Keshishian, H. | bioRxiv Science

bioRxiv2020

Thousands of novel unannotated proteins expand the MHC I immunopeptidome in cancer

Tumor epitopes - peptides that are presented on surface-bound MHC I proteins - provide targets for cancer immunotherapy and have been identified extensively in the annotated protein-coding regions of the genome. Motivated by the recent discovery of translated novel unannotated open reading frames (nuORFs) using ribosome profiling (Ribo-seq), we hypothesized that cancer-associated processes could generate nuORFs that can serve as a new source of tumor antigens that harbor somatic mutations or show tumor-specific expression. To identify cancer-specific nuORFs, we generated Ribo-seq profiles for 29 malignant and healthy samples, developed a sensitive analytic approach for hierarchical ORF prediction, and constructed a high-confidence database of translated nuORFs across tissues. Peptides from 3,555 unique translated nuORFs were presented on MHC I, based on analysis of an extensive dataset of MHC I-bound peptides detected by mass spectrometry, with >20-fold more nuORF peptides detected in the MHC I immunopeptidomes compared to whole proteomes. We further detected somatic mutations in nuORFs of cancer samples and identified nuORFs with tumor-specific translation in melanoma, chronic lymphocytic leukemia and glioblastoma. NuORFs thus expand the pool of MHC I-presented, tumor-specific peptides, targetable by immunotherapies.

cancer biology↗

bioRxiv2019

A conserved translation factor is required for optimal synthesis of a membrane protein family in mycobacteria

Ribosomes require the activity of associated GTPases to synthesize proteins. Despite strong evolutionary conservation, the roles of many of these remain unknown. For example, LepA (also known as elongation factor 4) is a ribosome-associated GTPase found in bacteria, mitochondria, and chloroplasts, yet its physiological contribution to cell survival is not clear. Recently, we found that loss of lepA in Mycobacterium smegmatis (Msm) altered tolerance to rifampin, a drug that targets a non-ribosomal cellular process. To uncover the determinants of LepA-mediated drug tolerance, we characterized the whole-cell proteomes and transcriptomes of a lepA deletion mutant relative to a wild-type strain. We find that LepA is important for the steady-state abundance of an outer membrane porin, which is integral to nutrient uptake and drug susceptibility. Loss of LepA leads to a decreased amount of porin in the membrane, resulting in the drug tolerance phenotype of the lepA mutant. LepA control requires a sequence motif in the 5 region of the porin transcript. Thus, LepA controls the abundance of specific proteins, likely through its activity during translation. ImportanceOur understanding of how ribosomes properly synthesis an entire cellular proteome, in all its complexity, is still evolving. Ribosomal GTPases are often highly conserved, but the roles of many are not well understood. For example, elongation factor 4, or LepA, is a ribosome-associated GTPase conserved across bacteria, mitochondria, and chloroplasts. Using whole-cell proteomics and RNA-sequencing of wild type and a lepA deletion mutant, we find that LepA improves translation of mycobacterial porins in a message-specific manner. As porins play a key role in cell wall permeability, loss of LepA produces a plethora of phenotypic changes. These findings underline the problem of building proteins into a complex cell wall, such as that of mycobacteria, and point to a solution in the use of GTPases such as LepA, that have evolved to aid in specific protein synthesis.

microbiology↗