Chemoproteomic profiling of lysine acetyltransferases highlights an expanded landscape of catalytic acetylation

Article abstract of DOI:10.1021/ja502372j

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. This article not subject to U.S. Published 2014 by the American Chemical Society.

Full text of DOI:10.1021/ja502372j

Article
pubs.acs.org/JACS
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
S
* 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.
transcription factor-related and orphan (sequence disparate)
KAT activities (Supplementary Figure S1). KAT family
members demonstrate significant intrafamily but little inter-
family sequence homology, hindering bioinformatics ap-
proaches to KAT discovery and classification. The functional
characterization of KATs is also limited by their regulation by
protein partners and PTMs, factors that are difficult to
recapitulate in vitro.12,13 Furthermore, while individual KATs
have been shown to be susceptible to inhibition by small
molecules and cellular acetyl-CoA/CoA ratio, methods for
comparing the selectivity of these perturbations among multiple
KATs in parallel do not exist.14,15 Thus, our ability to discover
and characterize acetylation-mediated signaling would be
greatly advanced by the development of new methods for the
global analysis of KAT activity in cellular contexts.
Chemoproteomic profiling provides a powerful alternative to
traditional biochemical assays for measuring enzyme activity in
complex biological settings. In this approach, also commonly
referred to as activity-based protein profiling (ABPP), active-
site probes for an enzyme class of interest are modified with
chemical handles enabling detection or affinity enrichment.
Covalent labeling or enrichment of an enzyme by the affinity
probe is then used as a proxy for enzyme activity.16
Chemoproteomic probes for KDAC enzymes have been used
to discover novel KDAC complexes17 and characterize inhibitor
selectivity in cell lysates.18 Similar approaches have also been
pursued to study KAT enzymes, utilizing electrophile-
INTRODUCTION
■
Lysine acetylation plays a critical role in the regulation of
transcription, metabolism, and other central biological
functions. Acetylation of lysine residues can impact protein
and genome function through multiple mechanisms, including
physical relaxation of histone−DNA interactions,1 recruitment
of bromodomain-containing effector proteins,2 covalent active-
site modification,3 and regulation of protein stability.4 Lysine
acetylation is a dynamic PTM that represents an equilibrium
between the activity of two opposing enzyme classes: lysine
acetyltransferase (KAT) enzymes, which impose the mark, and
lysine deacetylases (KDACs), which remove it.5 While KDACs
have been extensively investigated as epigenetic drug targets,
several analyses indicate KATs can also drive cellular
transformation and cancer progression in a tissue-specific
manner. For example, fusion of the KAT enzyme MOZ to TIF2
is the primary genetic lesion associated with a subset of
leukemias and imbues differentiated red blood cells with cancer
stem cell-like properties.6 Nonmutant KAT activities can also
support oncogenic gene expression programs, functioning as
essential coactivators for transcription factors such as c-Myc
and E2A-PBX gene fusions in cancer.7
Proteome-wide studies have revealed lysine acetylation is a
prevalent PTM, rivaling phosphorylation in terms of substrate
diversity with ∼4700 human acetylation sites identified to
date.8−10 However, in contrast to the hundreds of known
protein kinases, a recent phylogenetic analysis identified only
18 KATs in the human genome.11 The majority of these
canonical KATs fall into four families: GCN5/PCAF, P300/
CBP, MYST, and NCOA, with the rest consisting of
Received: March 8, 2014
Published: May 17, 2014
This article not subject to U.S. Copyright.
Published 2014 by the American Chemical
Society
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with both the substrate and cofactor binding sites of KAT
enzymes.22 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),
necessary for covalent labeling and detection.23,24 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-
CoA-BPyne; 3) (Figure 1; Supplementary Scheme S1).22,25
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-
containing “parent” inhibitor scaffolds (4−6; Supplementary
Figure S2). Assayed at a single concentration (1 μM), probes
1−3 demonstrate inhibitory potencies and selectivities that
closely mimic those of parent inhibitor scaffolds 4−6
(Supplementary Figures S3 and S4). Dose-response analysis
of Lys-CoA-BPyne (1) demonstrated an IC50 of 26.7 nM
toward p300 (95% confidence interval [CI95] = 11.75−60.64),
Figure 1. Cofactor-based affinity probes for the analysis of KAT
activity. (a) Clickable photoaffinity labeling scheme. (b) Structures of
KAT probes 1−3. Ahx = 6-aminohexanoic acid.
containing analogues of the CoA cofactor.19,20 However, these
probes have not been widely applied to profile KAT activity,
owing to the fact that most KATs do not utilize mechanisms
involving active-site nucleophiles.21
within error of the inhibition by parent compound 4 (IC50
=
Here we report a general strategy for chemoproteomic
profiling of KAT activity (Figure 1a). Bisubstrate inhibitors
targeting three phylogenetically distinct KAT families were
converted to clickable photoaffinity probes to enable KAT
labeling and detection. Cofactor-based affinity probes quanti-
tatively report on KAT-inhibitor interactions, are applied to
identify a previously unknown acyltransferase activity possessed
by the canonical KAT enzyme Gcn5, and report on KAT
activity in cell lysates. Affinity purification and unbiased LC−
MS/MS profiling of probe targets led to the identification of
two noncanonical KAT enzymes, highlighting the existence of
several orphan lysine acetyltransferases present in the human
genome. In addition to providing insight into the global
selectivity and sensitivity of CoA-based chemical proteomic
probes that will guide future applications, these studies
demonstrate the ability of chemical tools for profiling KAT
activity to provide new insights into KATs and their molecular
interactions in complex biological contexts.
34.5 nM, CI95 = 17.5−67.8 nM; Supplementary Figure S5).
Similar results are seen upon comparison of H3K14-CoA-
BPyne 2 and parent bisubstrate 5. Together, these results
suggest the BPyne subunit has minimal effects on KAT active-
site recognition.
Selective In Vitro Labeling of KAT Enzymes. Next, we
evaluated the utility of 1−3 as KAT labeling reagents in vitro.
KAT probes 1−3 were incubated with purified recombinant
KATs, photoirradiated at 365 nm, and subjected to Cu-
catalyzed [3 + 2] cycloaddition (“click chemistry”) with a
fluorescent azide tag.26 SDS-PAGE analysis of labeling reactions
demonstrated dose-dependent fluorescent labeling of KATs at
probe concentrations between 1 and 10 μM. Notably, each
probe showed sensitive detection of the KAT enzyme family it
was designed to target, i.e., KAT probe 1 reacted effectively
with p300 (P300/CBP), probe 2 with pCAF (GCN5/
PCAF),22 and probe 3 with Mof (MYST) (Figure 2a).25
Fluorescent labeling required probe, photo-cross-linking, and
Cu2+; omission of any single component abolished labeling
(Supplementary Figure S6). KAT probes 2 and 3, which are ∼3
kDa, caused a significant gel shift upon photoirradiation that
was visible upon Coomassie staining (Figure 2a, Supplementary
Figure S6). This property allowed us to quantify and optimize
our photo-cross-linking conditions using gel densitometry. In
our optimized photo-cross-linking protocol, we found that
∼33−40% of pCAF was covalently labeled by 2 after 60 min of
irradiation at 365 nm on ice. These conditions are consistent
RESULTS AND DISCUSSION
■
Synthesis and Evaluation of Chemoproteomic Probes
for KAT Enzymes. We envisioned a general strategy for
chemical profiling of KAT activity based on combining
molecular recognition elements from KAT bisubstrate inhib-
itors with a clickable photoaffinity tag for covalent cross-linking
and detection (Figure 1a). Pioneered by Cole and co-workers,
KAT bisubstrate inhibitors link CoA with the ε-amino group of
a lysine-containing peptide to form high affinity interactions
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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).
For all three probes, selectivities were found to be reduced at
higher probe concentrations (10 μM), where 1−3 exhibit
considerable cross-reactivity (Supplementary Figure S8). Given
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
for applications requiring a general chemoproteomic reporter of
KAT activity. In contrast, when applied at suitably low
concentrations, peptidyl-KAT probes 2 and 3 may be better
suited for applications that require more selective labeling of
specific KAT families or as components of KAT chemo-
proteomic probe cocktails designed to achieve broad super-
family coverage.28
Chemoproteomic Probes Report on KAT−Small
Molecule and KAT−Cofactor Interactions. Having dem-
onstrated the ability of our probes to label three classes of
KATs, we next sought to investigate their ability to report on
changes in KAT activity resulting from exposure to diverse
molecular stimuli. First we investigated their ability to report on
the affinity and selectivity of small molecule inhibitors.18,29 Co-
incubation of pCAF with the known KAT inhibitor garcinol
decreased labeling by KAT probe 2 in a dose-dependent
manner (Figure 4). Quantification of fluorescent pCAF labeling
Figure 2. (a) Concentration dependence of probe labeling for
preferred KAT partners. (b) Structure of fluorescent KAT photo-
affinity probe 7. (c) Relative labeling of pCAF by clickable (left) and
fluorescent (right) photoaffinity probes at low (0.1 μM) and high (1
μM) probe concentrations.
with the literature,27 and cross-linking yields were not found to
increase with extended photoirradiation times.
The alkyne handle of probes 1−3 was chosen for its minimal
footprint and versatility toward conjugation of diverse azide
reporters. However, it is not strictly necessary for ex vivo
affinity profiling applications. In order to evaluate the utility of
our bioorthogonal detection strategy, we compared the labeling
of pCAF by clickable probe 2 with a fluorescent KAT
photoaffinity probe, TAMRA-H3K14-CoA 7 (Figure 2b).
Both probes facilitate fluorescent detection of pCAF at high
probe concentrations (1 μM), suggesting fluorescent bisub-
strates may be useful profiling agents for KAT enzymes.
However, clickable probe 2 exhibits visibly greater labeling than
fluorescent probe 7 at low probe concentrations (0.1 μM;
Figure 2b). This suggests click chemistry detection strategies
improve probe sensitivity, possibly by abrogating negative
interactions of the fluorescent TAMRA reporter on probe-
protein recognition that reduce photo-cross-linking.28
Moving toward more complex settings, we assessed the
specificity and selectivity of each KAT probe (1−3) using a
cocktail composed of p300, pCAF, and the MYST family
acetyltransferase Mof. Specific probe labeling events were
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-
CoA-BPyne 2 demonstrated a clear preference for specific
labeling of pCAF and weaker, but detectable, labeling of Mof
(Figure 3/Supplementary Figure S8). H4K16-CoA-BPyne 3
most strongly labeled the MYST family member Mof and
exhibited fainter labeling of p300 and pCAF (Figure 3). In
by gel densitometry yielded an IC50 of 4.5
1.2 μM for
garcinol, consistent with the literature IC50 value of 5 μM.30
Similarly, labeling of p300 by Lys-CoA BPyne 1 was sensitive to
inhibition by the small molecule inhibitor C646 (Supplemen-
tary Figure S9).31
Figure 4. Competitive profiling of KAT active-site occupancy. (a)
Fluorescent and Coomassie gels from pCAF-garcinol competition
experiment. Conditions: 2 (1 μM), garcinol (0, 0.08, 0.16, 0.33, 0.63,
1.26, 2.5, 3.75, 5, 10, 20, 40 μM). (b) Dose-response analysis of
competitive labeling generated via gel densitometry analysis of
fluorescent labeling by KAT probe 2.
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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
suggested KAT activity may be sensitive to changes in cellular
acyl-CoA pools, providing a potential mechanism to link
changes in the metabolic state of the cell to differential histone
acylations and epigenetic control of gene expression.15,32 To
test whether chemoproteomic probes could provide insight into
these mechanisms, we investigated the ability of four different
acyl-CoAs (acetyl, propionyl, butyryl, and crotonyl-CoA) to
compete with 2 for the active site occupancy of Gcn5, a KAT
whose activity has been proposed to be metabolically regulated
(Figure 5).15,33 We found that high concentrations of acetyl-
CoA efficiently competed labeling by probe 2, consistent with
its role as a universal KAT cofactor. Propionyl-CoA also
antagonized labeling, while butyryl-CoA was a partial
antagonist, and crotonyl-CoA did not impede labeling (Figure
5b). While the ability of Gcn5 to utilize propionyl- and butyrl-
CoA as cofactors has not been previously explored, our results
are consistent with a previous biochemical analysis of human
pCAF (which has a highly homologous KAT domain). These
studies indicated a kinetic preference for acyl group donors of
acetyl-CoA (kcat/Km ≈ 535 s−1 M−1) > propionyl-CoA (kcat/Km
≈ 92 s−1 M−1) ≫ butyrl-CoA and that malonyl-CoA was not
utilized as a substrate.34 Indeed, we confirmed Gcn5 utilized
propionyl- and butyrl-CoA as substrates via LC−MS/MS
analysis (Supplementary Figure S10).
Figure 6. Labeling of KATs in cell lysates. (a) Labeling of pCAF in
HEK-293 overexpression extracts. Comp. = competitor. (b) Proteins
identified in LC−MS/MS experiments as targets of H3K14-CoA-
BPyne 2 in pCAF transfected HEK-293 extracts. Control = competitor
treated lane, EV = extract derived from HEK-293 cells transfected with
empty vector. (c) Limit of detection of recombinant pCAF spiked into
HeLa cell proteome. Conditions: 2 (1 μM), proteome (7 μg),
recombinant pCAF (0, 6.25, 5, 3.75, 2.5, 1.25 pmol).
by fluorescence (Figure 6a). Notably, labeling was sensitive to
competition by H3K14-CoA, and immunoblotting confirmed
comigration with pCAF. To further verify labeling, we
subjected proteins labeled by 2 in pCAF overexpression
extracts to click chemistry with biotin azide, followed by
affinity purification, tryptic digest, and LC−MS/MS protein
identification. Notably, pCAF peptides were specifically
identified in overexpression extracts treated with 2 (Figure
6b), but not in extracts pretreated with excess competitor 5 or
lysates derived from HEK-293 cells transfected with an empty
vector control (Figure 6a, bottom, Supplementary Table S1). In
addition to overexpression extracts, clickable photoaffinity
probes 1 and 2 are capable of detecting p300 and pCAF,
respectively, when spiked into HeLa cell proteomes (Supple-
mentary Figure S11). We used this characteristic to assess the
sensitivity of probe 2 and estimate a lower limit at which
GCN5/PCAF family enzymes may be detected. This is
especially relevant since our inability to enrich pCAF from
empty vector transfected HEK-293 lysates indicates expression
level may be a limiting factor for KAT identification. We found
that probe 2 could detect as low as 2.5 pmols of pCAF in a
standard gel-based experiment against a proteomic background
(Figure 6b). These findings calibrate the ability of chemo-
proteomic probes to monitor KAT activity in model systems
and cell lysates and may be useful for the development of
chemoproteomic approaches to screen for selective inhibitors
of KATs and KAT-containing multiprotein complexes.36,37
Chemoproteomic Profiling of KAT Activity: Probe
Reactivity and Orphan KATs. Having demonstrated the
utility of chemoproteomic probes for the targeted study of KAT
activity in cellular contexts, we last sought to explore their
utility for the discovery of potentially novel KAT activities
directly from cancer cell proteomes. Labeling of whole cell
extracts by 1−3 demonstrated a distinct pattern of specific
protein labeling events for each probe, with KAT probe 1
targeting the largest number of proteins (Figure 7). To identify
the proteomic targets of broad-spectrum KAT probe 1, HeLa
cells were lysed, photo-cross-linked in the presence of 1, and
These data suggest competitive affinity profiling provides a
useful approach to rapidly gain new insights into KAT inhibitor
and substrate selectivity.
Chemical Affinity Profiling of KAT Activity in Cell
Lysates. The activity of KAT enzymes such as Gcn5 and
pCAF is ideally studied in cellular settings. Recent commentary
has suggested the failure of conventional high-throughput
screening campaigns to yield selective KAT inhibitors may be
due to the inability of these screens to interrogate the ability of
small molecules to disrupt native KATs, which can exist as
multiprotein complexes.11 Therefore, methods to monitor KAT
activity directly from cell extracts are potentially valuable for
next-generation inhibitor discovery efforts. Overexpression
extracts of epitope-labeled ATAC (Ada Two-A containing)
complex constitute an advanced model system applied for the
study of GCN5 family KATs in their endogenous setting.35 We
thus asked whether we could use chemoproteomic probe 2 to
directly detect KAT activity in these systems, without the need
for prefractionation, affinity purification, or antibodies.
Accordingly, pCAF overexpression extracts from HEK-293
cells were treated with 2, followed by photo-cross-linking and
click chemistry. Weak but detectable labeling of a protein
corresponding to the molecular weight of pCAF was observed
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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 a druggable target for the treatment of
Hutchinson−Gilford progeria syndrome.41,42 Naa50 (Nat13)
is the catalytic component of the NatE acetyltransferase
complex that is required for proper sister chromatid adhesion
and chromatin condensation in vivo.43,44 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
protein acetylation of N-terminal Met residues.45,46 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 1.47 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.
The proteomic identification of two orphan KAT activities by
affinity probe 1 led us to re-evaluate the lysine acetyltransferase
literature and consider whether other KAT activities were also
missing from the list of 18 canonical human KATs
(Supplementary Figure S1). This analysis identified 14 proteins,
including Nat10 and Naa50, not in the list of 18 canonical
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
acetyltransferase proteins, divided into canonical (P300/CBP,
GCN5/PCAF, MYST, NCOA) and orphan KATs (Figure 8).
Notably, many KATs from the initial list of 18 (ATAT1,
TF3C4, ELP3; Supplementary Figure S1) cluster more closely
with orphan KATs, indicative of greater sequence similarity.
Together, these proteins encompass the most comprehensive
list of human KATs assembled to date. However, we take care
to point out that the experimental evidence supporting the
activity of all 32 KATs, canonical and orphan, ranges widely. In
particular, this list contains two proteins (Oga/Ncoat, an
orphan KAT, and Src1, a canonical KAT) for which conflicting
observations of KAT activity have been made.48,49 These
discrepancies support the need for universal methodologies
capable of directly assaying KAT activity in endogenous cells,
toward which our current study provides an initial step. In the
meantime, our chemoproteomics-inspired census of KAT
activity provides fertile ground for functional investigation of
orphan KAT enzymes using traditional structural and
biochemical approaches, studies that may facilitate a more
complete understanding of lysine acetylation in living systems.
Figure 7. Cofactor-based affinity profiling of endogenous KAT activity
in a cancer cell proteome. (a) Labeling of HeLa cell proteomes by
KAT probes 1−3 (10 μM). Specific labeling events show sensitivity to
competition by parent bisubstrates (100 equiv). (b) Proteins identified
in LC−MS/MS experiments as targets of Lys-CoA-BPyne 1. Values
represent the average spectral counts of two biological replicates.
Green, acetyltransferases; Red, CoA-binding proteins. (c) Affinity
labeling of recombinant protein verifies Acly and Naa50 as targets of 1.
subjected to click chemistry with biotin-azide followed by
streptavidin enrichment, tryptic digest, and LC−MS/MS
analysis. We identified 16 proteins that were abundant (>5
spectral counts) and showed >5-fold preferential enrichment in
the absence of competitor (Figure 7b, Supplementary Table
S2). Peptides matching canonical KAT p300 and CBP were not
observed in our MS/MS data set, likely due to their low
abundance in whole cell lysates (confirmed by Western blot;
Supplementary Figure S12). Of the 16 proteins, 2 were
acetyltransferases (green), 5 were CoA-binding proteins related
to primary metabolism (red), with the remaining hits
composed of highly abundant proteins and members of the
proteasome regulatory complex. To validate LC−MS/MS
identifications, we performed probe labeling experiments of
two overexpressed and purified targets of 1: ATP-citrate lyase
(Acly), the highest abundance pulldown target of 1, and N-α-
acetyltransferase 50 (Naa50), an acetyltransferase. Both
enzymes exhibited competitor-sensitive labeling, indicative of
specific molecular recognition by KAT probe 1 (Figure 7c).
Conserved domain analysis indicated that each protein
specifically labeled by 1 either contains or is closely associated
with a protein containing a binding site for an adenine
nucleotide-containing cofactor (CoA, ATP, or NAD(P); Figure
7c). The off-target engagement of adenosine-binding proteins
by KAT probe 1 parallels the widespread reactivity that has
been observed for ATP- and GTP-containing chemical
proteomic probes in biological settings.38−40 This is likely to
be a general limitation of chemical proteomic probes
incorporating adenine cofactor-based chemical scaffolds and
highlights opportunities for design improvements as well as the
requirement for orthogonal validation strategies to support
chemical proteomic KAT discovery efforts.
CONCLUSION
■
In summary, here we have described a suite of probes for
cofactor-based affinity profiling of KAT activity. We have
defined key structural features necessary for the covalent
labeling and detection of three families of KAT enzymes and
demonstrated the utility of these probes to monitor KAT
activity in settings ranging from purified enzymes to KAT
overexpression extracts to native proteomes. Chemoproteomic
probes of KAT activity provided new insights into KAT
inhibitor and cofactor selectivity and highlighted the existence
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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.52 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
(Biotek).
Figure 8. Expanded phylogenetic tree of KAT enzymes, including
canonical KATs and orphan KAT activities observed in this study or
annotated in the literature. KATs are denoted by gene name and
relevant pseudonyms. Uniprot accession numbers and literature
references for KAT activity are provided in the Supporting
Information (Table S3).
KAT Inhibition Assays. Recombinant KAT activity was measured
by electrophoretic mobility shift assay (EMSA) as previously
reported.53 This assay measures the separation of FITC-labeled KAT
substrate peptides (Histone H3 5-23 QTARKSTGGKAPRKQLATK-
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
Supplementary Figure S14. P300 and pCAF assays were performed in
30 μL of reaction buffer (50 mM HEPES, pH 7.5, 50 mM NaCl, 2 mM
EDTA, 0.05% Tween 20, 10 μg/mL BSA) with KAT (p300 [50 nM]
or pCAF [10 nM]) and FITC-peptide (FITC-H4 for P300; FITC-H3
for pCAF; 1 μM). Acetylation of FITC-H3/H4 peptide by p300 and
pCAF was confirmed by LC−MS. Reactions were plated in 384-well
plates, allowed to equilibrate at room temperature for 10 min, and
initiated by addition of acetyl-CoA (final concentration = 5 μM).
Plates were then transferred to a Lab-Chip EZ-Reader at ambient
temperature and analyzed by microfluidic electrophoresis. Optimized
separation conditions were downstream voltage of −400 V, upstream
voltage of −2900 V, and a pressure of −2.0 psi for FITC-H3 and
downstream voltage of −500 V, upstream voltage of −1500 V and a
pressure of −2.0 psi for FITC-H4. Percent conversion is calculated by
ratiometric measurement of substrate/product peak heights. Percent
activity represents the percent conversion of KAT reactions treated
with inhibitors 1−6 relative to untreated control KAT reactions,
measured in triplicate, and corrected for nonenzymatic acetylation.
Mof showed low activity toward FITC-H3/H4 peptide substrates and
was monitored by fluorogenic KAT assay using an unlabeled H4
substrate peptide as previously reported.54 Dose-response analysis of
p300 and pCAF inhibition by KAT probes 1 and 2 and parent
inhibitors 4 and 5 were performed in triplicate and analyzed by
of several noncanonical orphan KAT activities that may
contribute to cellular acetylation signaling pathways. In addition
to these advances, it is also important to call attention to the
limitations of our initial study. For example, while we
demonstrated the ability of KAT affinity probes to report on
pCAF activity in HEK-293 overexpression extracts, we were
unable to detect canonical KAT enzymes such as CBP and
Gcn5 directly from HeLa proteomes. This may be due to the
low abundance of these KATs in whole cell lysates
(Supplementary Figure S13) or the low cross-linking yields of
our clickable photoaffinity probes, which covalently label only
∼33−40% of recombinant pCAF in vitro. This challenge may
be addressed in future studies through scale-up, nuclear
prefractionation, or utilization of multidimensional protein
identification technology (MuDPIT) to increase LC−MS/MS
detection sensitivity.50 Alternatively, chemical proteomic
studies of kinase and KDAC activity have shown that
noncovalent affinity probe resins can enable enrichment of
specific enzyme classes without the need for photo-cross-
linking,18,51 providing another potential route for the analysis of
low abundance KATs. Furthermore, while the activity of KAT
complexes have been shown to be preserved in cell extracts,
these activities would be ideally studied in living cells and
probes 1−3 are not cell-permeable. Cell-penetrating peptides
have been used to promote uptake of KAT bisubstrate
inhibitors, and similar approaches may facilitate live cell
profiling of KAT activity.23 These improvements will be
important to expand the scope of chemical proteomic analyses
of KAT activity. Regardless, the ability of our current suite of
chemoproteomic probes to highlight an expanded landscape of
catalytic lysine acetylation provides an example of the power of
this approach as currently constituted and sets the stage for the
development of chemoproteomic strategies to identify KAT
inhibitors and the functional characterization of canonical and
nonlinear least-squares regression fit to Y = 100/(1 + 10∧(Log IC50
X)*H), where H = Hill slope (variable). IC50 values represent the
concentration that inhibits 50% of KAT activity. All calculations were
performed using Prism 6 (GraphPad) software.
−
Fluorescent Labeling of KAT Enzymes for SDS-PAGE
Analysis. Purified KAT enzymes (0.5−5 μg) or whole cell proteomes
(20 μg) were incubated with KAT probes 1−3 (1 μM probe for
recombinant labelings; 10 μM probe for proteomic labelings) in PBS
(pH 7.0) for 1 h. Control experiments to correct for nonspecific cross-
linking were treated with 1−3 in the presence of 100 equiv of
competitors 4−6. Following equilibration, samples were photo-cross-
linked on ice for 1 h using a 365 nm UV light in a FB-UVXL-1000 UV
cross-linker. Probe labeling was detected by Cu(I)-catalyzed [3 + 2]
cycloaddition (“click chemistry”). Click reactions were initiated by
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sequential addition of TAMRA-azide 8 (100 μM; 5 mM stock solution
in DMSO, structure given in Supplementary Figure S15), TCEP (1
mM; 100 mM stock in H2O), tris(benzyltriazolylmethyl)amine ligand
(TBTA; 100 μM; 1.7 mM stock in DMSO/tert-butanol 1:4), and
CuSO4 (1 mM; 50 mM stock in H2O). Samples were vortexed and
incubated at room temperature for 1 h. Cycloaddition reactions were
quenched by addition of 5x SDS-loading buffer (strongly reducing)
and subjected to SDS-PAGE (22 μL per well). Excess probe
fluorescence was removed by destaining in a solution of 50%
MeOH/40% H2O/10% AcOH overnight. Gels were then washed
with water and fluorescently visualized using a ImageQuant Las4010
(GE Healthcare) with green LED excitation (λmax 520−550 nm) and a
575DF20 filter. For KAT probes 2 and 3, a characteristic intense low
molecular weight fluorescence signal ∼3 kDa was observed
(corresponding to the fluorescently labeled KAT probe [2, 3]),
indicative of a high-yielding click chemistry reaction.
Cell Culture and Isolation of Whole-Cell Lysates. HeLa S3
cells (ATCC; Manassas VA) were cultured at 37 °C under 5% CO2
atmosphere in a culture medium of DMEM supplemented with 10%
FBS and glutamine. HEK-293 cells were obtained from the NCI
Tumor Cell Repository. For isolation of whole cell proteomes, HeLa
cells were grown to 80−90% confluency, washed 3× with ice-cold
PBS, scraped, and pelleted by centrifugation (1400g × 3 min, 4 °C).
After removal of PBS cell pellets were stored at −76 °C or
immediately processed. Cell pellets were resuspended in 1−2 mL of
ice-cold PBS (10−20 × 106 cells/mL) and lysed by sonication
(QSonica XL2000 100 W sonicator, 3 × 10 s pulse, 50% power, 60 s
between pulses). Lysates were pelleted by centrifugation (14,000g ×
30 min, 4 °C) and quantified on a Qubit 2.0 Fluorometer using a
Qubit Protein Assay Kit. Proteomes were diluted to 2 mg/mL and
stored in 1 mg aliquots at −76 °C until further processing.
Western Blotting. SDS-PAGE gels were transferred to nitro-
cellulose membranes (Novex, Life Technologies) by electroblotting at
30 V for 1 h using a XCell II Blot Module (Novex). Membranes were
blocked using StartingBlock (PBS) Blocking Buffer (Thermo
Scientific) for 20 min and then incubated overnight at 4 °C in a
solution containing the primary antibody of interest (anti-Gcn5
[3305], anti-CBP [3378], Cell Signaling, 1:1000 dilution) in the above
blocking buffer with 0.05% Tween 20. The membranes were next
washed with TBST buffer and incubated with a secondary HRP-
conjugated antibody (anti-rabbit IgG, HRP-linked [7074], Cell
Signaling, 1:1000 dilution) for 1.5 h at room temperature. The
membranes were again washed with TBST, treated with chemilumi-
nescence reagents (Western Blot Detection System, Cell Signaling) for
1 min, and imaged for chemiluminescent signal using an ImageQuant
Las4010 Digitial Imaging System (GE Healthcare).
Enrichment of KAT Enzymes for Proteomic Analysis. Whole
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.
HEK-293 enrichments utilized 0.5 mg of proteome as starting material,
while HeLa enrichments utilized 1 mg of proteome. Control samples
to correct for nonspecific cross-linking were preincubated with each
probe’s cognate competitor (4 or 5; 100 equiv). Following
equilibration, samples were split into 5 × 200 μL aliquots and
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
recombined and subjected to Cu(I)-catalyzed [3 + 2] cycloaddition
with TAMRA biotin-azide 9 (Supplementary Figure S15) as previously
described. Final concentrations for click reactions were as follows:
HeLa proteome (1 mg/mL in PBS), probe 1 (10 μM), TAMRA
biotin-azide (40 μM), TCEP (1 mM), TBTA (100 μM), tert-butanol
(4.8%), and CuSO4 (1 mM). Samples were vortexed and incubated at
room temperature for 1 h. Ice-cold 4:1 MeOH/CHCl3 (2.5 mL) was
then added directly to the reaction mixture and mixed vigorously by
vortexing. The biphasic solution was centrifuged (4000g × 20 min, 4
°C), and protein precipitated at the interface as a solid disk. Liquid
layers were carefully discarded, and the resulting precipitate was
resuspended in ice-cold 1:1 MeOH/CHCl3 (1 mL), sonicated on ice
to resuspend, and repelleted by centrifugation (14,000g × 10 min, 4
°C). This wash step was repeated with ice-cold MeOH (1 mL). The
resulting cell pellet was air-dried to remove excess methanol and
redissolved in 1.2% SDS (1 mL) using iterative cycles of heating (95
°C) and sonication. Redissolved protein was allowed to cool to room
temperature and added to 5 mL of PBS to give a final SDS
concentration of 0.2%. Samples were then treated with 100 μL of
streptavidin-agarose resin (prewashed 3× with 1 mL of PBS) and
rotated for 1 h at room temperature. Streptavidin-agarose bound
samples were then washed sequentially with 0.2% SDS in PBS (3 × 10
mL) and PBS (3 × 10 mL). Samples were then prepared for on-bead
digest by reduction with 10 mM tris(2-carboxyethyl)phosphine
(TCEP) and alkylation with 12 mM iodoacetamide. Samples were
diluted to 2 M urea with 50 mM Tris-Cl pH 8.0 (400 μL total
volume), followed by addition of trypsin and 2 mM CaCl2. Digests
were allowed to proceed overnight at 37 °C. After extraction, tryptic
peptide samples were acidified to a final concentration of 5% formic
acid and frozen at −80 °C for LC−MS/MS analysis.
Liquid Chromatography−Mass Spectrometry and Data
Analysis. Tryptic peptides enriched by probe 1 were loaded onto a
reverse phase capillary column and analyzed by LC separation in
combination with tandem MS. Peptides were eluted using a gradient of
5−42% over 40 min with the flow rate through the column set at 0.20
μL/min. Data was collected in a dual-pressure linear ion trap mass
spectrometer (ThermoFisher LTQ VelosPro) set in a data-dependent
acquisition mode. The 15 most intense molecular ions in the MS scan
were sequentially and dynamically selected for subsequent collision-
induced dissociation (CID) using a normalized collision energy of
35%. Tandem mass spectra were searched against UniProt H. sapiens
protein database (01-13 release) using SEQUEST (ThermoFisher).
Search parameters were fixed as follows: (i) enzyme specificity:
trypsin; (ii) variable modification: methionine oxidation and cysteine
carbamidomethylation; (iii) precursor mass tolerance 1.40 amu; and
(iv) fragment ion mass tolerance 0.5 amu. Only those tryptic
peptides with up to two missed cleavage sites meeting a specific
SEQUEST scoring criteria (Delta Correlation (ΔCn) ≥ 0.08 and
charge state dependent cross correlation (Xcorr) ≥ 1.9 for [M + H]1+,
≥ 2.2 for [M + 2H]2+, and ≥3.1 for [M + 3H]3+) were considered as
legitimate identifications. Spectral count values depicted in Figure 7
represent an average of two biological replicates. Raw spectral counts
for biological duplicates of probe-enriched and control experiments are
provided in Supplementary Table S2.
Phylogenetic Analysis. Amino acid sequences for canonical and
orphan lysine acetyltransferases were obtained from Uniprot.
Accession numbers are provided in Table S3 (Supporting
Information). A pairwise alignment was generated using Clustal
Omega,55 and a phylogenic tree was constructed using the neighbor-
joining method. All phylogenetic trees were displayed in hyperbolic
space using Hypertree, with branches of the tree designated by
different colors and labeled by name where appropriate.56
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic materials and methods, characterization data, and
supplementary figures and schemes. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
jordan.meier@nih.gov
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Dr. Ming Zhou (Laboratory of Proteomics
and Analytical Technology) for LC−MS/MS analyses, Dr.
Hans Luecke (NIDDK) for the pCAF overexpression plasmid,
Dr. Brian Lewis (NCI) for helpful discussions, and Dr. Michael
Giano of the Schneider lab for assistance with peptide synthesis.
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