Dataset Information

Tryptophan depletion provokes the production and presentation of in-frame tryptophan to phenylalanine substitutions

ABSTRACT: Activated T cells secrete interferon-gamma (IFN) that triggers an intra-cellular tryptophan shortage through the activation of the Indoleamine 2,3-Dioxygenase 1 (IDO1) enzyme. Surprisingly, we here show that despite tryptophan depletion, in-frame protein synthesis bypassing tryptophan codons continues. Using mass spectrometry, we identified tryptophan to phenylalanine (W>F) substitution as the major event that facilitates in-frame protein synthesis in IFN-treated and tryptophan-depleted cells. We validated their expression using in vitro reporter assays, and pinpoint tryptophanyl-tRNA synthetase (WARS1) as the candidate gene that accounts for this event. We named these mRNA-translation-driven aberrations ‘substitutants’ to distinguish them from somatic genetic variants. To uncover their biological relevance, we interrogated cancer proteomes and phosphoproteomes of breast, lung and kidney cancers. Interestingly, we specifically identified a high level of tryptic peptides containing W>F substitutants with strong association to IDO1 expression, T cell activity, p53 mutations, and oncogenic MAPK signaling, indicating a role of infiltrating T cells in their production and highlighting cancer relevance. We further show that substitutants can impair protein function and be processed and presented at the cell surface to elicit immune cell recognition. Indeed, immunopeptidomics data from of colorectal-cancer organoids and glioblastoma cell lines revealed a large number of W>F substitutant-peptides being presented on HLA class I molecules following IFN treatment. Thus, W>F substitutants are a novel form of non-genetic mutations induced by tumor-infiltrating T cells with potential impact on gene function and on the repertoire of neopeptides presented by cancer cells.

INSTRUMENT(S): Orbitrap Fusion, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Melanocyte Of Skin, Cell Culture

DISEASE(S): Skin Cancer

SUBMITTER: Onno Bleijerveld

LAB HEAD: Reuven Agami

PROVIDER: PXD028921 | Pride | 2021-12-23

REPOSITORIES: Pride

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Publications

Tryptophan depletion results in tryptophan-to-phenylalanine substitutants.

Pataskar Abhijeet A Champagne Julien J Nagel Remco R Kenski Juliana J Laos Maarja M Michaux Justine J Pak Hui Song HS Bleijerveld Onno B OB Mordente Kelly K Navarro Jasmine Montenegro JM Blommaert Naomi N Nielsen Morten M MM Lovecchio Domenica D Stone Everett E Georgiou George G de Gooijer Mark C MC van Tellingen Olaf O Altelaar Maarten M Joosten Robbie P RP Perrakis Anastassis A Olweus Johanna J Bassani-Sternberg Michal M Peeper Daniel S DS Agami Reuven R

Nature 20220309 7902

Activated T cells secrete interferon-γ, which triggers intracellular tryptophan shortage by upregulating the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme<sup>1-4</sup>. Here we show that despite tryptophan depletion, in-frame protein synthesis continues across tryptophan codons. We identified tryptophan-to-phenylalanine codon reassignment (W>F) as the major event facilitating this process, and pinpointed tryptophanyl-tRNA synthetase (WARS1) as its source. We call these W>F peptides 'substitutants ...[more]

PMID: 35264796

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Project description:Many tumors escape by activating multiple cellular pathways that induce immunosuppression. One pivotal immune-suppressive mechanism is the production of tryptophan metabolites along the kynurenine pathway by IFNγ-induced IDO1 enzyme production 4-8. Phase III clinical trials using chemical inhibition of IDO1 in combination with PD1 pathway blockade, however, failed to improve melanoma treatment 9-12. This points at an incomplete understanding of the role of IDO1 and the consequent tryptophan degradation on mRNA translation and cancer progression. Here, we investigated the effects of prolonged IFNγ treatment on mRNA translation in melanoma cells by ribosome profiling. Surprisingly, we observed a massive accumulation of ribosomes ~20 amino acids downstream of tryptophan codons (termed here as W-Bumps) along with their expected stalling at the tryptophan codon itself. This indicated ribosomal bypass of the tryptophan codons in the absence of tryptophan. Detailed examination of W-Bumps position and its corresponding peptide sequences pinpointed towards ribosomal frameshifting events and their effects in the ribosome exit tunnel. In particular, W-Bumps strength was associated with the disorderedness level of potential out-of-frame peptides predicted downstream of tryptophan codons. Indeed, reporter assays demonstrated the induction of ribosomal frameshifting, and the generation of trans-frame proteins and their presentation at the cell surface after IFNγ treatment. Proteomics and immunopeptidomics analyses verified the production of IFNγ-induced trans-frame and out-of-frame aberrant peptides and their presentation on HLA class I molecules. Priming of naïve T cells from healthy donors with aberrant peptides resulted in identification of reactive, peptide-specific T cells. Altogether, our results suggest that IFNγ-induced IDO1-mediated tryptophan depletion plays a role in the immune recognition of melanoma cells by contributing to the diversity of the peptidome landscape, and by inducing the presentation of aberrant peptides.

Dataset Information

Tryptophan depletion provokes the production and presentation of in-frame tryptophan to phenylalanine substitutions

Publications

Tryptophan depletion results in tryptophan-to-phenylalanine substitutants.

Similar Datasets

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