bims-micpro 2023-06-11 papers

bioRxiv. 2023 May 18. pii: 2023.05.16.541049. [Epub ahead of print]

What can Ribo-seq and proteomics tell us about the non-canonical proteome?

John R Prensner, Jennifer G Abelin, Leron W Kok, Karl R Clauser, Jonathan M Mudge, Jorge Ruiz-Orera, Michal Bassani-Sternberg, Eric W Deutsch, Sebastiaan van Heesch.

Ribosome profiling (Ribo-seq) has proven transformative for our understanding of the human genome and proteome by illuminating thousands of non-canonical sites of ribosome translation outside of the currently annotated coding sequences (CDSs). A conservative estimate suggests that at least 7,000 non-canonical open reading frames (ORFs) are translated, which, at first glance, has the potential to expand the number of human protein-coding sequences by 30%, from ∼19,500 annotated CDSs to over 26,000. Yet, additional scrutiny of these ORFs has raised numerous questions about what fraction of them truly produce a protein product and what fraction of those can be understood as proteins according to conventional understanding of the term. Adding further complication is the fact that published estimates of non-canonical ORFs vary widely by around 30-fold, from several thousand to several hundred thousand. The summation of this research has left the genomics and proteomics communities both excited by the prospect of new coding regions in the human genome, but searching for guidance on how to proceed. Here, we discuss the current state of non-canonical ORF research, databases, and interpretation, focusing on how to assess whether a given ORF can be said to be "protein-coding".
In brief: The human genome encodes thousands of non-canonical open reading frames (ORFs) in addition to protein-coding genes. As a nascent field, many questions remain regarding non-canonical ORFs. How many exist? Do they encode proteins? What level of evidence is needed for their verification? Central to these debates has been the advent of ribosome profiling (Ribo-seq) as a method to discern genome-wide ribosome occupancy, and immunopeptidomics as a method to detect peptides that are processed and presented by MHC molecules and not observed in traditional proteomics experiments. This article provides a synthesis of the current state of non-canonical ORF research and proposes standards for their future investigation and reporting.
Highlights: Combined use of Ribo-seq and proteomics-based methods enables optimal confidence in detecting non-canonical ORFs and their protein products.Ribo-seq can provide more sensitive detection of non-canonical ORFs, but data quality and analytical pipelines will impact results.Non-canonical ORF catalogs are diverse and span both high-stringency and low-stringency ORF nominations.A framework for standardized non-canonical ORF evidence will advance the research field.

DOI: https://doi.org/10.1101/2023.05.16.541049

PLoS One. 2023 ;18(6): e0286422

A short peptide LINC00665_18aa encoded by lncRNA LINC00665 suppresses the proliferation and migration of osteosarcoma cells through the regulation of the CREB1/RPS6KA3 interaction.

Junwei Pan, Ming Liu, Xiaozong Duan, Dan Wang.

Long noncoding RNAs (lncRNAs) encompass short open reading frames (sORFs) that can be translated into small peptides. Here, we investigated the encoding potential of lncRNA LINC00665 in osteosarcoma (OS) cells. Bioinformatic analyses were utilized to predict the lncRNAs with encoding potential in human U2OS cells. Protein expression was assessed by an immunoblotting or immunofluorescence method. Cell viability was assessed by cell counting Kit-8 (CCK-8). Cell proliferation was detected by 5-ethynyl-2'-deoxyuridine (EdU) assay. Cell migration was gauged by transwell assay. The downstream effectors of the short peptide were verified using qualitative proteome analysis after immunoprecipitation (IP) experiments. The effect of the short peptide on protein interactions were confirmed by Co-Immunoprecipitation (CoIP) assays. We found that lncRNA LINC00665 encoded an 18-amino acid (aa)-long short peptide (named LINC00665_18aa). LINC00665_18aa suppressed the viability, proliferation, and migration of human MNNG-HOS and U2OS OS cells in vitro and diminished tumor growth in vivo. Mechanistically, LINC00665_18aa impaired the transcriptional activity, nuclear localization, and phosphorylation of cAMP response element-binding protein 1 (CREB1). Moreover, LINC00665_18aa weakened the interaction between CREB1 and ribosomal protein S6 kinase A3 (RPS6KA3, RSK2). Additionally, increased expression of CREB1 reversed the inhibitory effects of LINC00665_18aa on OS cell proliferation and migration. Our findings show that the short peptide LINC00665_18aa exerts a tumor-inhibitory function in OS, providing a new basis for cancer therapeutics through the functions of the short peptides encoded by lncRNAs.

DOI: https://doi.org/10.1371/journal.pone.0286422

NAR Cancer. 2023 Jun;5(2): zcad024

CrypticProteinDB: an integrated database of proteome and immunopeptidome derived non-canonical cancer proteins.

Ghofran Othoum, Christopher A Maher.

Translated non-canonical proteins derived from noncoding regions or alternative open reading frames (ORFs) can contribute to critical and diverse cellular processes. In the context of cancer, they also represent an under-appreciated source of targets for cancer immunotherapy through their tumor-enriched expression or by harboring somatic mutations that produce neoantigens. Here, we introduce the largest integration and proteogenomic analysis of novel peptides to assess the prevalence of non-canonical ORFs (ncORFs) in more than 900 patient proteomes and 26 immunopeptidome datasets across 14 cancer types. The integrative proteogenomic analysis of whole-cell proteomes and immunopeptidomes revealed peptide support for a nonredundant set of 9760 upstream, downstream, and out-of-frame ncORFs in protein coding genes and 12811 in noncoding RNAs. Notably, 6486 ncORFs were derived from differentially expressed genes and 340 were ubiquitously translated across eight or more cancers. The analysis also led to the discovery of thirty-four epitopes and eight neoantigens from non-canonical proteins in two cohorts as novel cancer immunotargets. Collectively, our analysis integrated both bottom-up proteogenomic and targeted peptide validation to illustrate the prevalence of translated non-canonical proteins in cancer and to provide a resource for the prioritization of novel proteins supported by proteomic, immunopeptidomic, genomic and transcriptomic data, available at https://www.maherlab.com/crypticproteindb.

DOI: https://doi.org/10.1093/narcan/zcad024

Neuro Oncol. 2023 Jun 06. pii: noad099. [Epub ahead of print]

Upstream Open Reading Frame-encoded MP31 Disrupts the Mitochondrial Quality Control Process and Inhibits Tumorigenesis in Glioblastoma.

Nunu Huang, Zhipeng Chen, Xuesong Yang, Yixin Gao, Jian Zhong, Yan Li, Feizhe Xiao, Xiuxing Wang, Yu Shi, Nu Zhang.

BACKGROUND: Mitochondrial hyperpolarization achieved by the elevation of mitochondrial quality control (MQC) activity is a hallmark of glioblastoma (GBM). Therefore, targeting the MQC process to disrupt mitochondrial homeostasis should be a promising approach for GBM therapy.
METHOD: We used two-photon fluorescence microscopy, FACS and confocal microscopy with specific fluorescent dyes to detect the mitochondrial membrane potential (MMP) and mitochondrial structures. Mitophagic flux was measured with mKeima.
RESULTS: MP31, a PTEN uORF-translated and mitochondria-localized micropeptide, disrupted the MQC process and inhibited GBM tumorigenesis. Re-expression of MP31 in patient-derived GBM cells induced MMP loss to trigger mitochondrial fission but blocked mitophagic flux, leading to the accumulation of damaged mitochondria in cells, followed by ROS production and DNA damage. Mechanistically, MP31 inhibited lysosome function and blocked lysosome fusion with mitophagosomes by competing with V-ATPase A1 for LDHB binding to induce lysosomal alkalinization. Furthermore, MP31 enhanced the sensitivity of GBM cells to TMZ by suppressing protective mitophay in vitro and in vivo, but showed no side effects on normal human astrocytes (NHAs) or microglia cells (MG).
CONCLUSION: MP31 disrupts cancerous mitochondrial homeostasis and sensitizes GBM cells to current chemotherapy, without inducing toxicity in NHA and MG. MP31 is a promising candidate for GBM treatment.

Keywords: GBM; MP31; MQC; V-ATPase A1; mitophagy

DOI: https://doi.org/10.1093/neuonc/noad099