ABSTRACT
The 26S proteasome is the major protease responsible for nonlysosomal protein degradation in eukaryotic cells. The enzyme is composed of two subparticles: the 20S proteasome, and a 19S regulatory particle (PA700) which binds to the ends of the 20S proteasome cylinder and accounts for ATP dependence and substrate specificity. Among the approximately 18 subunits of PA700 regulator, six are ATPases. The ATPases presumably recognize, unfold, and translocate substrates into the interior of the 26S proteasome. It is generally believed that the ATPases form a hexameric ring. By means of chemical cross-linking, immunoprecipitation, and blotting, we have determined that the ATPases are organized in the order S6-S6'-S10b-S8-S4-S7. Additionally, we found cross-links between the ATPase S10b and the 20S proteasome subunit alpha6. Together with the previously known interaction between S8 and alpha1 and between S4 and alpha7, these data establish the relative orientations of ATPases with respect to the 20S proteasome.
Subject(s)
Adenosine Triphosphatases/chemistry , Cross-Linking Reagents/pharmacology , Cysteine Endopeptidases/chemistry , Multienzyme Complexes/chemistry , Peptide Hydrolases/chemistry , Adenosine Triphosphatases/metabolism , HeLa Cells , Humans , Microscopy, Electron , Precipitin Tests , Proteasome Endopeptidase Complex , Protein Binding , Protein Structure, Quaternary , Sodium Dodecyl Sulfate/pharmacologyABSTRACT
Proteasomes are complex multisubunit proteases which play a critical role in intracellular proteolysis. Immunoproteasomes, which contain three gamma-interferon-inducible subunits, are a subset of proteasomes which have a specialized function in antigen processing for presentation by the MHC class I pathway. Two of the gamma-interferon inducible subunits, LMP2 and LMP7, are encoded within the MHC class II region adjacent to the two TAP (transporter associated with antigen presentation) genes. We have investigated the localization of immunoproteasomes using monoclonal antibodies to LMP2 and LMP7. Immunoproteasomes were strongly enriched around the endoplasmic reticulum as judged by double-immunofluorescence experiments with anti-calreticulin antibodies, but were also present in the nucleus and throughout the cytosol. In contrast, proteasome subunit C2, which is present in all proteasomes, was found to be evenly distributed throughout the cytoplasm and in the nucleus, as was the delta subunit, which is replaced by LMP2 in immunoproteasomes. gamma-Interferon increased the level of immunoproteasomes, but had no effect on their distribution. Our results provide the first direct evidence that immunoproteasomes are strongly enriched at the endoplasmic reticulum, where they may be located close to the TAP transporter to provide efficient transport of peptides into the lumen of the endoplasmic recticulum for association with MHC class I molecules.
Subject(s)
Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Endoplasmic Reticulum/chemistry , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Antibodies, Monoclonal , Cell Line , Cell Nucleus/chemistry , Cell Nucleus/drug effects , Cytoplasm/chemistry , Cytoplasm/drug effects , Endoplasmic Reticulum/drug effects , Fluorescent Antibody Technique , HeLa Cells , Humans , Interferon-gamma/pharmacology , Lung/cytology , Lung/drug effects , Lung/embryology , Proteasome Endopeptidase Complex , Protein Subunits , Protein Transport/drug effects , Proteins/metabolismABSTRACT
We investigated the expression of standard proteasomes, immunoproteasomes, and their regulators, PA28, and PA700, in rat tissues. Immunoproteasomes (with subunits LMP2, LMP7, and MECL1) were abundant in the spleen but almost absent in the brain. In contrast, standard proteasomes (with X, Y, and Z) were highly expressed in the brain but not in the spleen. Both proteasome types were present in the lung and the liver. PA700 subunits (p112, S5a, and p45) were found in all tissues. PA28alpha, PA28beta, and PA28gamma were also expressed in all tissues, except for the brain which contained very little PA28beta. The results did not depend on rat sex or age. The cleavage specificity for peptide substrates differed greatly between brain and spleen proteasomes. Hybrid proteasomes, containing both PA28alphabeta and PA700, were not present in the brain but in all other tissues examined.
Subject(s)
Cysteine Endopeptidases/biosynthesis , Cysteine Endopeptidases/immunology , Multienzyme Complexes/biosynthesis , Multienzyme Complexes/immunology , Protein Biosynthesis , Proteins , Age Factors , Animals , Brain/metabolism , Cell Cycle Proteins , Cysteine Endopeptidases/metabolism , Electrophoresis, Polyacrylamide Gel , Female , Immunoglobulin G/metabolism , Interferon-gamma/metabolism , Liver/metabolism , Lung/metabolism , Male , Multienzyme Complexes/metabolism , Peptide Hydrolases/metabolism , Precipitin Tests , Proteasome Endopeptidase Complex , Protein Binding , Rats , Rats, Wistar , Sex Factors , Spleen/metabolism , Tissue Distribution , Viral Matrix Proteins/biosynthesisABSTRACT
Eukaryotic cells contain various types of proteasomes. Core 20 S proteasomes (abbreviated 20 S below) have two binding sites for the regulatory particles, PA700 and PA28. PA700-20 S-PA700 complexes are known as 26 S proteasomes and are ATP-dependent machines that degrade cell proteins. PA28 is found both in previously described complexes of the type PA28-20 S-PA28 and in complexes that also contain PA700, as PA700-20 S-PA28. We refer to the latter as "hybrid proteasomes." The relative amounts of the various types of proteasomes in HeLa extracts were determined by a combination of immunoprecipitation and immunoblotting. Hybrid proteasomes accounted for about a fourth of all proteasomes in the extracts. Association of PA28 and proteasomes proved to be ATP-dependent. Hybrid proteasomes catalyzed ATP-dependent degradation of ornithine decarboxylase (ODC) without ubiquitinylation, as do 26 S proteasomes. In contrast, the homo-PA28 complex (PA28-20 S-PA28) was incapable of degrading ODC. Intriguingly, a major immunomodulatory cytokine, interferon-gamma, appreciably enhanced the ODC degradation in HeLa and SW620 cells through induction of the hybrid proteasome, which may also be responsible for the immunological processing of intracellular antigens. Taken together, we report here for the first time the existence of two types of ATP-dependent proteases, the 26 S proteasome and the hybrid proteasome, which appear to share the ATP-dependent proteolytic pathway in mammalian cells.
Subject(s)
Adenosine Triphosphate/metabolism , Cysteine Endopeptidases/biosynthesis , Interferon-gamma/pharmacology , Multienzyme Complexes/biosynthesis , Muscle Proteins , Amino Acid Sequence , Catalysis , Cysteine Endopeptidases/metabolism , Enzyme Induction , HeLa Cells , Humans , Hydrolysis , Molecular Sequence Data , Multienzyme Complexes/metabolism , Ornithine Decarboxylase/metabolism , Proteasome Endopeptidase ComplexABSTRACT
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.
Subject(s)
Cysteine Endopeptidases/analysis , Cysteine Endopeptidases/chemistry , Multienzyme Complexes/analysis , Multienzyme Complexes/chemistry , Adenosine Triphosphate/pharmacology , Animals , Antibodies, Monoclonal/immunology , Biological Transport/drug effects , Blotting, Western , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/enzymology , Cysteine Endopeptidases/immunology , Cysteine Endopeptidases/metabolism , Cytosol/drug effects , Cytosol/enzymology , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/enzymology , Endoplasmic Reticulum/metabolism , Enzyme Stability/drug effects , Fluorescent Antibody Technique , Humans , Interferon-gamma/pharmacology , Liver/cytology , Liver/drug effects , Liver/enzymology , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Molecular Weight , Multienzyme Complexes/immunology , Multienzyme Complexes/metabolism , Peptide Hydrolases/analysis , Peptide Hydrolases/chemistry , Peptide Hydrolases/immunology , Peptide Hydrolases/metabolism , Proteasome Endopeptidase Complex , RatsABSTRACT
20 S Proteasomes are large proteinase complexes found in eukaryotic cells where they degrade cell proteins in an ATP-dependent manner. Proteasomes consist of 14 different subunits. One of them, zeta, was found in HeLa cells at a concentration of 890 microg per g of cell protein. A large proportion of zeta was found in the free state rather than incorporated into proteasomes, namely 28% in HeLa cells and 37% in BSC-1 cells. Free zeta was found in both nuclei and cytoplasm. In HeLa cells free zeta had a t1/2 of 2.8 h, compared to 5 d for proteasomes, and did not exchange with zeta in proteasomes. We confirmed (Petit F et al.: Biochem. J. 326: 93-98 (1997)) that both 20 S proteasomes and free zeta subunits possess RNase activity though the activities were very low: 4 mMoles and 0.6 mMoles of tobacco mosaic virus RNA degraded per mole of enzyme per min, respectively. The physiological function of the relatively abundant zeta monomers is not known.
Subject(s)
Cysteine Endopeptidases/chemistry , Multienzyme Complexes/chemistry , Ribonucleases/metabolism , Cysteine Endopeptidases/isolation & purification , Cysteine Endopeptidases/metabolism , Electrophoresis, Polyacrylamide Gel , Erythrocytes/enzymology , HeLa Cells , Humans , Multienzyme Complexes/isolation & purification , Multienzyme Complexes/metabolism , Proteasome Endopeptidase ComplexABSTRACT
The arrangement of subunits in human 20S proteasomes was recently determined by us by immunoelectron microscopy and chemical cross-linking. The positions of 4 of the 14 subunits differed from those found in the yeast proteasome by X-ray crystallography. Double labeling of human 20S proteasomes with antibodies to subunits C2 and C5 has now shown that these subunits are nearest neighbors. The result contradicts our published model for the human proteasome but is in accordance with the subunit arrangement in yeast proteasomes, suggesting that yeast and human proteasomes most probably have identical subunit arrangements. Immunoelectron microscopy also showed that the C-terminal extension at the human C2 subunit is flexible but takes up a well-defined position in the proteasome.
Subject(s)
Cysteine Endopeptidases/chemistry , Multienzyme Complexes/chemistry , Antibody Specificity , Cysteine Endopeptidases/immunology , Cysteine Endopeptidases/metabolism , Cysteine Endopeptidases/ultrastructure , Electrophoresis, Gel, Two-Dimensional , Epitopes/metabolism , Humans , Microscopy, Immunoelectron , Molecular Sequence Data , Multienzyme Complexes/immunology , Multienzyme Complexes/metabolism , Multienzyme Complexes/ultrastructure , Placenta/chemistry , Proteasome Endopeptidase Complex , Protein Conformation , Saccharomyces cerevisiaeABSTRACT
Proteasomes interact with a variety of macromolecular ligands that modulate their ability to degrade peptide and protein substrates. The effector PA28 increases the peptidase activities of proteasomes whereas HSP90 and alpha-crystallin inhibit a peptide-hydrolyzing activity. Four monoclonal antibodies were used as probes to detect conformational changes of proteasome subunits. Conformational changes in alpha- or beta-subunits were found upon binding PA28, HSP90, alpha-crystallin, and the substrate casein but not with the peptide substrate analogs calpain inhibitor 1 (Ac-Leu-Leu-norleucinal), calpain inhibitor 2 (Ac-Leu-Leu-methioninal), or MG 132 (N-Cbz-Leu-Leu-leucinal).
Subject(s)
Antibodies, Monoclonal/immunology , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Muscle Proteins , Animals , Antibody Affinity , Binding Sites, Antibody , Caseins/metabolism , Crystallins/metabolism , Cysteine Endopeptidases/immunology , Cysteine Proteinase Inhibitors/metabolism , Enzyme-Linked Immunosorbent Assay , Glycoproteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Humans , Leupeptins/metabolism , Ligands , Multienzyme Complexes/immunology , Oligopeptides/metabolism , Proteasome Endopeptidase Complex , Protein Conformation , Proteins/metabolismABSTRACT
We have employed cDNA cloning to deduce the complete primary structure of a new subunit, designated p27, of the modulator trimer complex that stimulates the association of the PA700 regulator with the catalytic 20S proteasome to form the ATP-dependent active 26S proteasome. We found two distinct cDNAs encoding two highly homologous proteins except in the C-terminal region, which are termed tentatively p27-1 and p27-2. The short p27-2 cDNA has a deletion of 65 bp near the 3'-end region of the long p27-1 cDNA, which encodes a large protein with an extended C-terminal region, designated p27-L, whereas the long p27-1 cDNA encodes a small protein named p27-S. The polypeptides of p27-L and p27-S consist of 223 and 209 amino acid residues with calculated molecular masses of 24,852 and 22,764 and isoelectric points of 6.50 and 5.28, respectively. Immunoblot analysis with anti-p27 antibody revealed that p27, together with two other ATPase components, TBP1 and p42, was associated with not only the modulator complex but also significantly with the 26S proteasome complex, suggesting that the three are common/sharing subunits in these two complexes. By the fluorescence in situ hybridization method, the p27 (PSMD9) gene was mapped to the q24.2-q24.3 band of human chromosome 12. Computer-assisted homology analysis revealed the high sequence similarities of p27-L with a possible counterpart in Caenorhabditis elegans and Saccharomyces cerevisiae whose function is yet unknown, the yeast gene that is here termed NAS2 (non-ATPase subunit 2). Disruption of NAS2 had no effect on cell viability, indicating that the subunit is not essential for proliferation of yeast cells.
Subject(s)
Chromosomes, Human, Pair 12 , Cysteine Endopeptidases , Multienzyme Complexes , Proteins/chemistry , Proteins/genetics , Saccharomyces cerevisiae Proteins , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Caenorhabditis elegans/genetics , Chromosome Mapping , Cloning, Molecular , DNA, Complementary , Fungal Proteins/chemistry , Fungal Proteins/genetics , Humans , Immunoblotting , In Situ Hybridization, Fluorescence , Molecular Sequence Data , Proteasome Endopeptidase Complex , Proteins/analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Saccharomyces cerevisiae/genetics , Sequence Analysis, DNAABSTRACT
Two activators, named PA700 and PA28, are known to bind to 20 S proteasomes, forming two different complexes. The PA700-proteasome complex, also known as the 26 S proteasome, can degrade intact proteins, whereas complexes with PA28 can degrade only peptides. Monoclonal antibodies to 20 S proteasomes or the p45 ATPase subunit (Trip1, Sug1) of PA700 precipitated the same set of proteins from HeLa extracts, including six different ATPase subunits of PA700. This shows that p45 is not present in other protein complexes and suggests that all 26 S proteasome particles contain the same set of ATPase subunits. Interferons alpha and gamma had no effect on the composition of the 26 S proteasome, except for the replacement of subunits delta, MB1 and Z with Lmp2, Lmp7 and MECL1 respectively. Surprisingly, antibodies to PA28 precipitated p42, a component of PA700. Conversely, anti-p45 antibodies precipitated not only 26 S proteasomes but also PA28 alpha, beta and gamma, indicating that 20 S proteasomes can simultaneously bind both PA700 and PA28. PA28 alpha beta is known to be involved in antigen presentation. Conceivably, intact substrate proteins are recognized by PA700 and fed into proteasomes whose cleavage specificity is optimized for antigen presentation on MHC class I by PA28 and three interferon inducible proteasome subunits.
Subject(s)
Cysteine Endopeptidases/metabolism , Multienzyme Complexes/metabolism , Muscle Proteins , Proteins/metabolism , Adenosine Triphosphatases/immunology , Adenosine Triphosphatases/metabolism , Antibodies, Monoclonal/immunology , Chromatography, Gel , Cysteine Endopeptidases/immunology , Electrophoresis, Gel, Two-Dimensional , Enzyme Activation , HeLa Cells , Humans , Interferon-alpha/metabolism , Interferon-gamma/metabolism , Multienzyme Complexes/immunology , Peptide Hydrolases/immunology , Peptide Hydrolases/metabolism , Proteasome Endopeptidase Complex , Protein BindingABSTRACT
We have identified two distinct subunits of 20 S proteasomes that are associated with RNase activity. Proteasome subunits zeta and iota, eluted from two-dimensional Western blots, hydrolysed tobacco mosaic virus RNA, whereas none of the other subunits degraded this substrate under the same conditions. Additionally, proteasomes were dissociated by 6 M urea, and subunit zeta, containing the highest RNase activity, was isolated by anion-exchange chromatography and gel filtration. Purified subunit zeta migrated as a single spot on two-dimensional PAGE with a molecular mass of approx. 28 kDa. Addition of anti-(subunit zeta) antibodies led to the co-precipitation of this proteasome subunit and nuclease activity. This is the first evidence that proteasomal alpha-type subunits are associated with an enzymic activity, and our results provide further evidence that proteasomes may be involved in cellular RNA metabolism.
Subject(s)
Cysteine Endopeptidases/metabolism , Multienzyme Complexes/metabolism , RNA, Viral/metabolism , Animals , Cattle , Cysteine Endopeptidases/isolation & purification , Hydrolysis , Liver/enzymology , Multienzyme Complexes/isolation & purification , Proteasome Endopeptidase Complex , Ribonucleases/metabolism , Tobacco Mosaic Virus/geneticsABSTRACT
We isolated and sequenced a cDNA encoding mouse proteasome subunit LMP3 from a macrophage cDNA library. The gene encodes a 264-amino-acid protein with a calculated molecular mass of 29.11 kDa and an isoelectric point (pI) of 5.44. Comparison of the predicted protein sequence with that of the human and rat homologues, N3, revealed 11 and eight changes, respectively, in the cleaved NH2-terminal presequence of the precursor protein (pre-LMP3), and six and 10 changes, respectively, in the processed product. To corroborate the predicted molecular mass and pI, we analyzed LMP3 by immunoprecipitation with a mAb to human N3 that crossreacts with mouse LMP3. Precursor and processed forms of LMP3 were identified by 2D NEPHGE-PAGE, and their mobilities suggest the Lmp3 clone encodes the entire protein sequence.
Subject(s)
Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Cross Reactions , Cysteine Endopeptidases/immunology , Electrophoresis, Polyacrylamide Gel/methods , Humans , Mice , Molecular Sequence Data , Multienzyme Complexes/immunology , Proteasome Endopeptidase Complex , Protein Precursors/chemistry , Protein Precursors/genetics , Protein Precursors/metabolism , Protein Processing, Post-Translational , Rats , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
In human 20S proteasomes two copies of each of seven different alpha-type and seven different beta-type subunits are assembled to form a stack of four seven-membered rings, giving the general structure alpha(1-7), beta(1-7), beta(1-7), alpha(1-7). By means of immunoelectron microscopy and chemical crosslinking of neighboring subunits, we have determined the positions of the individual subunits in the proteasome. The topography shows that for the trypsin-like, the chymotrypsin-like, and the postglutamyl cleaving activities, the pairs of beta type subunits, which are thought to form active sites, are nearest neighbors.
Subject(s)
Cysteine Endopeptidases/chemistry , Multienzyme Complexes/chemistry , Amino Acid Sequence , Cysteine Endopeptidases/metabolism , Humans , Microscopy, Immunoelectron , Molecular Sequence Data , Multienzyme Complexes/metabolism , Proteasome Endopeptidase Complex , Protein Conformation , Structure-Activity RelationshipABSTRACT
Molecular cloning of cDNA for a new regulatory subunit, designated p97, of the human 26S proteasome showed that the polypeptide consists of 908 amino acid residues with a calculated molecular mass of 100184 Da and an isoelectric point of 4.94. Computer analysis showed that p97 is very similar to type-1 tumor-necrosis-factor (TNF)-receptor-associated protein (TRAP)-2 and 55.11, both of which were identified recently as binding proteins of the cytoplasmic domain of type-1 TNF receptor by yeast two-hybrid screening. This finding suggests that the 26S proteasome might serve as a mediator molecule in the TNF signaling pathway in cells. Computer-assisted similarity analysis also revealed the high sequence similarity of p97 with a yeast protein whose function is yet unknown, the gene for which is here termed NAS1 (non-ATPase subunit 1). Disruption of NAS1 resulted in several phenotypes, including lethality and temperature-sensitive growth, depending on the genetic background of the cells used. The human p97 cDNA suppressed the growth defect of nas1 disruptant cells, when expressed from single-copy or multi-copy vectors, indicating that p97 is functionally equivalent to yeast Nas1p. Culturing of the temperature-sensitive nas1 cells at the restrictive temperature promoted the accumulation polyubiquitinated cellular proteins, implying that the 26S proteasome requires a functional Nas1p subunit for ubiquitin-dependent proteolysis. These results indicate that p97/Nas1p plays an important regulatory role in the function of the 26S proteasome.
Subject(s)
Carrier Proteins , Cysteine Endopeptidases/genetics , Multienzyme Complexes/genetics , Saccharomyces cerevisiae Proteins , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , Fungal Proteins/genetics , Genetic Complementation Test , Humans , Molecular Sequence Data , Proteasome Endopeptidase Complex , Proteins/genetics , RNA, Messenger/analysis , Receptors, Tumor Necrosis Factor/genetics , Saccharomyces cerevisiae/genetics , Sequence Analysis , Sequence Homology, Amino Acid , TNF Receptor-Associated Factor 2 , Tissue DistributionABSTRACT
Most antigenic peptides presented on major histocompatibility complex class I molecules are generated by proteasomes. Interferon-gamma, which stimulates antigen presentation, induces new proteasome beta-subunits LMP2 and LMP7, which replace the homologous beta-subunits Y (delta) and X (epsilon). As a result, the capacity of the proteasome to cleave model peptides increases after hydrophobic and basic residues and falls after acidic residues. To clarify the function of these subunits, we examined the effects of overexpressing subunits X (delta) and Y (epsilon). Transfection of the Y gene into HeLa cells stimulated the proteasomal cleavage after acidic residues without altering other peptidase activities. This effect was proportional to the amount of the Y subunits and opposite to the effect of its homolog, LMP2. Y appears to promote cleavages after acidic residues. Furthermore, in mutants lacking the LMP genes (in contrast to wild-type cells), interferon-gamma treatment increased the proteasome content of Y subunits and enhanced postacidic cleavages. Transfection with cDNA for the X subunit reduced hydrolysis after hydrophobic and basic residues, an effect opposite to transfection of LMP2 and LMP7. Surprisingly, transfection of X increased the amounts not only of X, but also of Y, while decreasing LMP2 content. Thus, the loss of the Y subunit upon interferon-gamma treatment or LMP2 transfection accounts for the suppression of postacidic cleavages, and the loss of X contributes to the increased hydrolysis after hydrophobic and basic residues. These adaptations should favor the production of the kinds of peptides that are presented on major histocompatibility complex class I molecules.
Subject(s)
Cysteine Endopeptidases/metabolism , Endopeptidases/biosynthesis , Endopeptidases/metabolism , Interferon-gamma/pharmacology , Multienzyme Complexes/metabolism , Proteins/metabolism , Amino Acid Sequence , B-Lymphocytes , Cell Line , Cysteine Endopeptidases/biosynthesis , Enzyme Induction , HeLa Cells , Humans , Kinetics , Macromolecular Substances , Molecular Sequence Data , Multienzyme Complexes/biosynthesis , Proteasome Endopeptidase Complex , Protein Biosynthesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Substrate Specificity , TransfectionABSTRACT
Mammalian proteasomes are composed of 14-17 different types of subunits, some of which, including major-histocompatibility-complex-encoded subunits LMP2 and LMP7, are non-essential and present in variable amounts. We have investigated the distribution of total proteasomes and some individual subunits in rat liver by quantitative immunoblot analysis of purified subcellular fractions (nuclei, mitochondria, microsomes and cytosol). Proteasomes were mainly found in the cytosol but were also present in the purified nuclear and microsomal fractions. In the nuclei, proteasomes were soluble or loosely attached to the chromatin, since they could be easily extracted by treatment with nucleases or high concentrations of salt. In the microsomes, proteasomes were on the outside of the membranes. Further subfractionation of the microsomes showed that the proteasomes in this fraction were associated with the smooth endoplasmic reticulum and with the cis-Golgi but were practically absent from the rough endoplasmic reticulum. Using monospecific antibodies for some proteasomal subunits (C8, C9, LMP2 and Z), the composition of proteasomes in nuclei, microsomes and cytosol was investigated. Although there appear not to be differences in proteasome composition in the alpha subunits (C8 and C9) in the different locations, the relative amounts of some beta subunits varied. Subunit Z was enriched in nuclear proteasomes but low in microsome-associated proteasomes, whereas LMP2, which was relatively low in nuclei, showed a small enrichment in the microsomes. These differences in subunit composition of proteasomes probably reflect differences in the function of proteasomes in distinct cell compartments.
Subject(s)
Cysteine Endopeptidases/analysis , Liver/enzymology , Multienzyme Complexes/analysis , Animals , Antibodies , Cell Fractionation , Cell Nucleus/enzymology , Cysteine Endopeptidases/immunology , Cytoplasm/enzymology , Electrophoresis, Polyacrylamide Gel , Endoplasmic Reticulum/enzymology , Fluorescent Antibody Technique , Golgi Apparatus , Immunoblotting , Immunoenzyme Techniques , Liver/ultrastructure , Microscopy, Electron , Microsomes, Liver/enzymology , Multienzyme Complexes/immunology , Proteasome Endopeptidase Complex , Protein Conformation , Rats , Trypsin/metabolismABSTRACT
The proteasome, a multimeric protease, plays an important role in nonlysosomal pathways of intracellular protein degradation. This study was undertaken to determine which subunits of mammalian proteasomes are phosphorylated and to investigate the possible role of phosphorylation in regulating proteasome activity and the association with regulatory components. Rat-1 fibroblasts were grown in the presence of [32P]phosphate and proteasomes were immunoprecipitated from cell lysates with proteasome-specific polyclonal antibodies. Subsequent analysis by two-dimensional polyacrylamide gel electrophoresis showed two radiolabeled proteasome subunits which were identified using monoclonal antibodies as C8 and C9. Treatment of human embryonic lung cells (L-132), under identical conditions, also showed the same two phosphorylated subunits. Phosphoamino acid analysis revealed phosphoserine to be present in both C8 and C9. Examination of the sequence of C9 showed a potential cGMP-dependent phosphorylation site (-Arg3-Arg-Tyr-Asp-Ser-Arg8-), whilst C8 contains several potential casein kinase II phosphorylation sites. Following immunoprecipitation by a monoclonal antibody and dephosphorylation by acid phosphatase, proteasomes were observed to have significantly lower activities when compared to phosphorylated proteasomes, implying that phosphorylation may be an important mechanism of regulating proteasome function. Free proteasomes were separated by gel-filtration from those complexed with regulatory complexes to form the 26S proteinase. The ratio of phosphorylation of C8 and C9 was found to be very similar in the two complexes but the level of phosphorylation was higher in the 26S proteinase than in free proteasomes.
Subject(s)
Cysteine Endopeptidases/metabolism , Multienzyme Complexes/metabolism , Peptide Hydrolases/metabolism , Acid Phosphatase/metabolism , Amino Acid Sequence , Animals , Antibodies/immunology , Cell Line , Chromatography, Gel , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/immunology , Electrophoresis, Gel, Two-Dimensional , Endopeptidases/metabolism , Humans , Lung/metabolism , Molecular Sequence Data , Multienzyme Complexes/chemistry , Multienzyme Complexes/immunology , Peptide Hydrolases/chemistry , Phosphopeptides/analysis , Phosphorylation , Phosphoserine/analysis , Precipitin Tests , Proteasome Endopeptidase Complex , RatsABSTRACT
We have studied the degradation of a set of long peptides (9-30 amino acids) from the nucleoprotein of influenza A. In common for all these peptides is the core sequence NH2-Ser-Arg-Tyr-Trp-Ala-Ile-Arg-Thr-Arg-COOH, NP383-391, known as an antigenic peptide specific for the HLA-B27 class I antigen. We show that this peptide is generated by enriched cytosolic proteasomes of two sizes, 20S and 12S. The 12S proteasome is the precursor, the preproteasome, to the 20S mature proteasome as shown by pulse-chase experiment and is most likely responsible for the proteolytic activity in the 12S region. Cleavage at the N-terminus is distinct and restricted to residue 383, independent of the N-terminal extension of the peptide. The C-terminus is generated via cleavage at three sites. Intermediate and final peptide products were identified by mass spectrometry. Finally, we show that the NP383-391 peptide generated by proteasomes in vitro is functional inasmuch as it possesses the ability to stimulate assembly of in vitro translated HLA-B27 antigens.
Subject(s)
HLA-B27 Antigen/chemistry , HLA-B27 Antigen/physiology , Influenza A virus/chemistry , Influenza A virus/immunology , Lymphocytes/chemistry , Microsomes/immunology , Microsomes/metabolism , Nucleoproteins/biosynthesis , Nucleoproteins/immunology , Nucleoproteins/physiology , Amino Acid Sequence , Blotting, Western , Cells, Cultured , Cloning, Molecular , Histocompatibility Antigens Class I/immunology , Humans , Molecular Sequence Data , Viral Proteins/biosynthesis , Viral Proteins/immunology , Viral Proteins/physiologyABSTRACT
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.
Subject(s)
Cysteine Endopeptidases/drug effects , Gene Expression Regulation, Enzymologic , Interferon-gamma/pharmacology , Multienzyme Complexes/drug effects , Amino Acid Sequence , Antigen Presentation , Base Sequence , Chromosome Banding , Chromosome Mapping , Cloning, Molecular , Cysteine Endopeptidases/biosynthesis , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/genetics , Down-Regulation , Humans , In Situ Hybridization, Fluorescence , Molecular Sequence Data , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Proteasome Endopeptidase Complex , Protein Biosynthesis , Protein Conformation , Protein Processing, Post-Translational , Sequence Homology, Amino AcidABSTRACT
Subunit HsN3 of the human proteasome is a beta-type subunit homologous to PRE4 from yeast, X1 beta from Xenopus and RN3 from the rat. Using electron microscopy, the binding sites of a monoclonal antibody with specificity for subunit HsN3 have been located in the two juxtaposed inner rings of the human proteasome. Subunit HsN3 was present in two copies, one in each ring, in accordance with our concept of two identical halves making up the complete human proteasome. The subunit is involved in the trypsin-like as well as the peptidylglutamyl-peptide cleavage activities.