Proteogenomic Discovery of Novel Open Reading Frames With HLA Immune Presentation on Human β-Cells.

Ribosome profiling (Ribo-seq) measures translational regulation and reveals novel or unannotated open reading frames (nuORFs) otherwise difficult to identify. Recent reports demonstrate that nuORFs regulate gene expression and immune recognition, highlighting their emerging biological roles. Pancreatic β-cells are critical for maintaining euglycemic conditions, and β-cell impairment contributes to diabetes development. Identification of nuORF and protein/peptide products in human β-cells could reveal novel mechanisms that regulate β-cell function during homeostatic and disease conditions. Here, we applied a proteogenomic approach to human β-cells to define previously unknown protein/peptide products. First, we applied cell type-specific Ribo-seq to map the translatome of human stem cell-derived β-cells (sBCs). Pathways crucial for β-cell function and antigen presentation were subject to translational regulation. We detected a recently described immunogenic neoantigen, INS-DRiP, presumably originating from a downstream start site in INS mRNA. Moreover, our analysis revealed 965 novel nuORFs in sBCs, with a majority showing protein-level support. Comparison with primary human islets further validated nuORF translation and highlighted β-cell specificity. We identified a novel, primate-specific regulatory upstream ORF within TYK2, which is crucial for β-cell function and interferon response and has many variants strongly associated with type 1 diabetes. Finally, we used immunopeptidomics, HLA-binding prediction models, and T-cell coculture assays to validate the presentation and immunogenicity of preproinsulin peptides and nuORFs. Our findings underscore the importance of translational regulation in β-cell function and provide an important resource to the diabetes research community. ARTICLE HIGHLIGHTS: We developed a cell type-specific proteogenomic approach to reveal novel or unannotated open reading frames (nuORFs) using transcriptomics, ribosomal profiling, and proteomic analysis of human pancreatic β-cells using stem cell-derived β-cells and/or cadaveric islets. Our analysis revealed translational regulation of β-cell-specific pathways during differentiation and identified 965 nuORFs, with a majority exhibiting protein support and substantial β-cell specificity. A primate-specific ORF located in the 5' untranslated region of the type 1 diabetes risk gene TYK2 may act as a translational activator. We provide HLA class I immunopeptidomic data from cytokine-stimulated human β-cells and demonstrate their utility in coculture assays with autoreactive T-cell transductants. Taken together, our results define the human β-cell translatome, an important resource to the research field.