No evidence for immunoproteasomes in chicken lymphoid organs and activated lymphocytes

Abstract

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.

Alternative haplotypes of antigen processing genes in zebrafish diverged early in vertebrate evolution

“…Accordingly, some of these genes, including psmb13, and allelic lineages, such as psmb8f, were subsequently lost in the mammalian branch of the tetrapod lineage. In certain vertebrate lineages, additional subsets of MHC pathway genes have been selectively lost, including MHCII genes in Atlantic cod (58) and nonconstitutive proteasome subunits in chickens (25). Nevertheless, immune systems within these species have apparently compensated for these genetic losses through other strategies, such as amplifying their MHCI gene number (58) or maintaining a larger number of MHCI and TAP alleles (26).…”

“…For example, rat MHCI genes are found more tightly linked with antigen processing genes than in other rodents, such as mice, consistent with rat haplotypes having more specialized antigen transport (TAP) alleles exhibiting either "restrictive" or "permissive" peptide repertoires (24). Chickens represent another interesting exception, because antigen processing genes from the first stage of the MHC pathway, the inducible proteasome subunits proteasome-β 8-10 (psmb8-10), are altogether missing (25). However, this lack of inducible proteasome subunits in chickens does not seem to have reduced the capacity for polymorphic antigen transport genes, encoding the TAP subunits, to coevolve distinctive peptide specificities with coinherited MHCI alleles (26).…”

Novel insights into renal d-amino acid oxidase accumulation: propiverine changes DAAO localization and peroxisomal size in vivo

“…Rat constitutive proteasome was obtained from R&D Systems (E-352-050). Proteasome activity was assessed as previously described, with minor variations (Erath and Groettrup 2014). Briefly, 85 ng of proteasome was incubated with 25 μL of propiverine, propiverine-N-oxide or 4 μL of MG132 (10 mM stock) for 30 min at 37 °C.…”

Coevolution of MHC genes (LMP/TAP/class Ia, NKT‐class Ib, NKp30‐B7H6): lessons from cold‐blooded vertebrates

“…Unlike the situation in all endotherms including birds (interestingly, birds have lost all of the immunoproteasome and thymic proteasome genes (β5t or psmb11), unusual creatures that they are, considering the loss of many other immune genes as well (see below), all cold‐blooded vertebrates studied to date have biallelic lineages of lmp7 (psmb8 or β5i). Lineages are clearly restricted to lmp7, not the other immunoproteasome members lmp2 (psmb9 or β2i) and mecl1 (psmb10 or β1i), where there is clearly no allelic variation among MHC haplotypes.…”