1. Volume 24, Issue 2, Pages 231-242 (February 2017)
Discovering Targets of Non-enzymatic Acylation by Thioester Reactivity Profiling 
Rhushikesh A. Kulkarni, Andrew J. Worth, Thomas T. Zengeya, Jonathan H. Shrimp, Julie M. Garlick, Allison M. Roberts, David C. Montgomery, Carole Sourbier, Benjamin K. Gibbs, Clementina Mesaros, Yien Che Tsai, Sudipto Das, King C. Chan, Ming Zhou, Thorkell Andresson, Allan M. Weissman, W. Marston Linehan, Ian A. Blair, Nathaniel W. Snyder, Jordan L. Meier 
Cell Chemical Biology 
Volume 24, Issue 2, Pages (February 2017)
DOI: /j.chembiol
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2. Cell Chemical Biology 2017 24, 231-242DOI: (10. 1016/j. chembiol. 2017
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3. Figure 1
Design of a Reactivity-Based Approach for Profiling Non-enzymatic Acylation
(A) Structure of acetyl-CoA bound to the prototypical lysine acetyltransferase GCN5L2 (PDB: 1Z4R). Yellow lines indicate hydrogen bonds made between Gcn5 and the acetyl-CoA cofactor.
(B) Reactivity-based profiling reagent 1.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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4. Figure 2
Assessing the Reactivity of a Thioester Reporter in Complex Proteomes
(A) Strategy for ex situ (lysate) and in situ (cellular) labeling using thioester 1.
(B) Dose-dependent ex situ labeling of cancer cell lysates by thioester 1 (A549, 15 hr; 0, 100, 200, 400, 800 μM).
(C) Labeling by 1 is competed by acetyl-CoA (1 hr pre-incubation with 0, 100, 200, 400, or 800 μM acetyl-CoA, then 400 μM 1 for 15 hr).
(D) Dose-dependent in situ labeling of cancer cell by thioester 1 (A549, 15 hr; 0, 100, 200, 400, 800 μM).
Cell Chemical Biology  , DOI: ( /j.chembiol )
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5. Figure 3
Assessing the Targets of a Thioester Reporter in Complex Proteomes
(A) Strategy for ex situ (lysate) and in situ (cellular) labeling of thioester 1 targets. Following click chemistry to biotin azide, tagged proteins are subjected to on-bead tryptic digest and identified by LC-MS/MS.
(B) Validation of targets by affinity capture/immunoblot. Left: in situ enrichment of Raji proteins by thioester 1 (15 hr; 0, 100, 400 μM). Right: ex situ enrichment of Raji proteins by thioester 1 is competed by acetyl-CoA (30 min, 1 mM acetyl-CoA; then 15 hr, 100 μM 1).
(C) Spectral count data for universally identified thioester-reactive proteins enriched by thioester probe 1 (15 hr, 400 μM). Data represent the output of individual LC-MS/MS experiments. Listed proteins were enriched ≥2-fold in each cancer cell line specified, using both in situ and ex situ labeling protocols, respectively. Functionally related proteins are grouped according to color. Complete lists of enriched targets are given in Table S1.
(D) Terms strongly enriched during gene ontology analysis of universally identified thioester-reactive proteins.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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6. Figure 4
Thioester Reactivity-Mediated Inhibition of Glycolytic Enzyme Activity
(A) Effect of cytosolic acyl-CoA thioesters (200 μM) on recombinant GAPDH activity after 30 min pre-incubation.
(B) Inhibition of recombinant GAPDH by malonyl-CoA (200 μM) is time dependent and correlates with increased lysine malonylation.
(C) Gel-based comparison of acetyl-CoA, malonyl-CoA, and succinyl-CoA reactivity. Acyl-CoAs (200 μM) were incubated with BSA at 37°C for 6 hr prior to western blotting.
Enzyme activity represents the average of at least three replicates, with significance analyzed by unpaired Student's t test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < ).
Cell Chemical Biology  , DOI: ( /j.chembiol )
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7. Figure 5
Exploring the Effects of Cytosolic Thioester Reactivity on Glycolytic Enzymes
(A) Strategies for direct (left) and indirect (right) manipulation of enzyme malonylation using malonyl-NAC and orlistat, respectively.
(B) Effects of malonyl-NAC (1 mM) and orlistat (25 μM) on cellular malonylation in A549 cells.
(C) Effects of malonyl-NAC (1 mM) and orlistat (25 μM) on GAPDH activity in A549 cells.
(D) Effects of malonyl-NAC (1 mM) on pyruvate kinase activity in A549 cells.
Enzyme activity represents the average of at least three replicates, with significance analyzed by unpaired Student's t test (**p < 0.01).
Cell Chemical Biology  , DOI: ( /j.chembiol )
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8. Figure 6
Exploring the Effects of Cytosolic Thioester Reactivity on Cellular Glucose Metabolism
(A) Schematic of glycolysis. Bold green and red arrows reflect expected changes in abundance of metabolites that lie upstream and downstream of glycolytic enzymes targeted by non-enzymatic acylation. Gene names in italic refer to high confidence targets of non-enzymatic acylation where malonyl-NAC may intervene.
(B) Effects of malonyl-NAC (1 mM, 24 hr) on glucose-6-phosphate (G6P) and fructose-1,6-bisphosphate (FBP) levels in A549 cells.
(C) Effects of malonyl-NAC (1 mM, 24 hr) on lactate levels in A549 cells.
(D) Effects of malonyl-NAC (1 mM, 24 hr) on glucose-derived acetyl-CoA (M+2) in A549 cells.
Metabolomic measurements represent the average of at least three replicates, with significance analyzed by unpaired Student's t test (*p < 0.05, **p < 0.01).
Cell Chemical Biology  , DOI: ( /j.chembiol )
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