Abstract
Despite the immense importance of enzyme-substrate reactions, there is a lack of generic and unbiased tools for identifying the molecular components participating in these reactions on a cellular level. Here we developed a universal method called System-wide Identification of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that enzymatic post-translational modification of substrate proteins changes their thermal stability, and applies the concept of specificity to reveal potential substrates. For selenoprotein thioredoxin reductase 1, SIESTA confirmed several known protein substrates and suggested novel candidates. For poly-(ADP-ribose) polymerase-10, SIESTA revealed a number of putative substrates, which were confirmed by targeted mass spectrometry and functional assays. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, and facilitate drug discovery.
One sentence summary Monitoring protein stability can reveal changes due to specific enzymatic post-translational modifications at the proteome level.