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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)

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

The specificity of the 20S proteasome, which degrades many intracellular proteins, is regulated by protein complexes that bind to one or both ends of the cylindrical proteasome structure. One of these regulatory complexes, the 11S regulator (known as REG or PA28), stimulates proteasome peptidase activity and enhances the production of antigenic peptides for presentation by class I molecules of the major histocompatibility complex (MHC). The three REG subunits that have been identified, REGalpha, REGbeta and REGgamma (also known as the Ki antigen), share extensive sequence similarity, apart from a highly variable internal segment of 17-34 residues which may confer subunit-specific properties. REGalpha and REGbeta preferentially form a heteromeric complex, although purified REGalpha forms a heptamer in solution and has biochemical properties similar to the heteromeric REGalpha/REGbeta complex. We have now determined the crystal structure of human recombinant REGalpha at 2.8 A resolution. The heptameric barrel-shaped assembly contains a central channel that has an opening of 20 A diameter at one end and another of 30 A diameter at the presumed proteasome-binding surface. The binding of REG probably causes conformational changes that open a pore in the proteasome alpha-subunits through which substrates and products can pass.
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PMID:Structure of the proteasome activator REGalpha (PA28alpha). 940 98

Proteasomes can be markedly activated by associating with 19S regulatory complexes to form the 26S protease or by binding 11S protein complexes known as REG or PA28. Three REG subunits, alpha, beta, and gamma, have been expressed in Escherichia coli, and each recombinant protein can activate human proteasomes. Combining PCR mutagenesis with an in vitro activity assay, we have isolated and characterized 36 inactive, single-site mutants of recombinant REGalpha. Most are monomers that produce functional proteasome activators when mixed with REGbeta subunits. Five REGalpha mutants that remain inactive in the mixing assay contain amino acid substitutions clustered between Arg-141 and Gly-149. The crystal structure of the REGalpha heptamer shows that this region forms a loop at the base of each REGalpha subunit. One mutation in this loop (N146Y) yields a REGalpha heptamer that binds the proteasome as tightly as wild-type REGalpha but does not activate peptide hydrolysis. Corresponding amino acid substitutions in REGbeta (N135Y) and REGgamma (N151Y) produce inactive proteins that also bind the proteasome and inhibit proteasome activation by their normal counterparts. Our studies clearly demonstrate that REG binding to the proteasome can be separated from activation of the enzyme. Moreover, the dominant negative REGs identified here should prove valuable for elucidating the role(s) of these proteins in antigen presentation.
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PMID:Identification of an activation region in the proteasome activator REGalpha. 950 Nov 56

The peptidase activities of eukaryotic proteasomes are markedly activated by the 11 S REG or PA28. The three identified REG subunits, designated alpha, beta, and gamma, differ significantly in sequence over a short span of 15-30 amino acids that we call homolog-specific inserts. These inserts were deleted from each REG to produce the mutant proteins REGalphaDeltai, REGbetaDeltai, and REGgammaDeltai. The purified recombinant proteins were then tested for their ability to oligomerize and activate the proteasome. Both REGalphaDeltai and REGgammaDeltai formed apparent heptamers and activated human red cell proteasomes to the same extent as their full-length counterparts. By contrast, REGbetaDeltai exhibited, at low protein concentrations, reduced proteasome activation when compared with the wild-type REGbeta protein. REGbetaDeltai was able to form hetero-oligomers with a single site, monomeric REGalpha mutant and with REGalphaDeltai. At low concentrations, the REGalphaDeltai/REGbetaDeltai hetero-oligomers stimulated the proteasome less than REGalpha/REGbeta oligomers formed from wild-type subunits, and the reduced activation by REGalphaDeltai/REGbetaDeltai was due to removal of the REGbeta insert, not the REGalpha insert. These studies demonstrate that the REGalpha and REGgamma inserts play virtually no role in oligomerization or in proteasome activation. By contrast, removal of REGbeta insert reduces binding of this subunit and REGalpha/REGbeta oligomers to proteasomes. On the whole, however, our findings show that REG inserts are not required for binding and activating the proteasome. We speculate that they serve to localize REG-proteasome complexes within cells, possibly by binding components in endoplasmic reticulum membranes.
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PMID:Proteasome activation by REG molecules lacking homolog-specific inserts. 954 78

The proteasome activator 11S REG or PA28 is a conical molecule composed of two homologous subunits, REG alpha and REG beta. Recombinant REG alpha forms a heptamer, whereas recombinant REG beta is a monomer. When mixed with REG beta, a monomeric REG alpha mutant (N50Y) forms an active hetero-oligomer in which the molar ratio of REG beta to REG alpha(N50Y) is close to 1.3. This apparent stoichiometry is consistent with the REG alpha(N50Y)/REG beta hetero-oligomer being a heptamer composed of three alpha and four beta subunits. Chemical cross-linking of the alpha/beta oligomers revealed the presence of REG alpha-REG beta and REG beta-REG beta dimers, but REG alpha-REG alpha dimers were not detected. The mass of the REG alpha(N50Y)/REG beta hetero-oligomer determined by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) is 194 871 +/- 40 Da in good agreement with the theoretical mass of 194 856 Da for an alpha 3 beta 4 heptamer. Hexamers were not observed in the mass spectrum. For wild-type REG subunits coexpressed in bacteria cells at an apparent beta/alpha molar ratio of approximately 1.2, the resulting hetero-oligomers observed by ESI-TOF MS were again predominantly alpha 3 beta 4 heptamers, with trace amounts of alpha 4 beta heptamers also present. On the other hand, the mass spectrum contained a mixture of alpha 7, alpha 6 beta 1, alpha 5 beta 2, and alpha 4 beta 3 heptamers when the REG beta/REG alpha ratio was 0.1. Thus, formation of heptamers is an intrinsic property of recombinant REG alpha and REG beta subunits. On the basis of these results, we propose that 11S REG purified directly from eukaryotic cells is also heptameric, likely alpha 3 beta 4 or a mixture of alpha 3 beta 4 and alpha 4 beta 3 species.
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PMID:Proteasome activator 11S REG or PA28: recombinant REG alpha/REG beta hetero-oligomers are heptamers. 1022 Mar 54

There are two immune responses in vertebrates: humoral immunity is mediated by circulating antibodies, whereas cytotoxic T lymphocytes (CTL) confer cellular immunity. CTL lyse infected cells upon recognition of cell-surface MHC Class I molecules complexed with foreign peptides. The displayed peptides are produced in the cytosol by degradation of host proteins or proteins from intracellular pathogens that might be present. Proteasomes are cylindrical multisubunit proteases that generate many of the peptides eventually transferred to the cell surface for immune surveillance. In mammalian proteasomes, six active sites face a central chamber. As this chamber is sealed off from the enzyme's surface, there must be mechanisms to promote entry of substrates. Two protein complexes have been found to bind the ends of the proteasome and activate it. One of the activators is the 19 S regulatory complex of the 26 S proteasome; the other activator is '11 S REG' [Dubiel, Pratt, Ferrell and Rechsteiner (1992) J. Biol. Chem. 267, 22369-22377] or 'PA28' [Ma, Slaughter and DeMartino (1992) J. Biol. Chem. 267, 10515-10523]. During the past 7 years, our understanding of the structure of REG molecules has increased significantly, but much less is known about their biological functions. There are three REG subunits, namely alpha, beta and gamma. Recombinant REGalpha forms a ring-shaped heptamer of known crystal structure. 11 S REG is a heteroheptamer of alpha and beta subunits. REGgamma is also presumably a heptameric ring, and it is found in the nuclei of the nematode work Caenorhabditis elegans and higher organisms, where it may couple proteasomes to other nuclear components. REGalpha and REGbeta, which are abundant in vertebrate immune tissues, are located mostly in the cytoplasm. Synthesis of REG alpha and beta subunits is induced by interferon-gamma, and this has led to the prevalent hypothesis that REG alpha/beta hetero-oligomers play an important role in Class I antigen presentation. In the present review we focus on the structural properties of REG molecules and on the evidence that REGalpha/beta functions in the Class I immune response.
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PMID:The proteasome activator 11 S REG (PA28) and class I antigen presentation. 1060 Jun 33

Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable gamma-interferon-inducible catalytic beta-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit specific antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core alpha-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some significant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immunofluorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following gamma-interferon treatment of cultured cells but gamma-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes.
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PMID:Subcellular localization of proteasomes and their regulatory complexes in mammalian cells. 1065 52

The REG homologs, alpha, beta and gamma, activate mammalian proteasomes in distinct ways. REGalpha and REGbeta activate the trypsin-like, chymotrypsin-like and peptidylglutamyl-preferring active sites, whereas REGgamma only activates the proteasome's trypsin-like subunit. The three REG homologs differ in carboxyl-terminal sequences that are located next to activation loops on their proteasome binding surface. To assess the importance of these carboxyl-terminal sequences in the activation of specific proteasome beta catalytic subunits, we characterized chimeras in which 8 or 12 residues were exchanged among the three proteins. Like the wild-type molecule, REGalpha chimeras activated all three proteasome catalytic subunits regardless of the carboxyl-terminal sequence. However, REGalpha-beta chimeras activated the proteasome at lower concentrations than wild-type REGalpha and higher levels of REGalpha-gamma chimeras were needed for maximal activation because exchanged carboxyl-terminal sequences can stabilize (REGalpha-beta) or destabilize (REGalpha-gamma) the REGalpha heptamer. REGgamma chimeras were equivalent to REGgamma in their activation properties, but they bound the proteasome less tightly than the wild-type molecule. REGbeta chimeras also bound the proteasome more weakly than wild-type REGbeta and were virtually unable to activate it. Our findings demonstrate that the carboxyl-terminal sequences of REG subunits can affect heptamer stability and proteasome affinity, but they do not determine which proteasome beta subunits become activated.
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PMID:The proteasome activator 11 S REG or PA28: chimeras implicate carboxyl-terminal sequences in oligomerization and proteasome binding but not in the activation of specific proteasome catalytic subunits. 1083 74

We report the cloning and characterization of a Drosophila proteasome 11 S REGgamma (PA28) homolog. The 28-kDa protein shows 47% identity to the human REGgamma and strongly enhances the trypsin-like activities of both Drosophila and mammalian 20 S proteasomes. Surprisingly, the Drosophila REG was found to inhibit the proteasome's chymotrypsin-like activity against the fluorogenic peptide succinyl-LLVY-7-amino-4-methylcoumarin. Immunocytological analysis reveals that the Drosophila REG is localized to the nucleus but is distributed throughout the cell when nuclear envelope breakdown occurs during mitosis. Through site-directed mutagenesis studies, we have identified a functional nuclear localization signal present in the homolog-specific insert region. The Drosophila PA28 NLS is similar to the oncogene c-Myc nuclear localization motif. Comparison between uninduced and innate immune induced Drosophila cells suggests that the REGgamma proteasome activator has a role independent of the invertebrate immune system. Our results support the idea that gamma class proteasome activators have an ancient conserved function within metazoans and were present prior to the emergence of the alpha and beta REG classes.
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PMID:Identification and characterization of a Drosophila nuclear proteasome regulator. A homolog of human 11 S REGgamma (PA28gamma ). 1102 88

Most of the non-lysosomal proteolysis that occurs in eukaryotic cells is performed by a nonspecific and abundant barrel-shaped complex called the 20S proteasome. Substrates access the active sites, which are sequestered in an internal chamber, by traversing a narrow opening (alpha-annulus) that is blocked in the unliganded 20S proteasome by amino-terminal sequences of alpha-subunits. Peptide products probably exit the 20S proteasome through the same opening. 11S regulators (also called PA26 (ref. 4), PA28 (ref. 5) and REG) are heptamers that stimulate 20S proteasome peptidase activity in vitro and may facilitate product release in vivo. Here we report the co-crystal structure of yeast 20S proteasome with the 11S regulator from Trypanosoma brucei (PA26). PA26 carboxy-terminal tails provide binding affinity by inserting into pockets on the 20S proteasome, and PA26 activation loops induce conformational changes in alpha-subunits that open the gate separating the proteasome interior from the intracellular environment. The reduction in processivity expected for an open conformation of the exit gate may explain the role of 11S regulators in the production of ligands for major histocompatibility complex class I molecules.
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PMID:Structural basis for the activation of 20S proteasomes by 11S regulators. 1108 19


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