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. 2021 Feb 26;12(1):1296.
doi: 10.1038/s41467-021-21540-6.

System-wide identification and prioritization of enzyme substrates by thermal analysis

Amir Ata Saei #  1   2 , Christian M Beusch #  3 , Pierre Sabatier  3 , Juan Astorga Wells  3 , Hassan Gharibi  3 , Zhaowei Meng  3 , Alexey Chernobrovkin  3   4 , Sergey Rodin  3   5 , Katja Näreoja  6 , Ann-Gerd Thorsell  6 , Tobias Karlberg  6 , Qing Cheng  7 , Susanna L Lundström  3 , Massimiliano Gaetani  3   8   9 , Ákos Végvári  3   10 , Elias S J Arnér  7 , Herwig Schüler  6 , Roman A Zubarev  11   12
Affiliations

System-wide identification and prioritization of enzyme substrates by thermal analysis

Amir Ata Saei et al. Nat Commun. .

Abstract

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. SIESTA workflow for unbiased proteome-wide identification and prioritization of enzyme substrates.
A master cell lysate is prepared by multiple freeze-thawing in a non-denaturing buffer. The cell lysate aliquots are treated with vehicle (control), cosubstrate, enzyme, or combination of enzyme with cosubstrate (both). After treatment, each aliquot is split into ten tubes, with each tube heated to a temperature point in the range from 37 to 67 °C. After removing unfolded proteins by ultracentrifugation, identical volumes of supernatants are digested with trypsin. The samples are then serially labeled with 10-plex tandem mass tag (TMT) reagents, pooled, cleaned, and fractionated by reversed-phase chromatography. After LC-MS/MS analyses of each fraction, protein IDs and abundances are determined, and sigmoid curves are fitted through an automated algorithm to determine the melting temperature (Tm) for each protein. For each non-vehicle treatment, the read-out is the protein’s ∆Tm shifts (of both signs) compared to control. Any protein shifting more upon addition of enzyme and cosubstrate compared to when they are added alone, are putative substrates of the enzyme under study. Such candidate protein substrates are subsequently confirmed by orthogonal verification methods, starting from the proteins exhibiting largest ∆Tm shifts and/or involved in relevant pathways.
Fig. 2
Fig. 2. Proof-of-principle SIESTA experiment revealed known TXNRD1 substrates and suggested novel candidates.
a Scatterplot of protein Tm differences upon addition of NADPH in lysate. Known proteins from UniProt are shown in red (n = 2 independent biological replicates; two-sided Student t-test; no adjustment for multiple comparisons was performed). b Representative stabilization of NADPH binding protein IDH1. c Scatterplot of Tm differences reveals the Tm shifts occurring only after simultaneous TXNRD1 + NADPH addition; these shifts are thus likely due to enzymatic modifications (yellow shaded area, known and putative substrates are shown as green circles). d Potential substrates (green circles) are mostly located close to the negative reference point (blue star) in an OPLS-DA model contrasting the TXNRD1 + NADPH Tm against the single treatments. Proteins shown in green are those identified as substrates in c. e Representative melting curves of GPX1, PRDX2, GULP1, and GSTO2 are shown. f Reduction of cysteines in the substrate proteins by incubation with TXNRD1 + NADPH (n = 3 independent biological replicates, one-sided Student t-test), measured by sequential iodoTMT labeling (Center line—median; box limits contain 50% of data; upper and lower quartiles, 75 and 25%; maximum—greatest value excluding outliers; minimum—least value excluding outliers; outliers—more than 1.5 times of the upper and lower quartiles) (NADPH nicotinamide adenine dinucleotide phosphate, Tm melting temperature, TXNRD1 thioredoxin reductase 1). Source data are provided as a Source Data file.
Fig. 3
Fig. 3. SIESTA identified known and putative substrates for AKT1 kinase.
a Scatterplot of Tm differences reveals the Tm shifts occurring only after simultaneous AKT1 + ATP addition (known and putative substrates are shown as green circles). b Representative melting curves for known and putative AKT1 substrates. c The reduction in the phosphorylation level of AKT1 substrate proteins by AKT1/2 inhibitor and ipatasertib treatment at 2.5 and 10 µM (all shown peptides are significant under all compound treatment conditions vs. DMSO condition (p < 0.05); n = 3 independent biological replicates, two-sided Student t-test). The phosphorylation levels of other phosphopeptides detected for the same proteins are compiled into one plot. N denotes the number of unchanged phosphopeptides for each protein. (Center line—median; box limits contain 50% of data; upper and lower quartiles, 75 and 25%; maximum—greatest value excluding outliers; minimum—least value excluding outliers; outliers—more than 1.5 times of the upper and lower quartiles) (AKT1 RAC-alpha serine/threonine-protein kinase or protein kinase B, ATP adenosine triphosphate, Tm melting temperature). Source data are provided as a Source Data file.
Fig. 4
Fig. 4. SIESTA identified and ranked known and novel PARP10 substrates.
a Scatterplot of Tm differences reveals the shifts occurring when PARP10 + NAD are incubated with cell lysate. Known and putative substrates are shown in green circles. b Representative melting curves of putative PARP10 substrates. c Mono-ADP-ribosylation on a glutamic acid residue Glu110 in the PDRG1 peptide with the highest sequence-fitting score revealed by targeted ETD MS/MS of 3+ molecular ions (M). The fragments carrying the modification are marked with an asterisk (Tm melting temperature, NAD nicotinamide adenine dinucleotide, PARP10 poly-(ADP-ribose) polymerase-10). Source data are provided as a Source Data file.
Fig. 5
Fig. 5. TPP identifies protein interaction partners of enzymes added to cell lysates.
a Proteins’ Tm shifting with addition of only enzyme to cell lysate. The known proteins interacting with AKT1 are shown in bold red (FUNCI, GET4, PHLPP2, and MAZ) (n = 2 independent biological replicates; two-sided Student t-test; no adjustment for multiple comparisons was performed). b Representative shifting proteins with each enzyme. DECR1 was also enriched in the affinity pulldown experiment with PARP10 (Tm melting temperature). Source data are provided as a Source Data file.
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