School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
Department of Anatomy, Physiology and Biochemistry, The Swedish University of Agricultural Sciences, Uppsala, Sweden.
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.
代谢物-蛋白质相互作用决定了代谢途径的输出，并调节许多细胞过程。尽管疾病通常表现为代谢过程的扭曲，但缺乏有效的方法来直接在细胞中发现和研究这些相互作用。我们开发了一种严格的全蛋白质组细胞热转移分析（CETSA）的实施方法，并将其应用于关键的细胞核苷酸，其中以前通过实验证实的蛋白质-核苷酸相互作用得到了很好的重现。我们发现了许多以前从未通过实验证实的、以及新的蛋白质-核苷酸相互作用。这些相互作用包括一系列不同的蛋白质家族，核苷酸在其中充当底物、产物、辅助因子或调节剂。在暴露于胸苷的细胞中，胸苷是 DNA 合成的限制前体，依次产生的胸苷代谢物的细胞内结合事件的剂量和时间依赖性被揭示出来。这些相互作用包括脱氧核苷酸代谢中的已知癌症靶点以及新的相互作用蛋白。这种基于严格 CETSA 的策略将适用于广泛的代谢物，因此将极大地促进对人类细胞中许多未被表征的代谢物的相互作用和特异性的发现和研究。
