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. Background & Summary Proteasomes are likely the most important proteases in eukaryotic cells. They destroy transcription factors, obsolete, damaged, wrongly transcribed proteins. Often the peptide fragments derived from this process are further trimmed by aminopeptidases to provide the amino acids needed for new protein synthesis. In some cases, their released protein/peptide fragments are actively involved in metabolic and immunological pathways. For instance, proteasomes can cleave p105, thereby generating a component of the transcription factor NF-κB, and osteopontin, thereby releasing peptides that promote cell migration 1-3. The most famous "active" peptide products released by proteasomes, however, are antigenic peptides bound to Human Leukocyte Antigen class I (HLA-I) complexes (i.e. the HLA-I immunopeptidome) and presented at the cell surface to CD8 + T cells 4. Eukaryotic cells can express various proteasome isoforms. The most active isoforms of proteasomes are 26S proteasomes, which have a core (20S proteasome) coupled to 19S regulatory complex and process poly-ubiquitinated substrates 5. 20S proteasomes, alone or coupled to other regulatory complexes such as PA28, can destroy proteins even in a ubiquitin-independent fashion 6. The various human 20S proteasome isoforms are all constituted by two α and two β heptameric rings. Standard proteasomes (s-proteasomes) contain the catalytic β1, β2, and β5 subunits, whereas immunoproteasomes (i-proteasomes) contain the β1i, β2i, and β5i subunits. The latter are constitutively present in immune cells as well as in cells exposed to an inflammatory milieu. Intermediate-type and subtype proteasomes have been also described. Furthermore, a third proteasome isoform,
Noncanonical epitopes presented by Human Leucocyte Antigen class I (HLA-I) complexes to CD8+ T cells attracted the spotlight in the research of novel immunotherapies against cancer, infection and autoimmunity. Proteasomes, which are the main producers of HLA-I-bound antigenic peptides, can catalyze both peptide hydrolysis and peptide splicing. The prediction of proteasome-generated spliced peptides is an objective that still requires a reliable (and large) database of non-spliced and spliced peptides produced by these proteases. Here, we present an extended database of proteasome-generated spliced and non-spliced peptides, which was obtained by analyzing in vitro digestions of 80 unique synthetic polypeptide substrates, measured by different mass spectrometers. Peptides were identified through invitroSPI method, which was validated through in silico and in vitro strategies. The peptide product database contains 16,631 unique peptide products (5,493 non-spliced, 6,453 cis-spliced and 4,685 trans-spliced peptide products), and a substrate sequence variety that is a valuable source for predictors of proteasome-catalyzed peptide hydrolysis and splicing. Potential artefacts and skewed results due to different identification and analysis strategies are discussed.
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