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Chemoproteomic Profiling of Lysine Acetyltransferases Highlights an Expanded Landscape of Catalytic Acetylation

Chemoproteomic Profiling of Lysine Acetyltransferases Highlights an Expanded Landscape of... Article pubs.acs.org/JACS Terms of Use Chemoproteomic Profiling of Lysine Acetyltransferases Highlights an Expanded Landscape of Catalytic Acetylation David C. Montgomery, Alexander W. Sorum, and Jordan L. Meier* Chemical Biology Laboratory, National Cancer Institute, Frederick, Maryland 21702, United States * Supporting Information ABSTRACT: Lysine acetyltransferases (KATs) play a critical role in the regulation of gene expression, metabolism, and other key cellular functions. One shortcoming of traditional KAT assays is their inability to study KAT activity in complex settings, a limitation that hinders efforts at KAT discovery, characterization, and inhibitor development. To address this challenge, here we describe a suite of cofactor-based affinity probes capable of profiling KAT activity in biological contexts. Conversion of KAT bisubstrate inhibitors to clickable photoaffinity probes enables the selective covalent labeling of three phylogenetically distinct families of KAT enzymes. Cofactor-based affinity probes report on KAT activity in cell lysates, where KATs exist as multiprotein complexes. Chemical affinity purification and unbiased LC−MS/MS profiling highlights an expanded landscape of orphan lysine acetyltransferases present in the human genome and provides insight into the global selectivity and sensitivity of CoA-based proteomic probes that will guide future applications. Chemoproteomic profiling provides a powerful method to study the molecular interactions of KATs in native contexts and will aid investigations into the role of KATs in cell state and disease. INTRODUCTION transcription factor-related and orphan (sequence disparate) KAT activities (Supplementary Figure S1). KAT family Lysine acetylation plays a critical role in the regulation of members demonstrate significant intrafamily but little inter- transcription, metabolism, and other central biological family sequence homology, hindering bioinformatics ap- functions. Acetylation of lysine residues can impact protein proaches to KAT discovery and classification. The functional and genome function through multiple mechanisms, including 1 characterization of KATs is also limited by their regulation by physical relaxation of histone−DNA interactions, recruitment 2 protein partners and PTMs, factors that are difficult to of bromodomain-containing effector proteins, covalent active- 12,13 3 4 recapitulate in vitro. Furthermore, while individual KATs site modification, and regulation of protein stability. Lysine have been shown to be susceptible to inhibition by small acetylation is a dynamic PTM that represents an equilibrium molecules and cellular acetyl-CoA/CoA ratio, methods for between the activity of two opposing enzyme classes: lysine comparing the selectivity of these perturbations among multiple acetyltransferase (KAT) enzymes, which impose the mark, and 14,15 5 KATs in parallel do not exist. Thus, our ability to discover lysine deacetylases (KDACs), which remove it. While KDACs and characterize acetylation-mediated signaling would be have been extensively investigated as epigenetic drug targets, greatly advanced by the development of new methods for the several analyses indicate KATs can also drive cellular global analysis of KAT activity in cellular contexts. transformation and cancer progression in a tissue-specific Chemoproteomic profiling provides a powerful alternative to manner. For example, fusion of the KAT enzyme MOZ to TIF2 traditional biochemical assays for measuring enzyme activity in is the primary genetic lesion associated with a subset of complex biological settings. In this approach, also commonly leukemias and imbues differentiated red blood cells with cancer referred to as activity-based protein profiling (ABPP), active- stem cell-like properties. Nonmutant KAT activities can also site probes for an enzyme class of interest are modified with support oncogenic gene expression programs, functioning as chemical handles enabling detection or affinity enrichment. essential coactivators for transcription factors such as c-Myc Covalent labeling or enrichment of an enzyme by the affinity and E2A-PBX gene fusions in cancer. probe is then used as a proxy for enzyme activity. Proteome-wide studies have revealed lysine acetylation is a Chemoproteomic probes for KDAC enzymes have been used prevalent PTM, rivaling phosphorylation in terms of substrate to discover novel KDAC complexes and characterize inhibitor diversity with ∼4700 human acetylation sites identified to 8−10 selectivity in cell lysates. Similar approaches have also been date. However, in contrast to the hundreds of known pursued to study KAT enzymes, utilizing electrophile- protein kinases, a recent phylogenetic analysis identified only 18 KATs in the human genome. The majority of these canonical KATs fall into four families: GCN5/PCAF, P300/ Received: March 8, 2014 CBP, MYST,and NCOA,withthe rest consisting of Published: May 17, 2014 This article not subject to U.S. Copyright. Published 2014 by the American Chemical 8669 dx.doi.org/10.1021/ja502372j | J. Am. Chem. Soc. 2014, 136, 8669−8676 Society Journal of the American Chemical Society Article with both the substrate and cofactor binding sites of KAT enzymes. These molecules inhibit KAT activity with nano- molar potencies, with selectivity for specific KATs encoded by the sequence of the bisubstrate peptide. On the basis of literature precedent, we hypothesized that modification of the N-termini of KAT bisubstrate inhibitors might be tolerated without a large loss in inhibitory potency. This provides a potential site for incorporation of the clickable photoaffinity element benzophenone-L-propargylglycine (BPyne; Figure 1), 23,24 necessary for covalent labeling and detection. To test the scope of this approach, we synthesized a suite of KAT probes based on bisubstrate scaffolds that have been shown to target three major families of KATs: P300/CBP (Lys-CoA-BPyne; 1), GCN5/PCAF (H3K14-CoA-BPyne; 2), and MYST (H4K16- 22,25 CoA-BPyne; 3) (Figure 1; Supplementary Scheme S1). KAT probes 1−3 were constructed from BPyne-peptide- bromoacetamide precursors, synthesized on Rink amide resin utilizing an orthogonal Lys-Dde protecting group and postcleavage HPLC purification. Nucleophilic displacement of BPyne-peptidyl-bromoacetamides, with commerical CoA, fol- lowed by final HPLC purification provided probes 1−3 on scales (1−100 μmol) sufficient for biological evaluation (Supplementary Scheme S1). To test the affect of our structural modifications on molecular recognition of KAT enzymes, we assayed probes 1−3 against recombinant p300, pCAF, and Mof (MYST1) and compared their inhibitory activity to that of non-BPyne- Figure 1. Cofactor-based affinity probes for the analysis of KAT containing “parent” inhibitor scaffolds (4−6; Supplementary activity. (a) Clickable photoaffinity labeling scheme. (b) Structures of Figure S2). Assayed at a single concentration (1 μM), probes KAT probes 1−3. Ahx = 6-aminohexanoic acid. 1−3 demonstrate inhibitory potencies and selectivities that closely mimic those of parent inhibitor scaffolds 4−6 19,20 containing analogues of the CoA cofactor. However, these (Supplementary Figures S3 and S4). Dose-response analysis probes have not been widely applied to profile KAT activity, of Lys-CoA-BPyne (1) demonstrated an IC of 26.7 nM owing to the fact that most KATs do not utilize mechanisms toward p300 (95% confidence interval [CI ] = 11.75−60.64), involving active-site nucleophiles. within error of the inhibition by parent compound 4 (IC = Here we report a general strategy for chemoproteomic 34.5 nM, CI = 17.5−67.8 nM; Supplementary Figure S5). profiling of KAT activity (Figure 1a). Bisubstrate inhibitors Similar results are seen upon comparison of H3K14-CoA- targeting three phylogenetically distinct KAT families were BPyne 2 and parent bisubstrate 5. Together, these results converted to clickable photoaffinity probes to enable KAT suggest the BPyne subunit has minimal effects on KAT active- labeling and detection. Cofactor-based affinity probes quanti- site recognition. tatively report on KAT-inhibitor interactions, are applied to Selective In Vitro Labeling of KAT Enzymes. Next, we identify a previously unknown acyltransferase activity possessed evaluated the utility of 1−3 as KAT labeling reagents in vitro. by the canonical KAT enzyme Gcn5, and report on KAT KAT probes 1−3 were incubated with purified recombinant activity in cell lysates. Affinity purification and unbiased LC− KATs, photoirradiated at 365 nm, and subjected to Cu- MS/MS profiling of probe targets led to the identification of catalyzed [3 + 2] cycloaddition (“click chemistry”) with a two noncanonical KAT enzymes, highlighting the existence of fluorescent azide tag. SDS-PAGE analysis of labeling reactions several orphan lysine acetyltransferases present in the human demonstrated dose-dependent fluorescent labeling of KATs at genome. In addition to providing insight into the global probe concentrations between 1 and 10 μM. Notably, each selectivity and sensitivity of CoA-based chemical proteomic probe showed sensitive detection of the KAT enzyme family it probes that will guide future applications, these studies was designed to target, i.e., KAT probe 1 reacted effectively demonstrate the ability of chemical tools for profiling KAT with p300 (P300/CBP), probe 2 with pCAF (GCN5/ activity to provide new insights into KATs and their molecular 22 25 PCAF), and probe 3 with Mof (MYST) (Figure 2a). interactions in complex biological contexts. Fluorescent labeling required probe, photo-cross-linking, and 2+ RESULTS AND DISCUSSION Cu ; omission of any single component abolished labeling (Supplementary Figure S6). KAT probes 2 and 3, which are ∼3 Synthesis and Evaluation of Chemoproteomic Probes kDa, caused a significant gel shift upon photoirradiation that for KAT Enzymes. We envisioned a general strategy for was visible upon Coomassie staining (Figure 2a, Supplementary chemical profiling of KAT activity based on combining Figure S6). This property allowed us to quantify and optimize molecular recognition elements from KAT bisubstrate inhib- our photo-cross-linking conditions using gel densitometry. In itors with a clickable photoaffinity tag for covalent cross-linking our optimized photo-cross-linking protocol, we found that and detection (Figure 1a). Pioneered by Cole and co-workers, KAT bisubstrate inhibitors link CoA with the ε-amino group of ∼33−40% of pCAF was covalently labeled by 2 after 60 min of a lysine-containing peptide to form high affinity interactions irradiation at 365 nm on ice. These conditions are consistent 8670 dx.doi.org/10.1021/ja502372j | J. Am. Chem. Soc. 2014, 136, 8669−8676 Journal of the American Chemical Society Article Figure 3. Selectivity of KAT labeling by probes 1−3 (1 μM) assayed in a mixture of proteins from the P300/CBP, GCN5/PCAF, and MYST family. Specific labeling events show sensitivity to competition by parent bisubstrate inhibitors (4−6; 200 equiv). addition to its parent bisubstrate, the Mof-3 interaction was competed by excess Lys-CoA (4), which had no similar effect on the interaction of pCAF with 2 (Supplementary Figure S8). Figure 2. (a) Concentration dependence of probe labeling for For all three probes, selectivities were found to be reduced at preferred KAT partners. (b) Structure of fluorescent KAT photo- higher probe concentrations (10 μM), where 1−3 exhibit affinity probe 7. (c) Relative labeling of pCAF by clickable (left) and considerable cross-reactivity (Supplementary Figure S8). Given fluorescent (right) photoaffinity probes at low (0.1 μM) and high (1 μM) probe concentrations. its combination of broad-spectrum reactivity and straightfor- ward synthesis of probe and competitor, these findings suggest Lys-CoA-BPyne 1 may be the most well-suited of the probes with the literature, and cross-linking yields were not found to for applications requiring a general chemoproteomic reporter of increase with extended photoirradiation times. KAT activity. In contrast, when applied at suitably low The alkyne handle of probes 1−3 was chosen for its minimal concentrations, peptidyl-KAT probes 2 and 3 may be better footprint and versatility toward conjugation of diverse azide suited for applications that require more selective labeling of reporters. However, it is not strictly necessary for ex vivo specific KAT families or as components of KAT chemo- affinity profiling applications. In order to evaluate the utility of proteomic probe cocktails designed to achieve broad super- our bioorthogonal detection strategy, we compared the labeling family coverage. of pCAF by clickable probe 2 with a fluorescent KAT Chemoproteomic Probes Report on KAT−Small photoaffinity probe, TAMRA-H3K14-CoA 7 (Figure 2b). Molecule and KAT−Cofactor Interactions. Having dem- Both probes facilitate fluorescent detection of pCAF at high onstrated the ability of our probes to label three classes of probe concentrations (1 μM), suggesting fluorescent bisub- KATs, we next sought to investigate their ability to report on strates may be useful profiling agents for KAT enzymes. changes in KAT activity resulting from exposure to diverse However, clickable probe 2 exhibits visibly greater labeling than molecular stimuli. First we investigated their ability to report on fluorescent probe 7 at low probe concentrations (0.1 μM; 18,29 the affinity and selectivity of small molecule inhibitors. Co- Figure 2b). This suggests click chemistry detection strategies incubation of pCAF with the known KAT inhibitor garcinol improve probe sensitivity, possibly by abrogating negative decreased labeling by KAT probe 2 in a dose-dependent interactions of the fluorescent TAMRA reporter on probe- manner (Figure 4). Quantification of fluorescent pCAF labeling protein recognition that reduce photo-cross-linking. by gel densitometry yielded an IC of 4.5 ± 1.2 μM for Moving toward more complex settings, we assessed the garcinol, consistent with the literature IC value of 5 μM. specificity and selectivity of each KAT probe (1−3) using a Similarly, labeling of p300 by Lys-CoA BPyne 1 was sensitive to cocktail composed of p300, pCAF, and the MYST family inhibition by the small molecule inhibitor C646 (Supplemen- acetyltransferase Mof. Specific probe labeling events were tary Figure S9). defined as those susceptible to competition by 50−100 equiv of parent KAT bisubstrate inhibitors 4−6 (Supplementary Figure S7), while selectivity refers to the subset of KAT enzymes labeled by each probe. Interestingly, each probe showed specific labeling of a unique subset of KAT enzymes at 1 μM, even in the presence of other KAT superfamily members. For example, Lys-CoA-BPyne 1 showed strong labeling of p300 and pCAF but did not significantly label the MYST family member Mof (Figure 3). The p300 signal was specifically competed in the presence of excess parent inhibitor, while a small portion of the pCAF signal remained, suggesting a combination of specific and nonspecific interactions between the 1-pCAF pair. H3K14- Figure 4. Competitive profiling of KAT active-site occupancy. (a) CoA-BPyne 2 demonstrated a clear preference for specific Fluorescent and Coomassie gels from pCAF-garcinol competition labeling of pCAF and weaker, but detectable, labeling of Mof experiment. Conditions: 2 (1 μM), garcinol (0, 0.08, 0.16, 0.33, 0.63, (Figure 3/Supplementary Figure S8). H4K16-CoA-BPyne 3 1.26, 2.5, 3.75, 5, 10, 20, 40 μM). (b) Dose-response analysis of most strongly labeled the MYST family member Mof and competitive labeling generated via gel densitometry analysis of exhibited fainter labeling of p300 and pCAF (Figure 3). In fluorescent labeling by KAT probe 2. 8671 dx.doi.org/10.1021/ja502372j | J. Am. Chem. Soc. 2014, 136, 8669−8676 Journal of the American Chemical Society Article Figure 5. (a) Scheme for competitive substrate profiling. (b) Fluorescent and Coomassie gels from Gcn5-acyl-CoA competition experiment. Conditions: 2 (10 μM), acyl-CoAs (1000 μM), garcinol (40 μM). In addition to small molecule inhibition, recent studies have Figure 6. Labeling of KATs in cell lysates. (a) Labeling of pCAF in suggested KAT activity may be sensitive to changes in cellular HEK-293 overexpression extracts. Comp. = competitor. (b) Proteins acyl-CoA pools, providing a potential mechanism to link identified in LC−MS/MS experiments as targets of H3K14-CoA- changes in the metabolic state of the cell to differential histone BPyne 2 in pCAF transfected HEK-293 extracts. Control = competitor 15,32 acylations and epigenetic control of gene expression. To treated lane, EV = extract derived from HEK-293 cells transfected with test whether chemoproteomic probes could provide insight into empty vector. (c) Limit of detection of recombinant pCAF spiked into these mechanisms, we investigated the ability of four different HeLa cell proteome. Conditions: 2 (1 μM), proteome (7 μg), acyl-CoAs (acetyl, propionyl, butyryl, and crotonyl-CoA) to recombinant pCAF (0, 6.25, 5, 3.75, 2.5, 1.25 pmol). compete with 2 for the active site occupancy of Gcn5, a KAT whose activity has been proposed to be metabolically regulated 15,33 (Figure 5). We found that high concentrations of acetyl- by fluorescence (Figure 6a). Notably, labeling was sensitive to CoA efficiently competed labeling by probe 2, consistent with competition by H3K14-CoA, and immunoblotting confirmed its role as a universal KAT cofactor. Propionyl-CoA also comigration with pCAF. To further verify labeling, we antagonized labeling, while butyryl-CoA was a partial subjected proteins labeled by 2 in pCAF overexpression antagonist, and crotonyl-CoA did not impede labeling (Figure extracts to click chemistry with biotin azide, followed by 5b). While the ability of Gcn5 to utilize propionyl- and butyrl- affinity purification, tryptic digest, and LC−MS/MS protein CoA as cofactors has not been previously explored, our results identification. Notably, pCAF peptides were specifically are consistent with a previous biochemical analysis of human identified in overexpression extracts treated with 2 (Figure pCAF (which has a highly homologous KAT domain). These 6b), but not in extracts pretreated with excess competitor 5 or studies indicated a kinetic preference for acyl group donors of lysates derived from HEK-293 cells transfected with an empty −1 −1 acetyl-CoA (k /K ≈ 535 s M ) > propionyl-CoA (k /K vector control (Figure 6a, bottom, Supplementary Table S1). In cat m cat m −1 −1 ≈ 92 s M ) ≫ butyrl-CoA and that malonyl-CoA was not addition to overexpression extracts, clickable photoaffinity utilized as a substrate. Indeed, we confirmed Gcn5 utilized probes 1 and 2 are capable of detecting p300 and pCAF, propionyl- and butyrl-CoA as substrates via LC−MS/MS respectively, when spiked into HeLa cell proteomes (Supple- analysis (Supplementary Figure S10). mentary Figure S11). We used this characteristic to assess the These data suggest competitive affinity profiling provides a sensitivity of probe 2 and estimate a lower limit at which useful approach to rapidly gain new insights into KAT inhibitor GCN5/PCAF family enzymes may be detected. This is and substrate selectivity. especially relevant since our inability to enrich pCAF from Chemical Affinity Profiling of KAT Activity in Cell empty vector transfected HEK-293 lysates indicates expression Lysates. The activity of KAT enzymes such as Gcn5 and level may be a limiting factor for KAT identification. We found pCAF is ideally studied in cellular settings. Recent commentary that probe 2 could detect as low as 2.5 pmols of pCAF in a has suggested the failure of conventional high-throughput standard gel-based experiment against a proteomic background screening campaigns to yield selective KAT inhibitors may be (Figure 6b). These findings calibrate the ability of chemo- due to the inability of these screens to interrogate the ability of proteomic probes to monitor KAT activity in model systems small molecules to disrupt native KATs, which can exist as and cell lysates and may be useful for the development of multiprotein complexes. Therefore, methods to monitor KAT chemoproteomic approaches to screen for selective inhibitors 36,37 activity directly from cell extracts are potentially valuable for of KATs and KAT-containing multiprotein complexes. next-generation inhibitor discovery efforts. Overexpression Chemoproteomic Profiling of KAT Activity: Probe extracts of epitope-labeled ATAC (Ada Two-A containing) Reactivity and Orphan KATs. Having demonstrated the complex constitute an advanced model system applied for the utility of chemoproteomic probes for the targeted study of KAT study of GCN5 family KATs in their endogenous setting. We activity in cellular contexts, we last sought to explore their thus asked whether we could use chemoproteomic probe 2 to utility for the discovery of potentially novel KAT activities directly detect KAT activity in these systems, without the need directly from cancer cell proteomes. Labeling of whole cell for prefractionation, affinity purification, or antibodies. extracts by 1−3 demonstrated a distinct pattern of specific Accordingly, pCAF overexpression extracts from HEK-293 protein labeling events for each probe, with KAT probe 1 cells were treated with 2, followed by photo-cross-linking and targeting the largest number of proteins (Figure 7). To identify click chemistry. Weak but detectable labeling of a protein the proteomic targets of broad-spectrum KAT probe 1, HeLa corresponding to the molecular weight of pCAF was observed cells were lysed, photo-cross-linked in the presence of 1, and 8672 dx.doi.org/10.1021/ja502372j | J. Am. Chem. Soc. 2014, 136, 8669−8676 Journal of the American Chemical Society Article In our data set, two acetyltransferases were enriched: Nat10 and Naa50. Nat10 is a noncanonical KAT (referred to here as orphan KATs) with a GNAT-related fold that displays histone and microtubule acetyltransferase activity in cells. Biologically, Nat10 has been observed to play a role in the regulation of telomerase function and nuclear shape and was recently identified as adruggabletargetfor thetreatment of 41,42 Hutchinson−Gilford progeria syndrome. Naa50 (Nat13) is the catalytic component of the NatE acetyltransferase complex that is required for proper sister chromatid adhesion 43,44 and chromatin condensation in vivo. The identification of Naa50 as a target of Lys-CoA-BPyne 1 initially struck us as paradoxical, as this enzyme belongs to the N-terminal acetyltransferase (NAT) family and has been shown to favor 45,46 protein acetylation of N-terminal Met residues. However, literature investigation revealed that, of the 7 NAT catalytic subunits encoded in the human genome, Naa50 is the only member to have biochemically characterized ε-lysine acetyl- transferase activity, providing a molecular rationale for its targeting by 1. The selective identification of Naa50 by 1 suggests that chemoproteomic profiling may have applications in identifying new KAT activities present in acetyltransferase families that are distinct in sequence from canonical KATs. Figure 7. Cofactor-based affinity profiling of endogenous KAT activity The proteomic identification of two orphan KAT activities by in a cancer cell proteome. (a) Labeling of HeLa cell proteomes by affinity probe 1 led us to re-evaluate the lysine acetyltransferase KAT probes 1−3 (10 μM). Specific labeling events show sensitivity to competition by parent bisubstrates (100 equiv). (b) Proteins identified literature and consider whether other KAT activities were also in LC−MS/MS experiments as targets of Lys-CoA-BPyne 1. Values missing from the list of 18 canonical human KATs represent the average spectral counts of two biological replicates. (Supplementary Figure S1). This analysis identified 14 proteins, Green, acetyltransferases; Red, CoA-binding proteins. (c) Affinity including Nat10 and Naa50, not in the list of 18 canonical labeling of recombinant protein verifies Acly and Naa50 as targets of 1. KATs for which evidence of lysine acetyltransferase activity has been observed. Sequence alignment and similarity analyses were used to construct an expanded phylogenetic tree of subjected to click chemistry with biotin-azide followed by acetyltransferase proteins, divided into canonical (P300/CBP, streptavidin enrichment, tryptic digest, and LC−MS/MS GCN5/PCAF, MYST, NCOA) and orphan KATs (Figure 8). analysis. We identified 16 proteins that were abundant (>5 Notably, many KATs from the initial list of 18 (ATAT1, spectral counts) and showed >5-fold preferential enrichment in TF3C4, ELP3; Supplementary Figure S1) cluster more closely the absence of competitor (Figure 7b, Supplementary Table with orphan KATs, indicative of greater sequence similarity. S2). Peptides matching canonical KAT p300 and CBP were not Together, these proteins encompass the most comprehensive observed in our MS/MS data set, likely due to their low list of human KATs assembled to date. However, we take care abundance in whole cell lysates (confirmed by Western blot; to point out that the experimental evidence supporting the Supplementary Figure S12). Of the 16 proteins, 2 were activity of all 32 KATs, canonical and orphan, ranges widely. In acetyltransferases (green), 5 were CoA-binding proteins related particular, this list contains two proteins (Oga/Ncoat, an to primary metabolism (red), with the remaining hits orphan KAT, and Src1, a canonical KAT) for which conflicting composed of highly abundant proteins and members of the 48,49 observations of KAT activity have been made. These proteasome regulatory complex. To validate LC−MS/MS discrepancies support the need for universal methodologies identifications, we performed probe labeling experiments of capable of directly assaying KAT activity in endogenous cells, two overexpressed and purified targets of 1: ATP-citrate lyase toward which our current study provides an initial step. In the (Acly), the highest abundance pulldown target of 1, and N-α- meantime, our chemoproteomics-inspired census of KAT acetyltransferase 50 (Naa50), an acetyltransferase. Both activity provides fertile ground for functional investigation of enzymes exhibited competitor-sensitive labeling, indicative of orphan KAT enzymes using traditional structural and specific molecular recognition by KAT probe 1 (Figure 7c). biochemical approaches, studies that may facilitate a more Conserved domain analysis indicated that each protein complete understanding of lysine acetylation in living systems. specifically labeled by 1 either contains or is closely associated with a protein containing a binding site for an adenine CONCLUSION nucleotide-containing cofactor (CoA, ATP, or NAD(P); Figure ■ 7c). The off-target engagement of adenosine-binding proteins In summary, here we have described a suite of probes for by KAT probe 1 parallels the widespread reactivity that has cofactor-based affinity profiling of KAT activity. We have been observed for ATP- and GTP-containing chemical defined key structural features necessary for the covalent 38−40 proteomic probes in biological settings. This is likely to labeling and detection of three families of KAT enzymes and be a general limitation of chemical proteomic probes demonstrated the utility of these probes to monitor KAT incorporating adenine cofactor-based chemical scaffolds and activity in settings ranging from purified enzymes to KAT highlights opportunities for design improvements as well as the overexpression extracts to native proteomes. Chemoproteomic requirement for orthogonal validation strategies to support probes of KAT activity provided new insights into KAT chemical proteomic KAT discovery efforts. inhibitor and cofactor selectivity and highlighted the existence 8673 dx.doi.org/10.1021/ja502372j | J. Am. Chem. Soc. 2014, 136, 8669−8676 Journal of the American Chemical Society Article orphan KAT activities in cellular settings. Such studies are currently underway, and will be reported in due course. EXPERIMENTAL DETAILS Biochemistry and Cell Biology. Recombinant pCAF, catalytic domain (aa 492−658) was obtained from Cayman Chemical. Recombinant p300, catalytic domain (aa 1284−1673) and recombi- nant Gcn5, catalytic domain (497−663), were obtained from Enzo. The plasmid encoding recombinant MOF, catalytic domain (aa 147− 449) was obtained from Addgene. His-tagged recombinant Mof was expressed in E. coli BL21 and purified via immobilized nickel affinity chromatography using standard conditions. Acly was obtained from US Biological. Naa50 was obtained from Origene. Garcinol and C646 were obtained from Cayman Chemical. Streptavidin-agarose was purchased from Pierce. SDS-PAGE was performed using Bis-Tris NuPAGE gels (4−12%) and MES running buffer in Xcell SureLock MiniCells (Invitrogen) according to the manufacturer’s instructions. SDS-PAGE fluorescence was visualized using an ImageQuant Las4010 Digitial Imaging System (GE Healthcare). Total protein content on SDS-PAGE gels was visualized by Blue-silver coomassie stain, made according to the published procedure. Separation-based assays for KAT activity were performed on a LabChip EZ Reader instrument (PerkinElmer) kindly provided by Dr. Jay Schneekloth. Fluorescence assays for the KAT enzyme Mof were analyzed on a Biotek Synergy 2 Figure 8. Expanded phylogenetic tree of KAT enzymes, including (Biotek). canonical KATs and orphan KAT activities observed in this study or KAT Inhibition Assays. Recombinant KAT activity was measured annotated in the literature. KATs are denoted by gene name and by electrophoretic mobility shift assay (EMSA) as previously relevant pseudonyms. Uniprot accession numbers and literature reported. This assay measures the separation of FITC-labeled KAT references for KAT activity are provided in the Supporting substrate peptides (Histone H3 5-23 QTARKSTGGKAPRKQLATK- Information (Table S3). Ahx-FITC; Histone H4 1-19 SGRGKGGKGLGKGGAKRHR-Ahx- FITC) from their acetylated products following incubation with recombinant KAT and acetyl-CoA. A model separation is shown in of several noncanonical orphan KAT activities that may Supplementary Figure S14. P300 and pCAF assays were performed in contribute to cellular acetylation signaling pathways. In addition 30 μL of reaction buffer (50 mM HEPES, pH 7.5, 50 mM NaCl, 2 mM to these advances, it is also important to call attention to the EDTA, 0.05% Tween 20, 10 μg/mL BSA) with KAT (p300 [50 nM] limitations of our initial study. For example, while we or pCAF [10 nM]) and FITC-peptide (FITC-H4 for P300; FITC-H3 demonstrated the ability of KAT affinity probes to report on for pCAF; 1 μM). Acetylation of FITC-H3/H4 peptide by p300 and pCAF activity in HEK-293 overexpression extracts, we were pCAF was confirmed by LC−MS. Reactions were plated in 384-well unable to detect canonical KAT enzymes such as CBP and plates, allowed to equilibrate at room temperature for 10 min, and initiated by addition of acetyl-CoA (final concentration = 5 μM). Gcn5 directly from HeLa proteomes. This may be due to the Plates were then transferred to a Lab-Chip EZ-Reader at ambient low abundance of these KATs in whole cell lysates temperature and analyzed by microfluidic electrophoresis. Optimized (Supplementary Figure S13) or the low cross-linking yields of separation conditions were downstream voltage of −400 V, upstream our clickable photoaffinity probes, which covalently label only voltage of −2900 V, and a pressure of −2.0 psi for FITC-H3 and ∼33−40% of recombinant pCAF in vitro. This challenge may downstream voltage of −500 V, upstream voltage of −1500 V and a be addressed in future studies through scale-up, nuclear pressure of −2.0 psi for FITC-H4. Percent conversion is calculated by prefractionation, or utilization of multidimensional protein ratiometric measurement of substrate/product peak heights. Percent identification technology (MuDPIT) to increase LC−MS/MS activity represents the percent conversion of KAT reactions treated detection sensitivity. Alternatively, chemical proteomic with inhibitors 1−6 relative to untreated control KAT reactions, measured in triplicate, and corrected for nonenzymatic acetylation. studies of kinase and KDAC activity have shown that Mof showed low activity toward FITC-H3/H4 peptide substrates and noncovalent affinity probe resins can enable enrichment of was monitored by fluorogenic KAT assay using an unlabeled H4 specific enzyme classes without the need for photo-cross- 18,51 substrate peptide as previously reported. Dose-response analysis of linking, providing another potential route for the analysis of p300 and pCAF inhibition by KAT probes 1 and 2 and parent low abundance KATs. Furthermore, while the activity of KAT inhibitors 4 and 5 were performed in triplicate and analyzed by complexes have been shown to be preserved in cell extracts, ∧ nonlinear least-squares regression fitto Y = 100/(1 + 10 (Log IC − these activities would be ideally studied in living cells and X)*H), where H = Hill slope (variable). IC values represent the probes 1−3 are not cell-permeable. Cell-penetrating peptides concentration that inhibits 50% of KAT activity. All calculations were have been used to promote uptake of KAT bisubstrate performed using Prism 6 (GraphPad) software. Fluorescent Labeling of KAT Enzymes for SDS-PAGE inhibitors, and similar approaches may facilitate live cell Analysis. Purified KAT enzymes (0.5−5 μg) or whole cell proteomes profiling of KAT activity. These improvements will be (20 μg) were incubated with KAT probes 1−3 (1 μM probe for important to expand the scope of chemical proteomic analyses recombinant labelings; 10 μM probe for proteomic labelings) in PBS of KAT activity. Regardless, the ability of our current suite of (pH 7.0) for 1 h. Control experiments to correct for nonspecific cross- chemoproteomic probes to highlight an expanded landscape of linking were treated with 1−3 in the presence of 100 equiv of catalytic lysine acetylation provides an example of the power of competitors 4−6. Following equilibration, samples were photo-cross- this approach as currently constituted and sets the stage for the linked on ice for 1 h using a 365 nm UV light in a FB-UVXL-1000 UV development of chemoproteomic strategies to identify KAT cross-linker. Probe labeling was detected by Cu(I)-catalyzed [3 + 2] inhibitors and the functional characterization of canonical and cycloaddition (“click chemistry”). Click reactions were initiated by 8674 dx.doi.org/10.1021/ja502372j | J. Am. Chem. Soc. 2014, 136, 8669−8676 Journal of the American Chemical Society Article sequential addition of TAMRA-azide 8 (100 μM; 5 mM stock solution resulting cell pellet was air-dried to remove excess methanol and in DMSO, structure given in Supplementary Figure S15), TCEP (1 redissolved in 1.2% SDS (1 mL) using iterative cycles of heating (95 mM; 100 mM stock in H O), tris(benzyltriazolylmethyl)amine ligand °C) and sonication. Redissolved protein was allowed to cool to room (TBTA; 100 μM; 1.7 mM stock in DMSO/tert-butanol 1:4), and temperature and added to 5 mL of PBS to give a final SDS concentration of 0.2%. Samples were then treated with 100 μLof CuSO (1 mM; 50 mM stock in H O). Samples were vortexed and 4 2 streptavidin-agarose resin (prewashed 3× with 1 mL of PBS) and incubated at room temperature for 1 h. Cycloaddition reactions were rotated for 1 h at room temperature. Streptavidin-agarose bound quenched by addition of 5x SDS-loading buffer (strongly reducing) and subjected to SDS-PAGE (22 μL per well). Excess probe samples were then washed sequentially with 0.2% SDS in PBS (3 × 10 fluorescence was removed by destaining in a solution of 50% mL) and PBS (3 × 10 mL). Samples were then prepared for on-bead MeOH/40% H O/10% AcOH overnight. Gels were then washed digest by reduction with 10 mM tris(2-carboxyethyl)phosphine with water and fluorescently visualized using a ImageQuant Las4010 (TCEP) and alkylation with 12 mM iodoacetamide. Samples were (GE Healthcare) with green LED excitation (λ 520−550 nm) and a diluted to 2 M urea with 50 mM Tris-Cl pH 8.0 (400 μL total max 575DF20 filter. For KAT probes 2 and 3, a characteristic intense low volume), followed by addition of trypsin and 2 mM CaCl . Digests molecular weight fluorescence signal ∼3 kDa was observed were allowed to proceed overnight at 37 °C. After extraction, tryptic peptide samples were acidified to a final concentration of 5% formic (corresponding to the fluorescently labeled KAT probe [2, 3]), acid and frozen at −80 °C for LC−MS/MS analysis. indicative of a high-yielding click chemistry reaction. Liquid Chromatography−Mass Spectrometry and Data Cell Culture and Isolation of Whole-Cell Lysates. HeLa S3 Analysis. Tryptic peptides enriched by probe 1 were loaded onto a cells (ATCC; Manassas VA) were cultured at 37 °C under 5% CO reverse phase capillary column and analyzed by LC separation in atmosphere in a culture medium of DMEM supplemented with 10% combination with tandem MS. Peptides were eluted using a gradient of FBS and glutamine. HEK-293 cells were obtained from the NCI 5−42% over 40 min with the flow rate through the column set at 0.20 Tumor Cell Repository. For isolation of whole cell proteomes, HeLa μL/min. Data was collected in a dual-pressure linear ion trap mass cells were grown to 80−90% confluency, washed 3× with ice-cold spectrometer (ThermoFisher LTQ VelosPro) set in a data-dependent PBS, scraped, and pelleted by centrifugation (1400g × 3 min, 4 °C). acquisition mode. The 15 most intense molecular ions in the MS scan After removal of PBS cell pellets were stored at −76 °Cor were sequentially and dynamically selected for subsequent collision- immediately processed. Cell pellets were resuspended in 1−2mLof induced dissociation (CID) using a normalized collision energy of ice-cold PBS (10−20 × 10 cells/mL) and lysed by sonication 35%. Tandem mass spectra were searched against UniProt H. sapiens (QSonica XL2000 100 W sonicator, 3 × 10 s pulse, 50% power, 60 s protein database (01-13 release) using SEQUEST (ThermoFisher). between pulses). Lysates were pelleted by centrifugation (14,000g × Search parameters were fixed as follows: (i) enzyme specificity: 30 min, 4 °C) and quantified on a Qubit 2.0 Fluorometer using a trypsin; (ii) variable modification: methionine oxidation and cysteine Qubit Protein Assay Kit. Proteomes were diluted to 2 mg/mL and carbamidomethylation; (iii) precursor mass tolerance ±1.40 amu; and stored in 1 mg aliquots at −76 °C until further processing. (iv) fragment ion mass tolerance ±0.5 amu. Only those tryptic Western Blotting. SDS-PAGE gels were transferred to nitro- peptides with up to two missed cleavage sites meeting a specific cellulose membranes (Novex, Life Technologies) by electroblotting at SEQUEST scoring criteria (Delta Correlation (ΔCn) ≥ 0.08 and 30 V for 1 h using a XCell II Blot Module (Novex). Membranes were 1+ charge state dependent cross correlation (Xcorr) ≥ 1.9 for [M + H] , blocked using StartingBlock (PBS) Blocking Buffer (Thermo 2+ 3+ ≥ 2.2 for [M + 2H] , and ≥3.1 for [M + 3H] ) were considered as Scientific) for 20 min and then incubated overnight at 4 °Cin a legitimate identifications. Spectral count values depicted in Figure 7 solution containing the primary antibody of interest (anti-Gcn5 represent an average of two biological replicates. Raw spectral counts [3305], anti-CBP [3378], Cell Signaling, 1:1000 dilution) in the above for biological duplicates of probe-enriched and control experiments are blocking buffer with 0.05% Tween 20. The membranes were next provided in Supplementary Table S2. washed with TBST buffer and incubated with a secondary HRP- Phylogenetic Analysis. Amino acid sequences for canonical and conjugated antibody (anti-rabbit IgG, HRP-linked [7074], Cell orphan lysine acetyltransferases were obtained from Uniprot. Signaling, 1:1000 dilution) for 1.5 h at room temperature. The Accession numbers are provided in Table S3 (Supporting membranes were again washed with TBST, treated with chemilumi- Information). A pairwise alignment was generated using Clustal nescence reagents (Western Blot Detection System, Cell Signaling) for Omega, and a phylogenic tree was constructed using the neighbor- 1 min, and imaged for chemiluminescent signal using an ImageQuant joining method. All phylogenetic trees were displayed in hyperbolic Las4010 Digitial Imaging System (GE Healthcare). space using Hypertree, with branches of the tree designated by Enrichment of KAT Enzymes for Proteomic Analysis. Whole different colors and labeled by name where appropriate. cell proteomes were adjusted to a final protein concentration of 1 mg/ mL and incubated with the indicated probe (1 or 2;10 μM) for 1 h. ASSOCIATED CONTENT HEK-293 enrichments utilized 0.5 mg of proteome as starting material, while HeLa enrichments utilized 1 mg of proteome. Control samples * Supporting Information to correct for nonspecific cross-linking were preincubated with each Synthetic materials and methods, characterization data, and probe’s cognate competitor (4 or 5; 100 equiv). Following supplementary figures and schemes. This material is available equilibration, samples were split into 5 × 200 μL aliquots and free of charge via the Internet at http://pubs.acs.org. photo-cross-linked on ice for 1 h using a 365 nm UV light in a FB- UVXL-1000 UV cross-linker. Cross-linked samples were then AUTHOR INFORMATION recombined and subjected to Cu(I)-catalyzed [3 + 2] cycloaddition with TAMRA biotin-azide 9 (Supplementary Figure S15) as previously Corresponding Author described. Final concentrations for click reactions were as follows: [email protected] HeLa proteome (1 mg/mL in PBS), probe 1 (10 μM), TAMRA Notes biotin-azide (40 μM), TCEP (1 mM), TBTA (100 μM), tert-butanol (4.8%), and CuSO (1 mM). Samples were vortexed and incubated at The authors declare no competing financial interest. room temperature for 1 h. Ice-cold 4:1 MeOH/CHCl (2.5 mL) was then added directly to the reaction mixture and mixed vigorously by ACKNOWLEDGMENTS vortexing. The biphasic solution was centrifuged (4000g × 20 min, 4 The authors thank Dr. Ming Zhou (Laboratory of Proteomics °C), and protein precipitated at the interface as a solid disk. Liquid and Analytical Technology) for LC−MS/MS analyses, Dr. layers were carefully discarded, and the resulting precipitate was Hans Luecke (NIDDK) for the pCAF overexpression plasmid, resuspended in ice-cold 1:1 MeOH/CHCl (1 mL), sonicated on ice Dr. Brian Lewis (NCI) for helpful discussions, and Dr. Michael to resuspend, and repelleted by centrifugation (14,000g × 10 min, 4 °C). This wash step was repeated with ice-cold MeOH (1 mL). The Giano of the Schneider lab for assistance with peptide synthesis. 8675 dx.doi.org/10.1021/ja502372j | J. Am. Chem. Soc. 2014, 136, 8669−8676 Journal of the American Chemical Society Article (30) Balasubramanyam, K.; Altaf, M.; Varier, R. A.; Swaminathan, V.; This work was supported by the Intramural Research Program Ravindran, A.; Sadhale, P. P.; Kundu, T. K. J. Biol. Chem. 2004, 279, of the NIH, National Cancer Institute, Center for Cancer Research (ZIA BC011488-01). (31) Bowers, E. M.; Yan, G.; Mukherjee, C.; Orry, A.; Wang, L.; Holbert, M. 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Biotechnol. 2003, 21, 687. 8676 dx.doi.org/10.1021/ja502372j | J. Am. Chem. Soc. 2014, 136, 8669−8676 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of the American Chemical Society Pubmed Central

Chemoproteomic Profiling of Lysine Acetyltransferases Highlights an Expanded Landscape of Catalytic Acetylation

Montgomery, David C.; Sorum, Alexander W.; Meier, Jordan L.
Journal of the American Chemical Society , Volume 136 (24) – May 17, 2014
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10.1021/ja502372j
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Abstract

Article pubs.acs.org/JACS Terms of Use Chemoproteomic Profiling of Lysine Acetyltransferases Highlights an Expanded Landscape of Catalytic Acetylation David C. Montgomery, Alexander W. Sorum, and Jordan L. Meier* Chemical Biology Laboratory, National Cancer Institute, Frederick, Maryland 21702, United States * Supporting Information ABSTRACT: Lysine acetyltransferases (KATs) play a critical role in the regulation of gene expression, metabolism, and other key cellular functions. One shortcoming of traditional KAT assays is their inability to study KAT activity in complex settings, a limitation that hinders efforts at KAT discovery, characterization, and inhibitor development. To address this challenge, here we describe a suite of cofactor-based affinity probes capable of profiling KAT activity in biological contexts. Conversion of KAT bisubstrate inhibitors to clickable photoaffinity probes enables the selective covalent labeling of three phylogenetically distinct families of KAT enzymes. Cofactor-based affinity probes report on KAT activity in cell lysates, where KATs exist as multiprotein complexes. Chemical affinity purification and unbiased LC−MS/MS profiling highlights an expanded landscape of orphan lysine acetyltransferases present in the human genome and provides insight into the global selectivity and sensitivity of CoA-based proteomic probes that will guide future applications. Chemoproteomic profiling provides a powerful method to study the molecular interactions of KATs in native contexts and will aid investigations into the role of KATs in cell state and disease. INTRODUCTION transcription factor-related and orphan (sequence disparate) KAT activities (Supplementary Figure S1). KAT family Lysine acetylation plays a critical role in the regulation of members demonstrate significant intrafamily but little inter- transcription, metabolism, and other central biological family sequence homology, hindering bioinformatics ap- functions. Acetylation of lysine residues can impact protein proaches to KAT discovery and classification. The functional and genome function through multiple mechanisms, including 1 characterization of KATs is also limited by their regulation by physical relaxation of histone−DNA interactions, recruitment 2 protein partners and PTMs, factors that are difficult to of bromodomain-containing effector proteins, covalent active- 12,13 3 4 recapitulate in vitro. Furthermore, while individual KATs site modification, and regulation of protein stability. Lysine have been shown to be susceptible to inhibition by small acetylation is a dynamic PTM that represents an equilibrium molecules and cellular acetyl-CoA/CoA ratio, methods for between the activity of two opposing enzyme classes: lysine comparing the selectivity of these perturbations among multiple acetyltransferase (KAT) enzymes, which impose the mark, and 14,15 5 KATs in parallel do not exist. Thus, our ability to discover lysine deacetylases (KDACs), which remove it. While KDACs and characterize acetylation-mediated signaling would be have been extensively investigated as epigenetic drug targets, greatly advanced by the development of new methods for the several analyses indicate KATs can also drive cellular global analysis of KAT activity in cellular contexts. transformation and cancer progression in a tissue-specific Chemoproteomic profiling provides a powerful alternative to manner. For example, fusion of the KAT enzyme MOZ to TIF2 traditional biochemical assays for measuring enzyme activity in is the primary genetic lesion associated with a subset of complex biological settings. In this approach, also commonly leukemias and imbues differentiated red blood cells with cancer referred to as activity-based protein profiling (ABPP), active- stem cell-like properties. Nonmutant KAT activities can also site probes for an enzyme class of interest are modified with support oncogenic gene expression programs, functioning as chemical handles enabling detection or affinity enrichment. essential coactivators for transcription factors such as c-Myc Covalent labeling or enrichment of an enzyme by the affinity and E2A-PBX gene fusions in cancer. probe is then used as a proxy for enzyme activity. Proteome-wide studies have revealed lysine acetylation is a Chemoproteomic probes for KDAC enzymes have been used prevalent PTM, rivaling phosphorylation in terms of substrate to discover novel KDAC complexes and characterize inhibitor diversity with ∼4700 human acetylation sites identified to 8−10 selectivity in cell lysates. Similar approaches have also been date. However, in contrast to the hundreds of known pursued to study KAT enzymes, utilizing electrophile- protein kinases, a recent phylogenetic analysis identified only 18 KATs in the human genome. The majority of these canonical KATs fall into four families: GCN5/PCAF, P300/ Received: March 8, 2014 CBP, MYST,and NCOA,withthe rest consisting of Published: May 17, 2014 This article not subject to U.S. Copyright. Published 2014 by the American Chemical 8669 dx.doi.org/10.1021/ja502372j | J. Am. Chem. Soc. 2014, 136, 8669−8676 Society Journal of the American Chemical Society Article with both the substrate and cofactor binding sites of KAT enzymes. These molecules inhibit KAT activity with nano- molar potencies, with selectivity for specific KATs encoded by the sequence of the bisubstrate peptide. On the basis of literature precedent, we hypothesized that modification of the N-termini of KAT bisubstrate inhibitors might be tolerated without a large loss in inhibitory potency. This provides a potential site for incorporation of the clickable photoaffinity element benzophenone-L-propargylglycine (BPyne; Figure 1), 23,24 necessary for covalent labeling and detection. To test the scope of this approach, we synthesized a suite of KAT probes based on bisubstrate scaffolds that have been shown to target three major families of KATs: P300/CBP (Lys-CoA-BPyne; 1), GCN5/PCAF (H3K14-CoA-BPyne; 2), and MYST (H4K16- 22,25 CoA-BPyne; 3) (Figure 1; Supplementary Scheme S1). KAT probes 1−3 were constructed from BPyne-peptide- bromoacetamide precursors, synthesized on Rink amide resin utilizing an orthogonal Lys-Dde protecting group and postcleavage HPLC purification. Nucleophilic displacement of BPyne-peptidyl-bromoacetamides, with commerical CoA, fol- lowed by final HPLC purification provided probes 1−3 on scales (1−100 μmol) sufficient for biological evaluation (Supplementary Scheme S1). To test the affect of our structural modifications on molecular recognition of KAT enzymes, we assayed probes 1−3 against recombinant p300, pCAF, and Mof (MYST1) and compared their inhibitory activity to that of non-BPyne- Figure 1. Cofactor-based affinity probes for the analysis of KAT containing “parent” inhibitor scaffolds (4−6; Supplementary activity. (a) Clickable photoaffinity labeling scheme. (b) Structures of Figure S2). Assayed at a single concentration (1 μM), probes KAT probes 1−3. Ahx = 6-aminohexanoic acid. 1−3 demonstrate inhibitory potencies and selectivities that closely mimic those of parent inhibitor scaffolds 4−6 19,20 containing analogues of the CoA cofactor. However, these (Supplementary Figures S3 and S4). Dose-response analysis probes have not been widely applied to profile KAT activity, of Lys-CoA-BPyne (1) demonstrated an IC of 26.7 nM owing to the fact that most KATs do not utilize mechanisms toward p300 (95% confidence interval [CI ] = 11.75−60.64), involving active-site nucleophiles. within error of the inhibition by parent compound 4 (IC = Here we report a general strategy for chemoproteomic 34.5 nM, CI = 17.5−67.8 nM; Supplementary Figure S5). profiling of KAT activity (Figure 1a). Bisubstrate inhibitors Similar results are seen upon comparison of H3K14-CoA- targeting three phylogenetically distinct KAT families were BPyne 2 and parent bisubstrate 5. Together, these results converted to clickable photoaffinity probes to enable KAT suggest the BPyne subunit has minimal effects on KAT active- labeling and detection. Cofactor-based affinity probes quanti- site recognition. tatively report on KAT-inhibitor interactions, are applied to Selective In Vitro Labeling of KAT Enzymes. Next, we identify a previously unknown acyltransferase activity possessed evaluated the utility of 1−3 as KAT labeling reagents in vitro. by the canonical KAT enzyme Gcn5, and report on KAT KAT probes 1−3 were incubated with purified recombinant activity in cell lysates. Affinity purification and unbiased LC− KATs, photoirradiated at 365 nm, and subjected to Cu- MS/MS profiling of probe targets led to the identification of catalyzed [3 + 2] cycloaddition (“click chemistry”) with a two noncanonical KAT enzymes, highlighting the existence of fluorescent azide tag. SDS-PAGE analysis of labeling reactions several orphan lysine acetyltransferases present in the human demonstrated dose-dependent fluorescent labeling of KATs at genome. In addition to providing insight into the global probe concentrations between 1 and 10 μM. Notably, each selectivity and sensitivity of CoA-based chemical proteomic probe showed sensitive detection of the KAT enzyme family it probes that will guide future applications, these studies was designed to target, i.e., KAT probe 1 reacted effectively demonstrate the ability of chemical tools for profiling KAT with p300 (P300/CBP), probe 2 with pCAF (GCN5/ activity to provide new insights into KATs and their molecular 22 25 PCAF), and probe 3 with Mof (MYST) (Figure 2a). interactions in complex biological contexts. Fluorescent labeling required probe, photo-cross-linking, and 2+ RESULTS AND DISCUSSION Cu ; omission of any single component abolished labeling (Supplementary Figure S6). KAT probes 2 and 3, which are ∼3 Synthesis and Evaluation of Chemoproteomic Probes kDa, caused a significant gel shift upon photoirradiation that for KAT Enzymes. We envisioned a general strategy for was visible upon Coomassie staining (Figure 2a, Supplementary chemical profiling of KAT activity based on combining Figure S6). This property allowed us to quantify and optimize molecular recognition elements from KAT bisubstrate inhib- our photo-cross-linking conditions using gel densitometry. In itors with a clickable photoaffinity tag for covalent cross-linking our optimized photo-cross-linking protocol, we found that and detection (Figure 1a). Pioneered by Cole and co-workers, KAT bisubstrate inhibitors link CoA with the ε-amino group of ∼33−40% of pCAF was covalently labeled by 2 after 60 min of a lysine-containing peptide to form high affinity interactions irradiation at 365 nm on ice. These conditions are consistent 8670 dx.doi.org/10.1021/ja502372j | J. Am. Chem. Soc. 2014, 136, 8669−8676 Journal of the American Chemical Society Article Figure 3. Selectivity of KAT labeling by probes 1−3 (1 μM) assayed in a mixture of proteins from the P300/CBP, GCN5/PCAF, and MYST family. Specific labeling events show sensitivity to competition by parent bisubstrate inhibitors (4−6; 200 equiv). addition to its parent bisubstrate, the Mof-3 interaction was competed by excess Lys-CoA (4), which had no similar effect on the interaction of pCAF with 2 (Supplementary Figure S8). Figure 2. (a) Concentration dependence of probe labeling for For all three probes, selectivities were found to be reduced at preferred KAT partners. (b) Structure of fluorescent KAT photo- higher probe concentrations (10 μM), where 1−3 exhibit affinity probe 7. (c) Relative labeling of pCAF by clickable (left) and considerable cross-reactivity (Supplementary Figure S8). Given fluorescent (right) photoaffinity probes at low (0.1 μM) and high (1 μM) probe concentrations. its combination of broad-spectrum reactivity and straightfor- ward synthesis of probe and competitor, these findings suggest Lys-CoA-BPyne 1 may be the most well-suited of the probes with the literature, and cross-linking yields were not found to for applications requiring a general chemoproteomic reporter of increase with extended photoirradiation times. KAT activity. In contrast, when applied at suitably low The alkyne handle of probes 1−3 was chosen for its minimal concentrations, peptidyl-KAT probes 2 and 3 may be better footprint and versatility toward conjugation of diverse azide suited for applications that require more selective labeling of reporters. However, it is not strictly necessary for ex vivo specific KAT families or as components of KAT chemo- affinity profiling applications. In order to evaluate the utility of proteomic probe cocktails designed to achieve broad super- our bioorthogonal detection strategy, we compared the labeling family coverage. of pCAF by clickable probe 2 with a fluorescent KAT Chemoproteomic Probes Report on KAT−Small photoaffinity probe, TAMRA-H3K14-CoA 7 (Figure 2b). Molecule and KAT−Cofactor Interactions. Having dem- Both probes facilitate fluorescent detection of pCAF at high onstrated the ability of our probes to label three classes of probe concentrations (1 μM), suggesting fluorescent bisub- KATs, we next sought to investigate their ability to report on strates may be useful profiling agents for KAT enzymes. changes in KAT activity resulting from exposure to diverse However, clickable probe 2 exhibits visibly greater labeling than molecular stimuli. First we investigated their ability to report on fluorescent probe 7 at low probe concentrations (0.1 μM; 18,29 the affinity and selectivity of small molecule inhibitors. Co- Figure 2b). This suggests click chemistry detection strategies incubation of pCAF with the known KAT inhibitor garcinol improve probe sensitivity, possibly by abrogating negative decreased labeling by KAT probe 2 in a dose-dependent interactions of the fluorescent TAMRA reporter on probe- manner (Figure 4). Quantification of fluorescent pCAF labeling protein recognition that reduce photo-cross-linking. by gel densitometry yielded an IC of 4.5 ± 1.2 μM for Moving toward more complex settings, we assessed the garcinol, consistent with the literature IC value of 5 μM. specificity and selectivity of each KAT probe (1−3) using a Similarly, labeling of p300 by Lys-CoA BPyne 1 was sensitive to cocktail composed of p300, pCAF, and the MYST family inhibition by the small molecule inhibitor C646 (Supplemen- acetyltransferase Mof. Specific probe labeling events were tary Figure S9). defined as those susceptible to competition by 50−100 equiv of parent KAT bisubstrate inhibitors 4−6 (Supplementary Figure S7), while selectivity refers to the subset of KAT enzymes labeled by each probe. Interestingly, each probe showed specific labeling of a unique subset of KAT enzymes at 1 μM, even in the presence of other KAT superfamily members. For example, Lys-CoA-BPyne 1 showed strong labeling of p300 and pCAF but did not significantly label the MYST family member Mof (Figure 3). The p300 signal was specifically competed in the presence of excess parent inhibitor, while a small portion of the pCAF signal remained, suggesting a combination of specific and nonspecific interactions between the 1-pCAF pair. H3K14- Figure 4. Competitive profiling of KAT active-site occupancy. (a) CoA-BPyne 2 demonstrated a clear preference for specific Fluorescent and Coomassie gels from pCAF-garcinol competition labeling of pCAF and weaker, but detectable, labeling of Mof experiment. Conditions: 2 (1 μM), garcinol (0, 0.08, 0.16, 0.33, 0.63, (Figure 3/Supplementary Figure S8). H4K16-CoA-BPyne 3 1.26, 2.5, 3.75, 5, 10, 20, 40 μM). (b) Dose-response analysis of most strongly labeled the MYST family member Mof and competitive labeling generated via gel densitometry analysis of exhibited fainter labeling of p300 and pCAF (Figure 3). In fluorescent labeling by KAT probe 2. 8671 dx.doi.org/10.1021/ja502372j | J. Am. Chem. Soc. 2014, 136, 8669−8676 Journal of the American Chemical Society Article Figure 5. (a) Scheme for competitive substrate profiling. (b) Fluorescent and Coomassie gels from Gcn5-acyl-CoA competition experiment. Conditions: 2 (10 μM), acyl-CoAs (1000 μM), garcinol (40 μM). In addition to small molecule inhibition, recent studies have Figure 6. Labeling of KATs in cell lysates. (a) Labeling of pCAF in suggested KAT activity may be sensitive to changes in cellular HEK-293 overexpression extracts. Comp. = competitor. (b) Proteins acyl-CoA pools, providing a potential mechanism to link identified in LC−MS/MS experiments as targets of H3K14-CoA- changes in the metabolic state of the cell to differential histone BPyne 2 in pCAF transfected HEK-293 extracts. Control = competitor 15,32 acylations and epigenetic control of gene expression. To treated lane, EV = extract derived from HEK-293 cells transfected with test whether chemoproteomic probes could provide insight into empty vector. (c) Limit of detection of recombinant pCAF spiked into these mechanisms, we investigated the ability of four different HeLa cell proteome. Conditions: 2 (1 μM), proteome (7 μg), acyl-CoAs (acetyl, propionyl, butyryl, and crotonyl-CoA) to recombinant pCAF (0, 6.25, 5, 3.75, 2.5, 1.25 pmol). compete with 2 for the active site occupancy of Gcn5, a KAT whose activity has been proposed to be metabolically regulated 15,33 (Figure 5). We found that high concentrations of acetyl- by fluorescence (Figure 6a). Notably, labeling was sensitive to CoA efficiently competed labeling by probe 2, consistent with competition by H3K14-CoA, and immunoblotting confirmed its role as a universal KAT cofactor. Propionyl-CoA also comigration with pCAF. To further verify labeling, we antagonized labeling, while butyryl-CoA was a partial subjected proteins labeled by 2 in pCAF overexpression antagonist, and crotonyl-CoA did not impede labeling (Figure extracts to click chemistry with biotin azide, followed by 5b). While the ability of Gcn5 to utilize propionyl- and butyrl- affinity purification, tryptic digest, and LC−MS/MS protein CoA as cofactors has not been previously explored, our results identification. Notably, pCAF peptides were specifically are consistent with a previous biochemical analysis of human identified in overexpression extracts treated with 2 (Figure pCAF (which has a highly homologous KAT domain). These 6b), but not in extracts pretreated with excess competitor 5 or studies indicated a kinetic preference for acyl group donors of lysates derived from HEK-293 cells transfected with an empty −1 −1 acetyl-CoA (k /K ≈ 535 s M ) > propionyl-CoA (k /K vector control (Figure 6a, bottom, Supplementary Table S1). In cat m cat m −1 −1 ≈ 92 s M ) ≫ butyrl-CoA and that malonyl-CoA was not addition to overexpression extracts, clickable photoaffinity utilized as a substrate. Indeed, we confirmed Gcn5 utilized probes 1 and 2 are capable of detecting p300 and pCAF, propionyl- and butyrl-CoA as substrates via LC−MS/MS respectively, when spiked into HeLa cell proteomes (Supple- analysis (Supplementary Figure S10). mentary Figure S11). We used this characteristic to assess the These data suggest competitive affinity profiling provides a sensitivity of probe 2 and estimate a lower limit at which useful approach to rapidly gain new insights into KAT inhibitor GCN5/PCAF family enzymes may be detected. This is and substrate selectivity. especially relevant since our inability to enrich pCAF from Chemical Affinity Profiling of KAT Activity in Cell empty vector transfected HEK-293 lysates indicates expression Lysates. The activity of KAT enzymes such as Gcn5 and level may be a limiting factor for KAT identification. We found pCAF is ideally studied in cellular settings. Recent commentary that probe 2 could detect as low as 2.5 pmols of pCAF in a has suggested the failure of conventional high-throughput standard gel-based experiment against a proteomic background screening campaigns to yield selective KAT inhibitors may be (Figure 6b). These findings calibrate the ability of chemo- due to the inability of these screens to interrogate the ability of proteomic probes to monitor KAT activity in model systems small molecules to disrupt native KATs, which can exist as and cell lysates and may be useful for the development of multiprotein complexes. Therefore, methods to monitor KAT chemoproteomic approaches to screen for selective inhibitors 36,37 activity directly from cell extracts are potentially valuable for of KATs and KAT-containing multiprotein complexes. next-generation inhibitor discovery efforts. Overexpression Chemoproteomic Profiling of KAT Activity: Probe extracts of epitope-labeled ATAC (Ada Two-A containing) Reactivity and Orphan KATs. Having demonstrated the complex constitute an advanced model system applied for the utility of chemoproteomic probes for the targeted study of KAT study of GCN5 family KATs in their endogenous setting. We activity in cellular contexts, we last sought to explore their thus asked whether we could use chemoproteomic probe 2 to utility for the discovery of potentially novel KAT activities directly detect KAT activity in these systems, without the need directly from cancer cell proteomes. Labeling of whole cell for prefractionation, affinity purification, or antibodies. extracts by 1−3 demonstrated a distinct pattern of specific Accordingly, pCAF overexpression extracts from HEK-293 protein labeling events for each probe, with KAT probe 1 cells were treated with 2, followed by photo-cross-linking and targeting the largest number of proteins (Figure 7). To identify click chemistry. Weak but detectable labeling of a protein the proteomic targets of broad-spectrum KAT probe 1, HeLa corresponding to the molecular weight of pCAF was observed cells were lysed, photo-cross-linked in the presence of 1, and 8672 dx.doi.org/10.1021/ja502372j | J. Am. Chem. Soc. 2014, 136, 8669−8676 Journal of the American Chemical Society Article In our data set, two acetyltransferases were enriched: Nat10 and Naa50. Nat10 is a noncanonical KAT (referred to here as orphan KATs) with a GNAT-related fold that displays histone and microtubule acetyltransferase activity in cells. Biologically, Nat10 has been observed to play a role in the regulation of telomerase function and nuclear shape and was recently identified as adruggabletargetfor thetreatment of 41,42 Hutchinson−Gilford progeria syndrome. Naa50 (Nat13) is the catalytic component of the NatE acetyltransferase complex that is required for proper sister chromatid adhesion 43,44 and chromatin condensation in vivo. The identification of Naa50 as a target of Lys-CoA-BPyne 1 initially struck us as paradoxical, as this enzyme belongs to the N-terminal acetyltransferase (NAT) family and has been shown to favor 45,46 protein acetylation of N-terminal Met residues. However, literature investigation revealed that, of the 7 NAT catalytic subunits encoded in the human genome, Naa50 is the only member to have biochemically characterized ε-lysine acetyl- transferase activity, providing a molecular rationale for its targeting by 1. The selective identification of Naa50 by 1 suggests that chemoproteomic profiling may have applications in identifying new KAT activities present in acetyltransferase families that are distinct in sequence from canonical KATs. Figure 7. Cofactor-based affinity profiling of endogenous KAT activity The proteomic identification of two orphan KAT activities by in a cancer cell proteome. (a) Labeling of HeLa cell proteomes by affinity probe 1 led us to re-evaluate the lysine acetyltransferase KAT probes 1−3 (10 μM). Specific labeling events show sensitivity to competition by parent bisubstrates (100 equiv). (b) Proteins identified literature and consider whether other KAT activities were also in LC−MS/MS experiments as targets of Lys-CoA-BPyne 1. Values missing from the list of 18 canonical human KATs represent the average spectral counts of two biological replicates. (Supplementary Figure S1). This analysis identified 14 proteins, Green, acetyltransferases; Red, CoA-binding proteins. (c) Affinity including Nat10 and Naa50, not in the list of 18 canonical labeling of recombinant protein verifies Acly and Naa50 as targets of 1. KATs for which evidence of lysine acetyltransferase activity has been observed. Sequence alignment and similarity analyses were used to construct an expanded phylogenetic tree of subjected to click chemistry with biotin-azide followed by acetyltransferase proteins, divided into canonical (P300/CBP, streptavidin enrichment, tryptic digest, and LC−MS/MS GCN5/PCAF, MYST, NCOA) and orphan KATs (Figure 8). analysis. We identified 16 proteins that were abundant (>5 Notably, many KATs from the initial list of 18 (ATAT1, spectral counts) and showed >5-fold preferential enrichment in TF3C4, ELP3; Supplementary Figure S1) cluster more closely the absence of competitor (Figure 7b, Supplementary Table with orphan KATs, indicative of greater sequence similarity. S2). Peptides matching canonical KAT p300 and CBP were not Together, these proteins encompass the most comprehensive observed in our MS/MS data set, likely due to their low list of human KATs assembled to date. However, we take care abundance in whole cell lysates (confirmed by Western blot; to point out that the experimental evidence supporting the Supplementary Figure S12). Of the 16 proteins, 2 were activity of all 32 KATs, canonical and orphan, ranges widely. In acetyltransferases (green), 5 were CoA-binding proteins related particular, this list contains two proteins (Oga/Ncoat, an to primary metabolism (red), with the remaining hits orphan KAT, and Src1, a canonical KAT) for which conflicting composed of highly abundant proteins and members of the 48,49 observations of KAT activity have been made. These proteasome regulatory complex. To validate LC−MS/MS discrepancies support the need for universal methodologies identifications, we performed probe labeling experiments of capable of directly assaying KAT activity in endogenous cells, two overexpressed and purified targets of 1: ATP-citrate lyase toward which our current study provides an initial step. In the (Acly), the highest abundance pulldown target of 1, and N-α- meantime, our chemoproteomics-inspired census of KAT acetyltransferase 50 (Naa50), an acetyltransferase. Both activity provides fertile ground for functional investigation of enzymes exhibited competitor-sensitive labeling, indicative of orphan KAT enzymes using traditional structural and specific molecular recognition by KAT probe 1 (Figure 7c). biochemical approaches, studies that may facilitate a more Conserved domain analysis indicated that each protein complete understanding of lysine acetylation in living systems. specifically labeled by 1 either contains or is closely associated with a protein containing a binding site for an adenine CONCLUSION nucleotide-containing cofactor (CoA, ATP, or NAD(P); Figure ■ 7c). The off-target engagement of adenosine-binding proteins In summary, here we have described a suite of probes for by KAT probe 1 parallels the widespread reactivity that has cofactor-based affinity profiling of KAT activity. We have been observed for ATP- and GTP-containing chemical defined key structural features necessary for the covalent 38−40 proteomic probes in biological settings. This is likely to labeling and detection of three families of KAT enzymes and be a general limitation of chemical proteomic probes demonstrated the utility of these probes to monitor KAT incorporating adenine cofactor-based chemical scaffolds and activity in settings ranging from purified enzymes to KAT highlights opportunities for design improvements as well as the overexpression extracts to native proteomes. Chemoproteomic requirement for orthogonal validation strategies to support probes of KAT activity provided new insights into KAT chemical proteomic KAT discovery efforts. inhibitor and cofactor selectivity and highlighted the existence 8673 dx.doi.org/10.1021/ja502372j | J. Am. Chem. Soc. 2014, 136, 8669−8676 Journal of the American Chemical Society Article orphan KAT activities in cellular settings. Such studies are currently underway, and will be reported in due course. EXPERIMENTAL DETAILS Biochemistry and Cell Biology. Recombinant pCAF, catalytic domain (aa 492−658) was obtained from Cayman Chemical. Recombinant p300, catalytic domain (aa 1284−1673) and recombi- nant Gcn5, catalytic domain (497−663), were obtained from Enzo. The plasmid encoding recombinant MOF, catalytic domain (aa 147− 449) was obtained from Addgene. His-tagged recombinant Mof was expressed in E. coli BL21 and purified via immobilized nickel affinity chromatography using standard conditions. Acly was obtained from US Biological. Naa50 was obtained from Origene. Garcinol and C646 were obtained from Cayman Chemical. Streptavidin-agarose was purchased from Pierce. SDS-PAGE was performed using Bis-Tris NuPAGE gels (4−12%) and MES running buffer in Xcell SureLock MiniCells (Invitrogen) according to the manufacturer’s instructions. SDS-PAGE fluorescence was visualized using an ImageQuant Las4010 Digitial Imaging System (GE Healthcare). Total protein content on SDS-PAGE gels was visualized by Blue-silver coomassie stain, made according to the published procedure. Separation-based assays for KAT activity were performed on a LabChip EZ Reader instrument (PerkinElmer) kindly provided by Dr. Jay Schneekloth. Fluorescence assays for the KAT enzyme Mof were analyzed on a Biotek Synergy 2 Figure 8. Expanded phylogenetic tree of KAT enzymes, including (Biotek). canonical KATs and orphan KAT activities observed in this study or KAT Inhibition Assays. Recombinant KAT activity was measured annotated in the literature. KATs are denoted by gene name and by electrophoretic mobility shift assay (EMSA) as previously relevant pseudonyms. Uniprot accession numbers and literature reported. This assay measures the separation of FITC-labeled KAT references for KAT activity are provided in the Supporting substrate peptides (Histone H3 5-23 QTARKSTGGKAPRKQLATK- Information (Table S3). Ahx-FITC; Histone H4 1-19 SGRGKGGKGLGKGGAKRHR-Ahx- FITC) from their acetylated products following incubation with recombinant KAT and acetyl-CoA. A model separation is shown in of several noncanonical orphan KAT activities that may Supplementary Figure S14. P300 and pCAF assays were performed in contribute to cellular acetylation signaling pathways. In addition 30 μL of reaction buffer (50 mM HEPES, pH 7.5, 50 mM NaCl, 2 mM to these advances, it is also important to call attention to the EDTA, 0.05% Tween 20, 10 μg/mL BSA) with KAT (p300 [50 nM] limitations of our initial study. For example, while we or pCAF [10 nM]) and FITC-peptide (FITC-H4 for P300; FITC-H3 demonstrated the ability of KAT affinity probes to report on for pCAF; 1 μM). Acetylation of FITC-H3/H4 peptide by p300 and pCAF activity in HEK-293 overexpression extracts, we were pCAF was confirmed by LC−MS. Reactions were plated in 384-well unable to detect canonical KAT enzymes such as CBP and plates, allowed to equilibrate at room temperature for 10 min, and initiated by addition of acetyl-CoA (final concentration = 5 μM). Gcn5 directly from HeLa proteomes. This may be due to the Plates were then transferred to a Lab-Chip EZ-Reader at ambient low abundance of these KATs in whole cell lysates temperature and analyzed by microfluidic electrophoresis. Optimized (Supplementary Figure S13) or the low cross-linking yields of separation conditions were downstream voltage of −400 V, upstream our clickable photoaffinity probes, which covalently label only voltage of −2900 V, and a pressure of −2.0 psi for FITC-H3 and ∼33−40% of recombinant pCAF in vitro. This challenge may downstream voltage of −500 V, upstream voltage of −1500 V and a be addressed in future studies through scale-up, nuclear pressure of −2.0 psi for FITC-H4. Percent conversion is calculated by prefractionation, or utilization of multidimensional protein ratiometric measurement of substrate/product peak heights. Percent identification technology (MuDPIT) to increase LC−MS/MS activity represents the percent conversion of KAT reactions treated detection sensitivity. Alternatively, chemical proteomic with inhibitors 1−6 relative to untreated control KAT reactions, measured in triplicate, and corrected for nonenzymatic acetylation. studies of kinase and KDAC activity have shown that Mof showed low activity toward FITC-H3/H4 peptide substrates and noncovalent affinity probe resins can enable enrichment of was monitored by fluorogenic KAT assay using an unlabeled H4 specific enzyme classes without the need for photo-cross- 18,51 substrate peptide as previously reported. Dose-response analysis of linking, providing another potential route for the analysis of p300 and pCAF inhibition by KAT probes 1 and 2 and parent low abundance KATs. Furthermore, while the activity of KAT inhibitors 4 and 5 were performed in triplicate and analyzed by complexes have been shown to be preserved in cell extracts, ∧ nonlinear least-squares regression fitto Y = 100/(1 + 10 (Log IC − these activities would be ideally studied in living cells and X)*H), where H = Hill slope (variable). IC values represent the probes 1−3 are not cell-permeable. Cell-penetrating peptides concentration that inhibits 50% of KAT activity. All calculations were have been used to promote uptake of KAT bisubstrate performed using Prism 6 (GraphPad) software. Fluorescent Labeling of KAT Enzymes for SDS-PAGE inhibitors, and similar approaches may facilitate live cell Analysis. Purified KAT enzymes (0.5−5 μg) or whole cell proteomes profiling of KAT activity. These improvements will be (20 μg) were incubated with KAT probes 1−3 (1 μM probe for important to expand the scope of chemical proteomic analyses recombinant labelings; 10 μM probe for proteomic labelings) in PBS of KAT activity. Regardless, the ability of our current suite of (pH 7.0) for 1 h. Control experiments to correct for nonspecific cross- chemoproteomic probes to highlight an expanded landscape of linking were treated with 1−3 in the presence of 100 equiv of catalytic lysine acetylation provides an example of the power of competitors 4−6. Following equilibration, samples were photo-cross- this approach as currently constituted and sets the stage for the linked on ice for 1 h using a 365 nm UV light in a FB-UVXL-1000 UV development of chemoproteomic strategies to identify KAT cross-linker. Probe labeling was detected by Cu(I)-catalyzed [3 + 2] inhibitors and the functional characterization of canonical and cycloaddition (“click chemistry”). Click reactions were initiated by 8674 dx.doi.org/10.1021/ja502372j | J. Am. Chem. Soc. 2014, 136, 8669−8676 Journal of the American Chemical Society Article sequential addition of TAMRA-azide 8 (100 μM; 5 mM stock solution resulting cell pellet was air-dried to remove excess methanol and in DMSO, structure given in Supplementary Figure S15), TCEP (1 redissolved in 1.2% SDS (1 mL) using iterative cycles of heating (95 mM; 100 mM stock in H O), tris(benzyltriazolylmethyl)amine ligand °C) and sonication. Redissolved protein was allowed to cool to room (TBTA; 100 μM; 1.7 mM stock in DMSO/tert-butanol 1:4), and temperature and added to 5 mL of PBS to give a final SDS concentration of 0.2%. Samples were then treated with 100 μLof CuSO (1 mM; 50 mM stock in H O). Samples were vortexed and 4 2 streptavidin-agarose resin (prewashed 3× with 1 mL of PBS) and incubated at room temperature for 1 h. Cycloaddition reactions were rotated for 1 h at room temperature. Streptavidin-agarose bound quenched by addition of 5x SDS-loading buffer (strongly reducing) and subjected to SDS-PAGE (22 μL per well). Excess probe samples were then washed sequentially with 0.2% SDS in PBS (3 × 10 fluorescence was removed by destaining in a solution of 50% mL) and PBS (3 × 10 mL). Samples were then prepared for on-bead MeOH/40% H O/10% AcOH overnight. Gels were then washed digest by reduction with 10 mM tris(2-carboxyethyl)phosphine with water and fluorescently visualized using a ImageQuant Las4010 (TCEP) and alkylation with 12 mM iodoacetamide. Samples were (GE Healthcare) with green LED excitation (λ 520−550 nm) and a diluted to 2 M urea with 50 mM Tris-Cl pH 8.0 (400 μL total max 575DF20 filter. For KAT probes 2 and 3, a characteristic intense low volume), followed by addition of trypsin and 2 mM CaCl . Digests molecular weight fluorescence signal ∼3 kDa was observed were allowed to proceed overnight at 37 °C. After extraction, tryptic peptide samples were acidified to a final concentration of 5% formic (corresponding to the fluorescently labeled KAT probe [2, 3]), acid and frozen at −80 °C for LC−MS/MS analysis. indicative of a high-yielding click chemistry reaction. Liquid Chromatography−Mass Spectrometry and Data Cell Culture and Isolation of Whole-Cell Lysates. HeLa S3 Analysis. Tryptic peptides enriched by probe 1 were loaded onto a cells (ATCC; Manassas VA) were cultured at 37 °C under 5% CO reverse phase capillary column and analyzed by LC separation in atmosphere in a culture medium of DMEM supplemented with 10% combination with tandem MS. Peptides were eluted using a gradient of FBS and glutamine. HEK-293 cells were obtained from the NCI 5−42% over 40 min with the flow rate through the column set at 0.20 Tumor Cell Repository. For isolation of whole cell proteomes, HeLa μL/min. Data was collected in a dual-pressure linear ion trap mass cells were grown to 80−90% confluency, washed 3× with ice-cold spectrometer (ThermoFisher LTQ VelosPro) set in a data-dependent PBS, scraped, and pelleted by centrifugation (1400g × 3 min, 4 °C). acquisition mode. The 15 most intense molecular ions in the MS scan After removal of PBS cell pellets were stored at −76 °Cor were sequentially and dynamically selected for subsequent collision- immediately processed. Cell pellets were resuspended in 1−2mLof induced dissociation (CID) using a normalized collision energy of ice-cold PBS (10−20 × 10 cells/mL) and lysed by sonication 35%. Tandem mass spectra were searched against UniProt H. sapiens (QSonica XL2000 100 W sonicator, 3 × 10 s pulse, 50% power, 60 s protein database (01-13 release) using SEQUEST (ThermoFisher). between pulses). Lysates were pelleted by centrifugation (14,000g × Search parameters were fixed as follows: (i) enzyme specificity: 30 min, 4 °C) and quantified on a Qubit 2.0 Fluorometer using a trypsin; (ii) variable modification: methionine oxidation and cysteine Qubit Protein Assay Kit. Proteomes were diluted to 2 mg/mL and carbamidomethylation; (iii) precursor mass tolerance ±1.40 amu; and stored in 1 mg aliquots at −76 °C until further processing. (iv) fragment ion mass tolerance ±0.5 amu. Only those tryptic Western Blotting. SDS-PAGE gels were transferred to nitro- peptides with up to two missed cleavage sites meeting a specific cellulose membranes (Novex, Life Technologies) by electroblotting at SEQUEST scoring criteria (Delta Correlation (ΔCn) ≥ 0.08 and 30 V for 1 h using a XCell II Blot Module (Novex). Membranes were 1+ charge state dependent cross correlation (Xcorr) ≥ 1.9 for [M + H] , blocked using StartingBlock (PBS) Blocking Buffer (Thermo 2+ 3+ ≥ 2.2 for [M + 2H] , and ≥3.1 for [M + 3H] ) were considered as Scientific) for 20 min and then incubated overnight at 4 °Cin a legitimate identifications. Spectral count values depicted in Figure 7 solution containing the primary antibody of interest (anti-Gcn5 represent an average of two biological replicates. Raw spectral counts [3305], anti-CBP [3378], Cell Signaling, 1:1000 dilution) in the above for biological duplicates of probe-enriched and control experiments are blocking buffer with 0.05% Tween 20. The membranes were next provided in Supplementary Table S2. washed with TBST buffer and incubated with a secondary HRP- Phylogenetic Analysis. Amino acid sequences for canonical and conjugated antibody (anti-rabbit IgG, HRP-linked [7074], Cell orphan lysine acetyltransferases were obtained from Uniprot. Signaling, 1:1000 dilution) for 1.5 h at room temperature. The Accession numbers are provided in Table S3 (Supporting membranes were again washed with TBST, treated with chemilumi- Information). A pairwise alignment was generated using Clustal nescence reagents (Western Blot Detection System, Cell Signaling) for Omega, and a phylogenic tree was constructed using the neighbor- 1 min, and imaged for chemiluminescent signal using an ImageQuant joining method. All phylogenetic trees were displayed in hyperbolic Las4010 Digitial Imaging System (GE Healthcare). space using Hypertree, with branches of the tree designated by Enrichment of KAT Enzymes for Proteomic Analysis. Whole different colors and labeled by name where appropriate. cell proteomes were adjusted to a final protein concentration of 1 mg/ mL and incubated with the indicated probe (1 or 2;10 μM) for 1 h. ASSOCIATED CONTENT HEK-293 enrichments utilized 0.5 mg of proteome as starting material, while HeLa enrichments utilized 1 mg of proteome. Control samples * Supporting Information to correct for nonspecific cross-linking were preincubated with each Synthetic materials and methods, characterization data, and probe’s cognate competitor (4 or 5; 100 equiv). Following supplementary figures and schemes. This material is available equilibration, samples were split into 5 × 200 μL aliquots and free of charge via the Internet at http://pubs.acs.org. photo-cross-linked on ice for 1 h using a 365 nm UV light in a FB- UVXL-1000 UV cross-linker. Cross-linked samples were then AUTHOR INFORMATION recombined and subjected to Cu(I)-catalyzed [3 + 2] cycloaddition with TAMRA biotin-azide 9 (Supplementary Figure S15) as previously Corresponding Author described. Final concentrations for click reactions were as follows: [email protected] HeLa proteome (1 mg/mL in PBS), probe 1 (10 μM), TAMRA Notes biotin-azide (40 μM), TCEP (1 mM), TBTA (100 μM), tert-butanol (4.8%), and CuSO (1 mM). Samples were vortexed and incubated at The authors declare no competing financial interest. room temperature for 1 h. Ice-cold 4:1 MeOH/CHCl (2.5 mL) was then added directly to the reaction mixture and mixed vigorously by ACKNOWLEDGMENTS vortexing. The biphasic solution was centrifuged (4000g × 20 min, 4 The authors thank Dr. Ming Zhou (Laboratory of Proteomics °C), and protein precipitated at the interface as a solid disk. Liquid and Analytical Technology) for LC−MS/MS analyses, Dr. layers were carefully discarded, and the resulting precipitate was Hans Luecke (NIDDK) for the pCAF overexpression plasmid, resuspended in ice-cold 1:1 MeOH/CHCl (1 mL), sonicated on ice Dr. Brian Lewis (NCI) for helpful discussions, and Dr. Michael to resuspend, and repelleted by centrifugation (14,000g × 10 min, 4 °C). This wash step was repeated with ice-cold MeOH (1 mL). The Giano of the Schneider lab for assistance with peptide synthesis. 8675 dx.doi.org/10.1021/ja502372j | J. Am. Chem. Soc. 2014, 136, 8669−8676 Journal of the American Chemical Society Article (30) Balasubramanyam, K.; Altaf, M.; Varier, R. A.; Swaminathan, V.; This work was supported by the Intramural Research Program Ravindran, A.; Sadhale, P. P.; Kundu, T. K. J. Biol. Chem. 2004, 279, of the NIH, National Cancer Institute, Center for Cancer Research (ZIA BC011488-01). (31) Bowers, E. M.; Yan, G.; Mukherjee, C.; Orry, A.; Wang, L.; Holbert, M. 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Published: May 17, 2014

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