Protein Acetylation: A Switch to Gene Expression
Acetylation is one of the most abundant post-translational modifications in biology
and is conserved in all kingdoms of life. As many as 90% of proteins involved in Salmonella
enterica metabolic pathways are acetylated [1], primarily at lysine residues, and
more than 80% of human proteins are N-terminally acetylated [2]. Analogous to phosphorylation,
attachment of an acetyl group to a protein amino acid is a reversible reaction catalyzed
by dedicated enzymes that have acetyl-transferase or deacetylase activity. Perhaps
the most widely studied and best documented example of acetylation affects histones,
basic proteins tightly bound to DNA to form chromatin. Acetylation of critical lysines
in histone tails decreases the overall positive charge, decreasing electrostatic interactions
with DNA [3]. This is thought to promote chromatin decondensation and to enhance accessibility
to RNA polymerase, thereby stimulating transcriptional activity. On the contrary,
histone deacetylation is usually associated with a reduction of transcriptional activity.
Thus, protein acetylation represents a versatile and reversible molecular switch of
vital importance in biology.
Sirtuins Are Ubiquitous NAD+-Dependent Deacetylases
There are four classes of histone deacetylases (HDAC) in eukaryotes, of which sirtuins
(or class III) are highly conserved phylogenetically. One of the defining characteristics
of sirtuins is the requirement of NAD+ as cofactor [4], a dependence that links sirtuin
function to the metabolic state of the cell. Caloric restriction studies in yeast,
worms and flies have shown increased longevity linked to the increased activity of
homologs of the Saccharomyces cerevisiae founder sirtuin, Sir2 [4,5]. The human genome
encodes seven sirtuins, named SIRT1–SIRT7 [5]. SIRT1 (also known as Sir2α) is the
closest homolog of yeast Sir2, and as in this model organism, its activity is also
increased due to caloric restriction [6,7]. SIRT1 has many important cellular targets,
such as p53 [8], NF-κB [9], FOXO [10] and PPARγ [11], making it an attractive target
for cancer therapeutics and longevity studies. At the structural level, all sirtuins
have a conserved catalytic core composed of a Rossmann fold for NAD+ binding, a Zn2+
binding module and a helical module containing an NAD+-binding loop. NAD+ and the
acetylated lysine substrate bind across this interface, with the acetylated ε-amino
group of the lysine adjacent to the ribose moiety of NAD+, in a hydrophobic tunnel
where catalysis occurs. The substrate is primarily bound by backbone interactions
and the formation of a three-stranded antiparallel “β-staple”. Physiological acetyl-peptides
are accommodated in SIRT1 active site mainly by hydrogen bonding with their main-chain
backbone, allowing deacetylation without sequence conservation [12]. There appears
to be no particular consensus sequence for substrate binding to sirtuins, but specificity
is dependent on the amino acid context of the acetylated lysine [12,13]. After catalysis,
the products of this reaction are 2′-O-acetyl-ADP-ribose (OAADPr), nicotinamide and
the deacetylated substrate.
Regulation of Human SIRT1 by Its C-Terminal Regulatory Segment
In addition to the conserved catalytic core, located from residue 230 to residue 500,
SIRT1 also presents N- and C-terminal extensions found in no other human sirtuin,
which are thought to play a regulatory function. Kang et al. identified a short stretch
at the C-terminus of murine SIRT1 (residues 631–655) that is essential for deacetylase
activity [14] and that competes with an endogenous inhibitory factor known as DBC1
(Deleted in Breast Cancer-1) for activation of SIRT1 catalytic core (SIRT1CAT) [15].
They proposed that SIRT1 C-terminal regulatory (CTR) region (SIRT1CTR) functions allosterically
to alter the affinity of the catalytic core for the acetylated substrate. Concurrently,
Pan et al. found that SIRT1CAT has very low enzymatic activity on its own and that
both the N-and C-term regions are able to enhance SIRT1CAT activity [16]. They proposed
that the N-terminal domain increases the rate of catalysis, while the C-terminal domain
(residues 584–665) increases binding to NAD+ and can function in trans. In this issue
of the Journal of Molecular Biology, Davenport et al. report two crystal structures
of human SIRT1 catalytic domain bound to its CTR (residues 641–665), crystallized
in the presence (closed state) and in the absence (open state) of cofactor NAD+ [17].
Unexpectedly, the authors identified a dramatic conformational change between the
two states. Without NAD+ (which is reacted in crystal to generate adenosine diphosphatase
ribose), SIRT1 adopts an open conformation with the Zn2+ binding and helical modules
rotated with respect to the NAD+-binding domain. In the presence of cofactor, the
SIRT1 helical module is tightly folded onto the catalytic domain, adopting a closed
conformation. Davenport et al. showed that interaction between SIRT1CAT and the CTR
greatly stabilizes the catalytic core, which is quite unstable at 37 °C (the temperature
at which most enzymatic studies are performed). This structural stabilization does
not increase enzymatic activity but rather dampens SIRT1 activity in vitro. However,
removing the last 12 residues of the CTR or disrupting a conserved salt bridge between
residues R276 and E656 relieves this attenuation and increases SIRT1 activity. This
begs the question as to whether the CTR regulates deacetylase activity only intramolecularly
(in cis) or if a more complex intermolecular mechanism of trans-activation occurs
in solution, as previously suggested [16]. The authors explore both alternatives,
although a conclusive answer will require more extensive investigations in vivo. In
solution, an excess of CTR can compete off bound CTR and form a complex with SIRT1CAT
that co-migrates on a gel-filtration column and the SIRT1CAT and CTR were co-crystallized,
suggesting a binding affinity at least in the micromolar range. However, an accurate
dissociation constant between SIRT1CAT and CTR could not be determined due to the
tendency of SIRT1CAT to aggregate in solution. To complicate the puzzle, the authors
also identified a substrate-mimetic peptide projecting from C-terminal residues 504–510
of a crystallographic mate “stapled” inside SIRT1 active site. The backbone conformation
adopted by this pseudo-substrate is superimposable to the p53 substrate peptide (HKKAcLMF)
previously analyzed in complex with an archaeal SIRT1 ortholog [18]. A unique leucine
in the pseudo-substrate occupies the position of the substrate acetyl-lysine directly
facing NAD+. This suggests that various regions in the regulatory domains could potentially
occupy the substrate-binding pocket and auto-inhibit the enzyme, reconciling the observation
that the isolated SIRT1CAT is essentially inactive in vitro [16]. Although not directly
shown in this paper, we speculate that binding of a CTR to SIRT1CAT in the presence
of N-terminal regulatory and CTR domains may contribute to removing a pseudo-substrate
from the active site, stimulating catalytic activity.
Perspectives and Future Directions
The studies of Davenport et al. provide structural evidence for a regulatory role
of SIRT1 C-terminal domain on deacetylase activity. This includes not only the previously
identified CTR but also the C-terminal pseudo-substrate spanning region 504–510 that
occupies the cargo-binding groove. Building upon this work, it is foreseeable that
at least two directions of research will be particularly interesting to explore. First,
to delve into the exact regulatory role of SIRT1CTR, it is essential to study its
properties in a physiological environment. Phosphorylation in the CTR [19] is likely
to play a pivotal role in modulating the interplay between potential intramolecular
auto-inhibition by pseudo-substrate moieties and availability of acetylated substrates
and hence promote temporal and spatial control of deacetylase activity. Second, as
elegantly suggested by this paper, the observation that a leucine side chain can functionally
replace the side chain of an acetyl-lysine provides a powerful framework to engineer
peptides (or peptide-mimetics) that could compete with acetyl-substrates and inhibit
SIRT1 activity. Given the broad physiological importance of acetylation and the pivotal
role of acetylation in turning genes on and off, potent and selective small molecules
to modulate SIRT1 activity would be at the forefront of fighting cancer and metastatic
proliferation.