Purification of 20S proteasomes.
Explore the source record for details and available documents.
Adenosine Triphosphatases↗
Biomedical subjects
J J Monaco
Publications and source records attributed to J J Monaco.
Identification of 40 genes on a 1-Mb contig around the IL-4 cytokine family gene cluster on mouse chromosome 11.
Five related cytokine genes, interleukin 3 (Il3), interleukin 4 (Il4), interleukin 5 (Il5), interleukin 13 (Il13), and granulocyte-macrophage colony-stimulating factor (Csfgm or Csf2), are tightly linked on mouse chromosome 11. We now describe a 1-Mb transcript map of this cytokine cluster. Genomic clones obtained by screening mouse bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) libraries were subcloned into the pSPL3 expression vector and transfected into COS7 cells for exon trapping. In total, 118 distinct, putative exons were sequenced and characterized, mapping up to 29 distinct genes to the mouse cluster, including Il4 and Csf2. Northern blot and RT-PCR analyses indicate that all of these genes are expressed. Analysis of 1 Mb of published sequence from the region of conserved synteny on human chromosome 5q31-q33 identified 45 gene candidates, including 35 expressed genes in the human IL-4 cytokine gene cluster. Probes for 20 human genes were tested for cross-hybridization to murine BAC and PAC clones, thereby mapping 11 additional genes to the mouse complex. Thus, a total of 40 genes including 6 cytokine genes have been physically mapped within 1 Mb of mouse chromosome 11. Gene order in this complex is similar, but not identical, between human and mouse. The integrated physical and transcript maps should prove valuable as a complement to genomic sequencing and expression-dependent transcript maps of this segment of the genome.
Amino Acid Sequence↗
Identification of proteassemblin, a mammalian homologue of the yeast protein, Ump1p, that is required for normal proteasome assembly.
We have identified a mammalian homologue of yeast Ump1p by searching for similar proteins in human and mouse expressed sequence tag (EST) databases. Ump1p is an accessory protein that is required for normal proteasome assembly in yeast (1). A mammalian homologue, which we refer to as "proteassemblin," is a constituent of proteasome assembly intermediates (preproteasomes), but not fully assembled 20S proteasomes, as is Ump1p in yeast. We also provide evidence that proteassemblin is a constituent of pre-immunoproteasomes that contain the precursor of the interferon-gamma-inducible subunit LMP2. By analogy with Ump1p, we hypothesize that proteassemblin is required for normal mammalian proteasome assembly.
Amino Acid Sequence↗
LMP2 expression and proteasome activity in NOD mice.
Explore the source record for details and available documents.
Animals↗
Up-regulation of the proteasome subunit LMP7 in tissues of endotoxemic rats.
The proteasome has been implicated in systemic responses to infection or inflammatory stimuli including catabolism of skeletal muscle. Cytokines including tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) are known to be elevated systemically and locally under these conditions. They are also known to be potent inducers of three peptide subunits of the proteasome, including LMP7, that replace constitutively expressed subunits and change enzymatic properties. To determine whether endotoxemia alters the expression of inducible proteasome subunits, we examined the levels of LMP7 in tissues from rats 3 days after the injection of lipopolysaccharide (LPS) or normal saline solution (NS). By both immunoblotting and immunohistochemistry, significant increases in levels of LMP7 were observed in the heart, kidney, and lung of animals given LPS as compared with results in NS-treated animals, whereas immunoblotting revealed no changes in LMP7 levels in skeletal muscle or brain. Increased expression of LMP7 was limited to certain subpopulations of cells and was further localized at the subcellular level. Decreases in organ weight were also documented for organs in which the expression of LMP7 was up-regulated. Systemic or local release of cytokines or other proinflammatory mediators is suggested as the most likely mechanism for changes in LMP7 expression during endotoxemia. Changes in LMP7 expression may have functional consequences that contribute to organ dysfunction during systemic responses to infection and inflammatory stimuli.
Animals↗
Burn injury upregulates the activity and gene expression of the 20 S proteasome in rat skeletal muscle.
There is evidence that burn injury stimulates ubiquitin-proteasome-dependent protein breakdown in skeletal muscle. In this proteolytic pathway, protein substrates are conjugated to multiple molecules of ubiquitin, whereafter they are recognized, unfolded and degraded by the multicatalytic 26 S protease complex. The 20 S proteasome is the catalytic core of the 26 S protease complex. The influence of burn injury on the expression and activity of the 20 S proteasome has not been reported. We tested the hypothesis that burn injury increases 20 S proteasome activity and the expression of mRNA for the 20 S proteasome subunits RC3 and RC7. Proteolytic activity of isolated 20 S proteasomes, assessed as activity against fluorogenic peptide substrates, was increased in extensor digitorum longus muscles from burned rats. Northern-blot analysis revealed that the expression of mRNA for RC3 and RC7 was increased by 100% and 80% respectively following burn injury. Increased activity and expression of the 20 S proteasome in muscles from burned rats support the concept that burn-induced muscle cachexia is at least, in part, regulated by the ubiquitin-proteasome proteolytic pathway.
Animals↗
Class II antigen processing defects in two H2d mouse cell lines are caused by point mutations in the H2-DMa gene.
The molecular nature of the defect in two mouse antigen processing-defective cell lines was examined. Both mutants were derived from the A20 (BALB/c, H2d) B cell line, and both were found to have defects in the H2-DMa gene. Mutant 3A5 exhibits severely reduced amounts of H2-DMa message, and no detectable DMalpha protein. cDNA sequence revealed a C-->T transition at nucleotide 118, introducing a premature stop codon in exon 2 of the H2-DMa gene. In contrast, mutant 2A2 exhibits reduced but detectable levels of H2-DMa message and DMalpha protein only after treatment with IL-4, which induces the expression of both the H2-DMa and the H2-DMb genes in B cells. In this mutant the cDNA sequence revealed a missense mutation in exon 3 resulting in the conversion of a conserved proline residue in the Ig-like domain to serine. Stable transfection with full-length H2-DMa cDNA reconstitutes the antigen processing capacity of both mutants, as demonstrated by the ability to present native antigen to T cell clones, and by restored class II SDS stability.
Animals↗
Isolation and mapping of the rabbit DM genes.
Proper peptide presentation by major histocompatibility complex (MHC)-encoded class II antigens is dependent on the products of the MHC DM loci. We identified the rabbit orthologues (RLA-DMA and -DMB) of human HLA-DMA and -DMB and found that they have 76.9% and 78.8% identity with HLA-DMA and -DMB, respectively. Like classical class II MHC genes, RLA-DM genes are more closely related to human HLA-DM genes than to mouse H2-DM. Among the DM family, there is a high degree of variability at the amino terminus of the DMa chains, and length variability in the cytoplasmic tails of both DMalpha and DMbeta. The rabbit DM genes are coexpressed with class II genes in lymphoid tissues, as are the DM genes of other mammals. The RLA-DM locus maps to the class II region of the rabbit MHC, and is flanked by the DP and DOB loci. Despite having some similarities to class II genes of bony fishes, the DM family represents a separate branch of the MHC class II family.
Amino Acid Sequence↗
The complete primary structure of mouse 20S proteasomes.
The proteasome is a large multicatalytic proteinase that plays a role in the generation of peptides for presentation by major histocompatibility complex class I molecules. The 20S proteolytic core of mammalian proteasomes is assembled from a group of 17 protein subunits that generate a distinctive pattern of spots upon two-dimensional gel electrophoresis. The genes for most of these subunits have been cloned from humans and rats. We isolated cDNA clones for the mouse orthologues of ten of the subunits [PSMA1 (C2), PSMA2 (C3), PSMA3 (C8), PSMA4 (C9), PSMA5 (ZETA), PSMA6 (IOTA), PSMA7 (C6-I), PSMB2 (C7-I), PSMB3 (C10-II), and PSMB5 (X)] to complete the cloning of all of the mouse subunits. Using antisera raised against these subunits or their orthologues, we verified the identity of these proteins by two-dimensional NEPHGE-PAGE.
Amino Acid Sequence↗
Bacterioferritin A modulates catalase A (KatA) activity and resistance to hydrogen peroxide in Pseudomonas aeruginosa.
We have cloned a 3.6-kb genomic DNA fragment from Pseudomonas aeruginosa harboring the rpoA, rplQ, katA, and bfrA genes. These loci are predicted to encode, respectively, (i) the alpha subunit of RNA polymerase; (ii) the L17 ribosomal protein; (iii) the major catalase, KatA; and (iv) one of two iron storage proteins called bacterioferritin A (BfrA; cytochrome b1 or b557). Our goal was to determine the contributions of KatA and BfrA to the resistance of P. aeruginosa to hydrogen peroxide (H2O2). When provided on a multicopy plasmid, the P. aeruginosa katA gene complemented a catalase-deficient strain of Escherichia coli. The katA gene was found to contain two translational start codons encoding a heteromultimer of approximately 160 to 170 kDa and having an apparent Km for H2O2 of 44.7 mM. Isogenic katA and bfrA mutants were hypersusceptible to H2O2, while a katA bfrA double mutant demonstrated the greatest sensitivity. The katA and katA bfrA mutants possessed no detectable catalase activity. Interestingly, a bfrA mutant expressed only approximately 47% the KatA activity of wild-type organisms, despite possessing wild-type katA transcription and translation. Plasmids harboring bfrA genes encoding BfrA altered at critical amino acids essential for ferroxidase activity could not restore wild-type catalase activity in the bfrA mutant. RNase protection assays revealed that katA and bfrA are on different transcripts, the levels of which are increased by both iron and H2O2. Mass spectrometry analysis of whole cells revealed no significant difference in total cellular iron levels in the bfrA, katA, and katA bfrA mutants relative to wild-type bacteria. Our results suggest that P. aeruginosa BfrA may be required as one source of iron for the heme prosthetic group of KatA and thus for protection against H2O2.
Amino Acid Sequence↗
Activity and expression of the 20S proteasome are increased in skeletal muscle during sepsis.
Recent studies suggest that sepsis stimulates ubiquitin-dependent protein breakdown in skeletal muscle. In this proteolytic pathway, ubiquitinated proteins are recognized, unfolded, and degraded by the multicatalytic 26S protease complex. The 20S proteasome is the catalytic core of the 26S protease complex. The role of the 20S proteasome in the regulation of sepsis-induced muscle proteolysis is not known. We tested the hypothesis that sepsis increases 20S proteasome activity and the expression of mRNA for various subunits of this complex. Proteolytic activity of isolated 20S proteasomes, assessed as activity against fluorogenic peptide substrates, was increased in extensor digitorum longus muscles from septic rats. The proteolytic activity was inhibited by specific proteasome blockers. Northern blot analysis revealed an approximately twofold increase in the relative abundance of mRNA for the 20S alpha-subunits RC3 and RC9 and the beta-subunit RC7. However, Western blot analysis did not show any difference in RC9 protein content between sham-operated and septic rats. The increased activity and expression of the 20S proteasome in muscles from septic rats lend further support for a role of the ubiquitin-proteasome-pathway in the regulation of sepsis-induced muscle proteolysis.
Animals↗
Antigen processing of two H2-IEd-restricted epitopes is differentially influenced by the structural changes in a viral glycoprotein.
The factors that influence the intracellular location(s) of MHC class II-restricted epitope loading remain poorly understood. We present evidence that two I-Ed-restricted epitopes of the influenza hemagglutinin (HA) molecule, termed site 1 (S1; encompassing amino acid residues 107-119) and site 3 (S3; encompassing amino acid residues 302-313), are generated in distinct endocytic compartments. By means of an epitope-specific mAb, we show that S1 becomes detectable in late endocytic/lysosomal vesicles; using a mutant cell line, we also show that the presentation of S1 is dependent upon H2-DM expression. In contrast, S3; presentation is H2-DM-independent and appears in early endosomes as a result of acid-induced structural changes in HA. Presentation of both epitopes can be made H2-DM-independent by denaturing HA and made H2-DM-dependent by preventing the acid-induced conformational changes from occurring. These findings indicate that the structural context of a given epitope can determine where it is processed.
Animals↗
Physical and functional association of the major histocompatibility complex class I heavy chain alpha3 domain with the transporter associated with antigen processing.
CD8+ T lymphocytes recognize antigens as short, MHC class I-associated peptides derived by processing of cytoplasmic proteins. The transporter associated with antigen processing translocates peptides from the cytosol into the ER lumen, where they bind to the nascent class I molecules. To date, the precise location of the class I-TAP interaction site remains unclear. We provide evidence that this site is contained within the heavy chain alpha3 domain. Substitution of a 15 amino acid portion of the H-2Db alpha3 domain (aa 219-233) with the analogous MHC class II (H-2IAd) beta2 domain region (aa 133-147) results in loss of surface expression which can be partially restored upon incubation at 26 degrees C in the presence of excess peptide and beta2-microglobulin. Mutant H-2Db (Db219-233) associates poorly with the TAP complex, and cannot present endogenously-derived antigenic peptides requiring TAP-dependent translocation to the ER. However, this presentation defect can be overcome through use of an ER targeting sequence which bypasses TAP-dependent peptide translocation. Thus, the alpha3 domain serves as an important site of interaction (directly or indirectly) with the TAP complex and is necessary for TAP-dependent peptide loading and class I surface expression.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Immunoproteasome assembly: cooperative incorporation of interferon gamma (IFN-gamma)-inducible subunits.
LMP2, LMP7, and MECL are interferon gamma-inducible catalytic subunits of vertebrate 20S proteasomes, which can replace constitutive catalytic subunits (delta, X, and Z, respectively) during proteasome biogenesis. We demonstrate that MECL requires LMP2 for efficient incorporation into preproteasomes, and preproteasomes containing LMP2 and MECL require LMP7 for efficient maturation. The latter effect depends on the presequence of LMP7, but not on LMP7 catalytic activity. This cooperative mechanism favors the assembly of homogeneous "immunoproteasomes" containing all three inducible subunits, suggesting that these subunits act in concert to enhance proteasomal generation of major histocompatibility complex class I-binding peptides.
Animals↗
How do endogenous proteins become peptides and reach the endoplasmic reticulum.
Explore the source record for details and available documents.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Sequence, linkage to H2-K, and function of mouse tapasin in MHC class I assembly.
Assembly of major histocompatibility complex (MHC) class I molecules in human cells is dependent on the accessory protein tapasin, which mediates their interaction with the transporters associated with antigen processing (TAP) and thereby ensures efficient peptide binding. Analysis of a mouse tapasin complementary DNA defined a conserved polypeptide sharing sequences diagnostic of a transmembrane protein related to the immunoglobulin superfamily, and an endoplasmic reticulum retention motif. The mouse tapasin gene was mapped about 70 kilobases from H2-K at the centromeric end of the mouse MHC. Expression of mouse tapasin in a tapasin-deficient human mutant cell line restored the normal assembly and expression of class I alleles. Thus, tapasin is a structurally and functionally conserved component of the MHC class I antigen processing pathway. Its genetic linkage to the class I and TAP subunit genes in the MHC may be of significance in the coordinate expression and functional coadaptation of the diverse gene products.
Amino Acid Sequence↗
DNA sequence, chromosomal localization, and tissue expression of the mouse proteasome subunit lmp10 (Psmb10) gene.
Proteasomes are nonlysosomal multicatalytic proteases involved in antigen processing. Three of the 10 mammalian proteasome beta subunits (LMP2, LMP7, and LMP10) are induced by IFN-gamma. Two of these (LMP2 and LMP7) are encoded in the major histocompatibility complex of both human (chromosome 6) and mouse (chromosome 17). However, the human homologue of Lmp10, MECL1, is found on chromosome 16. Here we show that in mice, Lmp10 is a single-copy gene localized to chromosome 8, in a region of conserved synteny with human chromosome 16. Sequencing of a 129/SvJ strain genomic clone revealed that the gene has eight exons spanning 2.3 kb. Characterization of a full-length mouse cDNA clone indicates that Lmp10 encodes a protein of 273 amino acids with a calculated molecular weight of 29 kDa and an isoelectric point of 6.86. Northern analysis of Lmp2, Lmp7, and Lmp10 showed expression in heart, liver, thymus, lung, and spleen, but not in brain, kidney, skeletal muscle, or testis.
Amino Acid Sequence↗
Intermediates in the formation of mouse 20S proteasomes: implications for the assembly of precursor beta subunits.
The assembly of individual proteasome subunits into catalytically active mammalian 20S proteasomes is not well understood. Using subunit-specific antibodies, we characterized both precursor and mature proteasome complexes. Antibodies to PSMA4 (C9) immunoprecipitated complexes composed of alpha, precursor beta and processed beta subunits. However, antibodies to PSMA3 (C8) and PSMB9 (LMP2) immunoprecipitated complexes made up of alpha and precursor beta but no processed beta subunits. These complexes possess short half-lives, are enzymatically inactive and their molecular weight is approximately 300 kDa. Radioactivity chases from these complexes into mature, long-lived approximately 700 kDa proteasomes. Therefore, these structures represent precursor proteasomes and are probably made up of two rings: one containing alpha subunits and the other, precursor beta subunits. The assembly of precursor proteasomes occurs in at least two stages, with precursor beta subunits PSMB2 (C7-I), PSMB3 (C10-II), PSMB7 (Z), PSMB9 (LMP2) and PSMB10 (LMP10) being incorporated before others [PSMB1 (C5), PSMB6 (delta), and PSMB8 (LMP7)]. Proteasome maturation (processing of the beta subunits and juxtaposition of the two beta rings) is accompanied by conformational changes in the (outer) alpha rings, and may be inefficient. Finally, interferon-gamma had no significant effect on the half-lives or total amounts of precursor or mature proteasomes.
Animals↗