Identification of Microprotein-Protein Interactions via APEX Tagging

Biochemistry. 2017 Jul 5;56(26):3299-3306. doi: 10.1021/acs.biochem.7b00265. Epub 2017 Jun 7.

Abstract

Microproteins are peptides and small proteins encoded by small open reading frames (smORFs). Newer technologies have led to the recent discovery of hundreds to thousands of new microproteins. The biological functions of a few microproteins have been elucidated, and these microproteins have fundamental roles in biology ranging from limb development to muscle function, highlighting the value of characterizing these molecules. The identification of microprotein-protein interactions (MPIs) has proven to be a successful approach to the functional characterization of these genes; however, traditional immunoprecipitation methods result in the enrichment of nonspecific interactions for microproteins. Here, we test and apply an in situ proximity tagging method that relies on an engineered ascorbate peroxidase 2 (APEX) to elucidate MPIs. The results demonstrate that APEX tagging is superior to traditional immunoprecipitation methods for microproteins. Furthermore, the application of APEX tagging to an uncharacterized microprotein called C11orf98 revealed that this microprotein interacts with nucleolar proteins nucleophosmin and nucleolin, demonstrating the ability of this approach to identify novel hypothesis-generating MPIs.

Publication types

  • Comparative Study
  • Validation Study

MeSH terms

  • Active Transport, Cell Nucleus
  • Amino Acid Sequence
  • Animals
  • Ascorbate Peroxidases / chemistry
  • Ascorbate Peroxidases / genetics
  • Ascorbate Peroxidases / metabolism*
  • Cell Nucleolus / metabolism*
  • Conserved Sequence
  • HEK293 Cells
  • HeLa Cells
  • Hemagglutinins / chemistry
  • Hemagglutinins / genetics
  • Hemagglutinins / metabolism
  • Humans
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Oligopeptides / chemistry
  • Oligopeptides / genetics
  • Oligopeptides / metabolism
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Phosphoproteins / chemistry
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Protein Engineering*
  • Protein Interaction Domains and Motifs
  • Proteomics / methods*
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Sequence Alignment

Substances

  • C11orf98 protein, human
  • Hemagglutinins
  • Nuclear Proteins
  • Oligopeptides
  • Peptide Fragments
  • Phosphoproteins
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins
  • nucleolin
  • nucleophosmin
  • FLAG peptide
  • Ascorbate Peroxidases