Reduced Immunoproteasome Formation and Accumulation of Immunoproteasomal Precursors in the Brains of Lymphocytic Choriomeningitis Virus-Infected Mice

Kremer, Marcel; Henn, Anja; Kolb, Cornelia; Basler, Michael; Moebius, Jacqueline; Guillaume, Benoı̂t; Leist, Marcel; Eynde, Benoı̂t J. Van den; Groettrup, Marcus

doi:10.4049/jimmunol.1001517

Cited by 59 publications

(61 citation statements)

References 42 publications

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“…1a, the different chicken proteasomes possess high similarity in the numbers and migratory positions of protein spots and the proteasome subunits are distributed over the same isoelectric point (pI) as for human and murine proteasome. In complete brain tissue of the mouse, only constitutive 20S proteasome is visible in Coomassie-stained 2D gels (Kremer et al 2010). In contrast to the chicken spleen, in the mouse spleen, about equal amounts of constitutive 20S proteasome and 20S immunoproteasome was found and the immunoproteasome subunits β1i (LMP2), β2i (MECL-1), and β5i (LMP7) were readily visible on Coomassie-stained 2D gels and easily distinguished from the constitutive subunits β1, β2, and β5 due to marked differences in molecular mass and/or isoelectric point (Groettrup et al 1996;Khan et al 2001;Kremer et al 2010).…”

Section: Identification Of Chicken Proteasome Subunits In Different Omentioning

confidence: 99%

“…In complete brain tissue of the mouse, only constitutive 20S proteasome is visible in Coomassie-stained 2D gels (Kremer et al 2010). In contrast to the chicken spleen, in the mouse spleen, about equal amounts of constitutive 20S proteasome and 20S immunoproteasome was found and the immunoproteasome subunits β1i (LMP2), β2i (MECL-1), and β5i (LMP7) were readily visible on Coomassie-stained 2D gels and easily distinguished from the constitutive subunits β1, β2, and β5 due to marked differences in molecular mass and/or isoelectric point (Groettrup et al 1996;Khan et al 2001;Kremer et al 2010). To identify the subunits of chicken 20S proteasome from the brain and spleen and to exclude that putative immunosubunits migrated equally as constitutive ones, 18 spots were excised from 2D gels and were analyzed by mass spectrometry.…”

Section: Identification Of Chicken Proteasome Subunits In Different Omentioning

confidence: 99%

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No evidence for immunoproteasomes in chicken lymphoid organs and activated lymphocytes

Erath

Groettrup

2014

Immunogenetics

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The proteasome is the main protein-degrading machine within the cell, producing ligands for MHC class I molecules. It is a cylindrical multicatalytic protease complex, and the catalytic activity is mediated by the three subunits β1, β2, and β5 which possess caspase-, trypsin-, and chymotrypsin-like activities, respectively. By stimulation with interferon (IFN)-γ the replacement of these subunits by β1i, β2i, and β5i is induced leading to formation of immunoproteasomes with altered proteolytic and antigen processing properties. The genes coding for these immunosubunits are restricted to jawed vertebrates but have so far not been found in the genomes of birds, e.g., chicken, turkey, quail, black grouse and zebra finch. However, the chicken genome sequences are not completely assigned; therefore, we investigated the presence of immunoproteasome on protein level. 20S proteasome was purified from the chicken brain, blood, spleen, and bursa of Fabricius, followed by separation via two-dimensional (2D) gel electrophoresis. We analyzed the protein spots derived from the spleen and brain by mass spectrometry and could identify all 14 proteasomal subunits, but there were no differences detectable in the spot patterns. Moreover, we stimulated the chicken spleen cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin aiming at the induction of immunoproteasome, but in spite of the induction of proliferation and IFN-γ, no evidence for immunoproteasome formation in chicken could be obtained. This result was substantiated by the finding that 20S proteasomes isolated from immune and non-immune tissues showed very similar peptidolytic activities. Taken together, our results indicate that chicken lack immunoproteasomes also on protein level.

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Section: Identification Of Chicken Proteasome Subunits In Different Omentioning

confidence: 99%

Section: Identification Of Chicken Proteasome Subunits In Different Omentioning

confidence: 99%

No evidence for immunoproteasomes in chicken lymphoid organs and activated lymphocytes

Erath

Groettrup

2014

Immunogenetics

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“…Membranes were washed again and proteins were visualized with enhanced chemiluminescence. Primary antibodies used were: anti-LMP7 and anti-LMP2 (Kremer et al, 2010), anti-␤5 (D1H6B, Cell Signaling Technology), anti-␤1 (clone E1K90, Cell Signaling Technology), anti-␣1 (clone IB5, K. Scherrer, Paris, France), anti-IB␣ (clone L35A5, Cell Signaling Technology), anti-␣-tubulin (clone AA13, Sigma).…”

Section: Sds Page and Western Blotmentioning

confidence: 99%

Immunoproteasome subunit deficiency has no influence on the canonical pathway of NF-κB activation

Bitzer

Basler

Krappmann

et al. 2017

Molecular Immunology

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a b s t r a c tActivation of the pro-inflammatory transcription factor NF-B requires signal-induced proteasomal degradation of the inhibitor of NF-B (IB) in order to allow nuclear translocation. Most cell types are capable of expressing two types of 20S proteasome core particles, the constitutive proteasome and immunoproteasome. Inducible under inflammatory conditions, the immunoproteasome is mainly characterized through an altered cleavage specificity compared to the constitutive proteasome. However, the question whether immunoproteasome subunits affect NF-B signal transduction differently from constitutive subunits is still up for debate. To study the effect of immunoproteasomes on LPS-or TNF-␣-induced NF-B activation, we used IFN-␥ stimulated peritoneal macrophages and mouse embryonic fibroblasts derived from mice deficient for the immunoproteasome subunits low molecular mass polypeptide (LMP) 2, or LMP7 and multicatalytic endopeptidase complex-like 1 (MECL-1). Along the canonical signaling pathway of NF-B activation no differences in the extent and kinetic of IB degradation were observed. Neither the nuclear translocation and DNA binding of NF-B nor the production of the NF-B dependent cytokines TNF-␣, IL-6, and IL-10 differed between immunoproteasome deficient and proficient cells. Hence, we conclude that immunoproteasome subunits have no specialized function for canonical NF-B activation.

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“…A total of 1 mg purified proteasomes was separated by SDS-PAGE (15% gel), blotted onto nitrocellulose (Schleicher & Schuell BioSciences, Dassel, Germany), blocked (PBS/5% w/v low-fat dry milk/0.2% Tween 20) for 1 h, and agitated overnight at 4˚C with an affinity-purified polyclonal rabbit Ab recognizing MECL-1 (22). The blots were washed three times and incubated for 2 h with peroxidase-conjugated anti-rabbit Ab.…”

Section: Western Blottingmentioning

confidence: 99%

The Antiviral Immune Response in Mice Devoid of Immunoproteasome Activity

Basler

Beck

Kirk³

et al. 2011

The Journal of Immunology

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The replacement of the catalytically active proteasome subunits β1, β2, and β5 by the immunoproteasome subunits low molecular mass polypeptide (LMP) 2 (β1i), multicatalytic endopeptidase complex-like–1 (MECL-1) (β2i), and LMP7 (β5i) is required for the production of numerous class I ligands. Hitherto, investigation of the immunoproteasome was confined to the analysis of mice deficient for one or two immunosubunits. In this study, we characterized LMP2−/−/MECL-1−/− double-deficient mice and used the well-defined LMP7-selective inhibitor ONX 0914 in these mice to generate mice lacking the activity of all immunoproteasome subunits. LMP2−/−/MECL-1−/− double-deficient mice had strongly reduced numbers of CD8+ T cells in the spleen. Nevertheless, infection with the lymphocytic choriomeningits virus induced a normal cytotoxic T cell response in these mice, although the T cell response to several class I epitopes was altered. Treatment of LMP2−/−/MECL-1−/− double-deficient mice with the LMP7-selective inhibitor ONX 0914 elicited a strong CTL response in lymphocytic choriomeningitis virus-infected mice. Thereby, the TCD8+ response to nucleoprotein 205–212, which is barely detectable in LMP2−/−/MECL-1−/− double-deficient mice, could be reverted to normal levels by LMP7 inhibition. Additional experiments could demonstrate that the increased CTL response to the nucleoprotein 205–212 in mice lacking functional immunoproteasome is due to an altered class I presentation of this epitope. Taken together, to our knowledge, this is the first study investigating viral infection in mice lacking activity of all three immunoproteasome subunits.

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Reduced Immunoproteasome Formation and Accumulation of Immunoproteasomal Precursors in the Brains of Lymphocytic Choriomeningitis Virus-Infected Mice

Abstract: Material

Cited by 59 publications

References 42 publications

No evidence for immunoproteasomes in chicken lymphoid organs and activated lymphocytes

No evidence for immunoproteasomes in chicken lymphoid organs and activated lymphocytes

Immunoproteasome subunit deficiency has no influence on the canonical pathway of NF-κB activation

The Antiviral Immune Response in Mice Devoid of Immunoproteasome Activity

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