Deletion of Immunoproteasome Subunits Imprints on the Transcriptome and Has a Broad Impact on Peptides Presented by Major Histocompatibility Complex I molecules

Proteasome-mediated proteolysis plays a crucial role in many basic cellular processes. In addition to constitutive proteasomes (CPs), which are found in all eukaryotes, jawed vertebrates also express immunoproteasomes (IPs). Evidence suggests that the key role of IPs may hinge on their impact on the repertoire of peptides associated to major histocompatibility complex (MHC) I molecules. Using a label-free quantitative proteomics approach, we identified 417 peptides presented by MHC I molecules on primary mouse dendritic cells (DCs). By comparing MHC I-associated peptides (MIPs) eluted from primary DCs and thymocytes, we found that the MIP repertoire concealed a cell type-specific signature correlating with cell function. Notably, mass spectrometry analyses of DCs expressing or not IP subunits MECL1 and LMP7 showed that IPs substantially increase the abundance and diversity of MIPs. Bioinformatic analyses provided evidence that proteasomes harboring LMP7 and MECL1 have specific cleavage preferences and recognize unstructured protein regions. Moreover, while differences in MIP repertoire cannot be attributed to potential effects of IPs on gene transcription, IP subunits deficiency altered mRNA levels of a set of genes controlling DC function. Regulated genes segregated in clusters that were enriched in chromosomes 4 and 8. Our peptidomic studies performed on untransfected primary cells provide a detailed account of the MHC I-associated immune self. This work uncovers the dramatic impact of IP subunits MECL1 and LMP7 on the MIP repertoire and their non-redundant influence on expression of immune-related genes. Proteasome-mediated proteolysis plays a crucial role in many basic cellular processes. In addition to constitutive proteasomes (CPs), which are found in all eukaryotes, jawed vertebrates also express immunoproteasomes (IPs). Evidence suggests that the key role of IPs may hinge on their impact on the repertoire of peptides associated to major histocompatibility complex (MHC) I molecules. Using a label-free quantitative proteomics approach, we identified 417 peptides presented by MHC I molecules on primary mouse dendritic cells (DCs). By comparing MHC I-associated peptides (MIPs) eluted from primary DCs and thymocytes, we found that the MIP repertoire concealed a cell type-specific signature correlating with cell function. Notably, mass spectrometry analyses of DCs expressing or not IP subunits MECL1 and LMP7 showed that IPs substantially increase the abundance and diversity of MIPs. Bioinformatic analyses provided evidence that proteasomes harboring LMP7 and MECL1 have specific cleavage preferences and recognize unstructured protein regions. Moreover, while differences in MIP repertoire cannot be attributed to potential effects of IPs on gene transcription, IP subunits deficiency altered mRNA levels of a set of genes controlling DC function. Regulated genes segregated in clusters that were enriched in chromosomes 4 and 8. Our peptidomic studies performed on untransfected primary cells provide a detailed account of the MHC I-associated immune self. This work uncovers the dramatic impact of IP subunits MECL1 and LMP7 on the MIP repertoire and their non-redundant influence on expression of immune-related genes. Proteasomes are the main proteases responsible for protein degradation and the production of major histocompatibility complex I (MHC I)1 ligands (1.Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules.Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2194) Google Scholar, 2.Wherry E.J. Golovina T.N. Morrison S.E. Sinnathamby G. McElhaugh M.J. Shockey D.C. Eisenlohr L.C. Re-evaluating the generation of a "proteasome-independent" MHC class I-restricted CD8 T cell epitope.J. Immunol. 2006; 176: 2249-2261Crossref PubMed Scopus (39) Google Scholar, 3.Loureiro J. Ploegh H.L. Antigen presentation and the ubiquitin-proteasome system in host-pathogen interactions.Adv. Immunol. 2006; 92: 225-305Crossref PubMed Scopus (132) Google Scholar, 4.Shastri N. Cardinaud S. Schwab S.R. Serwold T. Kunisawa J. All the peptides that fit: the beginning, the middle, and the end of the MHC class I antigen-processing pathway.Immunol. Rev. 2005; 207: 31-41Crossref PubMed Scopus (95) Google Scholar). Proteasomes are much more ancient than MHC molecules. Whereas proteasomes are found in all eukaryotes, the MHC appeared only in jawed vertebrates. Proteasomal degradation regulates many basic cellular processes such as cell cycle and division, differentiation and development, response to stress and extracellular effectors, modulation of cell surface receptors, DNA repair, transcriptional regulation and biogenesis of organelles (5.Glickman M.H. Ciechanover A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction.Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3341) Google Scholar, 6.Deshaies R.J. Joazeiro C.A. RING domain E3 ubiquitin ligases.Annu. Rev. Biochem. 2009; 78: 399-434Crossref PubMed Scopus (1876) Google Scholar). The 20S proteolytic core of the proteasome is hollow and provides an enclosed cavity open at both ends in which proteins are degraded (7.Kloetzel P.M. Antigen processing by the proteasome.Nat. Rev. Mol. Cell Biol. 2001; 2: 179-187Crossref PubMed Scopus (494) Google Scholar). The eukaryotic 20S particle is composed of 14 different subunits organized in a barrel-shaped complex with the stoichiometry α7β7β7α7. Three subunits of the two inner β-rings (β1, β2, and β5) participate directly in peptide bond cleavage. While all eukaryotes express the above-described constitutive proteasome (CP), gnathostomes (jawed vertebrates) also express another form of proteasome, the immunoproteasome (IP). In IPs, the three catalytic β-subunits expressed in CPs are replaced by three interferon-γ–inducible homologues (immunosubunits): low molecular weight protein (LMP)-2 (or β1i) for β1, multicatalytic endopeptidase complex-like (MECL)-1 (or β2i) for β2, and LMP7 (or β5i) for β5. In gnathostomes, most cells express only CPs under steady state conditions and harbor IPs when exposed to interferon-γ (8.Heink S. Ludwig D. Kloetzel P.M. Kruger E. IFN-γ-induced immune adaptation of the proteasome system is an accelerated and transient response.Proc. Natl. Acad. Sci. 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