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Query: EC:3.4.25.1 (proteasome)
 28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytotoxic T cells recognize viral proteins as peptide fragments which are produced in the cytosol and transported on major histocompatibility complex (MHC) class I proteins to the cell surface. Viral peptides that meet the stringent binding characteristics of class I proteins are generated by the 20S proteasome. The interferon (IFN)-gamma-inducible activator of the 20S proteasome, PA28, strongly influences the proteasomal cleavage pattern in vitro. This led us to investigate whether changes in cellular levels of PA28 affect the efficiency of viral antigen processing. A mouse fibroblast line expressing the murine cytomegalovirus pp89 protein was transfected with either the human or murine gene encoding the PA28alpha subunit, which is sufficient to activate the peptide-hydrolysing activity of the 20S proteasome in vitro. Here we report that enhanced expression of PA28alpha at a level similar to that obtained after IFN-gamma induction resulted in a marked enhancement of recognition by pp89-specific cytotoxic T cells; the presentation of influenza nucleoprotein was also significantly improved. These results demonstrate a fundamental in vivo function for PA28alpha in antigen processing.
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PMID:A role for the proteasome regulator PA28alpha in antigen presentation. 861 16

A proteasome regulator, termed PA28, has been shown to modulate peptidase activities of the proteasomes in vitro. Two different but homologous PA28 molecules, designated as PA28alpha and PA28beta, have been cloned. Both alpha and beta polypeptides of PA28 are found in PA28 complexes isolated from cells, indicating that both are constituents of functional PA28 complexes. Using antisera specific to PA28alpha, PA28beta, and epitope-tagged PA28 molecules, we show that expression of PA28alpha and PA28beta is coordinately induced by various cytokines in different cell lines and that PA28 subunits and proteasomes have almost identical half-lives. In addition, we show that PA28 complexes are associated with 20 S but not 26 S proteasomes in vivo. Moreover, we demonstrate that PA28 complex is a heterohexamer composed of both alpha and beta subunits with a stoichiometry of alpha3beta3 in an alternating order.
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PMID:In vivo characterization of the proteasome regulator PA28. 866 20

PA28 is a protein activator of the 20S proteasome. It has a native molecular weight of approximately 200,000 and is composed of six 28,000-dalton subunits arranged in a ring-shaped complex. Purified preparations of PA28 contain two polypeptides, alpha and beta, which are about 50% identical in primary structure. It has been unclear whether native PA28 consists of two distinct homohexameric proteins or of a single protein containing both alpha and beta subunits. To distinguish between these possibilities, we prepared antibodies that reacted specifically with either the alpha or beta subunit and used these subunit-specific antibodies in two types of experiments designed to elucidate PA28 quaternary structure. In the first experiment, the alpha and beta subunits were completely co-immunoprecipitated by each subunit-specific antibody, indicating that both subunits were part of a single protein complex. In the second experiment, PA28 was chemically cross-linked using bis(sulfosuccinimidyl)suberate. When the cross-linked products were immunoblotted after SDS-polyacrylamide gel electrophoresis, indistinguishable patterns were obtained with each subunit-specific antibody. These results confirm that the alpha and beta subunits were part of the same protein complex. The pattern of cross-linked products also provided insight as to the relative abundance and arrangement of the subunits within the PA28 complex and indicated that the ring-shaped PA28 hexamer may be composed of alternating alpha and beta subunits with a stoichiometry of (alphabeta)3. PA28 was inactivated by treatment with carboxypeptidase Y, which cleaved Tyr and Ile residues from the carboxyl terminus of the alpha subunit but had very little effect on the beta subunit. This selective and limited proteolysis prevented binding of both alpha and beta subunits to the proteasome and therefore provides additional evidence of the heterodimeric nature of PA28. These results indicate that a short carboxyl-terminal sequence of the alpha subunit is critical for binding of native PA28 to the proteasome. To learn about the relative functions of the alpha and beta subunits, PA28alpha was expressed in Escherichia coli and purified to homogeneity. Purified PA28alpha stimulated proteasome activity but required 5-10-fold greater concentrations than the heterodimeric PA28 to achieve a given level of activity. These results suggest that the heterodimeric structure of PA28 is required for maximal proteasome activation.
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PMID:A model for the quaternary structure of the proteasome activator PA28. 882 98

Using reverse transcription polymerase chain reaction, we cloned and sequenced full-length mouse cDNAs for the two homologous subunits of the proteasome activator PA28 (PA28alpha and PA28beta), as well as for the related protein Ki. These proteins are highly conserved among species. Northern blot analysis of PA28a, PA28b, and Ki mRNA demonstrated broad tissue distribution. Although single transcripts were detected for PA28a and PA28b, two different sized transcripts were detected for mouse Ki, suggesting either alternative splicing or alternate polyadenylation sites. The levels of these transcripts increased in response to interferon-gamma (IFN-gamma) treatment in mouse H6 hepatoma cells, although PA28a and PA28b were induced to a greater extent than Ki, and the effect of IFN-gamma stimulation on Ki expression was transient. Southern blot analysis suggests that both PA28a and PA28b are multiple-copy genes, while Ki is a single-copy gene.
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PMID:Sequence and expression of mouse proteasome activator PA28 and the related autoantigen Ki. 916 94

The proteasome activator PA28 or 11S regulator is a protein complex composed of two different but homologous polypeptides, termed PA28alpha and PA28beta. The purified activator protein (approximately 200 kDa) is a ring-shaped heteromultimer containing the two polypeptides, possibly with an (alpha3beta3 stoichiometry. The activator, which by itself shows no hydrolytic activity elicits activation of the proteasome's multiple peptidase activities by binding to the terminal rings of the proteinase. In vitro, active PA28 can be reconstituted from isolated alpha and beta subunits, yielding two different oligomers: with the single alpha subunit, PA28alpha homomultimers with moderate stimulatory activity toward 20S proteasomes are obtained whereas isolated beta-subunits are unable to form oligomers and are devoid of stimulatory activity. However, in the presence of both subunits, alphabeta heteromultimers form, concomitant with restoration of full stimulatory activity. The recent finding that PA28 modulates the proteasome-catalyzed production of antigenic peptides presented to the immune system on MHC class I molecules indicates a cellular function of the activator in antigen processing.
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PMID:Structural and functional properties of proteasome activator PA28. 922 87

The development of small molecule peptide-based activators of the 20S proteasome or multicatalytic proteinase complex was initiated. The enhancement of antigen presentation by transfection of the protein activator PA28alpha into a mouse fibroblast cell line [10] supports the potential use of small molecule activators in stimulating the immune response. Four classes of peptide-based activators were synthesized, i.e. peptidyl alcohols, esters, p-nitroanilides and nitriles. These compounds markedly and reversibly stimulated the hydrolysis of suc-LLVY-MCA, Z-LLE-NA and Z-GPALG-p-aminobenzoate as well as hydrolysis of the decapeptide angiotensin I. Stimulation was due to a decrease in the Km and increase in the Vmax of the substrate. In general, the EC50 for activation ranged from 50-150 mM and maximal stimulation varied from 3 to 15 fold depending on the activity measured. Z-IE(Ot-Bu)AL-p-nitroanilide, a proteasome substrate, markedly stimulated the hydrolysis of Z-GPALG-pAB by binding to a saturable high affinity site distinct from its binding site as substrate. Since all effective activators contain hydrophobic groups in positions P1-P5, low aqueous solubility is a limitation of these compounds. Competition experiments suggest that these activators bind to the same site as PA28.
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PMID:Synthetic peptide-based activators of the proteasome. 922 92

We have cloned the mouse PA28 proteasome activator cDNAs. Northern blot demonstrates high PA28 mRNA levels in liver, kidney and lung. mRNA levels are low in thymus, spleen and brain. In contrast, PA28 protein levels vary little between these tissues. Immunocytological analysis and cell fractionation experiments demonstrate that both subunits are almost equally distributed between the cytoplasm and the nucleus. Interestingly, PA28alpha spares nucleoli, while PA28beta is strongly enhanced in the nucleolus. This indicates for the first time that the PA28alpha and PA28beta subunits may serve nuclear functions which may be different from and independent of each other.
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PMID:Expression and subcellular localization of mouse 20S proteasome activator complex PA28. 928 11

Full-length cDNAs for three human proteasome activator subunits, called REGalpha, REGbeta, and REGgamma, have been expressed in Escherichia coli, and the purified recombinant proteins have been characterized. Recombinant alpha or gamma subunits form heptameric species; recombinant beta subunits are found largely as monomers or small multimers. Each recombinant REG stimulates cleavage of fluorogenic peptides by human red cell proteasomes. The pattern of activated peptide hydrolysis is virtually identical for REGalpha and REGbeta. These two subunits, alone or in combination, stimulate cleavage after basic, acidic, and most hydrophobic residues in many peptides. Recombinant alpha and beta subunits bind each other with high affinity, and the REGalpha/beta heteromeric complex activates hydrolysis of LLVY-methylcoumaryl-7-amide (LLVY-MCA) and LLE-beta-nitroanilide (LLE-betaNA) more than REGalpha or REGbeta alone. Using filter binding and gel filtration assays, recombinant REGgamma subunits were shown to bind themselves but not alpha or beta subunits. REGgamma differs from REGalpha and REGbeta in that it markedly stimulates hydrolysis of peptides with basic residues in the P1 position but only modestly activates cleavage of LLVY-MCA or LLE-betaNA by the proteasome. REGgamma binds the proteasome with higher affinity than REGalpha or REGbeta yet with lower affinity than complexes containing both REGalpha and REGbeta. In summary, each of the three REG homologs is a proteasome activator with unique biochemical properties.
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PMID:Characterization of recombinant REGalpha, REGbeta, and REGgamma proteasome activators. 932 61

Genomic clones were obtained for the genes encoding the beta subunits of the human proteasome and for the associated proteasome activators PA28alpha and beta (PSME1 and PSME2, respectively). Fluorescence in situ hybridization was used to map the gene encoding the beta subunit PSMB3 (beta3 hs, HsC10-II) to chromosome band 2q35, PSMB2 (beta4 hs, HsC7-I) to band 1p34.2, and PSMB4 (beta7 hs, HSBpros 26) to band 1q21. Genes encoding the alpha and beta subunits of the PA28 complex were found closely linked on chromosome band 14q11.2, within 1 Mb of the beta proteasome locus PSMB5 (beta5 hs, MB1, X). These data complete the mapping of the human proteasome beta subunit loci. With the exception of the genes encoding the PSMB9 and PSMB8 (LMP2 and LMP7, respectively) subunits, the beta genes were not closely linked in the human genome. Both PSMB2 and PSMB4 mapped to a region of chromosome 1 that is proposed to be paralogous to other regions of the human genome where beta proteasome genes map: chromosome 6 containing the major histocompatibility complex (MHC) and chromosome 9. The independent regulation of expression of all of these genes, implied by this study, is consistent with a key role for proteasome assembly in coordination of the complex.
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PMID:Genetic relationships of the genes encoding the human proteasome beta subunits and the proteasome PA28 complex. 934 61

PA28 is a 180,000-dalton protein that activates hydrolysis of small nonubiquitinated peptides by the 20 S proteasome. PA28 is composed of two homologous subunits, alpha and beta, arranged in alternating positions in a ring-shaped oligomer with a likely stoichiometry of (alphabeta)3. Our previous work demonstrated that the carboxyl terminus of the alpha subunit was necessary for PA28 to bind to and activate the proteasome. The goals of this work were to define the exact structural basis for this effect and to determine the relative roles of the alpha and beta subunits in proteasome activation. Each subunit and various mutants of the alpha subunit were expressed in Escherichia coli and purified. PA28alpha stimulated the proteasome, but had a much greater Kact than native heteromeric PA28. In contrast, PA28beta was unable to stimulate the proteasome. Mutants of the alpha subunit in which the carboxyl-terminal tyrosine residue was deleted or substituted with charged amino acids could neither bind to nor activate the proteasome. However, substitution of the carboxyl-terminal tyrosine with other amino acids resulted in proteins which could stimulate the proteasome to various extents. Tryptophan mutants stimulated the proteasome as well as did native PA28, whereas serine or phenylalanine mutants stimulated the proteasome much poorer than did wild type PA28alpha. Deletion of the "KEKE" motif, a 28-amino acid domain near the amino terminus of PA28alpha, had no effect on proteasome stimulatory activity. Hetero-oligomeric PA28 proteins were reconstituted from isolated wild type and mutant subunits. PA28 reconstituted from wild type subunits had structural and functional properties that were indistinguishable from those of the native hetero-oligomeric protein. PA28 molecules reconstituted from inactive alpha subunits and wild type beta subunits remained inactive. However, PA28 molecules reconstituted from suboptimally active alpha mutants and wild type beta subunits had the same activity as native heteromeric PA28. These results indicate that the beta subunit modulates PA28 activity, perhaps by influencing the affinity of PA28 for the proteasome.
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PMID:Relative functions of the alpha and beta subunits of the proteasome activator, PA28. 934 51


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