Abstract
Proteasomes are the main producers of antigenic peptides presented to CD8+ T cells. They can cut proteins and release their fragments or recombine non-contiguous fragments thereby generating novel sequences, i.e. spliced peptides. Understanding which are the driving forces and the sequence preferences of both reactions can streamline target discovery in immunotherapies against cancer, infection and autoimmunity. Here, we present a large database of spliced and non-spliced peptides generated by proteasomes in vitro, which is available as simple CSV file and as a MySQL database. To generate the database, we performed in vitro digestions of 55 unique synthetic polypeptide substrates with different proteasome isoforms and experimental conditions. We measured the samples using three mass spectrometers, filtered and validated putative peptides, identified 22,333 peptide product sequences (15,028 spliced and 7,305 non-spliced product sequences). Our database and datasets have been deposited to the Mendeley (doi:10.17632/nr7cs764rc.1) and PRIDE (PXD016782) repositories. We anticipate that this unique database can be a valuable source for predictors of proteasome-catalyzed peptide hydrolysis and splicing, with various future translational applications.
Highlights
Background & SummaryProteasomes are likely the most important proteases in eukaryotic cells
All peptides were synthesized using Fmoc solid phase chemistry. 20S s- and i-proteasomes were purified from peripheral blood (s-proteasomes), T2 cell line (s-proteasomes) or EBV-immortalized B lymphocytes (i-proteasomes) as follows: (i) either 10 ml human peripheral blood or 2 × 109 cells were homogenized, lysed and centrifuged; (ii) the supernatant was fractionated by ammonium sulphate precipitation (35% and 75%); (iii) the latter pellet was fractioned by chromatography on DEAE-Sephacel; (iv) the selected fractions were separated by 10–40% sucrose gradient and followed by (v) anion exchange chromatography on Mono Q in an Akta-FPLC system; (vi) the selected fractions (2–4 ml) were further purified by DEAE-Affi-gel-blue chromatography
In each of the (ii-vi) steps, the fractions were monitored by degradation assays of standard short fluorogenic substrate Suc-LLVY-AMC, which is a specific substrate for proteasome proteolytic activity[12]
Summary
Proteasomes are likely the most important proteases in eukaryotic cells. They destroy transcription factors, obsolete, damaged, wrongly transcribed proteins. I.e. the thymoproteasome, is expressed in thymic cortex and regulates T cell repertoire[4] These proteasome isoforms have different proteolytic dynamics and sequence preferences, it is still a matter of debate whether they generate a distinct set of peptides[7,8,9,10,11,12,13,14]. Understanding which are the driving forces and the sequence preferences of both proteasome-catalyzed peptide hydrolysis and peptide splicing can streamline target discovery in novel immunotherapies. Both proteasome-generated spliced and non-spliced epitopes can trigger an immune response against cancer, infection and autoimmune-relevant antigens and this response can lead, for example, to tumor regression and to cure patients[25,26,27,28,29,30]. We here propose a large database containing spliced and non-spliced peptides produced in vitro by proteasome isoforms in different conditions, derived from 55 synthetic polypeptide substrates and measured with different MS equipment multiple times
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