepsin L (221 residues). Rat cathepsin L shows 94% amino acid identity with mouse cysteine proteinase. Amino acid sequence homologies of rat cathepsin L with rat cathepsins H and B are 45 and 25%, respectively. These facts indicate that mouse cysteine proteinase is probably mouse cathepsin L and that cathepsin L is more closely related to cathepsin H than cathepsin B.

Amino Acid Sequence↗
PubMed1986

Diacylglycerol amplifies the induction in vivo of tyrosine aminotransferase and ornithine decarboxylase by glucocorticoid.

In adrenalectomized rats, diacylglycerol, a potent activator of protein kinase C, specifically enhanced the induction of tyrosine aminotransferase and ornithine decarboxylase by even maximally effective doses of dexamethasone phosphate, but itself had no effect on these enzyme inductions in the absence of glucocorticoid. The amplifications of enzyme induction by diacylglycerol was dose-dependent and the time courses of the amplified inductions were similar to those of the inductions by dexamethasone phosphate alone. Since diacylglycerol did not affect the induction of these enzymes by glucagon and insulin, its amplifying effect seemed to be specific for induction by glucocorticoids.

Adrenalectomy↗