Highlights d A standardized, ultra-high-throughput clinical platform for serum and plasma proteomics d Platform enables high precision quantification of 180 human proteomes per day at low cost d 27 biomarkers are differentially expressed between WHO severity grades for COVID-19 d Biomarkers include proteins not previously associated with COVID-19 infection

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 ...

Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100mel47–52/40–42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100mel47–52/40–42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8+ T cell response. Importantly, we demonstrate that different gp100mel-derived spliced epitopes are generated and presented to CD8+ T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100mel-derived spliced epitopes trigger activation of CD8+ T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes.

The assembly of ribosomal subunits is an essential prerequisite for protein biosynthesis in all domains of life. Although biochemical and biophysical approaches have advanced our understanding of ribosome assembly, our mechanistic comprehension of this process is still limited. Here, we perform an in vitro reconstitution of the Escherichia coli 50S ribosomal subunit. Late reconstitution products were subjected to high-resolution cryo-electron microscopy and multiparticle refinement analysis to reconstruct five distinct precursors of the 50S subunit with 4.3-3.8 Å resolution. These assembly intermediates define a progressive maturation pathway culminating in a late assembly particle, whose structure is more than 96% identical to a mature 50S subunit. Our structures monitor the formation and stabilization of structural elements in a nascent particle in unprecedented detail and identify the maturation of the rRNA-based peptidyl transferase center as the final critical step along the 50S assembly pathway.