Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
Nat Commun. 2021 Feb 26;12(1):1296. doi: 10.1038/s41467-021-21540-6.
Despite the immense importance of enzyme-substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, 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. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery.
尽管酶-底物反应极其重要，但目前缺乏通用且无偏的工具来识别和优先考虑在结构水平上被酶修饰的底物蛋白。在这里，我们描述了一种称为通过热分析系统鉴定和优先考虑酶底物的高通量无偏蛋白质组学方法（SIESTA）。该方法假设底物蛋白的酶翻译后修饰很可能改变其热稳定性。在我们的概念验证研究中，SIESTA 成功地鉴定了几种已知和新型的硒蛋白硫氧还蛋白还原酶 1、蛋白激酶 B（AKT1）和聚（ADP-核糖）聚合酶-10 系统的底物候选物。SIESTA 的更广泛应用可以增强我们对酶在体内平衡和疾病中的作用的理解，为研究翻译后修饰对信号转导的影响并促进药物发现提供了机会。
