bims-micpro 2024-10-06 papers

bims-micpro

Biomed News

on Discovery and characterization of microproteins

Issue of 2024–10–06
six papers selected by
Thomas Farid Martínez, University of California, Irvine

Discovery of a conserved translationally repressive upstream open reading frame within the iron-deficiency response regulator IDEF2.
Deciphering the ghost proteome in ovarian cancer cells by deep proteogenomic characterization.
A peptide encoded by upstream open reading frame of MYC binds to tropomyosin receptor kinase B and promotes glioblastoma growth in mice.
An intronic variant in LAMB3 contributes to junctional epidermolysis bullosa and enamel hypoplasia via translational attenuation.
Detection of host cell microprotein impurities in antibody drug products.
Translational profiling of stress-induced small proteins uncovers an unexpected connection among distinct signaling systems.

BMC Plant Biol. 2024 Sep 30. 24(1): 891

Discovery of a conserved translationally repressive upstream open reading frame within the iron-deficiency response regulator IDEF2.

Oscar Carey-Fung, Jesse T Beasley, Ronan C Broad, Roger P Hellens, Alexander A T Johnson.

   BACKGROUND: Iron (Fe) deficiency affects 30-50% of the world's population. Genetic biofortification of staple crops is a promising strategy for improving human nutrition, but the number of effective precision breeding targets for Fe biofortification is small. Upstream open reading frames (uORFs) are cis-regulatory elements within the 5' leader sequence (LS) of genes that generally repress translation of the main open reading frame (mORF).
RESULTS: We aligned publicly available rice (Oryza sativa L.) ribo-seq datasets and transcriptomes to identify putative uORFs within important Fe homeostasis genes. A dual luciferase assay (DLA) was used to determine whether these uORFs cause repression of mORF translation and pinpoint LS regions that can be mutated for mORF derepression. A translationally repressive uORF region was identified in two positive regulators of the Fe-deficiency response: IDEF1 and IDEF2. The IDEF2-uORF peptide was highly conserved among monocots and a mutation series in the 5' LS of the wheat (Triticum aestivum L.) TaIDEF2-A1 gene demonstrated variable mORF derepression.
CONCLUSIONS: Together these results reveal a possible regulatory mechanism by which IDEF2 transcription factors modulate the Fe deficiency response in monocots, and highlight novel precision breeding targets to improve crop nutrition and abiotic stress tolerance.

Keywords:  Biofortification; CPuORF; Dual luciferase; IDEF1; Rice; Transcription factor; Wheat; uORF

DOI:  https://doi.org/10.1186/s12870-024-05473-y
Cell Death Dis. 2024 Sep 30. 15(9): 712

Deciphering the ghost proteome in ovarian cancer cells by deep proteogenomic characterization.

Diego Fernando Garcia-Del Rio, Mehdi Derhourhi, Amelie Bonnefond, Sébastien Leblanc, Noé Guilloy, Xavier Roucou, Sven Eyckerman, Kris Gevaert, Michel Salzet, Tristan Cardon.

Proteogenomics is becoming a powerful tool in personalized medicine by linking genomics, transcriptomics and mass spectrometry (MS)-based proteomics. Due to increasing evidence of alternative open reading frame-encoded proteins (AltProts), proteogenomics has a high potential to unravel the characteristics, variants, expression levels of the alternative proteome, in addition to already annotated proteins (RefProts). To obtain a broader view of the proteome of ovarian cancer cells compared to ovarian epithelial cells, cell-specific total RNA-sequencing profiles and customized protein databases were generated. In total, 128 RefProts and 30 AltProts were identified exclusively in SKOV-3 and PEO-4 cells. Among them, an AltProt variant of IP_715944, translated from DHX8, was found mutated (p.Leu44Pro). We show high variation in protein expression levels of RefProts and AltProts in different subcellular compartments. The presence of 117 RefProt and two AltProt variants was described, along with their possible implications in the different physiological/pathological characteristics. To identify the possible involvement of AltProts in cellular processes, cross-linking-MS (XL-MS) was performed in each cell line to identify AltProt-RefProt interactions. This approach revealed an interaction between POLD3 and the AltProt IP_183088, which after molecular docking, was placed between POLD3-POLD2 binding sites, highlighting its possibility of the involvement in DNA replication and repair.

DOI: https://doi.org/10.1038/s41419-024-07046-1
Sci Transl Med. 2024 Oct 02. 16(767): eadk9524

A peptide encoded by upstream open reading frame of MYC binds to tropomyosin receptor kinase B and promotes glioblastoma growth in mice.

Fanying Li, Kailin Yang, Xinya Gao, Maolei Zhang, Danling Gu, Xujia Wu, Chenfei Lu, Qiulian Wu, Deobrat Dixit, Ryan C Gimple, Yongping You, Stephen C Mack, Yu Shi, Tiebang Kang, Sameer A Agnihotri, Michael D Taylor, Jeremy N Rich, Nu Zhang, Xiuxing Wang.

MYC promotes tumor growth through multiple mechanisms. Here, we show that, in human glioblastomas, the variant MYC transcript encodes a 114-amino acid peptide, MYC pre-mRNA encoded protein (MPEP), from the upstream open reading frame (uORF) MPEP. Secreted MPEP promotes patient-derived xenograft tumor growth in vivo, independent of MYC through direct binding, and activation of tropomyosin receptor kinase B (TRKB), which induces downstream AKT-mTOR signaling. Targeting MPEP through genetic ablation reduced growth of patient-derived 4121 and 3691 glioblastoma stem cells. Administration of an MPEP-neutralizing antibody in combination with a small-molecule TRKB inhibitor reduced glioblastoma growth in patient-derived xenograft tumor-bearing mice. The overexpression of MPEP in surgical glioblastoma specimens predicted a poor prognosis, supporting its clinical relevance. In summary, our results demonstrate that tumor-specific translation of a MYC-associated uORF promotes glioblastoma growth, suggesting a new therapeutic strategy for glioblastoma.

DOI: https://doi.org/10.1126/scitranslmed.adk9524
Arch Oral Biol. 2024 Sep 27. pii: S0003-9969(24)00222-X. [Epub ahead of print]169 106101

An intronic variant in LAMB3 contributes to junctional epidermolysis bullosa and enamel hypoplasia via translational attenuation.

Yi Yang, Yao Wang, Man Qin, Yuming Zhao, Cristina Has, Xin Wang.

   OBJECTIVES: This study aimed to investigate the genetic etiology of a family affected by junctional epidermolysis bullosa (JEB) and generalized enamel hypoplasia, and to explore how an intronic variant influenced the 5' untranslated region (5'UTR), thereby affecting LAMB3 expression and contributing to the pathogenesis of the disease.
DESIGN: Whole-exome and whole-genome sequencing were used to screen for genetic defects in the patient. Mutational consequences were characterized through luciferase assays, splice assay, in silico analyses, and verification using the patient's gingival sample.
RESULTS: A nonsense variant (c.2983 C>T; p.Gln995*) and an intronic variant (c.-38+2 T>C) of LAMB3 were identified. In vitro assays demonstrated that the intronic variant activated a cryptic splice site, resulting in a 120 bp intronic inclusion. This splicing alteration significantly reduced the translation efficiency of the downstream coding sequence, while overall mRNA expression remained unaffected. Bioinformatic analysis unveiled the creation of three upstream AUG codons, leading to the presence of two upstream open reading frames (uORFs) and one overlapping ORF. The longer uORF's AUG exhibited a moderate Kozak strength similar to that of the main ORF's AUG. Structural analysis of the mutant 5'UTR sequence revealed a more complex secondary structure, characterized by a large branch loop and a stem-loop preceding the coding sequence's start codon.
CONCLUSION: This study suggests that variants affecting the 5'UTR may contribute to the genetic etiology of JEB. These findings could help enhance the diagnostic accuracy and efficiency in JEB patients.

Keywords:  5’ untranslated region; Amelogenesis imperfecta; Enamel; Intronic variant; Junctional epidermolysis bullosa; LAMB3

DOI:  https://doi.org/10.1016/j.archoralbio.2024.106101
Nat Commun. 2024 Oct 04. 15(1): 8605

Detection of host cell microprotein impurities in antibody drug products.

Ioanna Tzani, Marina Castro-Rivadeneyra, Paul Kelly, Lisa Strasser, Lin Zhang, Martin Clynes, Barry L Karger, Niall Barron, Jonathan Bones, Colin Clarke.

Chinese hamster ovary (CHO) cells are used to produce almost 90% of therapeutic monoclonal antibodies (mAbs) and antibody fusion proteins (Fc-fusion). The annotation of non-canonical translation events in these cellular factories remains incomplete, limiting our ability to study CHO cell biology and detect host cell protein (HCP) impurities in the final antibody drug product. We utilised ribosome footprint profiling (Ribo-seq) to identify novel open reading frames (ORFs) including N-terminal extensions and thousands of short ORFs (sORFs) predicted to encode microproteins. Mass spectrometry-based HCP analysis of eight commercial antibody drug products (7 mAbs and 1 Fc-fusion protein) using the extended protein sequence database revealed the presence of microprotein impurities. We present evidence that microprotein abundance varies with growth phase and can be affected by the cell culture environment. In addition, our work provides a vital resource to facilitate future studies of non-canonical translation and the regulation of protein synthesis in CHO cell lines.

DOI: https://doi.org/10.1038/s41467-024-51870-0
bioRxiv. 2024 Sep 16. pii: 2024.09.13.612970. [Epub ahead of print]

Translational profiling of stress-induced small proteins uncovers an unexpected connection among distinct signaling systems.

Sangeevan Vellappan, Junhong Sun, John Favate, Pranavi Jagadeesan, Debbie Cerda, Premal Shah, Srujana S Yadavalli.

Signaling networks in bacteria enable sensing and adaptation to challenging environments by activating specific genes that help counteract stressors. Small proteins (≤ 50 amino acids long) are a rising class of bacterial stress response regulators. Escherichia coli encodes over 150 small proteins, most of which lack known phenotypes and their biological roles remain elusive. Using magnesium limitation as a stressor, we investigate small proteins induced in response to stress using ribosome profiling, RNA sequencing, and transcriptional reporter assays. We uncover 17 small proteins with increased translation initiation, a majority of which are transcriptionally upregulated by the PhoQ-PhoP two-component signaling system, crucial for magnesium homeostasis. Next, we describe small protein-specific deletion and overexpression phenotypes, which underscore the physiological significance of their expression in low magnesium stress. Most remarkably, our study reveals that a small membrane protein YoaI is an unusual connector of the major signaling networks - PhoR-PhoB and EnvZ-OmpR in E. coli , advancing our understanding of small protein regulators of cellular signaling.
Highlights: Ribo-RET identifies 17 small proteins induced under low Mg 2+ stress in E. coli Many of these proteins are transcriptionally activated by PhoQP signaling systemHalf of the stress-induced small proteins localize to the membraneDeletion or overexpression of specific small proteins affects growth under stressSmall protein YoaI connects PhoR-PhoB and EnvZ-OmpR signaling networks.
Graphical abstract:

DOI: https://doi.org/10.1101/2024.09.13.612970