Proteasome isoforms exhibit only quantitative differences in cleavage and epitope generation

Abstract

Immunoproteasomes are considered to be optimised to process Ags and to alter the peptide repertoire by generating a qualitatively different set of MHC class I epitopes. Whether the immunoproteasome at the biochemical level, influence the quality rather than the quantity of the immuno-genic peptide pool is still unclear. Here, we quantified the cleavage-site usage by human standard-and immunoproteasomes, and proteasomes from immuno-subunit-deficient mice, as well as the peptides generated from model polypeptides. We show in this study that the different proteasome isoforms can exert significant quantitative differences in the cleavage-site usage and MHC class I restricted epitope production. However, independent of the proteasome isoform and substrates studied, no evidence was obtained for the abolishment of the specific cleavage-site usage, or for differences in the quality of the peptides generated. Thus, we conclude that the observed differences in MHC class I restricted Ag presentation between standard-and immunoproteasomes are due to quantitative differences in the proteasome-generated antigenic peptides.Keywords: Antigen presentation r Immunoproteasome r MHC class I restricted epitopes r Proteasome r Proteolysis See accompanying Commentary by Zanker and ChenAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe 20S proteasome is the central proteolytic machinery of the ubiquitin proteasome system, being responsible for the main Correspondence: Dr. Michele Mishto e-mail: michele.mishto@charite.de part of extra-lysosomal protein degradation and generation of MHC class I restricted epitopes [1]. During evolution, the 20S proteasome retained a conserved structure of four stacked seven membered rings (α 7 β 7 β 7 α 7 ). In each β ring, the 20S standard proteasome has three catalytic standard subunits (i.e. β1s, β2s and β5s) that carry an N-terminal threonine residue as a reactive nucleophile. Based on the analysis of yeast 20S proteasome active site mutants with short fluorogenic peptide substrates C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2014. 44: 3508-3521 Antigen processing 3509 chymotryptic-, tryptic-and caspase-like activities were assigned to the β5, β2 and β1 subunits, respectively [2]. Larger polypeptide substrates bind with their residues surrounding the cleavage site, that is residues in position P4 to P1 (cleavage site) and P1 to P4 , to the non-primed and primed substrate binding sites [3] of the proteasome, respectively. This provides the stability and the orientation of the substrate, thereby determining the cleavage-site usage within a protein substrate [4]. In mammalia, the cytokine IFN-γ induces the expression of three active sites carrying alternative β1i/LMP2, β2i/MECL1 and β5i/LMP7 immuno-subunits, and in consequence the formation of the immunoproteasome isoforms [5].Since the β1i and β5i immuno-subunits are encoded within the MHC class II region in close neighbourhood to ...

The T210M Substitution in the HLA-a*02:01 gp100 Epitope Strongly Affects Overall Proteasomal Cleavage Site Usage and Antigen Processing

“…1). In agreement with our previous results (14), the peptide pools produced by both s-or i-proteasomes perfectly overlapped with respect to numbers and sequences of the products (data not shown). 56 products (2 spliced peptides, i.e.…”

“…The T2 cell line is a human T cell leukemia/B cell line hybrid defective in TAP1/ TAP2 (transporter associated with antigen presentation) and ␤1i/␤5i subunits. The T2 cell line contains only standard proteasome (s-proteasomes), whereas the lymphoblastoid cell line possesses proteasomes carrying mainly immuno-subunits, and it is named here as immunoproteasome (i-proteasome) (14,25).…”

“…Intermediate-type proteasomes are expressed in most tumor cells and in many tissues of the human body under normal physiological nutrition and growth conditions (12). It has been recently shown that the active subunit composition of 20S proteasomes in principle does not affect the quality of proteasome-generated peptides (5,13,14). Nevertheless, proteasomal subunit composition can strongly affect cleavage site usage within a given substrate and hence the relative quantity of non-spliced or spliced peptides produced.…”

“…Nevertheless, proteasomal subunit composition can strongly affect cleavage site usage within a given substrate and hence the relative quantity of non-spliced or spliced peptides produced. Such quantitative differences in the generation of cleavage products can strongly affect cell surface presentation of MHC class I-peptide complexes and in consequence the efficacy of a peptide-specific CD8 ϩ T cell response (5,(13)(14)(15). Although sequence requirements for proteasomal cleavage site usage are difficult to predict, there exists frequent evidence that seemingly minor alteration in the primary sequence of a protein substrate can have an impact on proteasomal processing and thereby positively or negatively affecting the liberation of antigenic peptides and concomitantly the CD8 ϩ T celldependent immune response (7,10,16).…”

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

“…Two decades of immunoproteasome research solidified the hypothesis of immunoproteasome particles shaping the immunopeptideome presented on MHC class I molecules (Basler et al, 2011;Basler et al, 2006;Basler et al, 2004;Kincaid et al, 2012;Mishto et al, 2014;Morel et al, 2000;Osterloh et al, 2006;Sijts et al, 2000;Toes et al, 2001). Moreover, it became evident that the immunoproteasome has, apart from antigen processing, Fig.…”