Constitutive and interferon-γ-induced expression of the human proteasome subunit multicatalytic endopeptidase complex-like 11

Biochimica et Biophysica Acta 1402 Ž1998. 17–28

Constitutive and interferon-g-induced expression of the human proteasome subunit multicatalytic endopeptidase complex-like 1 1 Grethe S. Foss, Frank Larsen, Jorun Solheim, Hans Prydz

)

Biotechnology Centre of Oslo, UniÕersity of Oslo, P.O. Box 1125 Blindern, N-0316 Oslo, Norway Received 19 September 1997; revised 1 December 1997; accepted 3 December 1997

Abstract Proteasomes generate peptides from intracellular endogenous and viral proteins for presentation by MHC class I molecules. During viral infection, interferon-g ŽIFN-g . acts as a cytokine altering the catalytic specificity of proteasomes by inducing the synthesis of the three proteasome subunits, low molecular weight protein ŽLMP. 2, LMP7 and multicatalytic endopeptidase complex-like 1 ŽMECL1.. LMP2 and LMP7 have been shown to favour the presentation of certain antigenic peptides. These subunits are constitutively expressed in cell lines related to the immune system and IFN-g-inducible in other cell lines. Less is known about MECL1. To reveal the extent of constitutive and IFN-g-induced expression of MECL1, we studied MECL1 in different cell lines by Northern and Western blotting. The two B cell lines IM9 and Reh showed high constitutive expression of MECL1, only slightly induced by IFN-g stimulation. The B cell line Daudi and the monocyte cell line THP-1 expressed MECL1 constitutively at an intermediate level. The MECL1 protein level in the THP-1 cells increased markedly in response to IFN-g . In cells unrelated to the immune system, a very low constitutive expression of MECL1 was detected, highly inducible by IFN-g . These results indicate that, similar to LMP2 and LMP7, MECL1 is constitutively expressed at high levels only in certain cell lines and can be induced by IFN-g in other cell lines. The differential expression of MECL1 may be of importance for which antigenic peptides are presented by different cells as well as by the same cells at different IFN-g levels. q 1998 Elsevier Science B.V. Keywords: Proteasome; Multicatalytic endopeptidase complex-like 1; Interferon-g ; Antigen processing

1. Introduction Antigenic peptides derived from intracellular proteins are continuously presented to the immune sys-

Abbreviations: LMP2r7, low molecular weight proteins 2r7; MECL1, multicatalytic endopeptidase complex-like 1; RACE, rapid amplification of cDNA ends; SFV, Semliki Forest virus ) Corresponding author. Fax: q47-22-69-41-30; E-mail: [email protected] 1 EMBL accession number for MECL1 cDNA: Y13640.

tem by MHC class I molecules on the surface of immune and nonimmune cells. The generation of most antigenic peptides is dependent on proteasomes w1x. As part of the pathway for degradation of ubiquitin-conjugated proteins, proteasomes are the major nonlysosomal proteolytic enzymes in the cell and degrade the bulk of cytosolic proteins w2x. Eukaryotic 20S proteasomes are multicatalytic particles with twofold symmetry composed of 14 different subunits occupying distinct positions in four superimposed rings w3,4x. The two inner rings are formed by the

0167-4889r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 8 9 Ž 9 7 . 0 0 1 5 2 - 3

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b-type subunits, which contain catalytically active sites w4x. Interferon-g ŽIFN-g ., a cytokine of major importance to the immune system during viral infection, alters the subunit composition of the proteasomes w5x forming so-called immunoproteasomes w6x. Upon stimulation of cells with IFN-g , the three constitutive subunits X, Y and Z are replaced by the IFN-g-inducible proteasome subunits low molecular weight protein ŽLMP. 7, LMP2 and multicatalytic endopeptidase complex-like 1 ŽMECL1., respectively w6–13x. These six subunits all contain a putative catalytically active N-terminal threonine w14x and are thereby potential contributors to the catalytic specificity of the proteasome. IFN-g is shown to modify the peptidase activities of proteasomes w11,15–18x in a way that could promote more efficient formation of peptides suitable for binding to MHC class I molecules w19x. While not crucial to antigen processing in general w20,21x, the importance of the IFN-g-inducible subunits LMP2 and LMP7 for supply of antigenic peptides has been demonstrated in knockout mice w22– 24x. The altered catalytic activity may not only affect the amount of antigenic peptides produced but also the specific sets of peptides generated from the different proteins in the cell. Indeed, processing and presentation of specific peptides have been shown to be favoured by the presence of LMP2 and LMP7 w22–25x. Moreover, in cell lines LMP2-containing proteasomes are required for presentation of specific influenza virus antigens w26x. Thus, the IFN-g-inducible proteasome subunits LMP2 and LMP7 are probably important for determining the different peptides generated for presentation by MHC class I molecules. The third IFN-g-inducible subunit, MECL1, has been studied in much less detail, but may well be of importance for the catalytic properties of proteasomes. While LMP2 and LMP7 are encoded in the MHC class II region w27,28x, the gene for MECL1 is part of a tight cluster of unrelated genes on human chromosome 16q22.1 w29x. In addition to being IFN-g inducible, MECL1 is constitutively expressed in several tissues w29x. The aim of the present study was to reveal the extent of constitutive and IFN-g-induced expression of MECL1 in different cell lines. Comparison of the constitutive expression with the IFN-g-induced expression of MECL1 may give clues to the

role of MECL1 in antigen processing, both during normal conditions and during viral infection.

2. Materials and methods 2.1. cDNA cloning An AMA cell cDNA library in l-ZAPII was screened for MECL1 cDNA. The cDNA clones of the library were size-fractionated to 1000–2400 bp, and cloned between the EcoRI and XhoI sites of the polylinker. The library was amplified using the E. coli Epicurion Coli SURE Ž Stratagene. , and 4 = 10 5 plaques were screened using the same host. After plaque-lifting and denaturing, the filters were probed with a MECL1 cDNA fragment from a previously screened cDNA library from Daudi cells. This incomplete cDNA clone had an intron disrupting the open reading frame, and was labelled with 32 P-nucleotides by random priming w30x. After secondary screening, positive clones were amplified and zapped using the helper virus R408, giving pBluescript SKy carrying the cDNA insert. The cDNA clones and derived deletion clones were sequenced from each end using pBluescript primers. The polylinker enzymes SacII and KpnI cutting around the middle of the cDNA, were used for making deletion clones. The cDNA was sequenced using an Automated Laser Fluorescent DNA Sequencer ŽPharmacia. and a redundancy of 3. 2.2. Methylation status of CpG island Genomic DNA extracted from white blood cells was restricted with the methylation sensitive enzymes AÕaI, BstUI, HhaI, MluI, SacII, SalI, SmaI, and XhoI, respectively, in addition to SacI, which cuts on both sides of the MECL1 CpG island. A 440-bp MluI fragment of the CpG island was used as probe for the Southern blots w29x. 2.3. Transcription start sites Transcription start of the MECL1 gene was determined by 5X RACE–PCR ŽRACE being rapid amplifi-

G.S. Foss et al.r Biochimica et Biophysica Acta 1402 (1998) 17–28

cation of cDNA ends; 5X RACE KIT, Clontech. . Two gene specific reverse primers were designed, the primer for cDNA synthesis in exon 4 Ž 5X-TAGCTCCATCTTGGACGCCACCATC-3X . and the nested PCR primer at the end of exon 3 Ž5X-GTAGATTTTGGGGGCGATGAAG-3X . . mRNA was isolated from unstimulated HL-60 cells with magnetic beads as described below, and the first strand was synthesised using AMV reverse transcriptase ŽPromega.. The PCR products were cloned in the pNEB193 vector Ž New England Biolabs. using the EcoRI-site in the anchor primer and the SmaI-site in exon 2, and analysed by DNA sequencing. 2.4. Expression of MECL1 in the SFV-system The Semliki Forest virus Ž SFV. system was used to express MECL1 cDNA in unstimulated ECV304 cells. MECL1 cDNA was cloned into pSFV1 vector, where the insert uses its own translation initiation signal w31x. The cDNA was excised from pBluescript with BamHI and XhoI, and cloned into the BamHIsite in pSFV1 using a BamHI-linker. Virus particles were produced and used for infection as described in the SFV Expression System manual. ECV304 cells were infected for 1 h by a 1:5 dilution of the virus stock solution, and subjected to lysis 20 h later. 2.5. Cell lines and preparation of cell extracts The human cervix carcinoma cell lines HeLa and HeLa S3 ŽATCC. and human breast adenocarcinoma cell line MCF7 were grown in DMEM ŽGibco BRL. with 10% FCS ŽGibco BRL. , and the human colon adenocarcinoma Caco-2 cells Ž ATCC. in DMEM with 15% FCS. The human endothelial cell line ECV304 ŽATCC. grew in Medium 199 with 10% FCS. The human B cells lines, IM9 Ž multiple myeloma; IgG secreting. , Daudi ŽBurkitt’s lymphoma; mature B cell. and Reh Ž acute lymphoblastic leukaemia; pre-B cell. cells and the human monocyte cell line THP-1 were all grown in RPMI 1640 Ž Gibco BRL. with 10% FCS. HL-60 leukaemia cells were maintained in RPMI 1640 using 20% FCS. A freshly obtained buffy coat from a healthy individual was used for preparation of human white blood cells. The mononuclear white blood cells were

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isolated using Lymphoprep Ž Nycomed. as recommended by the manufacturers. An adherent fraction of the cells, corresponding to monocytes, was obtained by incubating the isolated cells on tissue culture plates for 2 h. Cells were lysed in lysis buffer Ž 50 mM Tris–HCl, pH 7.6 at 08C, 150 mM NaCl, 1% NP40. on ice for 30 min. No protease inhibitors were added. Extracts were centrifuged at 13 000 rpm for 10 min and the supernatant frozen at y208C for later SDS–PAGE. 2.6. Cytokine stimulation The IFN-g stimulation conditions were examined using 0, 25, 50, 100, 200, 400 and 800 Urml for 48 h and 200 Urml for 0, 12, 24, 36, 48 and 72 h. In all the further experiments, ECV304 cells were stimulated with 200 Urml IFN-g for 48 h prior to lysis. Other cytokines were examined for stimulating effect on MECL1 in ECV304 and Caco-2 cells: IL-1 Ž100 Urml., IL-6 Ž400 Urml., TGFb Ž3 ngrml. and TNFa Ž0.9 m grml. all for 6 and 24 h. ECV304, Caco-2, HeLa and HeLa S3 cells were submitted to heat shock for 0.5, 1, 2 and 4 h at 428C. 2.7. Proteasome isolation Proteasomes were isolated by affinity chromatography using monoclonal antibodies as described by Hendil and Uerkvitz w32x. Monoclonal anti-proteasome antibodies were produced from the MCP-21 hybridoma cells w32x kindly provided by Dr. K. Hendil ŽUniversity of Copenhagen.. The antibody was precipitated from the cell culture supernatant by 55% ammonium sulphate, purified on Protein A–Sepharose with high salt and high pH and coupled to CNBr-activated Sepharose 4B ŽPharmacia. w33x. Cultured cells Žca. 2 = 10 8 cells., either unstimulated or stimulated with IFN-g , were scraped off and homogenised with a Potter–Elvehjem homogeniser in two volumes of 5 mM Tris–HCl, pH 7.6 at 08C, 1 mM EDTA, 5 mM mercaptoethanol. Cellular proteins precipitated by 10% Žwrv. PEG 6000 were subjected to the affinity column and isolated as described w32x. The eluted proteasomes were desalted on a Sephadex G-25 Fine ŽPharmacia. column with 25 mM Tris– HCl, pH 7.6 at 08C, concentrated using Centricon-10 ŽAmicon. to 2 mgrml and frozen at y208C.

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2.8. Antibodies Rabbit oligoclonal antibodies were raised against a peptide from the MECL1 sequence: SYSRLPFTALGSGQD Ž position 157–171.. The peptide was synthesised using t-BOC chemistry on an ABI instrument and coupled to keyhole limpet haemocyanine before injection. The rabbit immunoglobulins were purified on a Protein A-coupled Sepharose Ž Pharmacia. column w33x, dissolved to 1 mgrml, and used for immunoblotting. 2.9. Northern blots mRNA was isolated directly from tissue culture cells using magnetic polyŽdT. beads ŽDynabeads. w34x. The cells were lysed and the DNA viscosity reduced by shearing DNA through a 21 gauge needle, before extracting the mRNA. Isolated mRNA was run on a denaturing agarose gel and blotted. The filters were probed with a cDNA fragment covering exon 1 to 5 and a 440-bp MluI fragment from the CpG island w29x, respectively, and washed using stringent conditions. The mRNA amounts were checked by hybridising to a glyceraldehyde-3-phosphate dehydrogenase cDNA probe. 2.10. SDS–PAGE and immunoblotting The gels Ž12%. were made according to Laemmli w35x, and after separation, electroblotted onto polyvinylidene fluoride membranes ŽImmobilon-P. . Biotinylated low molecular weight standards Ž Amersham. were used. The membranes, except for lanes with molecular weight standards, were blocked with 5% ECL blocking reagent Ž Amersham. at 48C for at least 1 h. The rabbit anti-MECL1-peptide antibodies were used at a predetermined dilution of 1:5000 overnight at 48C. The ECL system ŽAmersham. was used for detection as recommended by the manufacturers. Horseradish peroxidase-linked donkey–anti-rabbit antibodies and streptavidin–horseradish peroxidase Žboth Amersham. were both used at a dilution of 1:5000 for 1–2 h at 48C.

3–10 L, 11 cm, and ExcelGel SDS gradient 8–18 ŽPharmacia.. The purified proteasome fractions Ž100 m g. were concentrated by N2-gas before loaded. The gels were blotted for 30 min in a semidry sandwich and silver stained. MECL1 was detected on the polyvinylidene fluoride membrane as described above.

3. Results 3.1. MECL1 cDNA confirms the predicted protein sequence MECL1 genomic DNA was first cloned in our laboratory w29x. To determine the exons of the MECL1 gene Žalso called PSMB10 . , we screened a human cDNA library from AMA cells. Three positive clones were obtained. Sequencing revealed two identical full length MECL1 cDNA clones Ž EMBL Y13640. and a third clone starting six basepairs downstream of the exon 1–exon 2 boundary. The full length cDNA clone was 996 bp with untranslated 5X and 3X regions of 88 and 54 bp, respectively, and a polyA-tail of 35 nucleotides. The open reading frame was 819 bp, corresponding to 273 amino acids. The amino acid translation of MECL1 cDNA confirmed the protein sequence predicted from the reported genomic sequence w29x ŽEMBL X71874; SwissProt P40306., containing the propeptide and the conserved residues for processing and proteolytic activity w14x. The 5X part of the MECL1 gene is shown in Fig. 1, including the first two exons encoding the propeptide and the processing site. MECL1 cDNA corresponds to eight small exons varying in size from 59 to 152 bp. The gene covers 2.3 kb, with introns sized between 85 and 471 bp. There were two base differences between the cDNA and the genomic sequence; one transition in the promoter ŽG to A, position 2298 of the genomic fragment. Ž Fig. 1. and another in exon 5 ŽT to C in position 3464.. The change in exon 5 does not alter the encoded amino acid ŽGly151..

2.11. Two-dimensional gel electrophoresis

3.2. Various transcription start sites in the unmethylated CpG island

The components of proteasomes were separated on 2-D IEFrSDS–PAGE using Immobiline DryStrip pH

A 1.7 kb long CpG island covers the first six exons of the MECL1 gene w29x. An unmethylated

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Fig. 1. The promoter and 5X part of the MECL1 gene. The MECL1 gene covers 2.3 kb and contains eight small exons. The exon–intron boundaries are here shown for the first two exons and introns as marked with open arrows. Only parts of the 208 bp long intron 1 are shown. The filled arrowhead indicates the start of the full length cDNA clone, and an open asterisk marks one of the two polymorphisms found in the cDNA sequence ŽG to A.. The derived MECL1 protein sequence is given in one-letter code beneath the nucleotide sequence starting from translation initiation code ŽATG. in position 2345. A vertical black bar indicates the conserved processing site of active b-type proteasome subunits, and a filled asterisk marks the resulting putative catalytically active N-terminal threonine. All CpG dinucleotides are underlined showing the first part of the CpG island. The 5X transcriptional start sites found using 5X RACE are shown with black arrows, the wider arrow marking the start site found in four of six analysed 5X RACE-clones. The SmaI site shown was used for cloning 5X RACE-products, and is also one of the sites used in analysing the methylation of CpG dinucleotides. Different sequences similar to promoter elements are boxed: Sp1 site w37x, GAS ŽIFN-g activation site; consensus TTNCNNNAA. w40x, ISRE Žinterferonstimulated response element; consensus GŽA.AAAŽGrC.ŽTrC.GAAAŽGrC.ŽTrC.. w41x, IRE Žinterferon response element; consensus CŽArT.ŽGrT.ŽGrT.ANNŽCrT.. w42x and GATE-like Ž68% identity in position 4–22 of the reported g-activated transcriptional element; 5X-CCGGAGAGAATTGAAACTTAGGG-3X . w43x. Another Sp1 binding sequence was found in intron 1 Žnot shown.. Base numbering is according to the 13.9 kb genomic sequence ŽEMBL X71874. w29x.

status of a CpG island is often associated with active transcription in the region. DNA from six different tissues has previously been shown to be unmethylated at the three CpG dinucleotides covered by the methylation sensitive enzyme HpaII w29x. By restriction of DNA from white blood cells using eight different methylation sensitive enzymes, we increased the number of examined CpGs to 16 of the 125 CpGs in the CpG island and found them all to be unmethylated Ždata not shown.. One additional site Ž position 3723–4. at the end of the island was partly methylated, which is not unusual at the borders of CpG islands w36x. To define the promoter region, the transcription start of MECL1 was examined by 5X RACE. mRNA

isolated from HL-60 cells was used, and six different clones of the PCR-product were sequenced. They were found to correspond to three different transcription start sites, all within the CpG island, resulting in 5X-untranslated mRNA sequences of 69, 81 and 101 bp, respectively ŽFig. 1.. A number of different transcription start sites are regularly seen for genes associated with unmethylated CpG islands. As often seen in these CpG rich promoters, there were no clear consensus sequences for TATA- or CAAT-boxes upstream of the transcription start sites. However, Sp1 binding sequences w37x were found ŽFig. 1. . The MECL1 promoter sequence was also examined for sequence elements associated with IFN-g stimulation. Induction of genes by IFN-g depends on the

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STAT1 protein Žsignal transducers and activators of transcription. w38,39x, which in its phosphorylated form can bind directly to IFN-g activation sites ŽGAS. w40x. GAS-like sequences were found not far from the transcription start region of MECL1 Ž Fig. 1.. Also, two sequences with homology to the interferon-stimulated response element Ž ISRE., to which members of the interferon regulatory factor ŽIRF. family will bind w41x, were observed close to the transcription start sites Ž Fig. 1. . Moreover, sequences homologous to the interferon response elements Ž IRE. reported in promoters of MHC class II genes w42x were present in the MECL1 promoter region. Recently, the IFN-g induction of the murine interferon-stimulated gene factor 3g Ž ISGF3grp48. gene was found to be mediated by novel factors binding a g-activated transcriptional element ŽGATE. w43x. A sequence similar to the GATE sequence was also found in the MECL1 promoter, covering both the major transcriptional start site and the beginning of the cDNA clone Ž Fig. 1.. The presence of these sequences in the MECL1 promoter suggests regulated transcription of the MECL1 gene. 3.3. ConstitutiÕe and IFN-g-induced transcription of MECL1 We have previously detected MECL1 transcription in each of eight human tissues examined w29x. The highest MECL1 mRNA level was found in spleen, suggesting high constitutive expression of MECL1 in immune-related cells. To study the constitutive MECL1 transcription and the effect of IFN-g stimulation on MECL1 expression, we tested three different B cell lines ŽIM9, Daudi and Reh. and two cell lines unrelated to the immune system ŽHeLa and MCF7. ŽFig. 2. . The level of MECL1 mRNA in unstimulated cells varied markedly between the different cell lines. In the unstimulated B cell lines IM9 and Reh, the level of MECL1 mRNA was considerably higher than in unstimulated HeLa and MCF7 cells. In response to IFN-g , MECL1 mRNA increased in both HeLa and MCF7 cells, in HeLa reaching the range of the constitutive mRNA level in unstimulated IM9 and Reh cells. The corresponding IFN-g stimulation of IM9 and Reh resulted in a relatively small increase of MECL1 mRNA. The Daudi cells behaved differently from the other cell

Fig. 2. Constitutive and IFN-g-induced MECL1 mRNA in different cell lines. MECL1 mRNA signals were detected on a Northern blot of mRNA isolated from different unstimulated Žy. and IFN-g stimulated Žq. cell lines by hybridising to a part of MECL1 cDNA Župper panel.. The cell lines were stimulated with IFN-g at a concentration of 200 Urml for 48 h. The same hybridisation pattern was obtained using a genomic probe from the MECL1 CpG island. The size of the MECL1 transcript was 1.2 kb. Hybridisation signals obtained using a glyceraldehyde-3phosphate dehydrogenase ŽGAPDH. specific probe on the same blot are shown in the lower panel.

lines, expressing MECL1 at an intermediate level and showing hardly any difference between unstimulated and stimulated cells ŽFig. 2.. These results suggest that the constitutive MECL1 transcription in IM9 and Reh cell lines is close to the maximum level of MECL1 transcription induced by IFN-g . 3.4. IFN-g-induction of MECL1 protein and posttranslational processing The induction of MECL1 protein by IFN-g was analysed using Western blotting. Rabbit antibodies were raised against a peptide in the MECL1 sequence. Different human cell lines were examined for IFN-g-induced increase of MECL1 protein. Induction of MECL1 protein from an undetectable level in unstimulated cells, was found in the cell lines HeLa, HeLa S3, Caco-2, and ECV304, as shown for ECV304 in Fig. 3 Žlanes 2 and 4. . Different IFN-g concentrations Ž 25 to 800 Urml. and stimulation periods Ž12 to 72 h. were tested on ECV304 cells. The amount of MECL1 protein showed a graded increase with both IFN-g concentration and stimulation time Ždata not shown. . For subsequent experiments we used conditions providing maximum induction Ž 200 Urml for 48 h.. The IFN-g-induced protein band was estimated by SDS–PAGE to correspond to

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Fig. 3. Processing and incorporation of IFN-g-induced MECL1 protein into proteasomes in ECV304 cells. Lane 1, biotinylated low molecular weight standard; lane 2, extract of unstimulated ECV304 cells; lane 3, extract of ECV304 cells infected with SFV–MECL1 cDNA; lane 4, extract of IFN-g-stimulated ECV304 cells; lane 5, proteasomes purified from unstimulated cells; lane 6, proteasomes purified from IFN-g-stimulated cells. ECV304 cells were stimulated with 200 Urml IFN-g for 48 h. 5 m l Ž8 m g protein. of each cell extract and 2 m g purified proteasomes were loaded on the gel. MECL1 protein bands were detected by polyclonal anti-peptide antibodies.

a molecular mass of 25.1 kDa ŽFig. 3, lane 4.. To compare the size of IFN-g-induced MECL1 with unprocessed MECL1, we expressed full-length MECL1 in ECV304 cells by the SFV expression system, where the endogenous protein synthesis is inhibited. In this system, MECL1 protein was detected by the antibodies at an apparent molecular mass of 28.8 kDa ŽFig. 3, lane 3.. These results suggest that the IFN-g-induced MECL1 protein was processed, consistent with the finding that MECL1 protein is processed at the processing site in the amino acid sequence ŽG-TT. w6x, reducing the theoretical mass of the protein from 28958 to 24589 Da.

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To investigate if the induction by IFN-g was specific, the effect of other cytokines and heat shock on MECL1 expression was tested in the low-expressing cell lines ECV304 and Caco-2. The cells were subjected to stimulation by IL-1, IL-6, TNFa , or TGFb , or to heat shock at 428C. No detectable MECL1 increase was found in the Western blots for any of these treatments Ždata not shown.. 3.5. Incorporation of IFN-g-induced MECL1 into proteasomes To show that IFN-g-induced MECL1 was incorporated into proteasome particles, we isolated 20S proteasomes from both unstimulated and IFN-g-treated ECV304 cells by affinity chromatography. Proteasomes purified from unstimulated cells contained small amounts of MECL1 ŽFig. 3, lane 5., not detectable in the whole cell extract ŽFig. 3, lane 2. . The proteasomes from IFN-g-stimulated cells showed a dramatic increase in MECL1 content ŽFig. 3, lane 6.. The purified proteasomes were also subjected to 2-D IEFrSDS–PAGE gels ŽFig. 4. . The position of MECL1 protein in the 2-D gel patterns ŽFig. 4A and B. was determined by immunoblotting Ž Fig. 4C and D.. The anti-MECL1 antibodies detected only one proteasome subunit ŽFig. 4C and D.. Corresponding to this position, a protein spot was seen after silver staining in proteasomes from IFN-g treated cells ŽFig. 4B. but not from untreated cells Ž Fig. 4A..

Fig. 4. MECL1 detection in proteasomes on 2-D gel. Proteasomes from unstimulated and IFN-g stimulated ECV304 cells were analysed on 2-D IEFrSDS–PAGE by silver staining ŽA and B. and anti-MECL1 antibodies ŽC and D.. Proteins separated from pH 4 Žleft. to pH 9 Žright. and from 32 Žtop. to 20 Žbottom. kDa are included.

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Thus, MECL1 protein content of proteasomes seemed to increase markedly after IFN-g stimulation. Other changes following IFN-g treatment could also be seen, but only MECL1 was subjected to identification.

3.6. ConstitutiÕe expression of MECL1 protein We then examined whether the constitutive MECL1 mRNA level seen in B cell lines ŽFig. 2. was expressed at the protein level. Reh and Daudi cells were stimulated with IFN-g , and the extracts of unstimulated and stimulated cells analysed by Western blotting Ž Fig. 5, lanes 1–4. . The IM9 cells showing similar MECL1 mRNA levels as Reh ŽFig. 2., were not tested for MECL1 protein. The Reh cells showed constitutive expression of MECL1 protein, increasing only slightly in response to IFN-g Ž Fig. 5, lanes 1 and 2.. Daudi cells also expressed MECL1 protein constitutively but at a lower level, showing no induction by IFN-g ŽFig. 5, lanes 3 and 4.. Thus, the MECL1 protein levels correspond well with mRNA levels in these two B cell lines. We also examined a monocyte cell line, THP-1, for constitutive and IFN-g-induced expression of

Fig. 5. Constitutive and IFN-g-induced MECL1 protein in different cell extracts. Lane 1, extract of unstimulated Reh cells; lane 2, IFN-g-stimulated Reh cells; lane 3, unstimulated Daudi cells; lane 4, IFN-g-stimulated Daudi cells; lane 5, unstimulated THP-1 cells; lane 6, IFN-g-stimulated THP-1 cells; lane 7, ECV304 cells grown in Reh-conditioned RPMI 1640 medium for 48 h; lane 8, ECV304 cells grown in RPMI 1640 containing IFN-g ; lane 9, freshly isolated human white blood cells; lane 10, a fraction of the white blood cells isolated by adhering to tissue culture plates. The position of the MECL1 band Žmarked by an arrow. was consistent with processed MECL1. 8 m g protein extract was loaded in each lane. IFN-g treatment was performed using 200 Urml for 48 h. MECL1 was detected in different cell extracts by anti-MECL1 antibodies. The band seen above the MECL1 band in the THP-1 cells Žlanes 5 and 6. corresponded to a strong cell line-specific protein band with estimated molecular weight similar to unprocessed MECL1, but may be unspecific.

MECL1 protein Ž Fig. 5, lanes 5 and 6.. In the unstimulated THP-1 cells MECL1 protein was present at an intermediate level Ž Fig. 5, lane 5. , similar to the level seen in Daudi cells Žlane 3.. IFN-g stimulation of the THP-1 cells resulted in high expression of MECL1 protein Ž Fig. 5, lane 6. , similar to that detected for Reh cells. In all these cell lines, the apparent molecular mass of MECL1 protein was similar to the molecular mass of the processed form of MECL1 found in proteasome particles from ECV304 cells ŽFig. 3.. As active b-type subunits are processed at assembly of the proteasome particles w4,44,45x, processed proteasome subunits that are not incorporated into proteasomes are normally not found in the cell. Thus, the constitutively expressed MECL1 protein in these cell lines is likely to be processed and incorporated into proteasome particles, in analogy with the incorporation of MECL1 into proteasomes of stimulated ECV304 cells. We can, therefore, assume that cell lines with high constitutive MECL1 expression contain more proteasomes with MECL1 than other cell lines. Conceivably, autocrine or paracrine factors released to the media by monocyte and B cell lines could explain the high constitutive expression of MECL1 in these cell lines. We tested this possibility by growing ECV304 cells in RPMI 1640 and Medium 199 media conditioned by Reh, Daudi and THP-1 cells, respectively. No induction of MECL1 was detected ŽFig. 5, lane 7 for Reh-conditioned RPMI 1640 medium.. As a positive control, ECV304 cells grown in RPMI 1640 medium showed normal induction of MECL1 by IFN-g ŽFig. 5, lane 8.. Therefore, the constitutive expression of MECL1 in Reh, Daudi and THP-1 cells could not be explained by a diffusible and transferable factor released by these cells to the medium, although we can not exclude that ECV304 may lack receptors for such factors. Finally, we examined human white blood cells for the level of MECL1 protein. Mononuclear cells were isolated from buffy coat of freshly donated blood. Both the white blood cells and the monocyte fraction of these, isolated by adhering to tissue culture plates, were subjected to Western blotting Ž Fig. 5, lanes 9 and 10.. Both fractions showed a strong MECL1 protein signal, suggesting that MECL1 is constitutively expressed also in normal human white blood cells.

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4. Discussion MECL1 was found to be expressed at different levels in different cell lines. Cell lines unrelated to the immune system showed generally very low constitutive levels but markedly increased expression upon stimulation with IFN-g . B cell lines showed high constitutive expression levels and small effect of IFN-g . A monocyte cell line showed both constitutive expression of MECL1 and IFN-g inducibility. The differences in constitutive and IFN-g-induced expression of MECL1 were demonstrated both at the mRNA and the protein level. It is therefore likely that the regulation of MECL1 in these cell lines is different and may be functionally important. The IFN-g-induction of MECL1 has previously been shown in various nonimmune cell lines, both in human cells Ž HeLa and two renal carcinoma cell lines. w6x and in the murine B8 fibroblast w12x and H6 hepatoma cells w13x. These cell lines all showed a low or not detectable constitutive level of MECL1, consistent with our results. A major question is whether the difference we observed for constitutive MECL1 expression is present only in cell lines or whether it is also valid for cells in the human body. Although this question was not addressed in detail in the present study, we obtained some evidence that at least some white blood cells in the human body show a high constitutive expression of MECL1. Our group previously reported the expression of MECL1 mRNA in various human tissues Ž adrenal gland, spleen, lung, ileum, jejunum, pancreas and liver. w29x. Possibly, some of the detected MECL1 mRNA may stem from aggregates of lymphoid cells contained in some of these tissues. This is supported by the recent reports of MECL1 being expressed at high levels in murine spleen, thymus and lymph nodes w46x and in bovine spleen w47x. The observed expression pattern of MECL1 may in part be explained by the promoter sequence of the MECL1 gene. The MECL1 gene contains an unmethylated CpG island which covers the first six of the eight exons ŽRef. w29x and this study. . Unmethylated CpG islands are often, but not exclusively, associated with ‘housekeeping’ genes, consistent with a wide constitutive expression at a low level. Close to the transcriptional start region, several sequences were

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identified that may contribute to the regulation of MECL1 gene expression. The IFN-g-induction of MECL1 could be mediated through STAT1 binding to the GAS-like sequences. However, this is unlikely because of the time course of induction. Rather, since MECL1 was induced only after hours of stimulation with IFN-g , other sequences might potentially be involved through cascades following STAT1 activation. For instance, an ISRE element was found to mediate the IFN-g induction of the immunoproteasome subunit LMP2 by binding the IFN-g-inducible IRF-1 w48,49x. In addition to being involved in the IFN-g induction pathway, STAT1 might also be responsible for the high constitutive expression of MECL1 found in B cell lines. In fact, STAT1 protein has been reported to be constitutively active in peripheral blood cells from leukaemia patients and in Epstein–Barr virus-related lymphoma cell lines, including Daudi cells w50x. However, whereas no constitutive STAT1-DNA binding activity was detected in nuclei from mononuclear cells of healthy individuals in that study w50x, we found high expression of MECL1 in peripheral blood cells, suggesting that other mechanisms may be responsible in normal blood cells. Interestingly, the two IRF family members interferon consensus sequence binding protein ŽICSBP. and lymphocyte-specific IRF Ž LSIRFrIRF4rPip. are expressed mainly in cells of the immune system w51–53x, with particularly high expression of LSIRFrIRF-4rPip in B cells w53x. In light of the various functional combinations of the IRF family members w54x, the lymphocyte-specific expression of certain IRFs might be relevant for the constitutive expression of MECL1 in B cell lines. Functional studies of the MECL1 promoter are needed, however, to reveal the regulatory mechanisms involved. Interestingly, MECL1 showed similar expression pattern as the other immunoproteasome subunits LMP2 and LMP7. As MECL1, these subunits are expressed at a low level in cells unrelated to the immune system and are induced by IFN-g in these cells w55–57x. Moreover, in tissues and cells related to the immune system LMP2 and LMP7 are found at high constitutive levels, as shown for murine spleen and thymus w58x, the B cell line 721.45 w18,59x, the murine T cell line RMA and the murine macrophage cell line RHW w58x. Thus, MECL1, LMP2 and LMP7 are all IFN-g-inducible proteasome subunits with par-

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ticularly high constitutive expression in different immune-related cells. Further studies are needed to elucidate the possible importance of the concerted regulation of MECL1, LMP2 and LMP7 in the functions of the immune system.

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Acknowledgements The AMA cell cDNA library was a kind gift from ˚ Dr. Henrik Leffers, Arhus University. SFV vectors were kindly donated by Dr. Henrik Garoff, Novum Research Centre, Sweden. We are grateful to Dr. Klavs Hendil for the MCP-21 hybridoma cells, and Liv Eide for DNA sequencing. We thank Dr. Ole Paulsen for useful comments on the manuscript. G.S.F. and F.L. were fellows of the Research Council of Norway and The Norwegian Cancer Society, respectively. This study was also supported by grants to H.P. from the Research Council of Norway and The Norwegian Cancer Society.

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References w1x K.L. Rock, C. Gramm, L. Rothstein, K. Clark, R. Stein, L. Dick, D. Hwang, A.L. Goldberg, Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules, Cell 78 Ž1994. 761–771. w2x A.J. Rivett, Proteasomes: multicatalytic proteinase complexes, Biochem. J. 291 Ž1993. 1–10. w3x F. Kopp, K.B. Hendil, B. Dahlmann, P. Kristensen, A. Sobek, W. Uerkvitz, Subunit arrangement in the human 20S proteasome, Proc. Natl. Acad. Sci. U.S.A. 94 Ž1997. 2939– 2944. w4x M. Groll, L. Ditzel, J. Lowe, D. Stock, M. Bochtler, H.D. ¨ Bertunik, R. Huber, Structure of 20S proteasome from yeast ˚ resolution, Nature 386 Ž1997. 463–471. at 2.4 A w5x Y. Yang, J.B. Waters, K. Fruh, ¨ P.A. Peterson, Proteasomes are regulated by interferon g : implications for antigen processing, Proc. Natl. Acad. Sci. U.S.A. 89 Ž1992. 4928–4932. w6x H. Hisamatsu, N. Shimbara, Y. Saito, P. Kristensen, K.B. Hendil, T. Fujiwara, E. Takahashi, N. Tanahashi, T. Tamura, A. Ichihara, K. Tanaka, Newly identified pair of proteasomal subunits regulated reciprocally by interferon-g , J. Exp. Med. 183 Ž1996. 1807–1816. w7x K. Akiyama, S. Kagawa, T. Tamura, N. Shimbara, M. Takashina, P. Kristensen, K.B. Hendil, K. Tanaka, A. Ichihara, Replacement of proteasome subunits X and Y by LMP7 and LMP2 induced by interferon-g for acquirement

w14x

w15x

w16x

w17x

w18x

w19x

w20x

w21x

of the functional diversity responsible for antigenic processing, FEBS Lett. 343 Ž1994. 85–88. K. Fruh, ¨ M. Gossen, K. Wang, H. Bujard, P.A. Peterson, Y. Yang, Displacement of housekeeping proteasome subunits by MHC-encoded LMPs: a newly discovered mechanism for modulating the multicatalytic proteinase complex, EMBO J. 13 Ž1994. 3236–3244. K. Akiyama, K. Yokota, S. Kagawa, N. Shimbara, T. Tamura, H. Akioka, H.G. Nothwang, C. Noda, K. Tanaka, A. Ichihara, cDNA cloning and interferon g down-regulation of proteasomal subunits X and Y, Science 265 Ž1994. 1231–1234. M.P. Belich, R.J. Glynne, G. Senger, D. Sheer, J. Trowsdale, Proteasome components with reciprocal expression to that of the MHC-encoded LMP proteins, Curr. Biol. 4 Ž1994. 769–776. M. Aki, N. Shimbara, M. Takashina, K. Akiyama, S. Kagawa, T. Tamura, N. Tanahashi, T. Yoshimura, K. Tanaka, A. Ichihara, Interferon-g induces different subunit organizations and functional diversity of proteasomes, J. Biochem. 115 Ž1994. 257–269. M. Groettrup, R. Kraft, S. Kostka, S. Standera, R. Stohwasser, P.-M. Kloetzel, A third interferon-g-induced subunit exchange in the 20S proteasome, Eur. J. Immunol. 26 Ž1996. 863–869. D. Nandi, H. Jiang, J.J. Monaco, Identification of MECL-1 ŽLMP-10. as the third IFN-g-inducible proteasome subunit, J. Immunol. 156 Ž1996. 2361–2364. E. Seemuller, A. Lupas, D. Stock, J. Lowe, R. Huber, W. ¨ ¨ Baumeister, Proteasome from Thermoplasma acidophilum: a threonine protease, Science 268 Ž1995. 579–582. J. Driscoll, M.G. Brown, D. Finley, J.J. Monaco, MHC-linked LMP gene products specifically alter peptidase activities of the proteasome, Nature 365 Ž1993. 262–264. M. Gaczynska, K.L. Rock, A.L. Goldberg, g-Interferon and expression of MHC genes regulate peptide hydrolysis by proteasomes, Nature 365 Ž1993. 264–267. B. Boes, H. Hengel, T. Ruppert, G. Multhaup, U.H. Koszinowski, P.-M. Kloetzel, Interferon g stimulation modulates the proteolytic activity and cleavage site preference of 20S mouse proteasomes, J. Exp. Med. 179 Ž1994. 901–909. M. Gaczynska, K.L. Rock, T. Spies, A.L. Goldberg, Peptidase activities of proteasomes are differentially regulated by the major histocompatibility complex-encoded genes for LMP2 and LMP7, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 9213–9217. H.-G. Rammensee, K. Falk, O. Rotzschke, Peptides natu¨ rally presented by MHC class I molecules, Annu. Rev. Immunol. 11 Ž1993. 213–244. D. Arnold, J. Driscoll, A. Androlewicz, E. Hughes, P. Cresswell, T. Spies, Proteasome subunits encoded in the MHC are not generally required for the processing of peptides bound by MHC class I molecules, Nature 360 Ž1992. 171–174. F. Momburg, V. Ortiz-Navarrete, J. Neefjes, E. Goulmy, Y. van de Wal, H. Spits, S.J. Powis, G.W. Butcher, J.C.

G.S. Foss et al.r Biochimica et Biophysica Acta 1402 (1998) 17–28

w22x

w23x

w24x

w25x

w26x

w27x

w28x

w29x

w30x

w31x

w32x

w33x w34x

w35x

w36x

Howard, P. Walden, G.J. Hammerling, Proteasome subunits ¨ encoded by the major histocompatibility complex are not essential for antigen presentation, Nature 360 Ž1992. 174– 177. H.J. Fehling, W. Swat, C. Laplace, R. Kuhn, ¨ K. Rajewsky, U. Muller, H. von Boehmer, MHC class I expression in ¨ mice lacking the proteasome subunit LMP-7, Science 265 Ž1994. 1234–1237. L. Van Kaer, P.G. Ashton-Rickardt, M. Eichelberger, M. Gaczynska, K. Nagashima, K.L. Rock, A.L. Goldberg, P.C. Doherty, S. Tonegawa, Altered peptidase and viral-specific T cell response in LMP2 mutant mice, Immunity 1 Ž1994. 533–541. R. Stohwasser, U. Kuckelkorn, R. Kraft, S. Kostka, P.-M. Kloetzel, 20S proteasomes from LMP7 knock out mice reveals altered proteolytic activities and cleavage site preferences, FEBS Lett. 383 Ž1996. 109–113. V. Cerundolo, A. Kelly, T. Elliott, J. Trowsdale, A. Townsend, Genes encoded in the major histocompatibility complex affecting the generation of peptides for TAP transport, Eur. J. Immunol. 25 Ž1995. 554–562. C. Sibille, K.G. Gould, K. Willard-Gallo, S. Thomson, A.J. Rivett, S. Powis, G.W. Butcher, P. De Baetselier, LMP2q proteasomes are required for the presentation of specific antigens to cytotoxic T lymphocytes, Curr. Biol. 5 Ž1995. 923–930. M.G. Brown, J. Driscoll, J.J. Monaco, Structural and serological similarity of MHC-linked LMP and proteasome Žmulticatalytic proteinase. complexes, Nature 353 Ž1991. 355–357. R. Glynne, S.H. Powis, S. Beck, A. Kelly, L.-A. Kerr, J. Trowsdale, A proteasome-related gene between the two ABC transporter loci in the class II region of the human MHC, Nature 353 Ž1991. 357–360. F. Larsen, J. Solheim, T. Kristensen, A.-B. Kolstø, H. Prydz, A tight cluster of five unrelated human genes on chromosome 16q22.1, Hum. Mol. Genet. 2 Ž1993. 1589– 1595. A.P. Feinberg, V. Vogelstein, A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal. Biochem. 132 Ž1983. 6–13. M. Kozak, Compilation and analysis of sequence from the translational start site in eukaryotic mRNAs, Nucleic Acids Res. 12 Ž1983. 857–872. K.B. Hendil, W. Uerkvitz, The human multicatalytic proteinase: affinity purification using a monoclonal antibody, J. Biochem. Biophys. Methods 22 Ž1991. 159–165. E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988. E. Hornes, L. Korsnes, Magnetic DNA hybridization properties of oligonucleotide probes attached to superparamagnetic beads and their use in the isolation of polyŽA. mRNA from eukaryotic cells, Genet. Anal. Tech. Appl. 7 Ž1990. 145–150. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 Ž1970. 680–685. A.-B. Kolstø, G. Kollias, V. Giguere, K.I. Isobe, H. Prydz,

w37x

w38x

w39x

w40x

w41x

w42x

w43x

w44x

w45x

w46x

w47x

w48x

w49x

w50x

27

F. Grosveld, The maintenance of methylation free islands in transgenic mice, Nucleic Acids Res. 14 Ž1986. 9667–9678. J.T. Kadonaga, K.A. Jones, R. Tjian, Promoter-specific activation of RNA polymerase II transcription by Sp1, Trends Biochem. Sci. 11 Ž1986. 20–23. M.A. Meraz, J.M. White, K.C.F. Sheehan, E.A. Bach, S.J. Rodig, A.S. Dighe, D.H. Kaplan, J.K. Riley, A.C. Greenlund, D. Campbell, K. Carver-Moore, R.N. DuBois, R. Clark, M. Aguet, R.D. Schreiber, Targeted disruption of the Stat1 gene in mice reveals unexpected physiological specificity in the JAK-STAT signaling pathway, Cell 84 Ž1996. 431–442. J.E. Durbin, R. Hackenmiller, M.C. Simon, D.E. Levy, Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease, Cell 84 Ž1996. 443–450. J.E. Darnell Jr., I.M. Kerr, G.R. Stark, Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signalling proteins, Science 264 Ž1994. 1415– 1421. N. Tanaka, T. Kawakami, T. Taniguchi, Recognition DNA sequences of interferon regulatory factor 1 ŽIRF-1. and IRF-2, regulators of cell growth and the interferon system, Mol. Cell. Biol. 13 Ž1993. 4531–4538. Z. Yang, M. Sugawara, P.D. Ponath, L. Wessendorf, J. Banerji, Y. Li, J.L. Strominger, Interferon g response region in the promoter of the human DPA gene, Proc. Natl. Acad. Sci. U.S.A. 87 Ž1990. 9226–9230. X. Weihua, V. Kolla, D.V. Kalvakolanu, Interferon g-induced transcription of the murine ISGF3g Žp48. gene is mediated by novel factors, Proc. Natl. Acad. Sci. U.S.A. 94 Ž1997. 103–108. P. Zwickl, F. Lottspeich, W. Baumeister, Expression of functional Thermoplasma acidophilum proteasomes in Escherichia coli, FEBS Lett. 312 Ž1992. 157–160. K. Fruh, ¨ Y. Yang, D. Arnold, J. Chambers, L. Wu, J.B. Waters, T. Spies, P.A. Peterson, Alternative exon usage and processing of the major histocompatibility complex-encoded proteasome subunits, J. Biol. Chem. 267 Ž1992. 22131– 22140. R. Stohwasser, S. Standera, I. Peters, P.-M. Kloetzel, M. Groettrup, Molecular cloning of the mouse proteasome subunits MC14 and MECL-1: reciprocally regulated tissue expression of interferon-g-modulated proteasome subunits, Eur. J. Immunol. 27 Ž1997. 1182–1187. A.M. Eleuteri, R.A. Kohanski, C. Cardozo, M. Orlowski, Bovine spleen multicatalytic proteinase complex Žproteasome., J. Biol. Chem. 272 Ž1997. 11824–11831. L.C. White, K.L. Wright, N.J. Felix, H. Ruffner, L.F.L. Reis, R. Pine, J.P.-Y. Ting, Regulation of LMP2 and TAP1 genes by IRF-1 explains the paucity of CD8q T cells in IRF-1yry mice, Immunity 5 Ž1996. 365–376. R. Pine, A. Canova, C. Schindler, Tyrosine phosphorylated p91 binds to a single element in the ISGF2rIRF-1 promoter to mediate induction by IFN a and IFNg , and is likely to autoregulate the p91 gene, EMBO J. 13 Ž1994. 158–167. R. Weber-Nordt, C. Egen, J. Wehinger, W. Ludwig, V.

28

w51x

w52x

w53x

w54x

G.S. Foss et al.r Biochimica et Biophysica Acta 1402 (1998) 17–28 Gouilleux-Gruart, R. Mertelsmann, J. Finke, Constitutive activation of STAT proteins in primary lymphoid and myeloid leukemia cells and in Epstein–Barr virus ŽEBV.-related lymphoma cell lines, Blood 88 Ž1996. 809–816. P.H. Driggers, D.L. Ennist, S.L. Gleason, W.-H. Mak, M.S. Marks, B.-Z. Levi, J.R. Flanagan, E. Appella, K. Ozato, An interferon g-regulated protein that binds the interferon-inducible enhancer element of major histocompatibility complex class I genes, Proc. Natl. Acad. Sci. U.S.A. 87 Ž1990. 3743–3747. T. Matsuyama, A. Grossman, H.-W. Mittrucker, D.P. ¨ Siderovski, K. Kiefer, T. Kawakami, C.D. Richardson, T. Taniguchi, S.K. Yoshinaga, T.W. Mak, Molecular cloning of LSIRF, a lymphoid-specific member of the interferon regulatory factor family that binds the interferon-stimulated response element ŽISRE., Nucleic Acids Res. 23 Ž1995. 2127–2136. C.F. Eisenbeis, H. Singh, U. Storb, Pip, a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator, Genes Dev. 9 Ž1995. 1377–1387. C. Bovolenta, P.H. Driggers, M.S. Marks, J.A. Medin, A.D. Politis, S.N. Vogel, D.E. Levy, K. Sakaguchi, E. Appella, J.E. Coligan, K. Ozato, Molecular interactions between interferon consensus sequence binding protein and members

w55x

w56x

w57x

w58x

w59x

of the interferon regulatory factor family, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 5046–5050. V. Ortiz-Navarrete, A. Seelig, M. Gernold, S. Frenzel, P.M. Kloetzel, G.J. Hammerling, Subunit of the ‘20S’ proteasome ¨ Žmulticatalytic proteinase. encoded by the major histocompatibility complex, Nature 353 Ž1991. 662–664. C.K. Martinez, J.J. Monaco, Homology of proteasome subunits to a major histocompatibility complex-linked LMP gene, Nature 353 Ž1991. 664–667. A. Kelly, S.H. Powis, R. Glynne, E. Radley, S. Beck, J. Trowsdale, Second proteasome-related gene in the human MHC class II region, Nature 353 Ž1991. 667–668. S. Frentzel, I. Kuhn-Hartmann, M. Gernold, P. Gott, ¨ A. Seelig, P.-M. Kloetzel, The major-histocompatibility-complex-encoded beta-type proteasome subunits LMP2 and LMP7. Evidence that LMP2 and LMP7 are synthesized as proproteins and that cellular levels of both mRNA and LMP-containing 20S proteasomes are differentially regulated, Eur. J. Biochem. 216 Ž1993. 119–126. V. Ustrell, G. Pratt, M. Rechsteiner, Effects of interferon g and major histocompatibility complex-encoded subunits on peptidase activities of human multicatalytic proteases, Proc. Natl. Acad. Sci. U.S.A. 92 Ž1995. 584–588.