Identification, purification, and characterization of a PA700-dependent activator of the proteasome

The activity of the intracellular protease, the proteasome, is modulated by a number of specific regulatory proteins. One such regulator, PA700, is a 700,000-Da multisubunit protein that activates hydrolytic activities of the proteasome via a mechanism that involves the ATP-dependent formation of a proteasome-PA700 complex. Four subunits of PA700 have been shown previously to be members of a protein family that contains a consensus sequence for ATP binding, and purified PA700 expresses ATPase activity. We report here the identification, purification, and initial characterization of a new modulator of the proteasome. The modulator has no direct effect on the activity of the proteasome, but enhances PA700 activation of the proteasome by up to 8-fold. This activation is associated with the formation of a proteasome/PA700-containing complex that is significantly larger than that formed in its absence. The modulator has a native Mr of approximately 300,000, as determined by gel filtration chromatography, and is composed of three electrophoretically distinct subunits with Mr values of 50,000, 42,000, and 27,000 (p50, p42, and p27, respectively). Amino acid sequence analysis of the subunits shows that p50 and p42 are members of the same ATP-binding protein family found in PA700. The p50 subunit is identical to TBP1, a protein previously reported to interact with human immunodeficiency virus Tat protein (Nelbock, P., Dillion, P. J., Perkins, A., and Rosen, C. A. (1990) Science 248, 1650-1653), while the p42 subunit seems to be a new member of the family. The p27 subunit has no significant sequence similarity to any previously described protein. Both p50 and p42, but not p27, were also identified as components of PA700, increasing the number of ATP-binding protein family members in this complex to six. Thus, p50 and p42 are subunits common to two protein complexes that regulate the proteasome. The PA700-dependent proteasome activator represents a new member of a growing list of proteins that regulate proteasome activity.     

Nucleotidase activities of the 26 S proteasome and its regulatory complex

The 26 S proteasome can be assembled from the multicatalytic protease (or 20 S proteasome) and a large multisubunit regulatory complex in an ATP-dependent reaction. The 26 S proteasome and its regulatory complex were purified from rabbit reticulocytes for characterization of their nucleotidase properties. Both particles hydrolyze ATP, CTP, GTP, and UTP to the corresponding nucleoside diphosphate and inorganic phosphate. The Km values for hydrolysis of specific nucleotides by the 26 S proteasome are 15 microM for ATP and CTP, 50 microM for GTP, and 100 microM for UTP; Km values for nucleotide hydrolysis by the regulatory complex are 2-4-fold higher for each nucleotide. Several ATPase inhibitors (erythro-9-[3-(2-hydroxynonyl)]adenine, oligomycin, ouabain, and thapsigargin) had no effect on ATP hydrolysis by either complex whereas known inhibitors of proteolysis by the 26 S enzyme (hemin, N-ethylmaleimide (NEM), and vanadate) significantly reduced ATP hydrolysis by both particles. Hydrolysis of all nucleotides was inhibited by 5 mM NEM but only GTP and UTP hydrolysis was significantly reduced at 50 microM NEM. The 15 microM Km for ATP hydrolysis by the 26 S proteasome is virtually identical to the observed Km of 12 microM ATP for Ub-conjugate degradation. Although nucleotide hydrolysis is required for protein degradation by the 26 S proteasome, nucleotide hydrolysis and peptide bond cleavage are not strictly coupled. Substrate specificity constants (kcat/Km) are similar for hydrolysis of each nucleotide, yet GTP and UTP barely supported Ub-conjugate degradation. Further evidence that nucleotide hydrolysis is not coupled to peptide bond cleavage was obtained using N-acetyl-leucyl-leucyl-norleucinal (LLnL). This compound inhibited peptide hydrolysis by the multicatalytic protease and Ub-conjugate degradation by the 26 S proteasome, but it had little effect on ATP or UTP hydrolysis by the 26 S enzyme.     

The COP9 complex is conserved between plants and mammals and is related to the 26S proteasome regulatory complex

The COP9 complex, genetically identified in Arabidopsis as a repressor of photomorphogenesis, is composed of multiple subunits including COP9, FUS6 (also known as COP11) and the Arabidopsis JAB1 homolog 1 (AJH1) ([1-3]; unpublished observations). We have previously demonstrated the existence of the mammalian counterpart of the COP9 complex and purified the complex by conventional biochemical and immunoaffinity procedures [4]. Here, we report the molecular identities of all eight subunits of the mammalian COP9 complex. We show that the COP9 complex is highly conserved between mammals and higher plants, and probably among most multicellular eukaryotes. It is not present in the single-cell eukaryote Saccharomyces cerevisiae, however. All of the subunits of the COP9 complex contain structural features that are also present in the components of the proteasome regulatory complex and the translation initiation factor eIF3 complex. Six subunits of the COP9 complex have overall similarity with six distinct non-ATPase regulatory subunits of the 26S proteasome, suggesting that the COP9 complex and the proteasome regulatory complex are closely related in their evolutionary origin. Subunits of the COP9 complex include regulators of the Jun N-terminal kinase (JNK) and c-Jun, a nuclear hormone receptor binding protein and a cell-cycle regulator. This suggests that the COP9 complex is an important cellular regulator modulating multiple signaling pathways.     

The cyclophilin-like domain mediates the association of Ran-binding protein 2 with subunits of the 19 S regulatory complex of the proteasome

The combination of the Ran-binding domain 4 and cyclophilin domains of Ran-binding protein 2 selectively associate with a subset of G protein-coupled receptors, red/green opsins, upon cis-trans prolyl isomerase-dependent and direct modification of opsin followed by association of the modified opsin isoform to Ran-binding domain 4. This effect enhances in vivo the production of functional receptor and generates an opsin isoform with no propensity to self-aggregate in vitro. We now show that another domain of Ran-binding protein 2, cyclophilin-like domain, specifically associates with the 112-kDa subunit, P112, and other subunits of the 19 S regulatory complex of the 26 S proteasome in the neuroretina. This association possibly mediates Ran-binding protein 2 limited proteolysis into a smaller and stable isoform. Also, the interaction of Ran-binding protein 2 with P112 regulatory subunit of the 26 S proteasome involves still another protein, a putative kinesin-like protein. Our results indicate that Ran-binding protein 2 is a key component of a macro-assembly complex selectively linking protein biogenesis with the proteasome pathway and, thus, with potential implications for the presentation of misfolded and ubiquitin-like modified proteins to this proteolytic machinery.     

Progression of familial adenomatous polyposis (FAP) colonic cells after transfer of the src or polyoma middle T oncogenes: cooperation between src and HGF/Met in invasion

We have employed cDNA cloning to deduce the complete primary structures of p44.5 and p55, two subunits of PA700, a 700-kDa multisubunit regulatory complex of the human 26S proteasome. These polypeptides consist of 422 and 456 amino acids with calculated molecular masses of 47463 and 52903, and isoelectric points of 6.06 and 7.56, respectively. Computer-assisted homology analysis revealed high sequence similarities of p44.5 and p55 with yeast proteins whose functions are yet unknown. Disruption of the yeast genes, termed NAS4 and NAS5 (non-ATPase subunits 4 and 5), resulted in lethality, indicating that each of the two subunits is essential for proliferation of yeast cells.     

Relative functions of the alpha and beta subunits of the proteasome activator, PA28

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.     

Molecular organization of the 20S proteasome gene family from Arabidopsis thaliana

The 20S proteasome is the proteolytic complex in eukaryotes responsible for degrading short-lived and abnormal intracellular proteins, especially those targeted by ubiquitin conjugation. The 700-kD complex exists as a hollow cylinder comprising four stacked rings with the catalytic sites located in the lumen. The two outer rings and the two inner rings are composed of seven different alpha and beta polypeptides, respectively, giving an alpha7/beta7/beta7/alpha7 symmetric organization. Here we describe the molecular organization of the 20S proteasome from the plant Arabidopsis thaliana. From an analysis of a collection of cDNA and genomic clones, we identified a superfamily of 23 genes encoding all 14 of the Arabidopsis proteasome subunits, designated PAA-PAG and PBA-PBG for Proteasome Alpha and Beta subunits A-G, respectively. Four of the subunits likely are encoded by single genes, and the remaining subunits are encoded by families of at least 2 genes. Expression of the alpha and beta subunit genes appears to be coordinately regulated. Three of the nine Arabidopsis proteasome subunit genes tested, PAC1 (alpha3), PAE1 (alpha5) and PBC2 (beta3), could functionally replace their yeast orthologs, providing the first evidence for cross-species complementation of 20S subunit genes. Taken together, these results demonstrate that the 20S proteasome is structurally and functionally conserved among eukaryotes and suggest that the subunit arrangement of the Arabidopsis 20S proteasome is similar if not identical to that recently determined for the yeast complex.     

Structural and functional effects of PA700 and modulator protein on proteasomes

Control and targeting of the proteolytic activity of the major intracellular protease, the proteasome, is accomplished by various regulatory protein complexes that may form higher-order assemblies with the proteasome. An activator of proteolytic activity, PA700, has been shown to have an ATP-dependent stimulatory effect on the peptidase activities of the proteasome, and another protein factor, the modulator, further enhances the effect of PA700. Here we show that the addition of PA700 endows the proteasome with the ability to cleave ubiquitinated proteins, a property associated with the previously isolated 26 S form of the proteasome. The modulator further stimulates this specific activity, without having any such effect on the proteasome alone. Using electron microscopy, we show that addition of PA700 causes the appearance of protein "caps" at one or both ends of proteasomes, forming structures that are indistinguishable from 26 S proteasomes. Quantitation of the numbers of uncapped, singly capped and doubly capped complexes indicates cooperativity in the association of PA700 with the two ends of the proteasome. Addition of modulator protein makes no further structural modification that is detectable by electron microscopy, but does cause an increase in the number of capped complexes visible at subsaturating concentrations of PA700. Hence PA700 converts the proteasome both functionally and structurally to the 26 S form, and the modulator promotes this transformation, apparently without stable association with the resulting complex.     

The active sites of the eukaryotic 20 S proteasome and their involvement in subunit precursor processing

The 26 S proteasome is the central protease involved in ubiquitin-mediated protein degradation and fulfills vital regulatory functions in eukaryotes. The proteolytic core of the complex is the 20 S proteasome, a cylindrical particle with two outer rings each made of 7 different alpha-type subunits and two inner rings made of 7 different beta-type subunits. In the archaebacterial 20 S proteasome ancestor proteolytically active sites reside in the 14 uniform beta-subunits. Their N-terminal threonine residues, released by precursor processing, perform the nucleophilic attack for peptide bond hydrolysis. By directed mutational analysis of 20 S proteasomal beta-type proteins of Saccharomyces cerevisiae, we identified three active site-carrying subunits responsible for different peptidolytic activities as follows: Pre3 for post-glutamyl hydrolyzing, Pup1 for trypsin-like, and Pre2 for chymotrypsin-like activity. Double mutants harboring only trypsin-like or chymotrypsin-like activity were viable. Mutation of two potentially active site threonine residues in the Pre4 subunit excluded its catalytic involvement in any of the three peptidase activities. The generation of different, incompletely processed forms of the Pre4 precursor in active site mutants suggested that maturation of non-active proteasomal beta-type subunits is exerted by active subunits and occurs in the fully assembled particle. This trans-acting proteolytic activity might also account for processing intermediates of the active site mutated Pre2 subunit, which was unable to undergo autocatalytic maturation.     

The regulatory particle of the Saccharomyces cerevisiae proteasome

The proteasome is a multisubunit protease responsible for degrading proteins conjugated to ubiquitin. The 670-kDa core particle of the proteasome contains the proteolytic active sites, which face an interior chamber within the particle and are thus protected from the cytoplasm. The entry of substrates into this chamber is thought to be governed by the regulatory particle of the proteasome, which covers the presumed channels leading into the interior of the core particle. We have resolved native yeast proteasomes into two electrophoretic variants and have shown that these represent core particles capped with one or two regulatory particles. To determine the subunit composition of the regulatory particle, yeast proteasomes were purified and analyzed by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resolution of the individual polypeptides revealed 17 distinct proteins, whose identities were determined by amino acid sequence analysis. Six of the subunits have sequence features of ATPases (Rpt1 to Rpt6). Affinity chromatography was used to purify regulatory particles from various strains, each of which expressed one of the ATPases tagged with hexahistidine. In all cases, multiple untagged ATPases copurified, indicating that the ATPases assembled together into a heteromeric complex. Of the remaining 11 subunits that we have identified (Rpn1 to Rpn3 and Rpn5 to Rpn12), 8 are encoded by previously described genes and 3 are encoded by genes not previously characterized for yeasts. One of the previously unidentified subunits exhibits limited sequence similarity with deubiquitinating enzymes. Overall, regulatory particles from yeasts and mammals are remarkably similar, suggesting that the specific mechanistic features of the proteasome have been closely conserved over the course of evolution.     

Rapamycin inhibits proteasome activator expression and proteasome activity

Rapamycin (RAPA) is a potent immunosuppressive drug, and certain of its direct or indirect targets might be of vital importance to the regulation of an immune response. In this study, we used differential hybridization to search for human genes whose expression was sensitive to RAPA. Seven RAPA-sensitive genes were found and one of them encoded a protein with high homology to the alpha subunit of a proteasome activator (PA28 alpha). This gene was later found to code for the beta subunit of the proteasome activator (PA28 beta). Activated T and B cells had up-regulated PA28 beta expression at the mRNA level. Such up-regulation could be suppressed by RAPA, FK506, and cyclosporin A. RAPA and FK506 also repressed the up-regulated PA28 alpha messages in phytohemagglutinin (PHA)-stimulated T cells. At the protein level, RAPA inhibited PA28 alpha and PA28 beta in the activated T cells according to immunoblotting and confocal microscopy. Probably as a consequence, there was a fourfold increase of proteasome activities in the peripheral blood mononuclear cell lysate after the PHA activation. RAPA could inhibit the enhanced part of the proteasome activity. Considering the critical role played by the proteasome in degrading regulatory proteins, our data suggest that the proteasome activator is a relevant and important downstream target of rapamycin, and that the immune response could be modulated through the activity of the proteasome.     

The sensitivity of the Baermann method for the diagnosis of primary Dictyocaulus viviparus infections in calves

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.     

Biochemical characterization of the 20S proteasome from the methanoarchaeon Methanosarcina thermophila

The 20S proteasome from the methanoarchaeon Methanosarcina thermophila was produced in Escherichia coli and characterized. The biochemical properties revealed novel features of the archaeal 20S proteasome. A fully active 20S proteasome could be assembled in vitro with purified native alpha ring structures and beta prosubunits independently produced in Escherichia coli, which demonstrated that accessory proteins are not essential for processing of the beta prosubunits or assembly of the 20S proteasome. A protein complex with a molecular mass intermediate to those of the alpha7 ring and the 20S proteasome was detected, suggesting that the 20S proteasome is assembled from precursor complexes. The heterologously produced M. thermophila 20S proteasome predominately catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residues Phe and Tyr (chymotrypsinlike activity) in short chromogenic and fluorogenic peptides. Low-level hydrolyzing activities were also detected carboxyl to the acidic residue Asp and the basic residue Arg (trypsinlike activity). Sodium dodecyl sulfate and divalent or monovalent ions stimulated chymotrypsinlike activity and inhibited postglutamyl activity, whereas ATP stimulated postglutamyl activity but had little effect on the chymotrypsinlike activity. The results suggest that the 20S proteasome is a flexible protein which adjusts to binding of substrates. The 20S proteasome also hydrolyzed large proteins. Replacement of the nucleophilic Thr1 residue with an Ala in the beta subunit abolished all activities, which suggests that only one active site is responsible for the multisubstrate activity. Replacement of beta subunit active-site Lys33 with Arg reduced all activities, which further supports the existence of one catalytic site; however, this result also suggests a role for Lys33 in polarization of the Thr1 N, which serves to strip a proton from the active-site Thr1 Ogamma nucleophile. Replacement of Asp51 with Asn had no significant effect on trypsinlike activity, enhanced postglutamyl and trypsinlike activities, and only partially reduced lysozyme-hydrolyzing activity, which suggested that this residue is not essential for multisubstrate activity.     

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.     

Modulation of the PA28alpha-20S proteasome interaction by a peptidyl alcohol

The peptidyl alcohol N-benzyloxycarbonyl-Ile-Glu(O-t-Bu)-Ala-leucinol is a mild activator of the chymotrypsin-like activity of the proteasome. When added to an incubation mixture of recombinant PA28alpha plus 20S proteasome the peptidyl alcohol antagonizes the stimulation of the chymotrypsin-like activity by PA28alpha in a dose-dependent manner (IC50 = 30 microM). This effect is selective for the chymotrypsin-like activity. Stimulation of the peptidyl-glutamyl peptide bond hydrolyzing activity of the proteasome by PA28alpha is not affected by the peptidyl alcohol. The ovalbumin immunodominant epitope SIINFEKL is hydrolyzed by the PA28alpha-activated 20S proteasome to SIINF and SIINFE in approximately equimolar amounts. Addition of the peptidyl alcohol to an incubation mixture of PA28alpha, 20S proteasome and SIINFEKL shifts the ratio of products in favor of SIINFE. A similar shift in favor of postglutamyl cleavages occurs with the extended peptide LEQLESIINFEKLTE. By altering the ratio of products produced by the PA28alpha-activated proteasome, the peptidyl alcohol acts as a proteasome modulator. Proteasome modulators represent a novel class of molecules with a potential for altering the processing of antigens by the PA28-proteasome complex for presentation by the MHC class I system.     

Platelet endothelial cell adhesion molecule-1 expression during mouse postimplantation development

The proteasome is an unusually large multisubunit proteolytic complex, consisting of a central catalytic machine (equivalent to the 20S proteasome) and two terminal regulatory subcomplexes, termed PA700 or PA28, that are attached to both ends of the central portion in opposite orientations to form the enzymatically active proteasome. Totally about 40 subunits with sizes of 20-110 kDa are assembled to form two types of the proteasomal complexes with the same catalytic core and different regulatory modules. To date, cDNAs or genes encoding almost all subunits of human and the budding yeast proteasomes have been isolated by molecular-biological techniques. In this minireview, I summarize briefly available information on the structure-function relationships of the proteasome acting as a protein death machinery.