Abstract
Despite the immense importance of enzyme-substrate reactions, there is a lack of generic and unbiased tools for identifying and prioritizing substrate proteins which are modulated in the structural and functional levels through modification. Here we describe a high-throughput unbiased proteomic method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that enzymatic post-translational modification of substrate proteins might change their thermal stability. SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems in up to a depth of 7179 proteins. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, open new opportunities in investigating the effect of PTMs on signal transduction, and facilitate drug discovery.
Footnotes
To show the universal applicability of SIESTA for different enzyme systems and post-translational modifications, we have now amended the manuscript with a deep experiment on protein kinase B (AKT1) enzyme, proving that SIESTA can discover and prioritize phosphorylation events. In addition, a number of potential substrates for thioredoxin reductase 1 (TXNRD1) and poly-(ADP-ribose) polymerase-10 (PARP10) systems were further validated. Figures updated, author affiliations updated, supplemental files updated.
