• 文部科学省
  • JSPS
  • 科研費
  • ユビキチンネオバイオロジー
  • ユビキチンネオバイオロジー
研究チーム

Nobumoto Watanabe

Screening for small molecule competitors of the phosphorylated substrates ofβ-TrCP and their application for the induction of targeted protein degradation

Nobumoto Watanabe

Nobumoto Watanabe, PhD

RIKEN-Max Planck Joint Research Division for Systems Chemical Biology, Bioprobe Application Research Unit, RIKEN Center for Sustainable Resource Science
Unit Leader
http://www.riken.jp/research/labs/csrs/riken_max_planck/bioprobe_app/

Research summary

β-TrCP is a F-box protein, the substrate recognition subunit in the SCF (Skp1-Cul1-F-box protein) E3 ubiquitin ligase complex. F-box proteins frequently recognize their substrates not only via primary amino acid sequence motifs but also as a result of phosphorylation. The interaction between β-TrCP and its substrates is one of the most extensively studied examples of the phosphorylation dependent substrate recognition mechanism of F-box proteins. β-TrCP recognizes a DSGXXS motif in the N-terminal region of IκBα, only when these serines are phosphorylated by the IκB kinase, IKK. β-TrCP also recognizes other substrates, including β-catenin, Cdc25A, human immunodeficiency virus Vpu protein, Emi1 and others, in a phosphorylation-dependent manner.
Previously, we have identified human Wee1A, a protein tyrosine kinase that phosphorylates and keeps Cdk1 inactive at G2 phase of the cell cycle before cells are ready for mitosis, as one of the substrates for β-TrCP. When cells are ready for mitosis, Cdk1 phosphorylates 123th Serine of Wee1A and this phosphorylated serine (pS123) triggers the phosphorylation cascades for the Wee1A ubiquitination. The pS123 not only directly interacts with basic residues in the WD40 repeat domain of β-TrCP, but also primes phosphorylation by two independent protein kinases, Polo like kinase 1 (Plk1) and CK2, to create two phosphodegrons on Wee1A. In the case of Plk1, S123 phosphorylation creates a Polo box domain (PBD) binding motif (SpSP) on Wee1A to accelerate phosphorylation of S53 by Plk1. CK2 can phosphorylate S121, but only if S123 is phosphorylated first, thereby generating the second β-TrCP binding site surrounding S121.
We also developed a high-throughput screening system to identify inhibitors of PBD-dependent binding and screened a chemical library. We isolated a benzotropolone-containing natural compound derived from nutgalls (purpurogallin (PPG)) that inhibited PBD-dependent binding in vitro and in vivo. PPG not only delayed the onset of mitosis, but also pro-longed the progression of mitosis in HeLa cells.
Using this system, we are going to isolate small molecule competitors of the phosphorylated substrates ofβ-TrCP in this project. When such small molecules are identified, we will generate a synthetic molecule by binding it to a substrate recognition ligand for a target protein. We expect such molecule will induce the degradation of the target protein throughβ-TrCP dependent ubiquitination.

Publications

  1. Wang L, Liu S, Xu J, Watanabe N, Mayo KH, *Li J, *Li X.
    Emodin inhibits aggregation of amyloid-β peptide 1-42 and improves cognitive deficits in Alzheimer's disease transgenic mice.
    J. Neurochem. 157, 1992-2007 (2021)
    PMID: 32799401
  2. Wilke J, Kawamura T, Xu H, Brause A, Friese A, Metz M, Schepmann D, Wünsch B, Artacho-Cordón A, Nieto FR, Watanabe N, Osada H, Ziegler S, *Waldmann H .
    Discovery of a σ1 receptor antagonist by combination of unbiased cell painting and thermal proteome profiling.
    Cell Chem. Biol. 28, 848-854.e5 (2021)
    PMID: 33567254
  3. Tan FHP, Ting ACJ, Leow BG, Najimudin N,Watanabe N, *Azzam G.
    Alleviatory effects of Danshen, Salvianolic acid A and Salvianolic acid B on PC12 neuronal cells and Drosophila melanogaster model of Alzheimer’s disease.
    J. Ethnopharmacology 279, 114389 (2021)
    PMID: 34217797
  4. Tan FHP, Hadri NAB, Najimudin N, Watanabe N, *Azzam G.
    Ethyl caffeate ameliorated Aβ42-associated toxicity in PC12 cells and Drosophila melanogaster.
    Geriatr Gerontol Int. 21, 1125-1130 (2021)
    PMID: 34699118
  5. Moon JY, Adams E, Miyazaki T, Kondoh Y, Muroi M, Watanabe N, Osada H, Shin R.
    Cesium tolerance is enhanced by a chemical which binds to BETA- GLUCOSIDASE 23 in Arabidopsis thaliana.
    Sci. Rep. 11, 21109 (2021)
    PMID: 34702872
  6. Kwong MMY, Lee JW, Samian MR, Wahab H, Watanabe N, Ong EBB.
    Identification of Tropical Plant Extracts That Extend Yeast Chronological Life Span.
    Cells 10, 2718 (2021)
    PMID: 34685698
  7. Lee JW, Ong TG, Samian MR, Teh AH, Watanabe N, Osada H, Ong EBB.
    Screening of selected ageing-related proteins that extend chronological life span in yeast Saccharomyces cerevisoae.
    Sci. Rep. 11, 24148 (2021)
    PMID: 34921163
  8. Zou X, Liu Y, Di J, Wei W, Watanabe N, Li J, Li X.
    ZMIZ2 promotes the development of triple-receptor negative breast cancer.
    Cancer Cell Int. 22, 52 (2022)
    PMID: 35101047
  9. Yoshioka H, Kawamura T, Muroi M, KondohY, Honda K, Kawatani M, Aono H, Waldmann H, Watanabe N, Osada H.
    Identification of a small molecule that enhances ferroptosis via inhibition of FSP1.
    ACS Chem. Biol. 17, 483-491 (2022)
    PMID: 35128925
  10. Wei W, Pathak J L, LIU X, Mao T, Watanabe N, Li X, Zhang M, Li J.
    Apigenin, a single active component of herbal extract, alleviates xerostomia via ERα-mediated upregulation of AQP5 activation.
    Front. Pharmacol. 13, 818116 (2022)
    PMID: 35264956

Former Publications

  1. Suvarna K, Honda K, Kondoh Y, Osada H, *Watanabe N.
    Identification of a small molecule ligand of β-arrestin1 as an inhibitor of stromal fibroblast cell migration accelerated by cancer cells.
    Cancer Med. 7, 883-893 (2018)
    PMID: 29380537
  2. Suvarna K, Honda K, Muroi M, Kondoh Y, Osada H, *Watanabe N.
    A small-molecule ligand of valosin-containing protein/p97 inhibits cancer cell-accelerated fibroblast migration.
    J. Biol. Chem. 294, 2988-2996 (2019)
    PMID: 30610116
  3. Subedi A, Muroi M, Futamura Y, Kawamura T, Aono H, Nishi M, Ryo A, *Watanabe N, *Osada H.
    A novel inhibitor of tumorspheres revealed activation of serine biosynthetic pathway upon mitochondrial inhibition.
    FEBS Letters 593, 763-776 (2019)
    PMID: 30874300
  4. Suvarna K, Honda K, Muroi M, Kondoh Y, Osada H, *Watanabe N.
    Measurement of ATPase activity of valosin-containing protein/p97.
    Bio-protocol 10, e3516 (2020)
  5. *Watanabe N, Osada H.
    Small molecule inhibitors of E3 ubiquitin ligases.
    Drug Discovery Series No. 78
    Protein-Protein Interaction Regulators (Roy, S. and Fu, H. eds.) Royal Society of Chemistry pp.109-123 (2020)