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A Molecular Basis for the Presentation of Phosphorylated Peptides by HLA-B Antigens.

ABSTRACT: As aberrant protein phosphorylation is a hallmark of tumor cells, the display of tumor-specific phosphopeptides by Human Leukocyte Antigen (HLA) class I molecules can be exploited in the treatment of cancer by T-cell-based immunotherapy. Yet, the characterization and prediction of HLA-I phospholigands is challenging as the molecular determinants of the presentation of such post-translationally modified peptides are not fully understood. Here, we employed a peptidomic workflow to identify 256 unique phosphorylated ligands associated with HLA-B*40, -B*27, -B*39, or -B*07. Remarkably, these phosphopeptides showed similar molecular features. Besides the specific anchor motifs imposed by the binding groove of each allotype, the predominance of phosphorylation at peptide position 4 (P4) became strikingly evident, as was the enrichment of basic residues at P1. To determine the structural basis of this observation, we carried out a series of peptide binding assays and solved the crystal structures of HLA-B*40 in complex with a phosphorylated ligand or its nonphosphorylated counterpart. Overall, our data provide a clear explanation to the common motif found in the phosphopeptidomes associated to different HLA-B molecules. The high prevalence of phosphorylation at P4 is dictated by the presence of the conserved residue Arg62 in the heavy chain, a structural feature shared by most HLA-B alleles. In contrast, the preference for basic residues at P1 is allotype-dependent and might be linked to the structure of the A pocket. This molecular understanding of the presentation of phosphopeptides by HLA-B molecules provides a base for the improved prediction and identification of phosphorylated neo-antigens, as potentially used for cancer immunotherapy.

SUBMITTER: Alpizar A

PROVIDER: S-EPMC5294207 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A subset of class I major histocompatibility complex (MHC)-bound peptides is produced from immature proteins that are rapidly degraded after synthesis. These defective ribosomal products (DRiPs) have been implicated in early alert of the immune system about impending infections. Interferons are important cytokines, produced in response to viral infection, that modulate cellular metabolism and gene expression patterns, increase the presentation of MHC molecules, and induce rapid degradation of proteins and cell-surface presentation of their derived MHC peptides, thereby contributing to the battle against pathogen infections. This study evaluated the role of interferons in the induction of rapid degradation of DRiPs to modulate the repertoire of DRiP-derived MHC peptides. Cultured human breast cancer cells were treated with interferons, and the rates of synthesis and degradation of cellular protein and their degradation products were determined by LC-MS/MS analysis, following the rates of incorporation of heavy stable isotope-labeled amino acids (dynamic stable isotope labeling by amino acids in cell culture, dynamic SILAC) at several time points after the interferon application. Large numbers of MHC peptides that incorporated the heavy amino acids faster than their source proteins indicated that DRiP peptides were abundant in the MHC peptidome; interferon treatment increased by about twofold their relative proportions in the peptidome. Such typical DRiP-derived MHC peptides were from the surplus subunits of the proteasome and ribosome, which are degraded because of the transition to immunoproteasomes and a new composition of ribosomes incorporating protein subunits that are induced by the interferon. We conclude that degradation of surplus subunits induced by the interferon is a major source for DRiP-MHC peptides, a phenomenon relevant to coping with viral infections, where a rapid presentation of MHC peptides derived from excess viral proteins may help alert the immune system about the impending infection.

Project description:Treatment of cancer cells with anti-cancer drugs often fails to achieve complete remission. Yet, such drug treatments may induce alteration in the tumorâs gene expression patterns, including those of Cancer/Testis Antigens (CTA). The degradation products of such antigens can be presented as HLA peptides on the surface of the tumor cells and be developed into anti-cancer immunotherapeutics. For example, the DNA methyl transferase inhibitor, 5-aza-2'-deoxycytidine (Decitabine) has limited anti-tumor efficacy, yet it induces the expression of many genes, including CTAs that are normally silenced in the healthy adult tissues. In this study, the presentation of many new HLA peptides derived from CTAs and induced by Decitabine was demonstrated in three human Glioblastoma cell lines. Such presentation of CTA-derived HLA peptides can be exploited for development of new treatment modalities, combining drug treatment with anti-CTA targeted immunotherapy. The Decitabine-induced HLA peptidomes include many CTAs that are not normally detected in healthy tissues or in cancer cells, unless treated with the drug. In addition, the study included large-scale analyses of the simultaneous effects of Decitabine on the transcriptomes, proteomes and HLA peptidomes of the human Glioblastoma cells. It demonstrates the poor correlations between these three levels of gene expression, both in their total levels and in their response to the drug. The transcriptomes, proteomes and HLA peptidomes of the U-87, T98G and LNT-229 GBM human cell lines were analyzed before and after treatment with Decitabine. Overall, the RNA-Seq transcriptome analyses resulted in the identification of above 26000 transcripts, the proteome analyses identified about 7500 proteins and the HLA class I peptidome analyses resulted in above 25000 identified HLA peptides. Two biological repetitions of the transcriptome, three of the proteome and three of the HLA peptidome were performed with each of the cell lines and treatment, resulting in highly reproducible datasets.

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A Molecular Basis for the Presentation of Phosphorylated Peptides by HLA-B Antigens.

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