PROMIS, global analysis of PROtein–metabolite interactions using size separation in Arabidopsis thaliana

Abstract

Small molecules not only represent cellular building blocks and metabolic intermediates, but also regulatory ligands and signaling molecules that interact with proteins. Although these interactions affect cellular metabolism, growth, and development, they have been largely understudied. Herein, we describe a method, which we named PROtein–Metabolite Interactions using Size separation (PROMIS), that allows simultaneous, global analysis of endogenous protein–small molecule and of protein–protein complexes. To this end, a cell-free native lysate from Arabidopsis thaliana cell cultures was fractionated by size-exclusion chromatography, followed by quantitative metabolomic and proteomic analyses. Proteins and small molecules showing similar elution behavior, across protein-containing fractions, constituted putative interactors. Applying PROMIS to an A. thaliana extract, we ascertained known protein–protein (PPIs) and protein–metabolite (PMIs) interactions and reproduced binding between small-molecule protease inhibitors and their respective proteases. More importantly, we present examples of two experimental strategies that exploit the PROMIS dataset to identify novel PMIs. By looking for similar elution behavior of metabolites and enzymes belonging to the same biochemical pathways, we identified putative feedback and feed-forward regulations in pantothenate biosynthesis and the methionine salvage cycle, respectively. By combining PROMIS with an orthogonal affinity purification approach, we identified an interaction between the dipeptide Tyr–Asp and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. In summary, we present proof of concept for a powerful experimental tool that enables system-wide analysis of PMIs and PPIs across all biological systems. The dataset obtained here comprises nearly 140 metabolites and 5000 proteins, which can be mined for putative interactors.

PROMIS: Global Analysis of PROtein‐Metabolite Interactions

“…To narrow down potential candidates, we advise (i) combining the predicting power of PROMIS with biochemical knowledge regarding the proteins involved in the biosynthesis pathways of a given metabolite, and (ii) the use of binding evidence for experimentally confirmed protein-proteinmetabolite interactions in other organisms, considering that molecular complexes are often evolutionarily conserved. This approach led us to the identification of putative feedback and feed-forward regulatory mechanisms in pantothenate and methylthioadenosine synthesis pathways, as we proved the interaction between pantothenate and ketopantoate hydroxymethyltransferase (KPHMT1) and between methylthioadenosine and methylthioribose-1-phosphate isomerase (MTI; Veyel et al, 2018).…”

“…PROMIS is the only available non‐targeted method that allows for metabolome‐ and proteome‐wide analysis of protein–metabolite interactions at close to in vivo protein and metabolite concentrations (Veyel et al., , ). It requires no modification of the protein or metabolite parts of the complex, as it relies on the assumption that components of the molecular complexes co‐migrate through an SEC column.…”

“…interactions at close to in vivo protein and metabolite concentrations (Veyel et al, , 2018. It requires no modification of the protein or metabolite parts of the complex, as it relies on the assumption that components of the molecular complexes co-migrate through an SEC column.…”

“…PROtein–metabolite interactions using size separation (PROMIS) is a biochemical method for studying protein–small molecule interactions on a proteome‐ and metabolome‐wide scale, based on the simple assumption that the small molecules or molecular complexes will exhibit similar chromatographic behavior (Veyel et al., , ). Despite a few systematic studies, the true extent of protein–small molecule interactions in biological systems remains unknown (Diether, Nikolaev, Allain, & Sauer, ; Gallego et al., ; Li, Gianoulis, Yip, Gerstein, & Snyder, ; Piazza et al., ).…”

Systems level profiling of arginine starvation reveals MYC and ERK adaptive metabolic reprogramming

“…Alternatively, activity-based proteomic profiling (ABPP) can evaluate changes in protein activity (Cravatt et al , 2008), kinase activity (Duncan et al , 2012), or ligand binding events (Luzarowski & Skirycz, 2019). Changes in protein expression or protein-protein interactions invariably contribute to ABPP as well (Wolfe et al , 2013; Piazza et al , 2018a; Veyel et al , 2018). Ultimately, ABPP integrates multiple informative proteomic parameters and provides a broad view of proteomic regulation.…”