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DOI: 10.1002/cbic.201200727
Dissecting the Roles of the N- and C-Flanking Residues of
Acetyllysine Substrates for SIRT1 Activity
Roman Meledin,[a] Ashraf Brik,*[b] and Amir Aharoni*[a]
The sirtuin enzyme family catalyze the NAD + -dependent deacetylation of acetyllysine residues within protein targets.[1]
The products of this enzymatic reaction include the deacetylated protein, nicotinamide and 2-O-acetyl-ADP-ribose.[2] The sirtuin protein family is conserved from bacteria to humans, with
overexpression of sirtuins having been shown to be associated
with increased life span in yeast[3] and other higher eukaryotes,
including worms,[4] flies,[5] and most recently, in mice.[6] The
human sirtuin family is composed of seven members (SIRT1–7)
exhibiting diverse cellular localization, catalytic activity and
substrate specificity.[7] The SIRT1 protein is the most studied of
the human sirtuins and was shown to be involved in numerous
biological processes and in a variety of diseases, including diabetes, cancer and inflammation.[8] In these instances, SIRT1 deacetylates several key protein substrates, leading to the modulation of their biological activities.[9] Despite extensive research
on SIRT1, the molecular basis for its substrate recognition and
how it discriminates between its many substrates remains unknown.
In the past decade, crystal structures of several different sirtuins in the ligated and unligated forms have been solved, providing new insight into the peptide- and NAD + - binding activities of these enzymes, as well as elucidating the catalytic
mechanism of sirtuin-mediated deacetylation.[10] In these structures, the target acetyllysine is bound at a highly conserved
hydrophobic tunnel in the enzyme, while the rest of the peptide assumes a b-sheet-like structure that is stabilized through
a main chain hydrogen-bond network. This lack of side-chainspecific sirtuin–peptide interactions led to the suggestion that
sirtuins possess very little substrate specificity.[1, 11] However,
structural examination of Thermatoga maritima Sir2 (Sir2Tm)
bound to several peptides that vary substantially in their sequence showed that residues N- or C-flanking the acetyllysine
target residue can confer sequence-specific interactions.[12]
Moreover, kinetic analysis of sirtuin activity has demonstrated
large differences in the catalytic efficiencies of the deacetylation reaction toward different substrates.[13] In addition, screening of a large peptide library for human SIRT1 substrates indi-
cated that SIRT1 can discriminate between peptide substrates
resulting in up to 20-fold differences in activity.[14] These experiments point to the ability of sirtuins to discriminate between
thousands of acetylated lysines in the human proteome, enabling specific deacetylation of physiologically important protein substrates. Despite such knowledge, it is still not clear
how SIRT1 realizes such specificity at the molecular level.
In this work, we analyzed the contribution of different residues in the SIRT1 peptide substrates to SIRT1 catalytic activity.
We focused on two central SIRT1 substrates, p53 (K382) and
H4 (K16), proteins that significantly differ in their sequence and
chemical properties. To assess the contributions of residues in
the regions N- and C-flanking the target acetyllysine to SIRT1
catalytic activity, we synthesized an array of 19 different p53
and H4 peptide variants (Figure 1). We synthesized peptide
[a] R. Meledin, Prof. A. Aharoni
Department of Life Sciences and
the National Institute for Biotechnology in the Negev (NIBN)
Ben-Gurion University of the Negev
Be’er Sheva 84105 (Israel)
E-mail: [email protected]
Figure 1. The sequences of p53 and H4 peptides examined in this study. The
acetylated lysines are labeled red, while mutated amino acids are bold.
[b] Prof. A. Brik
Department of Chemistry3, Ben-Gurion University of the Negev
Be’er Sheva 84105 (Israel)
E-mail: [email protected]
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201200727.
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
variants that are truncated either before or after the acetyllysine as well as peptides containing substitutions at residues
flanking either side of the target acetyllysine residue (Figure 1).
To determine the catalytic activity of SIRT1 toward these peptides, we used a continuous spectroscopic assay for measuring
sirtuin activity based on a coupled assay monitoring the
release of nicotinamide during the deacetylation reaction (FigChemBioChem 0000, 00, 1 – 5
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Figure 2. Michaelis–Menten plots describing the deacetylation of p53 (A, B) and H4 (C, D) peptide variants by SIRT1. Additional Michaelis–Menten plots are
shown in Figures S2–5. The kinetic parameters derived from the fits are presented in Table 1.
ure S1 in the Supporting Information).[15] This assay allowed us
to perform steady-state kinetic analysis of SIRT1 activity at different peptide concentrations and derive the Michaelis–
Menten parameters for each peptide (Figures 2, 3, S2–S5 and
Table 1).
Analysis of SIRT1 activity with p53 and H4 peptides lacking
the N-flanking region (p53-N-D and H4-N-D) showed a dramatic
decrease of ~ 30- and ~ 14-fold in catalytic efficiency (kcat/KM),
relative to the native peptides, respectively (Figures 2 and 3
and Table 1). We found that substitution of the three residues
N-flanking the acetyllysine to alanine in p53 and substitutions
of the two residues in H4 (p53-N-3M and H4-N-2M, Figure 1)
led to a decrease of up to approximately fourfold in catalytic
efficiency (Figures 2 and 3, Table 1). These results highlight the
importance of the N-flanking region of p53 and H4 for SIRT1
activity. To analyze the level of specificity of SIRT1 toward the
p53 and H4 peptides, we synthesized hybrid p53-H4-H or H4p53-H peptides in which p53 N-flanking residues (RHK) are replaced with the equivalent residues of the H4 peptide (GGA)
and vice versa (Figure 1). Kinetic analysis of SIRT1 activity
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
toward these hybrid peptides showed a 30–40 % decrease in
catalytic efficiency, relative to the native peptides (Figures 2
and 3 and Table 1). To further identify specific N-flanking residues that may affect SIRT1 activity, we synthesized p53 and H4
mutant peptides containing a single alanine substitution at position 1, 2 or 3 in p53 (p53-N-1A, p53-N-2A and p53-N-3A,
Figure 1) and substitution at positions 2 or 3 in H4 (H4-N2A and H4-N-3A, Figure 1), with respect to the target acetyllysine residue. Position 1 with respect to the acetyllysine residue in p53 was previously shown to play an important role in
the Sirt2Tm–peptide interaction.[12] Our kinetic analysis of SIRT1
acting on the p53-N-1A peptide revealed a relatively modest
decrease of ~ 30 % in catalytic efficiency, a value that is significantly lower than that for the triple mutant (p53-N-3M, Figures 2 and 3, Table 1). In contrast, we found that substitution
at positions 2 or 3 in p53 and position 3 in H4 led to
a significant increase in SIRT1 catalytic efficiency of up to 1.9fold, relative to the native peptides (Figure S4, Table 1). These
results highlight the sub-optimality of these positions to SIRT1
activity and show the synergism between the three N-flanking
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Figure 3. The catalytic efficiency of SIRT1 deacetylation (kcat/KM) toward
A) p53 and B) H4 peptide variants. Peptide sequences are shown in Figure 1,
catalytic parameters are presented in Table 1.
Table 1. Kinetic parameters of SIRT1 activity against p53 and H4 peptide
variants.
Peptide
kcat
[min 1]
KM
[mm]
p53-WT
P53-N-D
p53-N-3M
p53-N-1A
p53-N-2A
p53-N-3A
p53-H4-H
p53-C-D
p53-C-3M
p53-C-2A
H4-WT
H4-N-D
H4-N-2M
H4-N-2A
H4-N-3A
H4-p53-H
H4-C-D
H4-C-3M
H4-C-2A
9.8(0.8)
1.7(0.1)
4.3(0.4)
7.6(0.4)
6.7(0.1)
3.5(0.1)
4.4(0.2)
4.2(0.1)
5.3(0.3)
8.2(0.6)
6.9(0.6)
1.2(0.1)
6.9(0.6)
3.6(0.9)
7.1(0.4)
4.3(0.5)
4.6(0.2)
5.3(0.2)
7.4(0.7)
167(31)
879(148)
249(43)
191(32)
72(5)
32(6)
120(15)
125(11)
179(21)
326(69)
171(36)
396(65)
345(72)
226(51)
95(17)
154(45)
309(35)
171(12)
158(37)
kcat/KM 104
[m 1 min 1]
5.9
0.2
1.7
4.0
9.3
11.0
3.7
3.3
2.9
2.5
4.1
0.3
2.0
3.6
7.5
2.8
1.5
3.1
4.7
Decrease in
activity[a] [%]
–
97
71
32
58
87
37
44
51
58
–
93
63
12
86
32
63
24
16
(29.5)
(3.5)
(1.5)
(1.6)[b]
(1.9)[b]
(1.6)
(1.8)
(2.0)
(2.4)
(13.7)
(2.1)
(1.1)
(1.9)[b]
(1.5)
(2.7)
(1.3)
(1.15)[b]
[a] Percentage of decreased SIRT1 catalytic efficiency (kcat/KM), relative to
the efficiency obtained with native peptides. The fold decreases in catalytic efficiency (kcat/KM), relative to the native peptides, are in parenthesis.
[b] Percentage of increased SIRT1 catalytic efficiency (kcat/KM), relative to
the native peptides. The fold increases in catalytic efficiency (kcat/KM), relative to the native peptides, are in parenthesis.
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
positions of p53 and H4 is needed to realize normal SIRT1 catalytic efficiency.
Analysis of SIRT1 activity against p53 and H4 peptides lacking the C-flanking region (p53-C-D, H4-C-D, Figure 1) and
against the C-flanking region triple-alanine mutant (p53-C-3M,
H4-C-3M) revealed a decrease of up to ~ 60 % in SIRT1 catalytic
efficiency, relative to the native peptides (Figures 2 and 3,
Table 1). These results clearly show that the C-flanking p53 and
H4 peptide residues are significantly less important for SIRT1
catalytic efficiency than are the corresponding N-flanking residues. To identify specific residues in the C-flanking region that
might contribute to SIRT1 activity, we synthesized peptide mutants in which the residue at position +2 is mutated to alanine
(p53-C-2A, H4-C-2A, Figure 1). Previously, structural and binding analysis of Sir2Tm in complex with different peptides
showed that this position plays an important role in Sir2Tm–
peptide interactions.[12] In agreement with this earlier structural
analysis, we found that SIRT1 catalytic efficiency is significantly
affected by the substitutions made. We found that the catalytic
efficiency of the enzyme toward the p53-C-2A peptide is similar to that observed with the p53-C-3M peptide (kcat/KM value
of 2.9 104 and 2.5 104 m 1 min 1, respectively, Table 1), highlighting the relative importance for this residue for SIRT1 activity. In contrast, we found that SIRT1 activity against H4-C-2A
moderately increased in terms of catalytic efficiency, relative to
the native peptide, highlighting the non-optimality of this residue for SIRT1 activity (Figures 2 and 3, Table 1). Since SIRT1 utilizes NAD + as a cofactor to catalyze the deacetylation reaction, we examined the effects of increasing the NAD + concentration on SIRT1 activity. In agreement with the results obtained by Smith et al.,[15] we found that the KM value for NAD +
is 234 mm, which is significantly lower than the concentration
of NAD + (500 mm) used for the SIRT1 kinetic analysis. To further verify that increases in NAD + levels did not dramatically
affect SIRT1 activity, we examined SIRT1 activity against p53-N2A peptide at NAD + concentrations of 0.5 and 1 mm. We
found no significant differences in SIRT1 activities (up to 10 %
difference in kcat/KM) measured in the presence of either NAD +
concentration (Figure S5).
Our kinetic analysis of SIRT1 with the p53 and H4 peptide
variants clearly shows the greater importance of the N-flanking
region, relative to the C-flanking region of the target acetyllysine for SIRT1 activity. These results encouraged us to examine whether the N-flanking region is more conserved in SIRT1
substrates than is the C-flanking region during the course of
evolution. Accordingly, we examined three different SIRT1 substrates, including p53,[16] Ku70,[17] and HSF1,[18] proteins in
which deacetylation of a specific acetyllysine residue was
shown to significantly affect function. We performed sequence
alignment of the relevant region in p53, Ku70 and HSF1 from
different species and found that indeed the N-flanking region
is much more conserved relative to the C-flanking region
(Table 2 and Figure S6). At the same time, due to high sequence conservation of the eukaryotic H4 histone protein, we
did not detect changes in the K16 acetyllysine N- or C-flanking
regions across evolution. These results thus suggest that the
N-flanking region of SIRT1 protein substrates is important for
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Table 2. The N-flanking residues of the p53, Ku70 and HSF1 target acetyllysine are more conserved than are the C-flanking residues. Shown is the
percentage change in the residues N- or C-flanking the acetyllysine in the
three substrates.
Protein substrate[a]
N-flanking residues[b]
C-flanking residues[b]
P53 (K382)
Ku70 (K539)
HSF1 (K80)
7%
10 %
13 %
28 %
46 %
74 %
[a] The target acetyllysine in each substrate is found in parentheses.
[b] Calculation of the percentage of sequence changes is based on the
multiple sequence alignment shown in Figure S6. For each substrate,
eight flanking residues were analyzed and the percentage of sequence
changes (Y) was calculated according to the following equation: Y =
(X/(8a)) 100, where X is the number of mutations in the N- or C-flanking residues (highlighted in yellow in Figure S6) and a represents the
number of substrate sequences that were analyzed.
SIRT1 activity in vivo and that SIRT1 can recognize other substrates, apart from p53 and H4, through the region N-flanking
the acetyllysine target residue.
Overall, our results show common properties in SIRT1 recognition of p53 and H4, despite dramatic sequence differences of
these two peptides. We found that the region N-flanking the
target acetyllysine in both peptides is significantly more important for SIRT1 activity than is the C-flanking region. This result
is particularly interesting since the p53 and H4 sequences
differ dramatically in their N-flanking regions. The p53 peptide
contains bulky and charged residues (RHK) at these positions,
whereas the H4 peptide contains very small residues (GGA)
that enable only main-chain interaction with SIRT1 while conferring high flexibility. The importance of the flexibility of the
GGA residues in H4 for SIRT1 activity is evident from the kinetic analysis of the GGA-to-AAA double mutant peptide (H4-N2M), where such mutation led to a ~ 60 % decrease in SIRT1
catalytic efficiency (Figures 2 and 3, Table 1). The importance of
the sequences flanking the target acetyllysine for SIRT1 activity
was also examined using two hybrid peptides (p53-H4-H and
H4-p53-H) in which the N-flanking regions were swapped between the peptides (Figure 1). The decrease in SIRT1 activity
toward these peptides, relative to the respective native peptides, demonstrates the sequence specificity of SIRT1 for its
substrates. Detailed analysis of the Michaelis–Menten parameters of the different mutated peptides indicates that most substitutions led to a reduction in kcat rather than having dramatic
effects on the KM (Table 1). These results suggest that SIRT1
can bind most of the mutated peptides with reasonable affinity, yet such binding is not optimal and thus compromises the
positioning of the acetylated lysine in the active site.
The importance of the N-flanking region of the acetyllysine
target relative to the C-flanking region is in good agreement
with the sequence analysis of different SIRT1 substrates. Our
analysis showed a much higher degree of sequence conservation in the N-flanking region, relative to the C-flanking region
in p53, Ku70 and HSF1 (Table 1). This higher degree of conservation suggests that the preference of SIRT1 for the N-flanking
region occurs in vivo in the context of the whole protein and,
moreover, that this tendency is true of several SIRT1 substrates.
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Finally, the importance of the N-flanking regions for SIRT1 activity supports the widespread usage of the Fluor de Lys assay
to analyze SIRT1 catalytic activity toward different substrates.[19]
This assay utilizes substrates containing an acetylated lysine
conjugated to 7-amino-4-methylcoumarin (AMC) and is based
on the coupling of SIRT1 activity to the activity of trypsin that
cleaves the peptide bond between the acetylated lysine and
AMC. In the case of the Fluor de Lys substrate, the fluorophore
is located C-terminally to the acetyllysine and thus its effect on
SIRT1 activity is likely smaller than that expected upon attachment of the fluorophore to the N-flanking region of the acetyllysine. This is assumed because the N-flanking region of the
acetyllysine substrate is more important for SIRT1 activity than
is the C-flanking region.
In summary, our work provides a detailed view on SIRT1 activity against p53 and H4 by revealing common features of
SIRT1–peptide recognition, despite dramatic difference in the
p53 and H4 sequences. The differences in SIRT1 peptide sequences suggest that the peptide conformations adopted
upon SIRT1 binding play a crucial role in allowing main chain
SIRT1–peptide interactions, thus facilitating high SIRT1 catalytic
efficiency. These peptide conformations can be dictated by the
primary sequence of the peptides and induced upon protein
binding. Since these conformations are extremely hard to predict based solely on sequence analysis, the discovery of new
SIRT1 substrates in the human proteome remains a great challenge. Our results could also have important implications in
the design of new peptide-based inhibitors for SIRT1, highlighting the importance of targeting SIRT1 regions interacting
with residues N-flanking the target acetyllysine residue of such
peptides.
Acknowledgements
A.A. was supported by the European Research Council “Ideas Program” (201177) the Israeli Science Foundation (ISF) and the
Deutsch–Israelische Projektkooperation (DIP) program.
Keywords: deacetylation
specificity
· peptides
·
sirt1
· substrate
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Received: November 21, 2012
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R. Meledin, A. Brik,* A. Aharoni*
&& – &&
Dissecting the Roles of the N- and CFlanking Residues of Acetyllysine
Substrates for SIRT1 Activity
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
SIRT1 specificity: The multispecific
SIRT1 enzyme catalyzes the deacetylation of acetyllysine residues within protein targets. However, little is known regarding the molecular basis for SIRT1
substrate recognition. Kinetic analysis of
SIRT1 with a panel of peptide substrates
shows the high importance of the
region N-flanking the target acetyllysine
and its high conservation through evolution.
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