Bioorthogonal chemical reporters for monitoring protein acetylation

Article abstract of DOI:10.1021/ja908871t

"Chemical equation presented" Protein acetylation is a key post-translational modification that regulates diverse biological activities in eukaryotes. Here we report bioorthogonal chemical reporters that enable direct in-gel fluorescent visualization and

Full text of DOI:10.1021/ja908871t

Published on Web 03/01/2010
Bioorthogonal Chemical Reporters for Monitoring Protein Acetylation
Yu-Ying Yang,† Janice M. Ascano,‡ and Howard C. Hang*,†
Laboratory of Chemical Biology and Microbial Pathogenesis and Laboratory of Biochemistry and Molecular
Biology, The Rockefeller UniVersity, New York, New York 10065
Received October 17, 2009; E-mail: hhang@mail.rockefeller.edu
Protein acetylation is a prevalent post-translational modification
(PTM) that modulates diverse biological activities in eukaryotes1a
as well as in bacterial pathogenesis.1b In particular, reversible
protein acetylation regulated by lysine acetyltransferases (KATs)
and lysine deacetylases (KDACs) plays key roles in controlling
gene expression and is misregulated in many diseases.1a Identifying
the protein substrates of specific KATs is crucial for elucidating
the function(s) of acetylation.2 Protein acetylation is primarily
visualized by employing radiolabeled acetate or acetyl-CoA,3 but
autoradiography exhibits low sensitivity and is hazardous to
implement. Alternatively, azide/alkyne-functionalized chemical
reporters in conjunction with bioorthogonal ligation methods have
Figure 1. (a) Two-step labeling strategy for detecting protein acetylation
in Vitro and in cells. (b) In-gel fluorescent detection of p300-catalyzed
histone H3 acylation. The Coomassie Blue (CB) gel shows the protein
afforded new opportunities for imaging and proteomic analysis of
PTMs.4 Herein, we report alkyne-derivatized chemical reporters
that enable rapid detection and identification of acetylated proteins
loading control.
in Vitro and in mammalian cells via Cu(I)-catalyzed azide-alkyne
cycloaddition (CuAAC)5 (Figure 1a).
p300-catalyzed acylation of histone H3 (Figure 1b). In the absence
We first investigated whether alkynyl-acetyl-CoA could be
utilized by KATs in Vitro. Three alkynyl-acetyl-CoA analogs,
3-butynoyl-CoA (1), 4-pentynoyl-CoA (2), and 5-hexynoyl-CoA
(3), were therefore synthesized and evaluated as acyl-CoA donors
for full-length p300-KAT.6 Mass spectrometry (MS) analysis of
p300-acylation reactions with 1-3 using H3 peptide as the acceptor
substrate revealed that 2 is readily utilized by p300 while 3 is a
less efficient acyl-donor substrate (Figure S1a). Compound 1 did
not function as a p300 substrate. Examining the stability of these
analogs revealed that half of the amount of 1 decomposed within
30 min, while 2 and 3 remained stable for up to 4 h under p300
reaction conditions (Figure S1b). We hypothesize that the relatively
acidic propargylic protons adjacent to the thioester of 1 render it
an unstable acetyl-CoA analog.
To verify that 2 and 3 were transferred onto lysine (Lys) residues,
we performed p300-catalyzed acylation with histone H3 protein
followed by in-gel trypsin digestion for MS/MS analysis (Figure
S1c,d). While alkynyl-acetyl-CoA analogs exhibited low levels of
chemical acylation in the absence of KAT, the addition of
4-pentynoyl and 5-hexanoyl groups from 2 and 3, respectively, onto
Lys residues significantly increased upon addition of p300 (Table
S1). These results suggest that 2 and 3 can be utilized by p300
with similar acceptor specificities in Vitro, albeit with reduced
efficiency for multiple acylation. These data are consistent with
the previous observations of p300 acetyl-donor promiscuity that
enables Lys propionylation and butyrylation of histones in Vitro
and in cells7a,b as well as yeast Gcn5-KAT utilization of chloro-
acetyl-CoA in Vitro.7c
of p300, only minimal fluorescent labeling of histone H3 was
observed. Quantifying the observed fluorescent intensities demon-
strated that p300 acylation with 2 was time- and enzyme-
concentration-dependent (Figure S1e,f). Using this fluorescence
assay and a standard curve for product formation, we estimated
the KM and kcat for 2 as 0.69 ( 0.12 µM and 2.10 ( 0.06 s-1
,
respectively (Figure S2). These values are comparable to those
determined for acetyl-CoA by radioactivity assay.9 These studies
demonstrate that 2 and CuAAC enables rapid fluorescence detection
of KAT activity with picomolar sensitivity within minutes, in
contrast to days or weeks as required for radioactivity.
To investigate whether the alkynyl-acetate analogs can metaboli-
cally label proteins in living cells, the alkynyl-acetate analogs
3-butynoate (4), 4-pentynoate (5), and 5-hexynoate (6) were
prepared as sodium salts (Figure S3) and examined for incorporation
into Jurkat T cells by CuAAC-fluorescent detection (Figure 1a).
The results showed that metabolic labeling with 4-6 was both dose-
and time-dependent and was optimal at 2.5-10 mM acetate
analogue for 6-8 h (Figure S4). The selective labeling of core
histones and histone H3 derived from metabolically labeled Jurkat
T cells demonstrate that 4-6 can be installed onto known Lys-
acetylated proteins (Figure 2a,b). Fluorescent profiling of total cell
lysates revealed many proteins labeled by 4-6 (Figure 2c). The
majority of the selectively labeled proteins were insensitive to
cycloheximide treatment and largely distinct from those targeted
by longer-chain alkynyl fatty acid analogs8 (Figure S5a,b). Co-
incubation with a KDAC inhibitor, suberoylanilide hydroxamic acid
(SAHA),10a slightly decreased the addition of 4-6 onto proteins
(Figure S5c), suggesting that blocking the removal of protein
acetylation prevents metabolic incorporation of these acetate
analogs. Cells treated with curcumin,10b a p300 inhibitor, reduced
the acetate-analog labeling of known p300 substrates such as core
histones (Figure S5d). Further analysis of the total cell lysates
showed that not all of the labeled protein signals were reduced by
We then evaluated fluorescent detection of KAT activity by
CuAAC of the in Vitro acylation reactions with azido-rhodamine8
(az-Rho). Both 2 and 3 served as sensitive reagents for visualizing
† Laboratory of Chemical Biology and Microbial Pathogenesis.
‡ Laboratory of Biochemistry and Molecular Biology.
9
3640 J. AM. CHEM. SOC. 2010, 132, 3640–3641
10.1021/ja908871t  2010 American Chemical Society

C O M M U N I C A T I O N S
observed in all of the MS/MS spectra. These experiments col-
lectively demonstrate that alkynyl-acetate analogs 2 and 5 function
as efficient chemical reporters for protein acetylation in Vitro and
in cells, respectively. Notably, fluorescent profiling of various
mammalian cell lines with 4, 5, and 6 revealed distinct and analog-
specific patterns of acetylomes in diverse cell types, which
highlights the generality and utility of these bioorthogonal chemical
reporters for protein acetylation detection (Figure S6).
Unraveling the functions of protein acetylation remains a
challenging task. The bioorthogonal chemical reporters presented
here provide readily accessible non-radioactive reagents for fluo-
rescence profiling and large-scale analysis of protein acetylomes.
Moreover, alkynyl-acetyl-CoA analogs enable rapid and sensitive
detection of KAT activities that should be useful for assigning
protein substrates in complex mixtures. This chemical approach
provides experimental tools complementary to anti-acetyl-Lys
antibodies,12a,b MS/MS,12a,b bioinformatic methods,12c and affinity-
based CoA probes.13 The functional analysis of KAT-regulated
acetylation will be essential going forward. The incorporation of
quantitative proteomic methods12b and bump-hole strategies14 in
the future should expand the utility of these chemical tools and
facilitate the functional analysis of protein acetylation in physiology
and disease.
Figure 2. Fluorescent detection of acetate reporter (4, 5, or 6)-labeled (a)
core histones, (b) purified histone H3, and (c) total cell lysates from Jurkat
T cells.
curcumin (Figure S5d), which suggests these acetate analogs may
also be utilized by other KATs or acyltransferases or incorporated
by chemical acylation. Nonetheless, these cellular experiments
suggest that 4, 5, and 6 can be converted into active alkynyl-acetyl-
CoA analogs in cells by promiscuous acetyl-CoA11 or acyl-CoA
synthetases and post-translationally installed onto proteins in living
cells.
To identify proteins metabolically labeled by 4, 5 and 6, Jurkat
T cell lysates were subjected to CuAAC with the cleavable azido-
diazo-biotin tag (Scheme S4) followed by affinity purification on
streptavidin beads (Figure S7). Subsequent treatment of the
streptavidin beads with sodium dithionite enabled efficient elution
of the captured proteins for gel-based proteomics using an LTQ-
Orbitrap mass spectrometer. A survey of the protein hits from the
cell lysates metabolically labeled with 4, 5, and 6 revealed many
reported (86%) as well as new candidate (14%) Lys-acetylated
proteins (Table S2). Our p300-acylation studies and cellular labeling
and proteomics data suggest that both 4-pentynoate derivatives (2
and 5) are the optimal chemical reporters for detecting protein
acetylation in Vitro and in cells. Though 4 and 6 can also
metabolically label Lys-acetylated proteins in cells, the CoA
derivative of 4, 3-butynoyl-CoA (2) is an unstable substrate for in
Vitro reactions, and 6 may also target long-chain fatty-acylated
proteins in cells (i.e., transferrin receptor, SNAP-23) as indicated
in the MS/MS-identified proteins lists (Table S2). We therefore
focused on 5 for additional proteomic studies. From three inde-
pendent proteomic experiments (Figures S7 and S8), we identified
approximately 194 4-pentynoate-labeled proteins from Jurkat T cells
(Tables S2 and S3), 86% of which were also identified by anti-
acetyl-Lys proteomic studies.12a,b We confirmed the enrichment of
several MS/MS-identified acetylated proteins, including Ku70,
moesin, cofilin, coronin-1A, Hsp90, HMG-1, and adenosine deami-
nase by Western blotting analysis of the affinity-enriched proteins
(Figure S8b). Bioinformatic analysis of our data set suggests that
the majority of acetylated proteins reside in the nucleus and
cytoplasm and are associated with diverse cellular functions ranging
from metabolism and signal transduction to gene expression (Figure
S8c).
Acknowledgment. We thank the members of the Hang labora-
tory for reagents and advice, Prof. Robert G. Roeder and Dr. Sohail
Malik for support, and the Rockefeller University Proteomics
Resource Center for MS analysis. This work was supported by the
Anderson Cancer Center Postdoctoral Fellowship at Rockefeller
University (Y.-Y.Y.) and NIH/NRSA (1F32DK082140-01A1 to
J.M.A.). H.C.H. acknowledges support from Rockefeller University
and NIH/NIDA (1R21DA025751-01).
Supporting Information Available: Experimental procedures and
supporting figures. This material is available free of charge via the
Internet at http://pubs.acs.org.
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