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N Shimbara

Publications and source records attributed to N Shimbara.

At least 19 recordsLinked to original sources
PubMed1999

The involvement of proteasome in myogenic differentiation of murine myocytes and human rhabdomyosarcoma cells.

The murine C2C12 myocytes terminally differentiate to myotubes in the mitogen-depletion, and a portion of the cells undergo apoptosis. In this study, a specific proteasome inhibitor lactacystin induced cell cycle withdrawal and precocious expression of myosin in C2C12 cells in mitogen-enriched medium, but these cells did not fuse to form myotubes. Mitogen-starved myocytes could not differentiate to myotubes under the proteasome inhibition. The genes for p21, MyoD, Myogenin and RB were activated, and p27 gene was repressed under the proteasome inhibition, suggesting the transcriptional regulation of these genes linked to the proteasome activity. The induction of p21 prior to MyoD may contribute to the incomplete myogenesis in the presence of lactacystin. In addition, lactacystin-treated C2C12 cells did not undergo apoptosis, while proteasome accumulated in the nuclei of apoptotic cells but not in those of myotubes during mitogen-depleted differentiation. Further, lactacystin induced similarly incomplete differentiation in human RD embryonal rhabdomyosarcoma cells. Our findings demonstrated that proteasome has an essential role in myogenesis, especially in transcriptional control of myogenic and cell cycle regulators, cell fusion forming myotubes, and apoptosis.

Acetylcysteine↗
PubMed1998

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.

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.

ATP-Dependent Proteases↗
PubMed1998

Contribution of proline residue for efficient production of MHC class I ligands by proteasomes.

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.

Amino Acid Sequence↗
PubMed1998

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.

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.

Amino Acid Sequence↗
PubMed1998

Chromosomal localization and immunological analysis of a family of human 26S proteasomal ATPases.

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.

Adenosine Triphosphatases↗
PubMed1997

SUG1, a component of the 26 S proteasome, is an ATPase stimulated by specific RNAs.

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.

Adenosine Triphosphatases↗
PubMed1997

Double-cleavage production of the CTL epitope by proteasomes and PA28: role of the flanking region.

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.

Amino Acid Sequence↗
PubMed1997

Molecular properties of the proteasome activator PA28 family proteins and gamma-interferon regulation.

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.

Amino Acid Sequence↗
PubMed1997

Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1.

Nin1p, a component of the 26S proteasome of Saccharomyces cerevisiae, is required for activation of Cdc28p kinase at the G1-S-phase and G2-M boundaries. By exploiting the temperature-sensitive phenotype of the nin1-1 mutant, we have screened for genes encoding proteins with related functions to Nin1p and have cloned and characterized two new multicopy suppressors, SUN1 and SUN2, of the nin1-1 mutation. SUN1 can suppress a null nin1 mutation, whereas SUN2, an essential gene, does not. Sun1p is a 268-amino acid protein which shows strong similarity to MBP1 of Arabidopsis thaliana, a homologue of the S5a subunit of the human 26S proteasome. Sun1p binds ubiquitin-lysozyme conjugates as do S5a and MBP1. Sun2p (523 amino acids) was found to be homologous to the p58 subunit of the human 26S proteasome. cDNA encoding the p58 component was cloned. Furthermore, expression of a derivative of p58 from which the N-terminal 150 amino acids had been removed restored the function of a null allele of SUN2. During glycerol density gradient centrifugation, both Sun1p and Sun2p comigrated with the known proteasome components. These results, as well as other structural and functional studies, indicate that both Sun1p and Sun2p are components of the regulatory module of the yeast 26S proteasome.

Amino Acid Sequence↗
PubMed1996

Functional maintenance of hepatocytes on collagen gel cultured with simple serum-free medium containing sodium selenite.

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.

Animals↗
PubMed1996

Newly identified pair of proteasomal subunits regulated reciprocally by interferon gamma.

Interferon (IFN) gamma induces replacements of the proteasomal subunits X and Y by LMP7 and LMP2, respectively, resulting in an alteration of the proteolytic specificity. We found a third pair of proteasome subunits expressed reciprocally in response to IFN-gamma. Molecular cloning of a cDNA encoding one subunit designated as Z, downregulated by IFN-gamma, showed that it is a novel proteasomal subunit with high homology to MECL1, which is markedly induced by IFN-gamma. Thus, IFN-gamma induces subunit replacements of not only X and Y by LMP7 and LMP2, respectively, but also of Z by MECL1, producing proteasomes responsible for immunological processing of endogenous antigens. When processed from their precursors, three pairs of the 10 homologous, but distinct, beta-type subunits of eukaryotic proteasomes, that is, X/LMP7, Y/LMP2, and Z/MECL1, have an NH2-terminal threonine residue, assumed to be part of a catalytic center. These findings suggest that the altered molecular organization of the proteasome induced by IFN-gamma may be responsible for acquisition of its functional change.

Amino Acid Sequence↗
PubMed1995

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.

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.

Amino Acid Sequence↗
PubMed1995

Nin1p, a regulatory subunit of the 26S proteasome, is necessary for activation of Cdc28p kinase of Saccharomyces cerevisiae.

The nin1-1 mutant of Saccharomyces cerevisiae cannot perform the G1/S and G2/M transitions at restrictive temperatures. At such temperatures, nin1-1 strains fail to activate histone H1 kinase after release from alpha factor-imposed G1 block and after release from hydroxyurea-imposed S block. The nin1-1 mutation shows synthetic lethality with certain cdc28 mutant alleles such as cdc28-IN. Two lines of evidence indicate that Nin1p is a component of the 26S proteasome complex: (i) Nin1p, as well as the known component of the 26S proteasome, shifted to the 26S proteasome peak in the glycerol density gradient after preincubation of crude extract with ATP-Mg2+, and (ii) nin1-1 cells accumulated polyubiquitinated proteins under restrictive conditions. These results suggest that activation of Cdc28p kinase requires proteolysis. We have cloned a human cDNA encoding a regulatory subunit of the 26S proteasome, p31, which was found to be a homolog of Nin1p.

Amino Acid Sequence↗
PubMed1995

cDNA cloning of p40, a regulatory subunit of the human 26S proteasome, and a homolog of the Mov-34 gene product.

The nucleotide sequence of a cDNA that encodes a new regulatory subunit, named p40, of the 26S proteasome of human hepatoblastoma HepG2 cells has been determined. The polypeptide predicted from the open reading frame consists of 324 amino acid residues with a calculated molecular mass of 37020 and isoelectric point of 6.03. A KEKE motif, consisting of a very hydrophilic domain rich in 'alternating' lysine (positive) and glutamate (negative) residues, is present in the C-terminus of p40. The overall structure of p40 is homologous to that of the mouse Mov-34 gene product, whose gene disruption by proviral integration results in a recessive embryonic lethality. Thus the p40/Mov-34 protein is a novel essential regulatory subunit of the human 26S proteasome.

Amino Acid Sequence↗
PubMed1995

cDNA cloning of a new putative ATPase subunit p45 of the human 26S proteasome, a homolog of yeast transcriptional factor Sug1p.

The nucleotide sequence of a cDNA that encodes a new regulatory subunit, named p45, of the 26S proteasome of human hepatoblastoma HepG2 cells has been determined. The polypeptide predicted from the open reading frame consists of 406 amino acid residues with a calculated molecular weight of 45770 and isoelectric point of 8.35. The sequences of several fragments of bovine p45, determined by protein chemical analyses, spanning 27% of the complete structure, were found to be in excellent accord with those deduced from the human cDNA sequence. Computer analysis showed that p45 belongs to a family of putative ATPases which includes regulatory components of 26S proteasomes. The overall structure of p45 was found to be homologous to that of yeast Sug1p, which has been identified as a transcriptional factor. It is closely similar, but not identical to the sequence reported for Trip1, a functional homolog of Sug1p in human tissues. These results are consistent with the possibility that Sug1-like proteins with distinct sequence function in transcription and protein degradation in human cells. However, the alternative hypothesis, that the same gene locus encodes both p45 and Trip1, cannot be excluded on the basis of such closely similar sequences. In either case, both proteins are likely to function equivalently well in either transcription or protein degradation.

Adenosine Triphosphatases↗
PubMed1994

cDNA cloning and interferon gamma down-regulation of proteasomal subunits X and Y.

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.

Amino Acid Sequence↗