45 results on '"Shimbara N"'
Search Results
2. Replacement of proteasome subunits X and Y by LMP7 and LMP2 induced by interferon-gamma for acquirement of the functional diversity responsible for antigen processing
- Author
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Akiyama, K., Kagawa, S., Tamura, Tomohiro, Shimbara, N., Takashima, M., Kristensen, Poul, Hendil, Klaus B., Tanaka, K., Ichihara, A., Akiyama, K., Kagawa, S., Tamura, Tomohiro, Shimbara, N., Takashima, M., Kristensen, Poul, Hendil, Klaus B., Tanaka, K., and Ichihara, A.
- Abstract
Engelsk
- Published
- 1994
3. The involvement of proteasome in myogenic differentiation of murine myocytes and human rhabdomyosarcoma cells.
- Author
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Mugita, N, primary, Honda, Y, additional, Nakamura, H, additional, Fujiwara, T, additional, Tanaka, K, additional, Omura, S, additional, Shimbara, N, additional, Ogawa, M, additional, Saya, H, additional, and Nakao, M, additional
- Published
- 1999
- Full Text
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4. Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1.
- Author
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Kominami, K, primary, Okura, N, additional, Kawamura, M, additional, DeMartino, G N, additional, Slaughter, C A, additional, Shimbara, N, additional, Chung, C H, additional, Fujimuro, M, additional, Yokosawa, H, additional, Shimizu, Y, additional, Tanahashi, N, additional, Tanaka, K, additional, and Toh-e, A, additional
- Published
- 1997
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5. Newly identified pair of proteasomal subunits regulated reciprocally by interferon gamma.
- Author
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Hisamatsu, H, primary, Shimbara, N, additional, Saito, Y, additional, Kristensen, P, additional, Hendil, K B, additional, Fujiwara, T, additional, Takahashi, E, additional, Tanahashi, N, additional, Tamura, T, additional, Ichihara, A, additional, and Tanaka, K, additional
- Published
- 1996
- Full Text
- View/download PDF
6. Nin1p, a regulatory subunit of the 26S proteasome, is necessary for activation of Cdc28p kinase of Saccharomyces cerevisiae.
- Author
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Kominami, K., primary, DeMartino, G.N., additional, Moomaw, C.R., additional, Slaughter, C.A., additional, Shimbara, N., additional, Fujimuro, M., additional, Yokosawa, H., additional, Hisamatsu, H., additional, Tanahashi, N., additional, and Shimizu, Y., additional
- Published
- 1995
- Full Text
- View/download PDF
7. cDNA Cloning of p40, a Regulatory Subunit of the Human 26S Proteasome, and a Homolog of the Mov-34 Gene Product
- Author
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Tsurumi, C., primary, Demartino, G.N., additional, Slaughter, C.A., additional, Shimbara, N., additional, and Tanaka, K., additional
- Published
- 1995
- Full Text
- View/download PDF
8. Regulation of gene expression of proteasomes (multi-protease complexes) during growth and differentiation of human hematopoietic cells.
- Author
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Shimbara, N, primary, Orino, E, additional, Sone, S, additional, Ogura, T, additional, Takashina, M, additional, Shono, M, additional, Tamura, T, additional, Yasuda, H, additional, Tanaka, K, additional, and Ichihara, A, additional
- Published
- 1992
- Full Text
- View/download PDF
9. Hybrid proteasomes. Induction by interferon-gamma and contribution to ATP-dependent proteolysis.
- Author
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Tanahashi, N, Murakami, Y, Minami, Y, Shimbara, N, Hendil, K B, and Tanaka, K
- Abstract
Eukaryotic cells contain various types of proteasomes. Core 20 S proteasomes (abbreviated 20 S below) have two binding sites for the regulatory particles, PA700 and PA28. PA700-20 S-PA700 complexes are known as 26 S proteasomes and are ATP-dependent machines that degrade cell proteins. PA28 is found both in previously described complexes of the type PA28-20 S-PA28 and in complexes that also contain PA700, as PA700-20 S-PA28. We refer to the latter as "hybrid proteasomes." The relative amounts of the various types of proteasomes in HeLa extracts were determined by a combination of immunoprecipitation and immunoblotting. Hybrid proteasomes accounted for about a fourth of all proteasomes in the extracts. Association of PA28 and proteasomes proved to be ATP-dependent. Hybrid proteasomes catalyzed ATP-dependent degradation of ornithine decarboxylase (ODC) without ubiquitinylation, as do 26 S proteasomes. In contrast, the homo-PA28 complex (PA28-20 S-PA28) was incapable of degrading ODC. Intriguingly, a major immunomodulatory cytokine, interferon-gamma, appreciably enhanced the ODC degradation in HeLa and SW620 cells through induction of the hybrid proteasome, which may also be responsible for the immunological processing of intracellular antigens. Taken together, we report here for the first time the existence of two types of ATP-dependent proteases, the 26 S proteasome and the hybrid proteasome, which appear to share the ATP-dependent proteolytic pathway in mammalian cells.
- Published
- 2000
10. A new SUMO-1-specific protease, SUSP1, that is highly expressed in reproductive organs.
- Author
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Kim, K I, Baek, S H, Jeon, Y J, Nishimori, S, Suzuki, T, Uchida, S, Shimbara, N, Saitoh, H, Tanaka, K, and Chung, C H
- Abstract
A full-length cDNA encoding a SUMO-1-specific protease, named SUSP1, was identified and cloned for the first time from the human brain. Nucleotide sequence analysis of the cDNA containing an open reading frame of 3336 base pairs revealed that the protease consists of 1112 amino acids with a calculated molecular mass of 126,116 Da. Like yeast Ulp1, SUSP1 is a cysteine protease containing the well conserved His/Asp/Cys catalytic triad. SUSP1 expressed in Escherichia coli cells efficiently released SUMO-1 from SUMO-1. beta-galactosidase fusion but not from other ubiquitin-like protein fusions, including Smt3.beta-galactosidase, suggesting its role in the generation of matured SUMO-1 specifically from its precursors. Interestingly, reproductive organs, such as testis, ovary, and prostate, contained much higher amounts of SUSP1 mRNA than colon and peripheral blood leukocyte, whereas other tissues, such as heart and spleen, had little or none. In addition, confocal microscopy using green fluorescent protein.SUSP1 fusion showed that SUSP1 is exclusively localized to the cytoplasm of NIH3T3 and HeLa cells. These results suggest that SUSP1 may play a role in the regulation of SUMO-1-mediated cellular processes particularly related to reproduction.
- Published
- 2000
11. Expression of random peptide fused to invasin on bacterial cell surface for selection of cell-targeting peptides
- Author
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Nakajima, H., Shimbara, N., Shimonishi, Y., Mimori, T., Niwa, S. i., and Saya, H.
- Published
- 2000
- Full Text
- View/download PDF
12. A new 30-kDa ubiquitin-related SUMO-1 hydrolase from bovine brain.
- Author
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Suzuki, T, Ichiyama, A, Saitoh, H, Kawakami, T, Omata, M, Chung, C H, Kimura, M, Shimbara, N, and Tanaka, K
- Abstract
SUMO-1 is a ubiquitin-like protein functioning as an important reversible protein modifier. To date there is no report on a SUMO-1 hydrolase/isopeptidase catalyzing the release of SUMO-1 from its precursor or SUMO-1-ligated proteins in mammalian tissues. Here we found multiple activities that cleave the SUMO-1 moiety from two model substrates, (125)I-SUMO-1-alphaNH-HSTVGSMHISPPEPESEEEEEHYC and/or GST-SUMO-1-(35)S-RanGAP1 conjugate, in bovine brain extracts. Of them, a major SUMO-1 C-terminal hydrolase had been partially purified by successive chromatographic operations. The enzyme had the ability to cleave SUMO-1 not only from its precursor but also from a SUMO-1-ligated RanGAP1 but did not exhibit any significant cleavage of the ubiquitin- and NEDD8-precursor. The activity of SUMO-1 hydrolase was almost completely inhibited by N-ethylmaleimide, but not by phenylmethanesulfonyl fluoride, EDTA, and ubiquitin-aldehyde known as a potent inhibitor of deubiquitinylating enzymes. Intriguingly, the apparent molecular mass of the isolated SUMO-1 hydrolase was approximately 30 kDa, which is significantly smaller than the recently identified yeast Smt3/SUMO-1 specific protease Ulp1. These results indicate that there are multiple SUMO-1 hydrolase/isopeptidases in mammalian cells and that the 30-kDa small SUMO-1 hydrolase plays a central role in processing of the SUMO-1-precursor.
- Published
- 1999
13. SUG1, a component of the 26 S proteasome, is an ATPase stimulated by specific RNAs.
- Author
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Makino, Y, Yamano, K, Kanemaki, M, Morikawa, K, Kishimoto, T, Shimbara, N, Tanaka, K, and Tamura, T
- Abstract
SUG1 is an integral component of the 26 S proteasome. Belonging to a novel putative ATPase family, it shares four conserved motifs characteristic of ATP-dependent DNA/RNA helicases. Recombinant rat SUG1 (rSUG1) produced in Escherichia coli was highly purified and characterized in terms of its biochemical properties. The rSUG1 exhibited a Mg2+-dependent ATPase activity. The Km for ATP and Vmax of rSUG1 were 35 microM and 7 pmol of ATP/min/microg of protein, respectively. Both ATPase activity to release [32P]monophosphate and [32P]ATP-labeling activity were coordinately affected by cold ATP severely, GTP and UTP moderately, and CTP little. Interestingly, the rSUG1 ATPase activity was stimulated by poly(U) and poly(C), but not by poly(A), poly(G), or by any forms of DNAs tested. A UV cross-linking assay also indicated poly(U)- and poly(C)-stimulated labeling of rSUG1 with [alpha-32P]ATP. Moreover, the ATPase activity was facilitated by cellular poly(A)+ RNA, but not by poly(A)- RNA. RNA transcribed in vitro from cDNA encoding a b-Zip protein could stimulate the ATPase activity. This is the first report to demonstrate a specific RNA requirement for ATPase with respect to the proteasomal ATPases. Our present work suggests that SUG1 can specifically interact with protein-coding RNA (mRNA) and play some roles in mRNA metabolism.
- Published
- 1997
14. Contribution of proline residue for efficient production of MHC class I ligands by proteasomes.
- Author
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Shimbara, N, Ogawa, K, Hidaka, Y, Nakajima, H, Yamasaki, N, Niwa, S, Tanahashi, N, and Tanaka, K
- Abstract
Proteasomes are processing enzymes capable of generating major histocompatibility complex (MHC) class I ligands, but the mechanism of how they excise ligands without destroying them is largely unknown. Previously, we reported that most products of ornithine decarboxylase degraded in vitro by the 26 S ATP-dependent proteasome, which contained one or two Pro residues (Tokunaga, F., Goto, T., Koide, T., Murakami, Y., Hayashi, S., Tamura, T., Tanaka, K., and Ichihara, A. (1994) J. Biol. Chem. 269,17382-17385), which implied that the Pro residue has a role in the escape from random cleavage by proteasomes. Here, we examine the role of the Pro residue in producing MHC class I ligands in vitro. Proteasomes generated two cytotoxic T lymphocyte-epitopic precursor peptides, SIIPGLPLSL and DMYPHFMPTNL, from the 29-mer and 25-mer peptides harboring these sequences, which are derived from the c-akt proto-oncogene and the pp89 protein of mouse cytomagalovirus, respectively. Replacement of the first or second Pro residue within these epitopes by Ala resulted in a marked reduction of this epitope-derived production or their random cleavage by proteasomes, irrespective of the presence of PA28, which greatly accelerates the generation of unmodified ligands. Moreover, replacement of a single amino acid residue other than Pro in both epitopic and flanking regions by Ala or Leu had no or little appreciable effect on the SIIPGLPLSL or its derivative production. Thus, Pro residue(s) within these epitopic sequences presumably contributes to efficient production of MHC class I ligands through prevention of their random cleavage by proteasomes.
- Published
- 1998
15. Effects of the cys mutations on structure and function of the ATP-dependent HslVU protease in Escherichia coli. The Cys287 to Val mutation in HslU uncouples the ATP-dependent proteolysis by HslvU from ATP hydrolysis.
- Author
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Yoo, S J, Kim, H H, Shin, D H, Lee, C S, Seong, I S, Seol, J H, Shimbara, N, Tanaka, K, and Chung, C H
- Abstract
To define the role of the Cys residues in the ATP-dependent HslVU protease, mutagenesis was performed to replace either Cys261 or Cys287 in HslU with Val and Cys159 in HslV with Ser or Ala. Whereas HslU/C261V could hydrolyze ATP and support the ATP-dependent proteolytic activity of HslV as well as the wild-type HslU, HslU/C287V could not hydrolyze ATP. Nevertheless, HslU/C287V could support the HslV-mediated proteolysis by forming the HslVU complex in the presence of ATP but not its absence, indicating that ATP binding but not its hydrolysis is essential for proteolysis. Whereas treatment of N-ethylmaleimide (NEM) resulted in dissociation of the oligomeric HslU into monomers, the C261V mutation, but not C287V, prevented the NEM effect. These results suggest that Cys261 is involved in oligomerization and that Cys287 is related to the ATPase function of HslU. NEM also dissociated the dodecameric HslV into monomers, and this effect could be prevented by either the C159S or C159A mutation, suggesting the involvement of Cys159 in oligomerization of HslV. Moreover, either mutation abolished both the basal and HslU-activated proteolytic activity of HslV and its ability to activate the HslU ATPase and to form the HslVU complex, indicating that Cys159 is essential for the proteolytic activity of HslV and its interaction with HslU. These results suggest that the Cys residues play an important role in maintaining the structure and function of the HslVU protease.
- Published
- 1998
16. cDNA cloning of a new putative ATPase subunit p45 of the human 26S proteasome, a homolog of yeast transcriptional factor Suglp
- Author
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Akiyama, K.-Y., Yokota, K.-Y., Kagawa, S., and Shimbara, N.
- Published
- 1995
- Full Text
- View/download PDF
17. A novel small-molecule inhibitor of NF-kappaB signaling.
- Author
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Nakajima H, Fujiwara H, Furuichi Y, Tanaka K, and Shimbara N
- Subjects
- Fluorescence Resonance Energy Transfer, I-kappa B Proteins antagonists & inhibitors, NF-KappaB Inhibitor alpha, Reproducibility of Results, Ubiquitin-Protein Ligases metabolism, Benzoates pharmacology, NF-kappa B antagonists & inhibitors, Pyrazoles pharmacology, Signal Transduction drug effects
- Abstract
The inducible transcription factor NF-kappaB regulates divergent signaling pathways including inflammatory response and cancer development. Selective inhibitors for NF-kappaB signaling are potentially useful for treatment of inflammation and cancer. NF-kappaB is canonically activated by preferential disposal of its inhibitory protein; IkappaB, which suppresses the nuclear translocation of NF-kappaB. IkappaBalpha (a major member of IkappaB family proteins) is phosphorylated with an IkappaB kinase (IKK) and subsequently polyubiquitylated by SCF(betaTrCP1) ubiquitin-ligase in the presence of E1 and E2 prior to proteasomal degradation. Here, we describe a novel inhibitor termed GS143, which suppressed IkappaBalpha ubiquitylation, but not IkappaBalpha phosphorylation, MDM2-directed p53 ubiquitylation, and proteasome activity in vitro. GS143 markedly suppressed the destruction of IkappaBalpha stimulated by TNFalpha and a set of downstream responses coupled to NF-kappaB signaling but not those of p53 and beta-catenin in vivo. Our results indicate that GS143 serves as an effective inhibitor of multiple pathways served by NF-kappaB signaling.
- Published
- 2008
- Full Text
- View/download PDF
18. Differential cytotoxicity of anticancer agents in pre- and post-immortal lymphoblastoid cell lines.
- Author
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Sawada K, Noda K, Nakajima H, Shimbara N, Furuichi Y, and Sugimoto M
- Subjects
- Cell Line, Transformed, Cell Transformation, Viral, Drug Screening Assays, Antitumor, Flow Cytometry, Herpesvirus 4, Human physiology, Humans, Antineoplastic Agents toxicity
- Abstract
We studied the cytotoxic effect of various anticancer agents on lymphoblastoid cell lines transformed by Epstein-Barr virus. Post-immortal N0005 (post-N0005) is an immortalized cell line derived from pre-immortal N0005 (pre-N0005) accompanied by increased telomerase activity, short-telomere, abnormal karyotypes, mutation of p53 gene, down regulation of p16/Rb and the ability to grow in soft agar medium. Compared with pre-N0005 cells, post-N0005 cells were significantly (p<0.001 by the Student t test) more resistant to the killing activity of seven DNA-modifying agents: camptothecin, etoposide, bleomycin, fluorouracil, thioguanine, melphalan and actinomycin D. However, both pre-N0005 and post-N0005 cells showed similar levels of cytotoxicity against four DNA-non-modifying agents: colchicine, paclitaxel, vincristine and methotrexate. DNA-modifying and DNA-non-modifying agents are distinguished by their different sensitivities with pre-N0005 and post-N0005. Based on these results, we propose that pre-N0005 and post-N0005 cell lines be used as a new method to assess and screen anticancer agents.
- Published
- 2005
- Full Text
- View/download PDF
19. Two distinct pathways mediated by PA28 and hsp90 in major histocompatibility complex class I antigen processing.
- Author
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Yamano T, Murata S, Shimbara N, Tanaka N, Chiba T, Tanaka K, Yui K, and Udono H
- Subjects
- Amino Acid Sequence, Animals, Antigen Presentation, Autoantigens, Base Sequence, Benzoquinones, Cells, Cultured, HSP90 Heat-Shock Proteins antagonists & inhibitors, In Vitro Techniques, Interferon-gamma pharmacology, Lactams, Macrocyclic, Mice, Mice, Knockout, Molecular Sequence Data, Ovalbumin chemistry, Ovalbumin immunology, Ovalbumin metabolism, Peptide Fragments chemistry, Peptide Fragments immunology, Peptide Fragments metabolism, Proteasome Endopeptidase Complex, Proteins genetics, Quinones pharmacology, Recombinant Proteins, HSP90 Heat-Shock Proteins metabolism, Histocompatibility Antigens Class I metabolism, Proteins metabolism
- Abstract
Major histocompatibility complex (MHC) class I ligands are mainly produced by the proteasome. Herein, we show that the processing of antigens is regulated by two distinct pathways, one requiring PA28 and the other hsp90. Both hsp90 and PA28 enhanced the antigen processing of ovalbumin (OVA). Geldanamycin, an inhibitor of hsp90, almost completely suppressed OVA antigen presentation in PA28alpha(-/-)/beta(-/-) lipopolysaccharide blasts, but not in wild-type cells, indicating that hsp90 compensates for the loss of PA28 and is essential in the PA28-independent pathway. In contrast, treatment of cells with interferon (IFN)-gamma, which induces PA28 expression, abrogated the requirement of hsp90, suggesting that IFN-gamma enhances the PA28-dependent pathway, whereas it diminishes hsp90-dependent pathway. Importantly, IFN-gamma did not induce MHC class I expressions in PA28-deficient cells, indicating a prominent role for PA28 in IFN-gamma-stimulated peptide supply. Thus, these two pathways operate either redundantly or specifically, depending on antigen species and cell type.
- Published
- 2002
- Full Text
- View/download PDF
20. NEDD8 recruits E2-ubiquitin to SCF E3 ligase.
- Author
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Kawakami T, Chiba T, Suzuki T, Iwai K, Yamanaka K, Minato N, Suzuki H, Shimbara N, Hidaka Y, Osaka F, Omata M, and Tanaka K
- Subjects
- Anaphase-Promoting Complex-Cyclosome, Cell Line, DNA-Binding Proteins metabolism, Humans, Ligases genetics, NEDD8 Protein, NF-KappaB Inhibitor alpha, SKP Cullin F-Box Protein Ligases, Ubiquitin-Protein Ligases, Ubiquitins metabolism, I-kappa B Proteins, Ligases metabolism, Peptide Synthases metabolism, Ubiquitin-Conjugating Enzymes, Ubiquitin-Protein Ligase Complexes, Ubiquitins physiology
- Abstract
NEDD8/Rub1 is a ubiquitin (Ub)-like post-translational modifier that is covalently linked to cullin (Cul)-family proteins in a manner analogous to ubiquitylation. NEDD8 is known to enhance the ubiquitylating activity of the SCF complex (composed of Skp1, Cul-1, ROC1 and F-box protein), but the mechanistic role is largely unknown. Using an in vitro reconstituted system, we report here that NEDD8 modification of Cul-1 enhances recruitment of Ub-conjugating enzyme Ubc4 (E2) to the SCF complex (E3). This recruitment requires thioester linkage of Ub to Ubc4. Our findings indicate that the NEDD8-modifying system accelerates the formation of the E2-E3 complex, which stimulates protein polyubiquitylation.
- Published
- 2001
- Full Text
- View/download PDF
21. Tissue and cell distribution of a mammalian proteasomal ATPase, MSS1, and its complex formation with the basal transcription factors.
- Author
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Yanagi S, Shimbara N, and Tamura Ta
- Subjects
- ATPases Associated with Diverse Cellular Activities, Adenosine Triphosphatases genetics, Adenosine Triphosphatases isolation & purification, Adenosine Triphosphatases metabolism, Animals, Cysteine Endopeptidases isolation & purification, DNA-Binding Proteins isolation & purification, HeLa Cells, Humans, Mammals, Molecular Sequence Data, Molecular Weight, Multienzyme Complexes isolation & purification, Open Reading Frames, Organ Specificity, Proteasome Endopeptidase Complex, RNA Polymerase II isolation & purification, RNA Polymerase II metabolism, Rats, Transcription Factors isolation & purification, Cysteine Endopeptidases genetics, Cysteine Endopeptidases metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Multienzyme Complexes genetics, Multienzyme Complexes metabolism, Transcription Factors metabolism
- Abstract
The proteasome is an eukaryotic multi-subunit protease complex composed of one 20S core component and two 19S regulatory complexes. The regulatory complex contains 6 putative ATPases. We investigated tissue and cell distribution of one of these ATPases, MSS1 (mammalian suppressor of sgv1). MSS1 was ubiquitously present in rat tissues as was the 20S core component of proteasome. However, the ratio of MSS1 to 20S varied greatly among tissues and MSS1 was concentrated in the thymus. Glycerol gradient sedimentation analysis revealed that MSS1 is included in protein complexes whose density is lighter than that of the proteasome. MSS1 was distributed in mammalian cells ubiquitously, while proteasome was rather concentrated in the nuclei. Hence, a novel molecular status of MSS1 distinct from proteasome is implicated. Interestingly, multiple basal transcription factors for RNA polymerase II, including TBP, TFIIB, TFIIH, and TFIIF, were found to be associated with MSS1. These results suggest that MSS1, in addition to proteolysis, plays a role in DNA metabolism including transcriptional regulation., (Copyright 2000 Academic Press.)
- Published
- 2000
- Full Text
- View/download PDF
22. Tissue distribution of constitutive proteasomes, immunoproteasomes, and PA28 in rats.
- Author
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Noda C, Tanahashi N, Shimbara N, Hendil KB, and Tanaka K
- Subjects
- Age Factors, Animals, Brain metabolism, Cell Cycle Proteins, Cysteine Endopeptidases metabolism, Electrophoresis, Polyacrylamide Gel, Female, Immunoglobulin G metabolism, Interferon-gamma metabolism, Liver metabolism, Lung metabolism, Male, Multienzyme Complexes metabolism, Peptide Hydrolases metabolism, Precipitin Tests, Proteasome Endopeptidase Complex, Protein Binding, Rats, Rats, Wistar, Sex Factors, Spleen metabolism, Tissue Distribution, Viral Matrix Proteins biosynthesis, Cysteine Endopeptidases biosynthesis, Cysteine Endopeptidases immunology, Multienzyme Complexes biosynthesis, Multienzyme Complexes immunology, Protein Biosynthesis, Proteins
- Abstract
We investigated the expression of standard proteasomes, immunoproteasomes, and their regulators, PA28, and PA700, in rat tissues. Immunoproteasomes (with subunits LMP2, LMP7, and MECL1) were abundant in the spleen but almost absent in the brain. In contrast, standard proteasomes (with X, Y, and Z) were highly expressed in the brain but not in the spleen. Both proteasome types were present in the lung and the liver. PA700 subunits (p112, S5a, and p45) were found in all tissues. PA28alpha, PA28beta, and PA28gamma were also expressed in all tissues, except for the brain which contained very little PA28beta. The results did not depend on rat sex or age. The cleavage specificity for peptide substrates differed greatly between brain and spleen proteasomes. Hybrid proteasomes, containing both PA28alphabeta and PA700, were not present in the brain but in all other tissues examined., (Copyright 2000 Academic Press.)
- Published
- 2000
- Full Text
- View/download PDF
23. Developmentally regulated, alternative splicing of the Rpn10 gene generates multiple forms of 26S proteasomes.
- Author
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Kawahara H, Kasahara M, Nishiyama A, Ohsumi K, Goto T, Kishimoto T, Saeki Y, Yokosawa H, Shimbara N, Murata S, Chiba T, Suzuki K, and Tanaka K
- Subjects
- Amino Acid Sequence, Animals, Conserved Sequence, Cyclin B metabolism, Evolution, Molecular, Mice, Molecular Sequence Data, RNA-Binding Proteins, Sequence Homology, Amino Acid, Ubiquitins metabolism, Alternative Splicing, Carrier Proteins genetics, Gene Expression Regulation, Developmental, Peptide Hydrolases genetics, Proteasome Endopeptidase Complex
- Abstract
The 26S proteasome is a multisubunit protein- destroying machinery that degrades ubiquitin-tagged proteins. To date only a single species of Rpn10, which possibly functions as a multiubiquitin chain-binding subunit, has been identified in various organisms. Here we report that mouse Rpn10 mRNAs occur in at least five distinct forms, named Rpn10a to Rpn10e, and that they are generated from a single gene by developmentally regulated, alternative splicing. Rpn10a is ubiquitously expressed, whereas Rpn10e is expressed only in embryos, with the highest levels of expression in the brain. Both forms of Rpn10 are components of the 26S proteasome, with an apparently similar affinity for multiubiquitylated [(125)I]lysozyme in vitro. However, they exert markedly divergent effects on the destruction of B-type cyclin in Xenopus egg extracts. Thus, the 26S proteasome occurs in at least two functionally distinct forms: one containing a ubiquitously expressed Rpn10a and the other a newly identified, embryo-specific Rpn10e. While the former is thought to perform proteolysis constitutively in a wide variety of cells, the latter may play a specialized role in early embryonic development.
- Published
- 2000
- Full Text
- View/download PDF
24. cDNA cloning, expression, and functional characterization of PI31, a proline-rich inhibitor of the proteasome.
- Author
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McCutchen-Maloney SL, Matsuda K, Shimbara N, Binns DD, Tanaka K, Slaughter CA, and DeMartino GN
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cattle, Circular Dichroism, Cloning, Molecular, Cysteine Endopeptidases metabolism, DNA, Complementary chemistry, Erythrocytes enzymology, Humans, Molecular Sequence Data, Multienzyme Complexes metabolism, Protein Conformation, Proteins metabolism, Cysteine Proteinase Inhibitors genetics, Muscle Proteins, Proteasome Endopeptidase Complex
- Abstract
The primary structure of PI31, a protein inhibitor of the 20 S proteasome, was deduced by cDNA cloning and sequencing. The human protein has a calculated molecular weight of 29,792, a value in excellent accord with 31,000, as estimated by SDS-polyacrylamide gel electrophoresis for purified bovine PI31, and is not similar to any other protein in current data bases. PI31 is a proline-rich protein, particularly within its carboxyl-terminal half where 26% of the amino acids are proline. Wild-type PI31 and various truncation mutants were expressed in Escherichia coli and purified to homogeneity. Recombinant wild-type PI31 displayed structural and functional properties similar to those of PI31 purified from bovine red blood cells and inhibited the hydrolysis of protein and peptide substrates by the 20 S proteasome. Analysis of truncation mutants demonstrated that proteasome inhibition was conferred by the carboxyl-terminal proline-rich domain of PI31, which appears to have an extended secondary structure. Inhibition of the 20 S proteasome by PI31 involved formation a proteasome-PI31 complex. In addition to its direct inhibition of the 20 S proteasome, PI31 inhibited the activation of the proteasome by each of two proteasome regulatory proteins, PA700 and PA28. These results suggest that PI31 plays an important role in control of proteasome function, including that in ubiquitin-dependent pathways of protein degradation.
- Published
- 2000
- Full Text
- View/download PDF
25. Covalent modification of all members of human cullin family proteins by NEDD8.
- Author
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Hori T, Osaka F, Chiba T, Miyamoto C, Okabayashi K, Shimbara N, Kato S, and Tanaka K
- Subjects
- Cell Cycle Proteins genetics, Gene Expression Regulation, Humans, NEDD8 Protein, RNA, Messenger genetics, RNA, Messenger metabolism, Ubiquitins genetics, Cell Cycle Proteins metabolism, Cullin Proteins, Ubiquitins metabolism
- Abstract
Recently we found that NEDD8, a ubiquitin-like protein, was linked covalently to human cullin-4A (abbreviated Cul-4A) by a new ubiquitin-related pathway that is analogous to but distinct from the ligating system for SUMO1, another ubiquitin-like protein. However, it remained unknown whether the other five members of the family of human cullin/Cdc53 proteins are modified by NEDD8. Here we report that all Hs-Cul family proteins, such as Cul-1, Cul-2, Cul-3, Cul-4B, and Cul-5, in addition to Cul-4A, were modified by covalent attachment of NEDD8 in rabbit reticulocyte lysates. Moreover, by comprehensive Northern-blot analyses, we examined multiple tissue distributions of the messages for all Cul-family proteins, NEDD8, and the NEDD8-ligating system consisting of APP-BP1/hUba3, and hUbc12, which function as E1- and E2-like enzymes, respectively. The expressions of Cul-1, Cul-2, and Cul-3 resembled each other and were apparently correlated to those of NEDD8 and the NEDD8-ligating system in various human cells and tissues. However, the mRNA levels of Cul-4A, Cul-4B, and Cul-5 differed considerably from each other as well as from other Cul-family proteins. The enhanced expression of all Cul-family proteins except Cul-5 was observed in a variety of tumor cell lines.
- Published
- 1999
- Full Text
- View/download PDF
26. Splice acceptor site mutation of the transporter associated with antigen processing-1 gene in human bare lymphocyte syndrome.
- Author
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Furukawa H, Murata S, Yabe T, Shimbara N, Keicho N, Kashiwase K, Watanabe K, Ishikawa Y, Akaza T, Tadokoro K, Tohma S, Inoue T, Tokunaga K, Yamamoto K, Tanaka K, and Juji T
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 2, ATP-Binding Cassette Transporters immunology, Antigen Presentation immunology, Female, Humans, Male, Severe Combined Immunodeficiency immunology, ATP-Binding Cassette Transporters genetics, Antigen Presentation genetics, Histocompatibility Antigens Class I immunology, Mutation, Severe Combined Immunodeficiency genetics
- Abstract
Expression of histocompatibility leukocyte antigen (HLA) class I molecules on the cell surface depends on the heterodimer of the transporter associated with antigen processing 1 and 2 (TAP1 and TAP2), which transport peptides cleaved by proteasome to the class I molecules. Defects in the TAP2 protein have been reported in two families with HLA class I deficiency, the so-called bare lymphocyte syndrome (BLS) type I. We have, to our knowledge, identified for the first time a splice site mutation in the TAP1 gene of another BLS patient. In addition, class I heavy chains (HCs) did not form the normal complex with tapasin in the endoplasmic reticulum (ER) of the cells of our patient.
- Published
- 1999
- Full Text
- View/download PDF
27. Characterization of the mouse PA28 activator complex gene family: complete organizations of the three member genes and a physical map of the approximately 150-kb region containing the alpha- and beta-subunit genes.
- Author
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Kohda K, Ishibashi T, Shimbara N, Tanaka K, Matsuda Y, and Kasahara M
- Subjects
- Amino Acid Sequence, Animals, Antigen Presentation, Base Sequence, Cysteine Endopeptidases physiology, Enzyme Activation, Histocompatibility Antigens Class I, Humans, Interferon-gamma pharmacology, Mice, Molecular Sequence Data, Multienzyme Complexes physiology, Promoter Regions, Genetic, Proteasome Endopeptidase Complex, Repetitive Sequences, Nucleic Acid, Chromosome Mapping, Cysteine Endopeptidases genetics, Multienzyme Complexes genetics, Proteins
- Abstract
The proteasome is a multisubunit protease responsible for the generation of peptides loaded onto MHC class I molecules. Recent evidence indicates that binding of an IFN-gamma-inducible PA28 activator complex to the 20S proteasome enhances the generation of class I binding peptides. The alpha- and beta-subunits, which constitute the PA28 activator complex in the form of an (alphabeta)3 heterohexamer, show significant amino acid sequence similarity to a protein, designated Ki or the gamma-subunit, that is capable of binding to the 20S proteasome. In this study, we describe the complete nucleotide sequences of the mouse genes, Psme1, Psme2, and Psme3, coding for the alpha-, beta-, and gamma-subunits, respectively. The overall exon-intron organizations of the three Psme genes are virtually identical, thus providing evidence that they are descended from a single ancestral gene. The promoter regions of the Psme1 and Psme2 genes contain sequence motifs that qualify as IFN-stimulated response elements, consistent with the observation that their expression is induced strongly by IFN-gamma. The Psme1 and Psme2 genes are located approximately 6 kb apart with their 3'-ends pointing toward each other on bands C2 to D1 of mouse chromosome 14, supporting the idea that they emerged by tandem duplication.
- Published
- 1998
28. Chromosomal localization and immunological analysis of a family of human 26S proteasomal ATPases.
- Author
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Tanahashi N, Suzuki M, Fujiwara T, Takahashi E, Shimbara N, Chung CH, and Tanaka K
- Subjects
- Adenosine Triphosphatases chemistry, Chromosome Mapping, Chromosomes, Human, Pair 11 genetics, Chromosomes, Human, Pair 12 genetics, Chromosomes, Human, Pair 17 genetics, Chromosomes, Human, Pair 19 genetics, Chromosomes, Human, Pair 7 genetics, Humans, Immunochemistry, In Situ Hybridization, Fluorescence, Peptide Hydrolases chemistry, Protein Conformation, Adenosine Triphosphatases genetics, Adenosine Triphosphatases immunology, Peptide Hydrolases genetics, Peptide Hydrolases immunology, Proteasome Endopeptidase Complex
- Abstract
The 26S proteasome is a eukaryotic ATP-dependent protease functioning as a protein death machine. It is a large multisubunit complex, consisting of a catalytic 20S proteasome and two regulatory modules, named PA700. The PA700 complex is composed of multiple subunits of 25-110 kDa, which are classified into two subgroups, a subgroup of at least 6 ATPases that consitute a unique multi-gene family encoding homologous polypeptides conserved during evolution and a subgroup of approximately 15 non-ATPase subunits, most of which are structurally unrelated to each other. In the present study, we report the chromosomal localization and immunological properties of six members of the human 26S proteasomal ATPase family. By use of the fluorescence in situ hybridization method, the S4 (PSMC1), MSS1 (PSMC2), TBP1 (PSMC3), TBP7 (PSMC4), p45 (PSMC5), and p42 (PSMC6) genes were mapped to human chromosomes 19p13.3, 7q22.1-q22.3, 11p11.2, 19q13.11-q13.13, 17q23.1-q23.3, and 12q15, respectively, indicating that the genes for multiple ATPases of the 26S proteasome are located on different chromosomes. Immunoblot analysis revealed that all these ATPases were associated with the purified 26S proteasome and that some of them showed striking heterogeneity in their electrical charges.
- Published
- 1998
- Full Text
- View/download PDF
29. Double-cleavage production of the CTL epitope by proteasomes and PA28: role of the flanking region.
- Author
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Shimbara N, Nakajima H, Tanahashi N, Ogawa K, Niwa S, Uenaka A, Nakayama E, and Tanaka K
- Subjects
- Amino Acid Sequence, Animals, Antigens, Neoplasm metabolism, Cell Cycle Proteins, Chromatography, High Pressure Liquid, Cysteine Endopeptidases isolation & purification, Immunodominant Epitopes metabolism, Major Histocompatibility Complex, Molecular Sequence Data, Multienzyme Complexes isolation & purification, Peptides genetics, Peptides immunology, Peptides metabolism, Proteasome Endopeptidase Complex, Proteins genetics, Proteins immunology, Proteins isolation & purification, Rats, Cysteine Endopeptidases metabolism, Epitopes, T-Lymphocyte metabolism, Multienzyme Complexes metabolism, Proteins metabolism, T-Lymphocytes, Cytotoxic immunology
- Abstract
Background: Proteasomes are known to produce major histocompatibility complex (MHC) class I ligands from endogenous antigens, and the gamma-interferon-inducible proteasome activator PA28 has been thought to play an important role in the generation of immunodominant MHC ligands by proteasomes. Several attempts have been made to show that proteasomes have the ability to yield cytotoxic T lymphocyte (CTL) epitopes effectively from model polypeptides derived from viral and intracellular proteins in vitro, but their antigen processing mechanism is poorly understood., Results: Proteasomes produce the tumour rejection antigen precursor peptide pRL1b (SIIPGLPLSL), but not pRL1a (IPGLPLSL), bound to the H-2Ld molecule, from synthetic peptides covering the CTL epitope. This double cleavage production of pRL1b by proteasomes seemed to depend on the length of the flanking regions adjacent to either end of the CTL epitope, in which their successive deletions caused the almost complete prevention of pRL1b excision. The newly identified PA28 collaborates with proteasomes for efficient production of pRL1b, by promoting not only single cleavage of all susceptible peptides, but also dual cleavage in some peptides harboring certain characteristic lengths., Conclusion: The flanking regions outside pRL1b of suitable length appear to be essential for the correct CTL epitope production, possibly functioning as anchors to trap target peptides for proteasomal degradation. We propose a novel mechanism for dual-cleavage excision of immunodominant epitopes by proteasomes and PA28.
- Published
- 1997
- Full Text
- View/download PDF
30. [Molecular mechanisms for processing of endogenous antigens].
- Author
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Tanaka K, Tanahashi N, and Shimbara N
- Subjects
- Animals, Interferon-gamma physiology, Major Histocompatibility Complex, Proteasome Endopeptidase Complex, Ubiquitins physiology, Antigens metabolism, Cysteine Endopeptidases physiology, Multienzyme Complexes physiology
- Published
- 1997
31. Molecular properties of the proteasome activator PA28 family proteins and gamma-interferon regulation.
- Author
-
Tanahashi N, Yokota K, Ahn JY, Chung CH, Fujiwara T, Takahashi E, DeMartino GN, Slaughter CA, Toyonaga T, Yamamura K, Shimbara N, and Tanaka K
- Subjects
- Amino Acid Sequence, Animals, Autoantigens, Blotting, Southern, Chromosome Mapping, Chromosomes, Human, Pair 17, Female, Humans, Male, Mice, Mice, Transgenic, Molecular Sequence Data, Nuclear Proteins immunology, Proteasome Endopeptidase Complex, Proteins immunology, RNA, Messenger genetics, Recombinant Proteins genetics, Recombinant Proteins immunology, Gene Expression Regulation physiology, Interferon-gamma physiology, Muscle Proteins, Proteins genetics
- Abstract
Background: Recent cDNA cloning of two homologous proteasome activators, PA28 alpha and PA28 beta, indicated the presence of a structurally related third protein, Ki antigen, but a functional relationship between Ki antigen and the two PA28 proteins is unknown. Accumulating evidence has implicated an important role for PA28 in the major histocompatibility complex (MHC) class I-restricted antigen processing pathway. Recently, an immunomodulatory cytokine gamma-interferon (gamma-IFN) was found to increase greatly the messages for PA28 alpha and PA28 beta, but not Ki antigen, in human cells., Results: Ki antigen was co-immunoprecipitated with the 20S proteasome by anti-proteasome antibody, and associated reversibly with the 20S proteasome, as observed for PA28 alpha and PA28 beta. Therefore, Ki antigen was renamed PA28 gamma. Anti-PA28 gamma antibody, however, did not immunoprecipitate PA28 alpha and PA28 beta. gamma-IFN caused an almost complete loss of the PA28 gamma protein in cells without affecting its mRNA level, whereas the levels of both mRNA and protein for PA28 alpha and PA28 beta were coordinately upregulated by gamma-IFN. Finally we showed that the human chromosomal genes of PA28 alpha and PA28 gamma were located on 14q11.2 and 17q21.32-21.33, respectively., Conclusion: PA28 gamma (equivalent to Ki antigen) is a new member of the PA28 family proteins. It exists as a unique homopolymer under non-denaturing conditions. gamma-IFN was found to induce the expression of PA28 alpha and PA28 beta, whereas it caused almost complete loss of the PA28 gamma protein in cells. The reciprocal expression of the PA28 family proteins may imply their involvement in distinct biological processes.
- Published
- 1997
- Full Text
- View/download PDF
32. Proteasomes and antigen processing.
- Author
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Tanaka K, Tanahashi N, Tsurumi C, Yokota KY, and Shimbara N
- Subjects
- ATP-Binding Cassette Transporters immunology, Amino Acid Sequence, Animals, Catalysis, Cysteine Endopeptidases chemistry, Cysteine Endopeptidases metabolism, Humans, Molecular Sequence Data, Multienzyme Complexes chemistry, Multienzyme Complexes metabolism, Proteasome Endopeptidase Complex, Structure-Activity Relationship, Ubiquitins immunology, Antigen Presentation, Cysteine Endopeptidases immunology, Multienzyme Complexes immunology
- Published
- 1997
- Full Text
- View/download PDF
33. Functional maintenance of hepatocytes on collagen gel cultured with simple serum-free medium containing sodium selenite.
- Author
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Nakajima H and Shimbara N
- Subjects
- Animals, Cells, Cultured, Collagen, Cytochrome P-450 Enzyme System metabolism, Enzyme Induction drug effects, Extracellular Matrix physiology, Gels, Male, Metals pharmacology, Rats, Rats, Wistar, Serum Albumin metabolism, Culture Media, Liver cytology, Sodium Selenite pharmacology
- Abstract
We found that simple serum-free medium containing sodium selenite (Se) is effective for long-term maintenance of functional hepatocytes cultured on a pepsin-digested collagen gel (DC-gel). The concentration of Se was important for maintenance of hepatocytes, and its optimal concentration was 10(-7) approximately 10(-6)M. The effect of Se was specific, as other metals did not have the same effect. The effect was equal to that of fetal bovine serum for maintenance of functional hepatocytes. Using this medium, we could obtained a high level of hepatocellular function including the production of albumin and transferrin, and activity of p450 throughout a long-term culture. Matrigel was almost equal to DC-gel for albumin secretion, but less effective for transferrin secretion, and P450 activity in long-term cultures. The growth of cells on DC-gel or matrigel was not observed, and cell morphology of a round-shaped form was similar on both substrata. These results indicate that serum-free medium containing Se in a DC-gel culture system provides a simple method for long-term culture of hepatocytes.
- Published
- 1996
- Full Text
- View/download PDF
34. Long-term culture of functional hepatocytes on chemically modified collagen gels.
- Author
-
Shimbara N, Atawa R, Takashina M, Tanaka K, and Ichihara A
- Abstract
For long-term maintenance of functional hepatocytes in primary culture, a new culture system with chemically modified type-I collagen gel was developed. Isolated hepatocytes spread as flat cells and rapidly lost their viability and functions when cultured on native collagen gel. In contrast, they survived for several weeks when cultured on collagen gels that had been modified by treatment with sodium-borohydride (NaBH(4)) or by digestion with pepsin, which resulted in destruction of crosslinking of collagen fibers and marked decrease in meachanical strength of the gels. These long-lived cells were round and aggregated and maintained high levels of various differentiated liver functions including albumin secretion and activities of tyrosine aminotransferase and P450. Moreover on collagen gels modified by treatment with NaBH(4) or pepsin, the cell showed less DNA synthesis in response to mitogenic stimulation than cells cultures on gel containing native collagen. Interestingly, crosslinking of these chemically modified gels with D-ribose resulted in changes in various phenotypes of hepatocytes cultures on them including shape, longevity, and functions expressed when the cells were cultured on native collagen gel, suggesting that the effect of modification of the collagen gel is reversible. Thus the structure of collagen gels, probably due to the degree of crosslinking, seems to affect the morphology, maintenance of differentiated functions, and growth of primary cultured hepatocytes.
- Published
- 1996
- Full Text
- View/download PDF
35. Rejection antigen peptides on BALB/c RL male 1 leukemia recognized by cytotoxic T lymphocytes: derivation from the normally untranslated 5' region of the c-akt proto-oncogene activated by long terminal repeat.
- Author
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Wada H, Matsuo M, Uenaka A, Shimbara N, Shimizu K, and Nakayama E
- Subjects
- Amino Acid Sequence, Animals, Antigens, Neoplasm biosynthesis, Base Sequence, Blotting, Northern, Exons, Gene Expression Regulation, Leukemic, Leukemia Virus, Murine genetics, Male, Mice, Mice, Inbred BALB C, Molecular Sequence Data, Polymorphism, Genetic, Proto-Oncogene Proteins biosynthesis, Proto-Oncogene Proteins c-akt, RNA, Messenger analysis, RNA, Messenger genetics, Antigens, Neoplasm genetics, Antigens, Neoplasm immunology, Leukemia, Experimental immunology, Protein Serine-Threonine Kinases, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins immunology, Repetitive Sequences, Nucleic Acid physiology, T-Lymphocytes, Cytotoxic immunology
- Abstract
Tumor antigen peptides on BALB/c leukemia RL male 1 that were recognized by cytotoxic T lymphocytes were shown to be derived from a normally untranslated region of the akt proto-oncogene (Uenaka, A. et al., J. Exp. Med., 180: 1599, 1994). We show here that the murine leukemia virus (MuLV) long terminal repeat (LTR) was inserted directly into the exon of c-akt in RL male 1 leukemia and that transcription started from the cap site of the LTR. Translation appeared to start from the ATG codon created in the six nucleotides of unknown origin, which were inserted into the LTR/akt junction. The deduced molecular size is approximately M(r) 59,000 due to the addition of 33 amino acid residues to the normally expressed c-AKT protein. Western blot analysis demonstrated the presence of M(r) 59,000 molecules in an RL male 1 lysate, and their expression at about ten times the level of normal AKT molecules of M(r) 56,000, which is consistent with the increased expression of akt mRNA demonstrated by Northern blot analysis. The findings show that the molecular alteration of AKT protein by insertion of MuLV LTR is the mechanism for creating rejection antigen peptides derived from the untranslated region of akt.
- Published
- 1995
36. cDNA cloning and interferon gamma down-regulation of proteasomal subunits X and Y.
- Author
-
Akiyama K, Yokota K, Kagawa S, Shimbara N, Tamura T, Akioka H, Nothwang HG, Noda C, Tanaka K, and Ichihara A
- Subjects
- Amino Acid Sequence, Base Sequence, Cloning, Molecular, DNA, Complementary genetics, Endopeptidases chemistry, Endopeptidases genetics, Humans, Major Histocompatibility Complex, Molecular Sequence Data, Proteins chemistry, Proteins metabolism, Sequence Homology, Amino Acid, Tumor Cells, Cultured, Cysteine Endopeptidases, Down-Regulation, Interferon-gamma pharmacology, Multienzyme Complexes, Proteasome Endopeptidase Complex, Proteins genetics
- Abstract
Proteasomes are the proteolytic complex responsible for major histocompatibility complex (MHC) class I-restricted antigen presentation. Interferon gamma treatment increases expression MHC-encoded LMP2 and LMP7 subunits of the proteasome and decreases expression of two proteasome subunits, named X and Y, which alters the proteolytic specificity of proteasomes. Molecular cloning of complementary DNAs encoding X and Y showed that their proteins are proteasomal subunits with high amino acid similarity to LMP7 and LMP2, respectively. Thus, interferon gamma may induce subunit replacements of X and Y by LMP7 and LMP2, respectively, producing proteasomes perhaps more appropriate for the immunological processing of endogenous antigens.
- Published
- 1994
- Full Text
- View/download PDF
37. Replacement of proteasome subunits X and Y by LMP7 and LMP2 induced by interferon-gamma for acquirement of the functional diversity responsible for antigen processing.
- Author
-
Akiyama K, Kagawa S, Tamura T, Shimbara N, Takashina M, Kristensen P, Hendil KB, Tanaka K, and Ichihara A
- Subjects
- Amino Acid Sequence, Cysteine Endopeptidases chemistry, Down-Regulation, Humans, Kidney enzymology, Liver enzymology, Molecular Sequence Data, Multienzyme Complexes chemistry, Proteasome Endopeptidase Complex, Sequence Homology, Amino Acid, Antigens metabolism, Cysteine Endopeptidases metabolism, Interferon-gamma pharmacology, Multienzyme Complexes metabolism, Proteins metabolism
- Abstract
Proteasomes catalyze the non-lysosomal, ATP-dependent selective breakdown of ubiquitinated proteins and are thought to be responsible for MHC class I-restricted antigen presentation. Recently, we reported that gamma interferon (IFN-gamma) induced not only marked synthesis of the MHC-encoded proteasome subunits LMP2 and LMP7, but also almost complete loss of two unidentified proteasome subunits tentatively designated as X and Y in various human cells. Here, we show that subunit X is a new proteasomal subunit highly homologous to LMP7, and that subunit Y is identical to the LMP2-related proteasomal subunit delta. Thus, IFN-gamma appears to induce subunit replacements of X and Y by LMP7 and LMP2, respectively, producing 'immuno-proteasomes' with the functional diversity responsible for processing of endogenous antigens.
- Published
- 1994
- Full Text
- View/download PDF
38. Interferon-gamma induces different subunit organizations and functional diversity of proteasomes.
- Author
-
Aki M, Shimbara N, Takashina M, Akiyama K, Kagawa S, Tamura T, Tanahashi N, Yoshimura T, Tanaka K, and Ichihara A
- Subjects
- Adenosine Triphosphate pharmacology, Amino Acid Sequence, Colonic Neoplasms, Cysteine Endopeptidases chemistry, Flow Cytometry, Histocompatibility Antigens Class I biosynthesis, Histocompatibility Antigens Class I genetics, Humans, Hydrolysis, Leukemia, Myeloid, Major Histocompatibility Complex drug effects, Molecular Sequence Data, Multienzyme Complexes chemistry, Nucleic Acid Hybridization, Proteasome Endopeptidase Complex, Proteins chemistry, Proteins metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Recombinant Proteins, Tumor Cells, Cultured, Ubiquitins pharmacology, Cysteine Endopeptidases metabolism, Interferon-gamma pharmacology, Multienzyme Complexes metabolism
- Abstract
To obtain information on the role of proteasomes in the immune system, we examined the effect of a major immunomodulatory cytokine, gamma interferon (IFN-gamma), on the expressions, structures, and functions of proteasomes. IFN-gamma greatly increased the levels of the mRNAs encoding LMP2 and LMP7, putative immuno-proteasome subunits encoded by genes within the class II MHC region, and these two subunits synthesized were assembled completely into the proteasomal multi-subunit complex in various types of human cells. The subunit organization of proteasome changed in response to IFN-gamma stimulation, due to assembly of newly synthesized subunits through up- and down-expressions of at least 6 proteasome genes including LMP2/LMP7 without change in the structure of pre-existing proteasomes. Interestingly, IFN-gamma dramatically stimulated the trypsin-like and chymotrypsin-like activities of the multifunctional proteasome and depressed the peptidylglutamyl-peptide-hydrolyzing activity, without affecting the activity for ATP-, ubiquitin-dependent proteolysis. These results indicate that IFN-gamma modifies not only the structural organization of the proteasome, but also its functions. Based on these findings, we discuss the role in the antigen processing/presentation pathway of proteasomes with functional diversity acquired through alteration of their subunit assembly in response to IFN-gamma stimulation.
- Published
- 1994
- Full Text
- View/download PDF
39. Down-regulation of ubiquitin gene expression during differentiation of human leukemia cells.
- Author
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Shimbara N, Sato C, Takashima M, Tanaka T, Tanaka K, and Ichihara A
- Subjects
- Down-Regulation, Humans, Leukemia, RNA, Messenger metabolism, Ribosomal Proteins genetics, Ribosomal Proteins metabolism, Transcription, Genetic, Tumor Cells, Cultured, Ubiquitins metabolism, Cell Differentiation genetics, Gene Expression Regulation, Ubiquitins genetics
- Abstract
Ubiquitin, which is ligated covalently to target proteins for their acquisition of a variety of functions, is encoded by multiple unique genes in human cells: two distinct poly-ubiquitin genes with tandemly repeated sequences of 3 or 9 moieties and two mono-ubiquitin genes fused with small and large ribosomal proteins. We found that all classes of ubiquitin genes as well as the two genes encoding the ribosomal proteins S17 and L31 were expressed at abnormally high levels in various hematopoietic malignant tumor cells. In contrast, in vitro terminal differentiation of various immature leukemic cell lines, such as HL-60 promyelocytic leukemia cells and K562 erythroleukemia cells into monocytic, granulocytic and erythroid cells, induced by various agents was found to cause rapid and marked down-regulation of ubiquitin expression, irrespective of the cell type, direction of differentiation or type of signal. These findings suggest that the expressions of the multiple ubiquitin genes, coordinated with those of the ribosomal protein genes, are in a dynamic state during growth and differentiation of leukemia cells.
- Published
- 1993
- Full Text
- View/download PDF
40. Regulation of proteasome expression in developing and transformed cells.
- Author
-
Ichihara A, Tanaka K, Andoh T, and Shimbara N
- Subjects
- Animals, Cell Division, Cell Transformation, Neoplastic, Female, Fetus enzymology, Gene Expression Regulation, Enzymologic, Liver embryology, Liver enzymology, Liver growth & development, Liver Neoplasms, Experimental enzymology, Pregnancy, Proteasome Endopeptidase Complex, RNA, Messenger genetics, RNA, Messenger metabolism, Rats, Rats, Wistar, Cysteine Endopeptidases genetics, Cysteine Endopeptidases metabolism, Multienzyme Complexes genetics, Multienzyme Complexes metabolism
- Abstract
The proteasome is a unique protease complex found in all eukaryotic cells and has multiple functions for essential activities. In this work we showed that it is expressed at high level in immature, rapidly growing cells, such as those in early embryonic tissues and cancer cells (Fig. 7). The increase of its expression is down-regulated on differentiation of the cells. However, lymphatic blastocytes grow rapidly and express high levels of proteasomes, but are differentiated. Therefore, the proteasome is not expressed at high levels only in immature cells, but is also involved specifically in nuclear activities of cells during rapid growth, possibly regulating proteinous factors in the cell cycle.
- Published
- 1993
- Full Text
- View/download PDF
41. Molecular cloning of cDNAs for rat proteasomes: deduced primary structures of four other subunits.
- Author
-
Tamura T, Shimbara N, Aki M, Ishida N, Bey F, Scherrer K, Tanaka K, and Ichihara A
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cloning, Molecular, DNA Probes, Genome, Liver Neoplasms, Experimental genetics, Macromolecular Substances, Molecular Sequence Data, Open Reading Frames, Proteasome Endopeptidase Complex, Rats, Sequence Homology, Amino Acid, Tumor Cells, Cultured, Cysteine Endopeptidases genetics, DNA, Neoplasm genetics, Multienzyme Complexes genetics
- Abstract
Proteasomes (multi-protease complexes) are composed of approximately 15 non-identical subunits of similar sizes (molecular weight = 21-32 kDa), but different charges (isoelectric point = 4-9). Previously, we deduced the primary structures of 6 subunits of rat proteasomes by recombinant DNA techniques. In this paper we report the nucleotide sequences of 4 other subunits, rIOTA, rZETA, rDELTA, and rRING12, determined from cDNA clones isolated by screening a rat H4TG hepatoma cell cDNA library with the cDNAs of their human counterparts as probes. The polypeptides deduced from their nucleotide sequences consisted of 246, 241, 202, and 219 amino acid residues with calculated molecular weights of 27,399, 26,391, 21,649, and 23,324, and calculated isoelectric points of 6.37, 4.65, 4.84, and 4.70, respectively. These results and previous findings indicate that the primary structures of the subunits of rat proteasomes show considerably high inter-subunit homology, but can be classified into apparently distinct sub-groups, suggesting that rat proteasome genes form a multi-gene family with the same evolutionary origin, but have diverged during evolution to acquire possibly subunit-specific functions.
- Published
- 1992
- Full Text
- View/download PDF
42. c-myc expression is down-regulated by cell-cell and cell-extracellular matrix contacts in normal hepatocytes, but not in hepatoma cells.
- Author
-
Shimbara N, Takashina M, Sato C, Iizuka M, Kobayashi S, Tanaka K, and Ichihara A
- Subjects
- Animals, Cell Line, Cells, Cultured, Kinetics, Male, RNA genetics, RNA isolation & purification, Rats, Rats, Inbred Strains, Cell Communication, DNA Replication, Extracellular Matrix physiology, Gene Expression Regulation, Gene Expression Regulation, Neoplastic, Genes, myc, Liver physiology, Liver Neoplasms, Experimental genetics
- Abstract
Primary culture of adult rat hepatocytes resulted in marked increase of c-myc expression within a few hours. The high level of c-myc mRNA was maintained throughout culture on collagen-coated dishes, but decreased greatly with time during culture on collagen-gel or matrigel. Expression of c-myc was also down-regulated at high cell density. The decrease in its expression appeared closely related to inhibitions of DNA synthesis and cell spreading. In contrast, hepatoma H4TG cells showed a high level of c-myc expression which was not affected by culture on any extracellular matrices examined or by the cell density. These results suggest that up-regulation of expression of the c-myc gene is linked to G0 to G1 transition during cell cycle progression, which in normal hepatocytes is strictly regulated by cell-cell and cell-extracellular matrix interactions, but that this control mechanism is defective in malignant hepatic tumor cells.
- Published
- 1992
- Full Text
- View/download PDF
43. cDNA cloning of rat proteasome subunit RC1, a homologue of RING10 located in the human MHC class II region.
- Author
-
Aki M, Tamura T, Tokunaga F, Iwanaga S, Kawamura Y, Shimbara N, Kagawa S, Tanaka K, and Ichihara A
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cloning, Molecular, Molecular Sequence Data, Molecular Weight, Open Reading Frames, Peptide Fragments chemistry, Peptide Fragments isolation & purification, Proteasome Endopeptidase Complex, Rats, Sequence Homology, Amino Acid, Cysteine Endopeptidases genetics, Genes, MHC Class II genetics, Multienzyme Complexes genetics
- Abstract
The nucleotide sequence of a cDNA that encodes a new subunit, named RCl, of rat proteasomes (multicatalytic proteinase complexes) has been determined. The polypeptide predicted from the open reading frame consisted of 208 amino acid residues with a calculated molecular mass of 23, 130, which is consistent with the size obtained by electrophoretic analysis of purified RCl. The partial amino acid sequences of several fragments of RCl, obtained by protein chemical analyses, were found to be in excellent accordance with those deduced from the cDNA sequence. Surprisingly, the overall structure of RCl was found to be almost identical to that of recently isolated RING10, whose gene is located in the class II region of the human MHC gene cluster. This finding suggests that RCl is a homologue of human RING10, supporting the proposal that proteasomes are involved in the antigen processing pathway.
- Published
- 1992
- Full Text
- View/download PDF
44. Changes in expressions of proteasome and ubiquitin genes in human renal cancer cells.
- Author
-
Kanayama H, Tanaka K, Aki M, Kagawa S, Miyaji H, Satoh M, Okada F, Sato S, Shimbara N, and Ichihara A
- Subjects
- Adult, Aged, Blotting, Northern, Cell Division, Female, Gene Expression, Humans, Immunohistochemistry, Kidney cytology, Kidney Neoplasms pathology, Male, Middle Aged, Proteasome Endopeptidase Complex, RNA, Messenger genetics, RNA, Neoplasm genetics, Tumor Cells, Cultured, Ubiquitins immunology, Cysteine Endopeptidases genetics, Kidney physiology, Kidney Neoplasms genetics, Multienzyme Complexes genetics, Ubiquitins genetics
- Abstract
Proteasomes and ubiquitin (Ub) are essential components of the energy-dependent, nonlysosomal proteolytic pathway. To clarify the physiological role of this proteasome/Ub-dependent pathway, we meaured the levels of expressions of proteasomes and Ub in human renal cancers by Northern blot and immunochemical analyses. The mRNAs for two of the multiple subunits of proteasomes, C2 and C9, were expressed at abnormally high levels in most neoplastic lesions of patients with various primary renal cell carcinomas and in all renal cancer cell lines examined. However, no significant difference was found by enzyme immunoassay in the proteasomal contents of cancerous and normal parts of the kidney. The levels of mRNAs for the subunits of proteasomes were high in rapidly proliferating renal cells and appeared to be correlated with the activities of these cells for proteasome synthesis, but the cellular contents of proteasomes in these cells were normal, suggesting rapid turnover of proteasomes in rapidly proliferating cancer cells. Consistent with the increased expressions of proteasomal mRNAs, the expressions of three Ub genes, mono-UbA80, mono-UbA52, and poly-UbC, were found to be greatly increased in these renal cancer cells. Immunohistochemical staining of normal kidney showed that the levels of both proteasomes and Ub were high in cells of renal tubules and collecting ducts, but low in the glomerulus. The levels of both proteins appeared to be considerably increased in the nuclei of granular and clear carcinoma cells of the kidney. Moreover, the profiles of cellular proteins conjugated with Ub in normal kidney tissues were different from those in cancerous parts of the kidney and in established renal cancer cells. These results suggest that the proteasome- and ubiquitin-mediated system is functionally involved in the cancerous state in human kidney.
- Published
- 1991
45. Radiolytic hydroxylation of 1-substituted thymines promoted by transition metal salts in N2- and N2O-saturated aqueous solutions.
- Author
-
Nishimoto S, Shimbara N, Kaneto M, Sakano K, and Kagiya T
- Subjects
- Hydroxylation, Indicators and Reagents, Metals, Thymine analogs & derivatives, Thymine radiation effects
- Abstract
The effect of high-valent transition metal salts on the radiation-induced hydroxylation of thymine (1a), 1-methylthymine (1b), and thymidine (1c) to the corresponding thymine glycol derivatives (2a-c) was studied at pH 7.0 in N2- and N2O-saturated aqueous solutions. The selectivities of (2a-c) based on converted (1a-c) increased to attain the maxima of 35-69% and then decreased, with increasing the one-electron reduction potential [E(M (n+1)+/Mn+)] of metal salts in the range of 0-1.0 V vs. NHE less than. Metal salts with E(M (n+1)+/Mn+) 0 or greater 1.0 V vs. NHE caused little change in the yields of (2a-c).
- Published
- 1985
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