Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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N-Terminal Modifications of Ubiquitin via Methionine Excision, Deamination, and Arginylation Expand the Ubiquitin Code

  • Nguyen, Kha The (Department of Life Sciences, Pohang University of Science and Technology) ;
  • Ju, Shinyeong (Center for Theragnosis, Korea Institute of Science and Technology) ;
  • Kim, Sang-Yoon (Department of Life Sciences, Pohang University of Science and Technology) ;
  • Lee, Chang-Seok (Department of Life Sciences, Pohang University of Science and Technology) ;
  • Lee, Cheolju (Center for Theragnosis, Korea Institute of Science and Technology) ;
  • Hwang, Cheol-Sang (Department of Life Sciences, Pohang University of Science and Technology)
  • Received : 2021.11.19
  • Accepted : 2022.01.12
  • Published : 2022.03.31
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Abstract

Ubiquitin (Ub) is post-translationally modified by Ub itself or Ub-like proteins, phosphorylation, and acetylation, among others, which elicits a variety of Ub topologies and cellular functions. However, N-terminal (Nt) modifications of Ub remain unknown, except the linear head-to-tail ubiquitylation via Nt-Met. Here, using the yeast Saccharomyces cerevisiae and an Nt-arginylated Ub-specific antibody, we found that the detectable level of Ub undergoes Nt-Met excision, Nt-deamination, and Nt-arginylation. The resulting Nt-arginylated Ub and its conjugated proteins are upregulated in the stationary-growth phase or by oxidative stress. We further proved the existence of Nt-arginylated Ub in vivo and identified Nt-arginylated Ub-protein conjugates using stable isotope labeling by amino acids in cell culture (SILAC)-based tandem mass spectrometry. In silico structural modeling of Nt-arginylated Ub predicted that Nt-Arg flexibly protrudes from the surface of the Ub, thereby most likely providing a docking site for the factors that recognize it. Collectively, these results reveal unprecedented Nt-arginylated Ub and the pathway by which it is produced, which greatly expands the known complexity of the Ub code.

Keywords

Acknowledgement

We are grateful to the current and former members of the Hwang and Lee laboratories for their assistance and advice. We also thank Edanz (www.edanz.com/ac) for editing a draft of this manuscript. This work was supported by grants from the Korean Government (MSIP) NRF-2020R1A3B2078127 and NRF2017R1A5A1015366 (C.-S.H.) and NRF-2020R1A2C2003685 (C.L.), and the BK21 Plus program (C.-S.H.).

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