Protein degradation by human 20S proteasomes elucidates the interplay between peptide hydrolysis and splicing.

If and how proteasomes catalyze not only peptide hydrolysis but also peptide splicing is an open question that has divided the scientific community. The debate has so far been based on immunopeptidomics, in vitro digestions of synthetic polypeptides as well as ex vivo and in vivo experiments, which could only indirectly describe proteasome-catalyzed peptide splicing of full-length proteins. Here we develop a workflow-and cognate software - to analyze proteasome-generated non-spliced and spliced peptides produced from entire proteins and apply it to in vitro digestions of 15 proteins, including well-known intrinsically disordered proteins such as human tau and α-Synuclein. The results confirm that 20S proteasomes produce a sizeable variety of cis-spliced peptides, whereas trans-spliced peptides are a minority. Both peptide hydrolysis and splicing produce peptides with well-defined characteristics, which hint toward an intricate regulation of both catalytic activities. At protein level, both non-spliced and spliced peptides are not randomly localized within protein sequences, but rather concentrated in hotspots of peptide products, in part driven by protein sequence motifs and proteasomal preferences. At sequence level, the different peptide sequence preference of peptide hydrolysis and peptide splicing suggests a competition between the two catalytic activities of 20S proteasomes during protein degradation.

InvitroSPI and a large database of proteasome-generated spliced and non-spliced peptides.

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.

Large database for the analysis and prediction of spliced and non-spliced peptide generation by proteasomes.

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.