Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes

Ebstein, Frédéric; Textoris-Taube, Kathrin; Keller, Christin; Golnik, Richard; Vigneron, Nathalie; Eynde, Benoı̂t J. Van den; Schuler‐Thurner, Beatrice; Schadendorf, Dirk; Lorenz, Felix K.M.; Uckert, Wolfgang; Urban, Sabrina; Lehmann, Andrea; Albrecht-Koepke, N; Janek, Katharina; Henklein, Petra; Niewienda, Agathe; Kloetzel, Peter M.; Mishto, Michele

doi:10.1038/srep24032

Cited by 89 publications

(138 citation statements)

References 35 publications

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“…Therefore, based on our results we conclude that spliced peptides not only represent one third of the HLA-I immunopeptidome variety but also approximately one fourth of the total amount of peptide molecules presented onto the HLA-I complex. In agreement with this finding we recently reported that the abundance of peptides of a small group of melanoma-associated spliced epitopes exposed onto the HLA-I complexes is comparable to that of non-spliced melanoma-associated epitopes (13).…”

supporting

confidence: 75%

A large fraction of HLA class I ligands are proteasome-generated spliced peptides

Liepe

Marino

Sidney

et al. 2016

Science

303

385

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catalyzed peptide splicing.Abbreviations: electron-transfer higher-energy collision dissociation (EThcD); higher-energy collision dissociation (HCD); false discovery rate (FDR); HLA class I (HLA-I); lymphoblastoid cell line (LCL); mass spectrometry (MS); molecular weight (MW); proteasome-catalyzed peptide splicing (PCPS). 2The proteasome generates the epitopes presented on HLA class I molecules that elicit CD8 + T cell responses. While reports of proteasome-generated spliced epitopes exist, they have been regarded as rare events. Here, however, we show that the proteasome-generated spliced peptide pool accounts for one third of the entire HLA class I immunopeptidome in terms of diversity and one fourth in terms of abundance. They also represent a unique set of antigens, possessing particular and distinguishing features. We validated this observation using a range of complementary experimental and bioinformatics approaches, and multiple cell types. The widespread appearance and abundance of proteasome-catalyzed peptide splicing events will inform immunobiology and autoimmunity theories, and may provide a previously untapped source of epitopes for uses in vaccines and cancer immunotherapy.3The presentation of epitopes on the cell surface is a key mechanism by which organisms identify the presence of pathogens, metabolic malfunctioning, or tumors. The HLA class I (HLA-I) immunopeptidome, i.e. the epitopes allocated onto the HLA-I molecules, impinges upon the CD8 + T cell repertoire and the cell-mediated immune response (1). HLA-I immunopeptidomes are usually investigated by sequence identification of peptides eluted from HLA-I molecules using LC-tandem mass spectrometry (MS) (Fig. S1). The key step for the transformation of a protein into HLA-Irestricted epitopes is usually processing by the proteasome (1), which cuts proteins into peptides;alternatively, the proteasome can also cut and paste peptide sequences, thereby releasing peptide antigens that do not correspond to the original protein sequence (2) (Fig. S2). This proteasomecatalyzed peptide splicing (PCPS) has long been considered to occur only very rarely; partly this has been because the screening of the HLA-I immunopeptidome for proteasome-generated spliced peptides was impeded by methodological challenges.To overcome these problems we developed an analytical strategy, which accounts for recent discoveries underpinning the PCPS mechanism, and which can handle the vast proteome-wide human spliced peptide database (Fig. S3). With this strategy we initially analyzed the HLA-I-eluted immunopeptidome of the GR lymphoblastoid cell line (GR-LCL) adopting, for a deeper coverage of the immunopeptidome, a 2D peptide pre-fractionation strategy, followed by a hybrid peptide fragmentation method, electron-transfer higher-energy collision dissociation (EThcD) for peptide identification (3, 4) ( Fig. S1), supplemented by an adapted target-decoy approach (Fig. S4). Our analysis led to the identification of 6592 non-spliced and 3417 spliced 9-12mer peptides ( Table S1)....

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supporting

confidence: 75%

A large fraction of HLA class I ligands are proteasome-generated spliced peptides

Liepe

Marino

Sidney

et al. 2016

Science

303

385

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“…The data led to the hypothesis that the catalytically active threonine residues in the standard proteasome form O ‐acyl intermediates with the N‐terminal peptide fragment which is then subject to nucleophilic attack by the N‐terminus of another peptide fragment, which forms the C‐terminal end of the spliced peptide (Figure ). This mechanism has since been supported by several other studies . In addition, Ebstein et al .…”

Section: Mechanisms and Motifssupporting

confidence: 68%

Shuffling peptides to create T‐cell epitopes: does the immune system play cards?

Mannering

Elso

et al. 2017

Immunol Cell Biol

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For a long time, immunologists have believed that classical CD4 and CD8 T cells recognize peptides (referred to as epitopes), derived from protein antigens presented by MHC/HLA class I or II. Over the past 10-15 years, it has become clear that epitopes recognized by CD8, and more recently CD4 T cells, can be formed by protein splicing. Here, we review the discovery of spliced epitopes recognized by tumor-specific human CD8 T cells. We discuss how these epitopes are formed and some of the unusual variants that have been reported. Now, over a decade since the first report, evidence is emerging that spliced CD8 T-cell epitopes are much more common, and potentially much more important, than previously imagined. Recent work has shown that epitopes recognized by CD4 T cells can also be formed by protein splicing. We discuss the recent discovery of spliced CD4 T-cell epitopes and their potential role as targets of autoimmune T-cell responses. Finally, we highlight some of the new questions raised from our growing appreciation of T-cell epitopes formed by peptide splicing.

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“…Because they observed that the peptide QLYPEW_RTK could be produced in vitro when incubating peptide fragment RTK and QLYPEW together with the proteasome, they suggested that the peptide could be produced in vitro through a proteasome-catalyzed condensation reaction; indeed the small and "final" size of those two fragments (RTK and QLYPEW) precludes the production of an acyl-enzyme intermediate that is required for the transpeptidation reaction. So far, however, the experimental evidence relative to the mechanism of condensation is limited to results obtained with in vitro digests or with cells expressing appropriately truncated constructs, but not with cells expressing full-length proteins (13). Moreover, the prominent role of transpeptidation over condensation was confirmed by mass spectrometry analysis of peptide digests performed in the presence of isotopically-labeled water (36).…”

Section: Minireview: Peptide Splicing By the Proteasomementioning

confidence: 99%

“…Interestingly, this peptide is composed of fragments that are identical to those contained in the previously described spliced peptide RTK_QLYPEW, but these fragments are spliced in reverse order. After stimulation of CD8 ϩ T cells with this peptide, Ebstein et al (13) isolated an HLA-A3-restricted CTL, which recognized the peptide and was able to kill gp100-expressing tumor cells. Because they observed that the peptide QLYPEW_RTK could be produced in vitro when incubating peptide fragment RTK and QLYPEW together with the proteasome, they suggested that the peptide could be produced in vitro through a proteasome-catalyzed condensation reaction; indeed the small and "final" size of those two fragments (RTK and QLYPEW) precludes the production of an acyl-enzyme intermediate that is required for the transpeptidation reaction.…”

Section: Minireview: Peptide Splicing By the Proteasomementioning

confidence: 99%

“…For a number of years, antigenic peptides recognized by CTL were believed to only derive from linear fragments of cellular proteins. This concept was challenged by the identification of several antigenic peptides, composed of two peptide fragments that were originally distant in the parental protein but are assembled together by the creation of a new peptide bond (7)(8)(9)(10)(11)(12)(13). This process, called peptide splicing, was shown to take place in the proteasome (8 -10).…”

mentioning

confidence: 99%

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Peptide splicing by the proteasome

Vigneron

Ferrari

Stroobant

et al. 2017

Journal of Biological Chemistry

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Edited by Peter CresswellThe proteasome is the major protease responsible for the production of antigenic peptides recognized by CD8؉ cytolytic T cells (CTL). These peptides, generally 8 -10 amino acids long, are presented at the cell surface by major histocompatibility complex (MHC) class I molecules. Originally, these peptides were believed to be solely derived from linear fragments of proteins, but this concept was challenged several years ago by the isolation of anti-tumor CTL that recognized spliced peptides, i.e. peptides composed of fragments distant in the parental protein. The splicing process was shown to occur in the proteasome through a transpeptidation reaction involving an acyl-enzyme intermediate. Here, we review the steps that led to the discovery of spliced peptides as well as the recent advances that uncover the unexpected importance of spliced peptides in the composition of the MHC class I repertoire.The immune system constantly monitors cellular integrity to restrain tumor development or viral infections. CD8 ϩ cytolytic T lymphocytes (CTL) 4 are essential players in this process, because of their ability to recognize and destroy cells expressing tumoral or viral proteins. CTL indeed recognize peptides derived from these proteins and displayed at the cell surface by major histocompatibility complex (MHC) class I molecules. Peptides recognized by CTL are produced in the cytosol by the proteasome, a large protease complex responsible for the bulk of cellular protein degradation (1). A fraction of the peptides released by the proteasome is thus transferred into the lumen of the ER by a specialized transporter called TAP (transporter associated with antigen processing) (2), where they can be further trimmed by ER-resident amino peptidases (ERAP1 and ERAP2) (3-5) and loaded onto MHC class I molecules. Stable MHC-peptide complexes then exit the ER and migrate through the secretory pathway to reach the cell surface, where they will be displayed for CTL recognition (6). For a number of years, antigenic peptides recognized by CTL were believed to only derive from linear fragments of cellular proteins. This concept was challenged by the identification of several antigenic peptides, composed of two peptide fragments that were originally distant in the parental protein but are assembled together by the creation of a new peptide bond (7-13). This process, called peptide splicing, was shown to take place in the proteasome (8 -10). In this minireview, we will recapitulate the discovery of this phenomenon and the recent developments that highlight the unforeseen importance of peptide splicing in the establishment of the MHC class I peptide repertoire.

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Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes

Cited by 89 publications

References 35 publications

A large fraction of HLA class I ligands are proteasome-generated spliced peptides

A large fraction of HLA class I ligands are proteasome-generated spliced peptides

Shuffling peptides to create T‐cell epitopes: does the immune system play cards?

Peptide splicing by the proteasome

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