Open AccessAn efficient proteome-wide strategy for discovery and characterization of cellular nucleotide-protein interactions
Author(s) -
Yan Ting Lim,
Nayana Prabhu,
Lingyun Dai,
Ka Diam Go,
Dan Chen,
Lekshmy Sreekumar,
Louise Egeblad,
S Eriksson,
Liyan Chen,
Saranya Veerappan,
Hsiangling Teo,
Chris Soon Heng Tan,
Johan Lengqvist,
Andreas Larsson,
Radoslaw M. Sobota,
P. Nordlund
Publication year - 2018
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0208273
Subject(s) - proteome , nucleotide , biology , protein–protein interaction , computational biology , cellular metabolism , deoxyribonucleotide , chemical biology , biochemistry , microbiology and biotechnology , metabolism , gene
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.