Proteomic and Biochemical Analyses Reveal a Novel Mechanism for Promoting Protein Ubiquitination and Degradation by UFBP1, a Key Component of Ufmylation

J Proteome Res. 2018 Apr 6;17(4):1509-1520. doi: 10.1021/acs.jproteome.7b00843. Epub 2018 Mar 22.

Abstract

Protein post-translational modification by ubiquitin-fold modifier 1, UFM1, regulates many biological processes such as response to endoplasmic reticulum stress and regulation of tumor progression. A recent study has indicated that the UFM1-binding and PCI domain-containing protein 1 (UFBP1) is required for the conjugation of UFM1 to a substrate. However, other biological functions of UFBP1 have not been explored. Here, we use immunoprecipitation and label-free quantitative proteomics to identify UFBP1-interacting proteins in a mammalian cell line. About 80 potential interacting proteins are obtained from MS analyses of three biological replicates. Bioinformatics analyses of these proteins suggest that UFBP1 may participate in the regulation of protein folding, stability, and trafficking. Biochemical experiments discover that UFBP1 expression downregulates the protein level and reduces the stability of several of its interacting proteins, while UFBP1 knockdown increases their protein levels. Protein synthesis inhibition and proteasomal inhibition experiments reveal that UFBP1 promotes their ubiquitination and degradation. Experiments using a model UFBP1-interacting protein ANT3 demonstrate that UFBP1 enhances the interaction between ANT3 and its E3 ligase and thus promotes its ubiquitination and degradation. Our work elucidates a novel molecular mechanism by which UFBP1 regulates protein ubiquitination and degradation.

Keywords: UFBP1; post-translational modification; protein stability; protein−protein interaction; quantitative proteomics; ubiquitination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism
  • Adaptor Proteins, Signal Transducing / physiology*
  • Adenine Nucleotide Translocator 3 / metabolism
  • Animals
  • Cell Line
  • Down-Regulation
  • HEK293 Cells
  • Humans
  • Mammals
  • Protein Binding
  • Protein Processing, Post-Translational*
  • Proteolysis*
  • Proteomics*
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitination*

Substances

  • Adaptor Proteins, Signal Transducing
  • Adenine Nucleotide Translocator 3
  • DDRGK1 protein, human
  • Ubiquitin-Protein Ligases