A systems chemoproteomic analysis of acyl-CoA/protein interaction networks

Abstract

CoA metabolite interactions, enabling the identification of biological processes susceptible to altered acetyl-CoA levels. Finally, we utilize systems-level analyses to assess the features of novel protein networks that may interact with acyl-CoAs and demonstrate a strategy for high-confidence annotation of direct acetyl-CoA binding proteins and AT enzymes in human proteomes. Overall our studies illustrate the power of integrating chemoproteomics and systems biology analysis methods and provide a novel resource for understanding the diverse signaling roles of acyl-CoAs in biology and disease. Results Validation of CATNIP for the global study of acyl-CoA/protein interactionsIn order to deeply sample acyl-CoA/protein interactions on a proteome-wide scale, we initially set out to integrate CoA-based protein capture methods with LC-MS/MS ( Fig. 2a). In this workflow, whole cell extracts are first incubated with Lys-CoA Sepharose. This affinity matrix enables active site-dependent enrichment of many different classes of CoA-binding proteins, 8 making it ideal for broad profiling studies. Next, enriched proteins are subjected to tryptic digest and analyzed using MudPIT (multidimensional protein identification technology), a proteomics platform that combines strong cation exchange and C18 reverse phase chromatography to pre-fractionate tryptic peptides, followed by ionization and data-dependent MS/MS. 9 The separation afforded by this approach significantly decreases sample complexity, allowing the identification of rare, low abundance peptides from complex proteomic mixtures. To facilitate the identification of acyl-CoA/protein interactions, competition experiments are performed in which proteomes are pre-incubated with a CoA metabolite prior to capture. 10 Decreased enrichment in competition samples compared to controls (as assessed by quantitative spectral counting) signifies that the CoA metabolite interacts with a protein of interest.These interacting proteins can then be further classified into pharmacological or biological networks using either conventional metrics (fold-change, gene ontology, etc) or systems-based analysis tools.As an initial model, we explored the utility of CATNIP to globally profile acetyl-CoA/protein interactions in unfractionated HeLa cell proteomes. Proteomes were pre-incubated with acetyl-CoA or vehicle (buffer) control, followed by enrichment using Lys-CoA Sepharose. These experiments assessed competition at 3, 30, and 300 µM acetyl-CoA, which spans the physiological concentration range of acetyl-CoA in the cytosol and mitochondria. Protein capture in each condition was quantified using distributed normalized spectral abundance factor (dNSAF), a label-free metric that normalizes spectral counts relative to overall protein length ( Fig. S1a-c , Table S1). 11 Each condition was analyzed in triplicate, constituting 12 experiments, >144 hours of instrument time, and over 1.1 million non-redundant peptide spectra collected. We limited our analysis to highconfidence protein identifications (>4...

Harnessing Ionic Selectivity in Acetyltransferase Chemoproteomic Probes

“…9−12 Recently, in the process of characterizing a histone acetyltransferase chemoproteomic probe, we discovered that CoA-based affinity resins exhibit broad-spectrum capture properties, enriching lysine acetyltransferases, N-terminal and metabolic acetyltransferases, and several CoA and NAD-based metabolic enzymes (e.g., oxidoreductases). 13,14 This indicates that CoA-based chemoproteomic probes may have utility in studying the pharmacological interactions of additional enzyme classes, beyond histone acetyltransferases. However, one challenge in applying this approach is how to differentiate a "true hit" from the background.…”

“…Of note, the use of TMT tagging represents a technical advance over our previous studies, which have demonstrated the utility of label-free spectral counting in analysis of acyl-CoA/protein interactions. 14 Applying our chemoproteomic probes in combination with a 4-plex TMTtagging strategy allowed comparative quantification of proteins enriched from HEK293T proteomes under four affinity capture conditions: (i) probe 1 (no competitor), (ii) probe 2 (no competitor), (iii) probe 1 (0.1 mM acetyl-CoA competitor), and (iv) probe 2 (0.1 mM acetyl-CoA competitor; Figure 2a). Three independent experiments were performed, and individual protein abundances were normal- ized relative to the overall protein intensity quantified in each tagged sample in order to account for any technical variations (i.e., sample loss) introduced prior to TMT tagging.…”

Chemoproteomics yields a selective molecular host for acetyl-CoA

“…4,5 Acetyl-CoA also plays an important role in epigenetic signaling, protein homeostasis, and protein synthesis due to its ability to serve as a cofactor used in the modifications of proteins [6][7] and RNA. 7 Over the last decade, substantial evidence indicates these processes can be regulated by subcellular acetyl-CoA levels, [8][9][10] highlighting the potential for acetyl-CoA to directly link metabolism and gene expression. Emphasizing this, a recent study demonstrated that concentrations of acetyl-CoA in the nucleus are distinct from those in other organelles.…”

“…1b). To assess whether this approach could be leveraged for selective sensing, we analyzed a previous chemoproteomic dataset which determined how capture of proteins by a CoA-based affinity resin were competed by acetyl-CoA or CoA, 10 fitting quantitative abundance data to a nonlinear regression model. Since CoA affinity resins can capture both direct and indirect interactors 23 we focused on 23 proteins known to directly bind to acetyl-CoA, 24 13 of which displayed dose-dependent competition (R 2 > 0.85) by both metabolites (Fig.…”

“…Our approach harnesses the observation that capture of NAA50 by chemoproteomic affinity resins is competed more efficiently by acetyl-CoA than CoA. 10,23 This enabled the development of a BRET-based indicator displacement assay that can differentiate acetyl-CoA and CoA in a narrow sensing window, whose absolute concentration range can be tuned via structural editing of the Cy3-CoA acceptor ligand, and which can be used for real-time measurements of histone acetyltransferase enzyme activity and small molecule inhibition.…”