Shin-biology regulated by protein lifetime

Grant-in-Aid for Transformative Research Areas (A) FY2023–FY2027, MEXT Japan

Planned Research

Development of Large-Scale Measurement Methods for Protein Lifetimes and Elucidation of Lifetime Control Principles
Yasushi Ishihama
Graduate School of Pharmaceutical Sciences, Kyoto University
Syujiro Okuda
Niigata University Graduate School of Medical and Dental Sciences
Koshi Imami
Proteome Homeostasis Research Unit, RIKEN Center for Integrative Medical Sciences

Using state-of-the-art proteomics, we will establish techniques to measure protein lifetimes quantitatively and comprehensively, especially degradation processes, and explore the correlation between proteoforms in various samples to elucidate the mechanisms of protein lifetime regulation. In addition, we will conduct exploratory research on protein lifetime regulators by analyzing information from large-scale public data.

1. Development of large-scale measurement approaches for protein lifetimes We will develop a proteome-wide protein lifetime measurement method that combines the SILAC method, in which stable isotope amino acids and non-natural amino acids are added during cell culture, with various selective protein and peptide enrichment methods, and apply it to various samples from various research groups.

2. Large-scale temporal analysis of various proteoforms during protein degradation We will examine differences in protein stability due to post-translational modifications, N- and C-terminal truncations, and isoforms in various samples on a proteome scale. We will also examine the effects of interacting proteins on protein stability.

3. Reanalysis and bioinformatics analysis of large public proteome data
Using jPOST repository, the Asian base of the ProteomeXchange Consortium, the world’s largest public repository of proteome data, we will reanalyze and standardize the proteome data, and then analyze protein stability in combination with RNA-Seq and Ribo-Seq data. We will also search for factors that regulate protein lifetime by integrating protein sequence, conformational, and post-translational modification information.

  1. *Imami K, Selbach M, Ishihama Y. Monitoring mitochondrial translation by pulse SILAC. J Biol Chem 299:102865 (2023).
  2. Tsai CF, Ogata K, Sugiyama N, *Ishihama Y. Motif-centric phosphoproteomics to target kinase-mediated signaling pathways. Cell Rep Methods. 2(1):100138 (2022).
  3. Uchiyama J, Roy R, Wang DO, Morikawa K, Kawahara Y, Iwasaki M, Yoshino C, Mishima Y, *Ishihama Y, *Imami K. pSNAP: Proteome-wide analysis of elongating nascent polypeptide chains. iScience 104516 (2022).
  4. Nishida H, *Ishihama Y One-Step Isolation of Protein C-Terminal Peptides from V8 Protease-Digested Proteins by Metal Oxide-Based Ligand-Exchange Chromatography. Anal Chem 94(2), 944–951 (2022).
  5. Chang C-H, Chang H-Y, Rappsilber J, *Ishihama Y, Isolation of Acetylated and Unmodified Protein N-Terminal Peptides by Strong Cation Exchange Chromatographic Separation of TrypN-Digested Peptides. Mol Cell Proteomics, 20, 100003 (2021).
  6. Tsumagari K, Chang CH, *Ishihama Y. Exploring the landscape of ectodomain shedding by quantitative protein terminomics. iScience 24, 102259 (2021)
  7. Sugiyama N, Imamura H, *Ishihama Y Large-scale Discovery of Substrates of the Human Kinome. Sci Rep 9 (1), 10503 (2019).
  8. Okuda S, Watanabe Y, Moriya Y, Kawano S, Yamamoto T, Matsumoto M, Takami T, Kobayashi D, Araki N, Yoshizawa AC, Tabata T, Sugiyama N, Goto S, *Ishihama Y, jPOSTrepo: an international standard data repository for proteomes, Nucleic Acids Res, 45 (D1), D1107-D1111 (2016).
  9. Lin M-H, Sugiyama N, *Ishihama Y, Systematic profiling of the bacterial phosphoproteome reveals bacterium-specific features of phosphorylation. Sci Signal 8 (394), rs10 (2015).
  10. Tsai C-F, Wang Y-T, Yen H-Y, Tsou C-C, Ku W-C, Lin P-Y, Chen H-Y, Nesvizhskii A I, *Ishihama Y, *Chen Y-J, Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics. Nat Commun 6, 6622 (2015).