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N Shimbara

Publications and source records attributed to N Shimbara.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1994

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.

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.

Amino Acid Sequence↗
PubMed1994

Interferon-gamma induces different subunit organizations and functional diversity of proteasomes.

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.

Adenosine Triphosphate↗
PubMed1993

Down-regulation of ubiquitin gene expression during differentiation of human leukemia cells.

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.

Cell Differentiation↗
PubMed1993

Regulation of proteasome expression in developing and transformed cells.

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.

Animals↗
PubMed1992

Regulation of gene expression of proteasomes (multi-protease complexes) during growth and differentiation of human hematopoietic cells.

We have reported that proteasomes are expressed at abnormally high levels in various hematopoietic tumor cells (Kumatori, A., Tanaka, K., Inamura, N., Sone, S., Ogura, T., Matsumoto, T., Tachikawa, T., Shin, S., and Ichihara, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7071-7075). In the present study, we examined changes in the expressions of proteasomes during growth of peripheral T-lymphocytes from healthy adults and differentiation of human leukemic cell lines. Up-regulation of mRNAs encoding multiple proteasome subunits was observed during proliferation of resting T-cells induced by mitogens such as phytohemagglutinin and interleukin-2. In contrast, in vitro terminal differentiation into monocytic, granulocytic, and erythroid cells of various immature leukemic cell lines, such as HL-60 promyelocytic leukemia cells and K562 erythroleukemia cells, by various inducing agents caused rapid and marked down-regulation of proteasomes expression, independently of the cell type, direction of differentiation, or type of signal. The syntheses of proteasome subunits of 21-31 kDa and their associated components of 35-110 kDa, measured by [35S]methionine incorporation, were much higher in mitogen-activated T-cells and unstimulated HL-60 cells, which grow rapidly, than in resting and differentiated cells, indicating apparent correlations of the mRNA levels of proteasomes with their translational activities. However, immunochemically, no detectable difference in the cellular contents of proteasomes was found in these cells in induced and uninduced states for proliferation and differentiation, suggesting accelerated turnover of proteasomes in rapidly proliferating cells. Inhibition of proteasome expression by an antisense oligodeoxynucleotide for the largest proteasome subunit, C2, caused partial arrest of cell cycle progression of T-lymphocytes, suggesting that up-regulation of proteasomes is indispensable for proliferation of the cells. We also observed that the nuclear fraction of proteasomes increased in proliferating T-cells and that proteasomes moved rapidly between the nucleus and cytoplasm during differentiation of HL-60 cells.

Base Sequence↗
PubMed1992

c-myc expression is down-regulated by cell-cell and cell-extracellular matrix contacts in normal hepatocytes, but not in hepatoma cells.

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.

Animals↗
PubMed1992

cDNA cloning of rat proteasome subunit RC1, a homologue of RING10 located in the human MHC class II region.

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.

Amino Acid Sequence↗