Proteasomes are the central cytosolic and nuclear proteases in eukaryotic cells. They degrade a broad range of proteins, mainly cytoplasmic, though they are also active in other cell compartments (1). The peptides produced during protein degradation can be further processed by cytosolic aminopeptidases to preserve amino acid availability for new protein synthesis. Not all proteasomal products are destinated for full degradation. For example, p105 is cleaved by proteasomes, thereby generating a component of the transcription factor NF-κB. Similarly, osteopontin seems to be processed by these proteases, which produce peptides that promote cell migration (2–4). However, the best studied peptide products produced by proteasomes are peptides destined for binding HLA-I complexes (i.e. the HLA-I immunopeptidome) for presentation at the cell surface to CD8+ T cells (5). HLA-I immunopeptidomes have widely been investigated in the context of cancer, radiotherapy, infection, autoimmunity and other conditions. These peptides can be used for detection of specific T cell immune responses and for vaccination purposes against infections and cancer. It is long established that proteases such as trypsin can both cleave peptide bonds but also ligate (repair) these peptide bonds (6, 7). The proteasome is no exception and can produce so-called spliced peptides through proteasome-catalyzed peptide splicing (PCPS). This latter process can occur by combining non-contiguous peptide fragments of either the same molecule – i.e. cis-PCPS – or of two distinct proteins - i.e. trans-PCPS ( Figure 1A ). This results in peptide sequences that are not defined by the genetic code. Cis-spliced peptide identification is challenging: depending on the method of identification used, their estimated frequency in HLA-I immunopeptidomes ranged from 1% to 34% (8). PCPS is not a random process and seems to be ruled by sequence motif preferences and driving forces, which may differ from peptide hydrolysis, although the exact rules are largely unknown (7, 9–11). Cis-spliced peptides seem to be routinely produced and presented by various cells, depicting peptide splicing as a standard activity of proteasomes (7, 9, 12–17). On average, they are produced and presented by HLA-I complexes in smaller amount than non-spliced peptides although exceptions have been described (10, 12, 15, 17). Cis-spliced peptides can target in vivo CD8+ T cell response against bacterial antigens otherwise neglected in a mouse model of Listeria monocytogenes infection (18). They can activate ex vivo CD8+ T cells specific to Listeria monocytogenes or HIV through cross-recognition (19, 20). Cis-spliced epitopes derived from melanoma-associated antigens are recognized by CD8+ T cells in peripheral blood of melanoma patients (13, 17). A melanoma patient with metastasis was cured through adoptive T cell therapy using an autologous tumor-infiltrating lymphocyte (TIL) clone, which was proved, in a later study, to be specific to a cis-spliced epitope rather than any non-spliced peptides derived from the melanoma-associated antigen tyrosinase (21, 22). Despite their theoretically vast sequence variability, cis-spliced peptides might not play a destabilizing role in central and peripheral tolerance and in pruning T cell repertoire (23), although they may be involved in autoreactive CD8+ T cell response in autoimmune diseases such as Type 1 Diabetes (T1D) (24, 25). Open in a separate window Figure 1 Proteasome-catalyzed peptide splicing and the investigated KRAS G12V cis-spliced and non-spliced peptides. (A) Proteasome-generated cis-spliced peptides can be formed by cis-PCPS, when the two splice-reactants, i.e. the non-contiguous peptide fragments ligated by proteasomes, derive from the same polypeptide molecule; the ligation can occur in normal order, i.e. following the orientation from N- to C-terminus of the parental protein (normal cis-PCPS), or in the reverse order (reverse cis-PCPS). (B) The KRAS5-6/8-14 G12V [KL][VVGAVGV] cis-spliced peptide and the KRAS5-13 G12V [KLVVVGAVG] non-spliced peptide, which are investigated in this study and derived from the KRAS2-35 G12V synthetic substrate polypeptide. The G12V mutation is marked in bold.