An efficient proteome-wide strategy for discovery and characterization of cellular nucleotide-protein interactions

PLoS One. 2018 Dec 6;13(12):e0208273. doi: 10.1371/journal.pone.0208273. eCollection 2018.


Metabolite-protein interactions define the output of metabolic pathways and regulate many cellular processes. Although diseases are often characterized by distortions in metabolic processes, efficient means to discover and study such interactions directly in cells have been lacking. A stringent implementation of proteome-wide Cellular Thermal Shift Assay (CETSA) was developed and applied to key cellular nucleotides, where previously experimentally confirmed protein-nucleotide interactions were well recaptured. Many predicted, but never experimentally confirmed, as well as novel protein-nucleotide interactions were discovered. Interactions included a range of different protein families where nucleotides serve as substrates, products, co-factors or regulators. In cells exposed to thymidine, a limiting precursor for DNA synthesis, both dose- and time-dependence of the intracellular binding events for sequentially generated thymidine metabolites were revealed. Interactions included known cancer targets in deoxyribonucleotide metabolism as well as novel interacting proteins. This stringent CETSA based strategy will be applicable for a wide range of metabolites and will therefore greatly facilitate the discovery and studies of interactions and specificities of the many metabolites in human cells that remain uncharacterized.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Nucleotides / genetics
  • Nucleotides / metabolism*
  • Protein Binding
  • Proteins / genetics
  • Proteins / metabolism*
  • Proteome / genetics
  • Proteome / metabolism*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism


  • DNA-Binding Proteins
  • Nucleotides
  • Proteins
  • Proteome
  • RNA-Binding Proteins

Grants and funding

This work was supported by Nanyang Technological University,, to PN, the Swedish Research Council (Vetenskapsrådet),, to PN, the Swedish Cancer Society (Cancerfonden),, to PN, Radiumhemmets research fund, to PN, the Knut and Alice Wallenberg foundation (Knut och Alice Wallenbergs Stiftelse),, to PN, Singapore Ministry of Health's National Medical Research Council,, MOHIAFCAT2/004/2015, to PN, RMS, and AL, and Agency for Science, Technology and Research Council (A-STAR BMRC) YIG2015 grant,, to RMS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.