Peptide splicing in the proteasome

Nathalie Vigneron

Peptide splicing was initially discovered in 2004, when Hanada et al. showed that a CD8+ cytolytic T lymphocyte, isolated from a patient with renal cell carcinoma, recognized a peptide composed of two non-contiguous fragments of the FGF-5 protein. Production of this peptide involved the removal of a 40-amino-acid intervening sequence and the creation of a new peptide bond between fragments to splice (Hanada et al., 2004). A few months later, we identified a second spliced peptide recognized by an anti-tumor CTL clone, which was isolated from a patient with melanoma (Vigneron et al., 2004). This second spliced peptide was composed of two fragments originating from the melanoma differentiation protein gp100 and spliced together after removal of a 4-amino-acid intervening sequence. The use of proteasome inhibitors and in vitro proteasome digests, showed that proteasome was performing this peptide splicing reaction through a process of transpeptidation involving the formation of an acyl-enzyme intermediate between the N-terminal peptide fragment and the hydroxyl group of the N-terminal threonine of the catalytic subunit of proteasome. The acyl-enzyme intermediate is then subjected to a nucleophilic attack by the N-terminus of another peptide fragment found in the proteasome chamber, leading to the creation of a new peptide bond between both peptide fragments. So far, six antigenic peptides produced by peptide splicing were identified and validated using specific anti-tumor CTLs. Among these, several contain fragments that are spliced together in the reverse order to that in which they occur in the parental protein (Dalet et al., 2011a; Ebstein et al., 2016; Michaux et al., 2014; Warren et al., 2006).

In contrast to the other examples of spliced peptides, which were composed of 3 to 6 amino-acid fragments, one of the spliced peptide identified contained an N-terminal splicing partner of 8 amino acids, to which a single arginine residue was added by transpeptidation. In vitro proteasome digestion experiments using pairs of peptides showed that the peptide causing the nucleophilic attack on the acyl-enzyme intermediate had a minimal length of 3-amino-acids. Therefore, a C-terminally extended spliced peptide is first produced and then further cleaved by the proteasome to form the final antigenic peptide (Michaux et al., 2014).

Four main subtypes of proteasomes exist: the standard proteasome, the immunoproteasome and intermediate proteasomes ß1-ß2-ß5i and ß1i-ß2-ß5i, which were previously identified by the Van den Eynde lab at de Duve Institute (Guillaume et al., 2010). Studying the production of spliced peptides by the four proteasome subtypes, we showed that all four proteasome subtypes are able to splice peptides, but some spliced peptides are better produced by the standard proteasome, while others are better produced by the immunoproteasome or the intermediate proteasomes (Dalet et al., 2011b; Ferrari et al., 2022). When studying the splicing by the four proteasome subtypes of the two reciprocal gp100 peptides RTK_QLYPEW and QLYPEW_RTK, we observed that, despite the fact that both peptides are composed of identical splicing partners, their production varies according to the proteasome subtype, indicating that the amount of splicing partner released by the proteasome is not the only factor driving peptide splicing (Ferrari et al., 2022). This suggests that peptide splicing efficiency might also rely on other factors such as the affinity of the C-terminal splice reactant for the primed binding site of the catalytic subunit. 

Interestingly, among the spliced peptide identified, one also contains two aspartate residues originating from the deglycosylation of genetically encoded, N-glycosylated asparagine residues (Dalet et al., 2011a). TILs targeting this peptide were used for adoptive T-cell transfer and shown to induce dramatic tumor rejection in the patient, highlighting the biological and clinical relevance of proteasomal spliced peptides (Robbins et al., 1994). The factors driving the efficiency of the peptide splicing reaction and the role played by spliced peptides in anti-tumor responses still need to investigated. 

List of publications

Dalet, A., P.F. Robbins, V. Stroobant, N. Vigneron, Y.F. Li, M. El-Gamil, K. Hanada, J.C. Yang, S.A. Rosenberg, and B.J. Van den Eynde. 2011a. An antigenic peptide produced by reverse splicing and double asparagine deamidation. Proc Natl Acad Sci U S A 108:E323-331.

Dalet, A., V. Stroobant, N. Vigneron, and B.J. Van den Eynde. 2011b. Differences in the production of spliced antigenic peptides by the standard proteasome and the immunoproteasome. Eur J Immunol 41:39-46.

Ebstein, F., K. Textoris-Taube, C. Keller, R. Golnik, N. Vigneron, B.J. Van den Eynde, B. Schuler-Thurner, D. Schadendorf, F.K. Lorenz, W. Uckert, S. Urban, A. Lehmann, N. Albrecht-Koepke, K. Janek, P. Henklein, A. Niewienda, P.M. Kloetzel, and M. Mishto. 2016. Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes. Sci Rep 6:24032.

Ferrari, V., V. Stroobant, J. Abi Habib, S. Naulaerts, B.J. Van den Eynde, and N. Vigneron. 2022. New Insights into the Mechanisms of Proteasome-Mediated Peptide Splicing Learned from Comparing Splicing Efficiency by Different Proteasome Subtypes. J Immunol 208:2817-2828.

Guillaume, B., J. Chapiro, V. Stroobant, D. Colau, B. Van Holle, G. Parvizi, M.P. Bousquet-Dubouch, I. Theate, N. Parmentier, and B.J. Van den Eynde. 2010. Two abundant proteasome subtypes that uniquely process some antigens presented by HLA class I molecules. Proc Natl Acad Sci U S A 107:18599-18604.

Hanada, K., J.W. Yewdell, and J.C. Yang. 2004. Immune recognition of a human renal cancer antigen through post-translational protein splicing. Nature 427:252-256.

Michaux, A., P. Larrieu, V. Stroobant, J.F. Fonteneau, F. Jotereau, B.J. Van den Eynde, A. Moreau-Aubry, and N. Vigneron. 2014. A spliced antigenic peptide comprising a single spliced amino acid is produced in the proteasome by reverse splicing of a longer peptide fragment followed by trimming. J Immunol 192:1962-1971.

Robbins, P.F., M. el-Gamil, Y. Kawakami, E. Stevens, J.R. Yannelli, and S.A. Rosenberg. 1994. Recognition of tyrosinase by tumor-infiltrating lymphocytes from a patient responding to immunotherapy. Cancer Res 54:3124-3126.

Vigneron, N., V. Stroobant, J. Chapiro, A. Ooms, G. Degiovanni, S. Morel, P. van der Bruggen, T. Boon, and B.J. Van den Eynde. 2004. An antigenic peptide produced by peptide splicing in the proteasome. Science 304:587-590.

Warren, E.H., N.J. Vigneron, M.A. Gavin, P.G. Coulie, V. Stroobant, A. Dalet, S.S. Tykodi, S.M. Xuereb, J.K. Mito, S.R. Riddell, and B.J. Van den Eynde. 2006. An antigen produced by splicing of noncontiguous peptides in the reverse order. Science 313:1444-1447.