Mechanism-based affinity capture of sirtuins

Article abstract of DOI:10.1039/c0ob00774a

The ability to probe for catalytic activities of enzymes and to detect their abundance in complex biochemical contexts has traditionally relied on a combination of kinetic assays and techniques such as western blots that use expensive reagents such as antibodies. The ability to simultaneously detect activity and isolate a protein catalyst from a mixture is even more difficult and currently impossible in most cases. In this manuscript we describe a chemical approach that achieves this goal for a unique family of enzymes called sirtuins using novel chemical tools, enabling rapid detection of activity and isolation of these protein catalysts. Sirtuin deacetylases are implicated in the regulation of many physiological functions including energy metabolism, DNA-damage response, and cellular stress resistance. We synthesized an aminooxy-derivatized NAD⁺ and a pan-sirtuin inhibitor that reacts on sirtuin active sites to form a chemically stable complex that can subsequently be crosslinked to an aldehyde-substituted biotin. Subsequent retrieval of the biotinylated sirtuin complexes on streptavidin beads followed by gel electrophoresis enabled simultaneous detection of active sirtuins, isolation and molecular weight determination. We show that these tools are cross reactive against a variety of human sirtuin isoforms including SIRT1, SIRT2, SIRT3, SIRT5, SIRT6 and can react with microbial derived sirtuins as well. Finally, we demonstrate the ability to simultaneously detect multiple sirtuin isoforms in reaction mixtures with this methodology, establishing proof of concept tools for chemical studies of sirtuins in complex biological samples.

Full text of DOI:10.1039/c0ob00774a

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Mechanism-based affinity capture of sirtuins†
Yana Cen,‡ Jessica N. Falco,‡ Ping Xu, Dou Yeon Youn and Anthony A. Sauve*
Received 24th September 2010, Accepted 3rd December 2010
DOI: 10.1039/c0ob00774a
The ability to probe for catalytic activities of enzymes and
to detect their abundance in complex biochemical contexts
has traditionally relied on a combination of kinetic assays
and techniques such as western blots that use expensive
reagents such as antibodies. The ability to simultaneously
detect activity and isolate a protein catalyst from a mixture is
even more difficult and currently impossible in most cases. In
this manuscript we describe a chemical approach that achieves
this goal for a unique family of enzymes called sirtuins using
novel chemical tools, enabling rapid detection of activity
and isolation of these protein catalysts. Sirtuin deacetylases
are implicated in the regulation of many physiological func-
tions including energy metabolism, DNA-damage response,
and cellular stress resistance. We synthesized an aminooxy-
derivatized NAD+ and a pan-sirtuin inhibitor that reacts on
sirtuin active sites to form a chemically stable complex that
can subsequently be crosslinked to an aldehyde-substituted
biotin. Subsequent retrieval of the biotinylated sirtuin com-
plexes on streptavidin beads followed by gel electrophoresis
enabled simultaneous detection of active sirtuins, isolation
and molecular weight determination. We show that these
tools are cross reactive against a variety of human sirtuin
isoforms including SIRT1, SIRT2, SIRT3, SIRT5, SIRT6 and
can react with microbial derived sirtuins as well. Finally,
we demonstrate the ability to simultaneously detect multiple
sirtuin isoforms in reaction mixtures with this methodology,
establishing proof of concept tools for chemical studies of
sirtuins in complex biological samples.
nuclear,8 whereas SIRT3, SIRT4 and SIRT5 are mitochondrial9
and SIRT2 is predominantly cytosolic although it can be nuclear
as well.10 Sirtuins are expressed at different levels in distinct
tissues, and stresses such as calorie restriction or fasting can affect
their abundance.11 Current methods to detect sirtuin biochemical
activity in complex mixtures such as lysates are cumbersome,
and detection and quantification of individual sirtuins in lysed
cells requires western blots. Problematically, current biochemical
assays do not distinguish between the activities of sirtuin isoforms.
Moreover, to detect each individual sirtuin, a distinct and relatively
expensive antibody is required. Importantly, the ability to detect
activity and determine identities of sirtuins in cells and tissues in
a manner that is straightforward, simultaneous and rapid could
provide accelerated investigation into sirtuin functions in a variety
of disease states and physiological conditions.
We sought to develop a technology that could simultaneously
detect sirtuin activity in complex mixtures and to isolate the
sirtuin thus detected. We required chemicals that could react
at a sirtuin active site using the enzyme mechanism (detection
of activity) to form a stable complex which could then be
harvested (isolation of sirtuin). In principle, such a technology
has the means to assess for isoform activity and could be
used for quantitation and identification of the isolated sirtuins,
by standard SDS-PAGE techniques. Interestingly, thioacetylated
peptides react with NAD+ on sirtuin active sites to form kinetically
stable thioimidate complexes,12 which have been characterized by
X-ray crystallography13,14 (Scheme 1). The reaction that forms
the thioimidate is analogous to the reactivity proposed for the
reactions of NAD+ with acetylated substrates13,15 which react on
sirtuin active sites to form reactive O-imidate complexes16,17 that
are intermediates in deacetylation chemistry (Scheme 2).16 In con-
sidering desired properties of the thioacetyl-peptide, we realized
that cross-reactivity against multiple sirtuin isoforms would be
beneficial, so that one reagent could work for multiple sirtuins.
The general reagent approach has been the basis of ‘activity-
based profiling’ as pioneered by Cravatt and co-workers.18–20
Interestingly, N-thioacetyl-lysine peptide analogues of sirtuin
substrates have already been reported to inhibit SIRT1, SIRT2 and
SIRT3 catalyzed deacetylations with affinities in the nanomolar to
low micromolar range.12,15,21 Thus, we were encouraged to identify
a simple peptide sequence that could achieve broad cross-reactivity
with human sirtuin isoforms.
The sirtuin enzymes are NAD+ dependent protein deacetylases
broadly distributed in biology and implicated in the regulation
of diverse processes within cells.1 Mammalian sirtuins SIRT1-
7 regulate apoptosis,2 proliferation,3 stress resistance,4,5 and
metabolism.6,7 Sirtuin localization in cells is compartmentalized.
For example SIRT1, SIRT6 and SIRT7 are thought to be
Department of Pharmacology, Weill Medical College of Cornell University,
1300 York Avenue, New York, NY 10065, USA. E-mail: aas2004@
med.cornell.edu; Tel: +1 212-746-6224
† Electronic supplementary information (ESI) available: Detailed ex-
perimentals of enzyme production and kinetic assays, sirtuin capture
assays, synthetic procedures and 1H and 13C NMR data. See DOI:
10.1039/c0ob00774a
The simple benzoylated tripeptide: Na-4-nitrobenzoyl-Ne-
thioacetyl-lysinyl-alaninyl-alanine methyl ester, 1, (Fig. 1, See
‡ These authors contributed equally to this work.
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Scheme 1 Mechanism for sirtuin inhibition by thioacetylpeptides and 1.
Scheme 2 Mechanism of sirtuin catalyzed deacetylation.
Table 1 Inhibition of sirtuin-catalyzed deacetylation by 1
synthesis in Supplementary Information) was found to have
suitable properties as a minimal structure that inhibits sirtuin
enzymes. In Table 1, we report data that shows it inhibits SIRT1,
SIRT2, SIRT3 with IC50 values in the low mM range although
the IC50 for SIRT3 is above 100 mM. The compound also inhibits
the Archaeoglobus fulgidus Af2Sir216 and Saccharomyces cerevisiae
Sir2 with IC50 values in the same range, indicating we have
identified a general sirtuin inhibitor (Table 1). These data provide
evidence that this simple structure can act as a general reagent
capable of reacting on sirtuin active sites, and inhibiting enzymatic
activity presumably via thioimidate formation (Scheme 1). Consis-
tent with this notion, inhibition by 1 was shown to be competitive
with an acetylated peptide substrate on SIRT1 with Ki = 611 nM
248 nM (Fig. S2).
The ability of 1 to react at the active site to form a stable
thioimidate complex on a variety of sirtuins accomplished a
key criterion for our technology, with the next requirement that
the thioimidate complexes could be cross-linked via peptide or
by another feature of the thioimidate complex to a pulldown
reagent, such as biotin which can be harvested by binding to
avidin beads. To achieve crosslinkable thioimidate complexes, a
chemically modified NAD+ was designed that could react to
Sirtuin
Acetylated substrate
IC50 (mM)
Af2Sir2
Yeast Sir2
SirT1
SirT2
SirT3
NBK(Ac)AAOMe
H3mer
p53mer
p53mer
H3mer
131 26.0
19.5 5.80
6.5 1.00
13.1 0.54
272 80.3
Assays were carried out at 37 ◦C for 30 to 60 min using purified sirtuin
enzyme, NAD+, inhibitor, and substrates as specified in the Supplementary
Information. Acetylated peptides are fully defined in the Supplementary
Information and NBK(Ac)AAOMe is the acetylated peptide isostructural
to 1. Data was plotted as percent enzyme activity remaining as a function
of log[1] in nanomolar. Curve fits were generated using the following
equation: v(%) = v(%)I - [v(%)I(10x)/(10x + IC50)]. v(%) represents percent
enzyme activity remaining and v(%)I represents the initial enzyme activity
of 100%. The variable x represents log[1] in nanomolar; where 10x is the
concentration of 1 in nanomolar. IC50 values were calculated from this
equation with relevant curves and data found in Fig. S1.
form the desired thioimidate, but could be chemically reacted
to biotin after thioimidate formation. A modified NAD+ with
a linker appended to the adenine ring at the 6 position was
designed which is abbreviated 6-AMX-NAD+ (2, Fig. 1). Synthesis
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Fig. 1 Structures of (1), 6-AMX-NAD+ (2) and N-biotinyl-6-aminohexanal (3).
Fig. 2 HPLC chromatograms showing 6-AMX-NAD+, 2, is a substrate for SirT1: (A) reaction containing 500 mM NAD+, 500 mM of p53mer in 100 mM
phosphate buffer pH 7.5 was initiated by addition of SirT1 to a final concentration of 10.7 mM. After incubation at 37 ◦C for 20 min, AADPR and NAM
were formed; (B) reaction performed as in (A), with addition of 250 mM of 1; (C) reaction containing 500 mM 2, 500 mM of p53mer in 100 mM phosphate
buffer pH 7.5 was initiated by addition of SirT1 to a final concentration of 10.7 mM. After incubation at 37 ◦C for 20 min, 6-AMX-AADPR and NAM
were formed; (D) reaction performed as in (C), with addition of 250 mM of 1.
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Scheme 3 Synthesis of 6-AMX-NAD+.
of this derivative is shown in Scheme 3. The presence of a
terminal aminooxy functionality on the linker provides a reactive
group that enables 6-AMX-NAD+ or thioimidate complexes
derived from it to be crosslinked to a biotin modified with a
reactive carbonyl. A condition for the linker is that it should not
interfere with NAD+ function as a substrate, and inspection of
several crystallographically determined sirtuins with NAD+22 or
thioimidate bound13 convinced us that the 6-exocyclic-N position
of the adenine ring of substrate is solvent accessible, and would
readily accommodate the linker.
In the imagined use of 6-AMX-NAD+, it would readily form
the thioimidate 1 to form a kinetically stable complex (Scheme 4)
that could be crosslinked to an aldehyde modified biotin (3,
Fig. 1).23 Conjugate formation involving aminooxy or hydrazino
groups24–26 has previously been used to interrogate functionalized
sugars bearing reactive carbonyls.24–26 Based on precedent, in situ
conjugate formation with an aminooxy group and an aldehyde
was concluded to enable a proof of concept test of the technology.
A specific advantage of aminooxy groups is their low predicted
pKa, 6–7, allowing us to cross link NAD+ to biotin at neutral to
slightly acidic pH, where chemistry on sirtuins occurs and where
the thioimidate complexes are predicted to be stable. Subsequent
capture of sirtuin-thioimidate-biotin conjugates could be obtained
by avidin beads (Scheme 4). Subsequent analysis on gels provides
molecular weights and abundance of captured sirtuins.
together. For example, evaluation of 6-AMX-NAD+ as a substrate
proved its ability to support sirtuin catalysis. 6-AMX-NAD+
was able to support SIRT1 catalyzed deacetylation as evident
from formation of 6-AMX-2¢-and 3¢-O-acetyl-ADPR (Fig. 2C),
which was further verified by isolation and MALDI-MS (Fig.
S3). Evaluation of steady state parameters for 6-AMX-NAD+ on
SIRT1 provided a Km value of 231 mM (Fig. S4), which compares
favorably with the Km value of NAD+, of 165 mM.27 Comparison of
reaction under identical concentrations, substrates and conditions
(Fig. 2A and 2C) established that 6-AMX-NAD+ reacts at a
rate of 0.91 relative to the rate of NAD+ for SIRT1 catalysed
deacetylation. Collectively, these data indicate that 6-AMX-NAD+
can act as a substrate in a manner that is highly similar to NAD+.
In addition, to acting as a substrate, we demonstrate that 6-AMX-
NAD+ supports full inhibition of SIRT1 by 1 (Fig. 2), consistent
with the effective ability of 6-AMX-NAD+ to form a stabilized
thioimidate complex. We further showed that 6-AMX-NAD+
and the thioacetyl-lysine inhibitor 1 form a thioimidate complex
on SIRT1, as shown by detection of the thioimidate species by
MALDI-TOF MS (Supplementary Information Fig. S6†).
‘Sirtuin capture’ using the described chemical tools was
demonstrated according to the sequence described in Scheme 4.
6-AMX-NAD+ was combined with 1 in a variety of reaction
mixtures containing recombinantly expressed sirtuins SIRT1,
SIRT3 and SIRT6 as shown in Fig. 3. Reaction conditions and
procedures are provided in the legend. Recovered beads were
loaded directly onto SDS-PAGE gels and proteins eluted from
To validate our tools, several control experiments were per-
formed to provide evidence that the constructs could function
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Scheme 4 Model of avidin-biotin affinity capture of sirtuin enzymes using compounds 1–3.
beads were visualized by silver staining. As evident from gel
images, only reaction mixtures containing 1 and 6-AMX-NAD+
yielded beads containing bound sirtuins, likely because of the
formation of stable thioimidates on the bound sirtuins. Acetylated
peptide substrates could not function as effective reagents,
presumably because O-imidate complexes are less kinetically
stable (Fig. 3). Similar results were obtained with SIRT2 and
SIRT5 (See Fig. S5). The same procedures were also able to capture
recombinant S. cerevisiae Sir2, and the A. fulgidus sirtuin, Af2Sir2
(See Fig. S5). These data provide the first demonstration, to our
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knowledge, of general reagents that can be used to detect
sirtuin biochemical reactivity and that can also capture the
reactive sirtuins via a biotin conjugation strategy.
A further demonstration of the versatility of the sirtuin capture
methodology is shown in the bottom gel of Fig. 3. In this instance,
multiple partially purified recombinant sirtuins SIRT1, SIRT2,
SIRT3, SIRT5 and SIRT6 were combined together and subjected
as a mixture to the thioimidate sirtuin capture procedure. As can
be seen, all relevant forms of sirtuins were recovered, including
what appear to be proteolysis-cleaved forms of sirtuins.
This paper describes the first chemical method to isolate
sirtuins intact from complex protein mixtures based on their
reactivity to a common sirtuin inhibitor, wherein the mechanism of
inhibition utilizes the catalytic mechanism to achieve stable com-
plex formation. This methodology provides important proof of
concept demonstration that it is possible to isolate multiple intact
sirtuins from impure protein mixtures. In theory this methodology
permits simultaneous assessment of reactivity, abundance, and
determination of molecular weight of multiple sirtuin isoforms
in a single assay. Conceivably the method could also be utilized
to re-interogate isolated sirtuin biochemical properties since the
sirtuin thioimidate complexes are thought to be only temporally
stable, and are likely to release free enzyme after extended
incubation in solution.15 As a demonstration of this possibility,
the capture and subsequent rerelease of Af2Sir2 from beads was
examined. Rereleased enzyme is active and recovery occurs in a
time-dependent manner (Supplementary Information, Fig. S7†)
consistent with slow reaction of the thioimidate to release enzyme.
Some improvements are still required, such as being able to
show that the method is quantitative in capturing distinct sirtuins
available in a reaction mixture, which would allow for isoform
quantitation. Improvements could include use of a more efficient
conjugation strategy, such as a clickable alkyne/azide pairing
which would form a stable bond in the conjugate, thus improving
quantitative recovery. The peptide could also be made to be
crosslinkable which could make the technology cell permanent
and more sirtuin specific. We anticipate that this technology could
allow increased understanding of sirtuin activities in cells and
could provide novel insights into enzymatic activity, regulation
and how sirtuin levels and activities differ in tissues, in various
physiological states and in pathologies. The isolation procedure
can also be potentially helpful to identify unique splice variants
or novelties such as post-translational modifications.
Fig. 3 Representative SDS-PAGE results for sirtuin capture by 1 and
2. (A) SirT1; (B) SirT3; (C) SirT6; (D) mixed sirtuins. Reactions were
typically performed in 300 mM phosphate buffer pH 7.5 in a total volume
of 50 mL. A typical reaction contained 800 mM of 2 and 400 mM of 1, three
control experiments were run at the same time: the first one contained
800 mM of 2 and 500 mM of an acetylated peptide substrate, p53mer
for SirT1, H3mer for SirT3, p53mer for SirT6 and p53mer for sirtuin
mixture (Sequences are found in Supplementary Information); the second
control sample contained 800 mM of 2 only; and the third control had
400 mM of 1 only. Reactions were initiated by addition of enzyme, final
concentrations 16.5 mM for SirT1, 11.5 mM for SirT3, 17.1 mM for SirT6.
For the sirtuin mixture, final enzyme concentrations were 16.5 mM for
SirT1, 12.5 mM for SirT2, 11.5 mM for SirT3, 13.6 mM for SirT5 and
17.1 mM for SirT6. Reactions were incubated at 37 ◦C for 30 min and then
adjusted to pH 6 by addition of 25 mL of 300 mM phosphate buffer pH 4.25.
Compound 3 was added to each sample to a final concentration of 1 mM,
incubations were continued at room temperature for one hour. Then 50 mL
of avidin agarose beads were added to each sample, and the reactions were
allowed to incubate at room temperature for another hour. After removal
of the supernatant, the beads were washed with 1 mM NAD+ in 300 mM
phosphate buffer (3 ¥ 100 mL), the washes were removed and saved for gel
analysis. The beads were boiled in SDS-PAGE sample buffer for 10 min
and subject to gel electrophoresis for analysis. s = supernatant; w3 = wash
3; b = beads; m = molecular weight marker, p = putative proteolyzed sirtuin
fragments.
Acknowledgements
This work was supported by R01 DK 73466 and Anthony A. Sauve
is an Ellison Medical Foundation New Scholar (2007).
References
1 M. C. Haigis and L. P. Guarente, Genes Dev., 2006, 20, 2913–2921.
2 H. Vaziri, S. K. Dessain, E. Ng Eaton, S. I. Imai, R. A. Frye, T. K.
Pandita, L. Guarente and R. A. Weinberg, Cell, 2001, 107, 149–159.
3 K. F. Chua, R. Mostoslavsky, D. B. Lombard, W. W. Pang, S. Saito,
S. Franco, D. Kaushal, H. L. Cheng, M. R. Fischer, N. Stokes, M. M.
Murphy, E. Appella and F. W. Alt, Cell Metab., 2005, 2, 67–76.
4 A. Brunet, L. B. Sweeney, J. F. Sturgill, K. F. Chua, P. L. Greer, Y. Lin,
H. Tran, S. E. Ross, R. Mostoslavsky, H. Y. Cohen, L. S. Hu, H. L.
Cheng, M. P. Jedrychowski, S. P. Gygi, D. A. Sinclair, F. W. Alt and
M. E. Greenberg, Science, 2004, 303, 2011–2015.
992 | Org. Biomol. Chem., 2011, 9, 987–993
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
5 D. B. Lombard, K. F. Chua, R. Mostoslavsky, S. Franco, M. Gostissa
and F. W. Alt, Cell, 2005, 120, 497–512.
6 B. Schwer, J. Bunkenborg, R. O. Verdin, J. S. Andersen and E. Verdin,
Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 10224–10229.
7 T. Nakagawa, D. J. Lomb, M. C. Haigis and L. Guarente, Cell, 2009,
137, 560–570.
8 E. Michishita, J. Y. Park, J. M. Burneskis, J. C. Barrett and I. Horikawa,
Mol. Biol. Cell, 2005, 16, 4623–4635.
9 J. Y. Huang, M. D. Hirschey, T. Shimazu, L. Ho and E. Verdin, Biochim.
Biophys. Acta, 1804, 1645–1651.
10 A. Vaquero, M. B. Scher, D. H. Lee, A. Sutton, H. L. Cheng, F. W.
Alt, L. Serrano, R. Sternglanz and D. Reinberg, Genes Dev., 2006, 20,
1256–1261.
11 D. Chen, J. Bruno, E. Easlon, S. J. Lin, H. L. Cheng, F. W. Alt and L.
Guarente, Genes Dev., 2008, 22, 1753–1757.
12 D. G. Fatkins, A. D. Monnot and W. Zheng, Bioorg. Med. Chem. Lett.,
2006, 16, 3651–3656.
13 W. F. Hawse, K. G. Hoff, D. G. Fatkins, A. Daines, O. V. Zubkova, V. L.
Schramm, W. Zheng and C. Wolberger, Structure, 2008, 16, 1368–1377.
14 L. Jin, W. Wei, Y. Jiang, H. Peng, J. Cai, C. Mao, H. Dai, W. Choy, J. E.
Bemis, M. R. Jirousek, J. C. Milne, C. H. Westphal and R. B. Perni,
J. Biol. Chem., 2009, 284, 24394–24405.
15 B. C. Smith and J. M. Denu, Biochemistry, 2007, 46, 14478–14486.
16 A. A. Sauve, I. Celic, J. Avalos, H. Deng, J. D. Boeke and V. L. Schramm,
Biochemistry, 2001, 40, 15456–15463.
17 A. A. Sauve, C. Wolberger, V. L. Schramm and J. D. Boeke, Annu. Rev.
Biochem., 2006, 75, 435–465.
18 G. C. Adam, B. F. Cravatt and E. J. Sorensen, Chem. Biol., 2001, 8,
81–95.
19 D. Kidd, Y. Liu and B. F. Cravatt, Biochemistry, 2001, 40, 4005–
4015.
20 Y. Liu, M. P. Patricelli and B. F. Cravatt, Proc. Natl. Acad. Sci. U. S. A.,
1999, 96, 14694–14699.
21 D. G. Fatkins and W. Zheng, Int. J. Mol. Sci., 2008, 9, 1–11.
22 K. G. Hoff, J. L. Avalos, K. Sens and C. Wolberger, Structure, 2006,
14, 1231–1240.
23 B. Allart, P. Lehtolainen, S. Yla-Herttuala, J. F. Martin and D. L.
Selwood, Bioconjugate Chem., 2003, 14, 187–194.
24 D. A. Nauman and C. R. Bertozzi, Biochim. Biophys. Acta, 2001, 1568,
147–154.
25 M. Howarth and A. Y. Ting, Nat. Protoc., 2008, 3, 534–545.
26 C. L. Jacobs, K. J. Yarema, L. K. Mahal, D. A. Nauman, N. W. Charters
and C. R. Bertozzi, Methods Enzymol., 2000, 327, 260–275.
27 A. A. Sauve, Biochim. Biophys. Acta, 1804, 1591–1603.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 987–993 | 993
©