bims-micpro 2023-07-02 papers

Biomolecules. 2023 06 12. pii: 979. [Epub ahead of print]13(6):

Real-Time Search-Assisted Multiplexed Quantitative Proteomics Reveals System-Wide Translational Regulation of Non-Canonical Short Open Reading Frames.

Hiroko Kozuka-Hata, Tomoko Hiroki, Naoaki Miyamura, Aya Kitamura, Kouhei Tsumoto, Jun-Ichiro Inoue, Masaaki Oyama.

Abnormal expression of histone deacetylases (HDACs) is reported to be associated with angiogenesis, metastasis and chemotherapy resistance regarding cancer in a wide range of previous studies. Suberoylanilide hydroxamic acid (SAHA) is well known to function as a pan-inhibitor for HDACs and recognized as one of the therapeutic drug candidates to epigenetically coordinate cancer cell fate regulation on a genomic scale. Here, we established a Real-Time Search (RTS)-assisted mass spectrometric platform for system-wide quantification of translated products encoded by non-canonical short open reading frames (ORFs) as well as already annotated protein coding sequences (CDSs) on the human transciptome and applied this methodology to quantitative proteomic analyses of suberoylanilide hydroxamic acid (SAHA)-treated human HeLa cells to evaluate proteome-wide regulation in response to drug perturbation. Very intriguingly, our RTS-based in-depth proteomic analysis enabled us to identify approximately 5000 novel peptides from the ribosome profiling-based short ORFs encoded in the diversified regions on presumed 'non-coding' nucleotide sequences of mRNAs as well as lncRNAs and nonsense mediated decay (NMD) transcripts. Furthermore, TMT-based multiplex large-scale quantification of the whole proteome changes upon differential SAHA treatment unveiled dose-dependent selective translational regulation of a limited fraction of the non-canonical short ORFs in addition to key cell cycle/proliferation-related molecules such as UBE2C, CENPF and PRC1. Our study provided the first system-wide landscape of drug-perturbed translational modulation on both canonical and non-canonical proteome dynamics in human cancer cells.

Keywords: cancer; histone deacetylase; proteomics; real-time search; short open reading frames

DOI: https://doi.org/10.3390/biom13060979

STAR Protoc. 2023 Jun 28. pii: S2666-1667(23)00347-7. [Epub ahead of print]4(3): 102380

Protocol to identify human subcellular alternative protein interactions using cross-linking mass spectrometry.

Diego Fernando Garcia-Del Rio, Isabelle Fournier, Tristan Cardon, Michel Salzet.

Since the start of mass-spectrometry-based proteomics, proteins from non-referenced open reading frames or alternative proteins (AltProts) have been overlooked. Here, we present a protocol to identify human subcellular AltProt and decipher some interactions using cross-linking mass spectrometry. We describe steps for cell culture, in cellulo cross-link, subcellular extraction, and sequential digestion. We then detail both liquid chromatography-tandem mass spectrometry and cross-link data analyses. The implementation of a single workflow allows the non-targeted identification of signaling pathways involving AltProts. For complete details on the use and execution of this protocol, please refer to Garcia-del Rio et al.1.

Keywords: Bioinformatics; Mass Spectrometry; Proteomics; Systems Biology

DOI: https://doi.org/10.1016/j.xpro.2023.102380

Plants (Basel). 2023 Jun 06. pii: 2232. [Epub ahead of print]12(12):

Dynamic Translational Landscape Revealed by Genome-Wide Ribosome Profiling under Drought and Heat Stress in Potato.

Hongju Jian, Shiqi Wen, Rongrong Liu, Wenzhe Zhang, Ziyan Li, Weixi Chen, Yonghong Zhou, Vadim Khassanov, Ahmed M A Mahmoud, Jichun Wang, Dianqiu Lyu.

The yield and quality of potatoes, an important staple crop, are seriously threatened by high temperature and drought stress. In order to deal with this adverse environment, plants have evolved a series of response mechanisms. However, the molecular mechanism of potato's response to environmental changes at the translational level is still unclear. In this study, we performed transcriptome- and ribosome-profiling assays with potato seedlings growing under normal, drought, and high-temperature conditions to reveal the dynamic translational landscapes for the first time. The translational efficiency was significantly affected by drought and heat stress in potato. A relatively high correlation (0.88 and 0.82 for drought and heat stress, respectively) of the fold changes of gene expression was observed between the transcriptional level and translational level globally based on the ribosome-profiling and RNA-seq data. However, only 41.58% and 27.69% of the different expressed genes were shared by transcription and translation in drought and heat stress, respectively, suggesting that the transcription or translation process can be changed independently. In total, the translational efficiency of 151 (83 and 68 for drought and heat, respectively) genes was significantly changed. In addition, sequence features, including GC content, sequence length, and normalized minimal free energy, significantly affected the translational efficiencies of genes. In addition, 28,490 upstream open reading frames (uORFs) were detected on 6463 genes, with an average of 4.4 uORFs per gene and a median length of 100 bp. These uORFs significantly affected the translational efficiency of downstream major open reading frames (mORFs). These results provide new information and directions for analyzing the molecular regulatory network of potato seedlings in response to drought and heat stress.

Keywords: drought stress; heat stress; potato; ribosome profiling; upstream open reading frames

DOI: https://doi.org/10.3390/plants12122232