PERSPECTIVES

but they all lacked a molecular handle to

P O S T- T R A N S L AT I O N A L M O D I F I C AT I O N S — T I M E L I N E
prove that this process had a regulatory role.
In 1964, soon after Phillips demonstrated
50 years of protein acetylation: the presence of acetyl groups in histones iso-
lated from calf thymus2, Allfrey showed that
from gene regulation to epigenetics, radio­labelled acetate (acetate‑2‑C14) was rap-
idly taken up and incorporated into histones
metabolism and beyond in isolated nuclei7 (FIG. 3). Surprisingly, this
incorporation was insensitive to the transla-
tion inhibitor puromycin, suggesting that it
Eric Verdin and Melanie Ott occurred after protein synthesis. The same
was observed using C14-methyl groups and
Abstract | In 1964, Vincent Allfrey and colleagues reported the identification of histone methylation7. Histones had recently
histone acetylation and with deep insight proposed a regulatory role for this protein been shown to inhibit RNA synthesis
modification in transcription regulation. Subsequently, histone acetyltransferases in nuclei and, in a remarkably prescient
(HATs), histone deacetylases (HDACs) and acetyl-Lys-binding proteins were experiment, Allfrey further showed that
acetylation of histones lowered their ability
identified as transcription regulators, thereby providing compelling evidence for
to inhibit RNA synthesis and carefully pro-
his daring hypothesis. During the past 15 years, reversible protein acetylation and posed a “dynamic and reversible mechanism
its modifying enzymes have been implicated in many cellular functions beyond for activation as well as repression of RNA
transcription regulation. Here, we review the progress accomplished during the synthesis” by reversible post-translational
past 50 years and discuss the future of protein acetylation. histone acetylation7. It would take another
30 years for this daring hypothesis to be
validated.
Post-translational modification (PTM) proteins; the link of histone acetylation
of proteins is emerging as a major regula- to epigenetics; and the connection found Laying the foundations
tory mechanism in all life forms. PTMs between acetylation and cellular metabo- After their initial discovery, Allfrey and
confer novel properties to the modified lism. We then discuss the effects that novel colleagues continued to work actively on
proteins, including changes in enzymatic proteomic approaches have had on the acetylation, further showing that histones
activity, subcellular localization, inter­action global identification of Lys acetylation sites, are acetylated on the ε‑amino group of Lys
partners, protein stability and DNA bind- and the advances made in developing drugs residues, identifying acetylation in other
ing. Although protein phosphorylation that target them. We end with an overview proteins — the high mobility group (HMG)
was discovered in 1959 (REF. 1) and protein of some future research directions and the chromatin proteins — and identifying
acetyl­ation in 1963 (REF. 2), the history of the challenges facing the field. histon­e deacetylase activities in nuclei.
two PTMs diverged dramatically over the In the mid‑1970s, Riggs and colleagues
following decades. Protein phosphorylation A daring hypothesis showed that induction of differentiatio­n
is the most studied PTM and occupies a The field of protein acetylation rests on of Friend erythroleukaemic cells into
central role in regulating signal transduc- work by Lipmann, Bloch and Lynen, all haemoglobi­n-synthesizing normoblast-like
tion pathways, metabolism and other cellu­ Nobel Prize winners for their discoveries of cells by the short fatty acid n‑butyrate was
lar processes. By contrast, acetylation was co­enzyme A (CoA) and acetyl-CoA. Not accompanied by strong histone hyperacetyl­
relatively ignored for the 30 years following only did they solve the structure of CoA and ation8. Following on this observation, the
its discovery (FIG. 1). A remarkable series of identify its coupling to acetate but they also groups of Allfrey and Davie reported that
congruent observations in the past 20 years highlighted the role of acetyl-CoA (which n‑butyrate was an inhibitor of HDACs9,10.
has brought protein acetylation and the used to be called ‘activated acetate’) as a key These papers showed for the first time that
enzymes that control it back to the forefront precursor to acetylation reactions3–6. small molecules targeting epigenetic regula-
of c­ellular regulatory mechanisms. In the early 1960s, at a time corre- tors can modify cellular function, including
This Timeline article marks the 50th sponding to the end of the golden age of the induction of cancer cell differentiation9,10.
anniversary of the discovery of protein metabolism discoveries, Vincent Allfrey Independently from this work,
acetylation. We describe the first steps made (FIG. 2), working with Alfred Mirsky, became tubuli­n was unexpectedly also found
in the field and highlight several key dis- interested in the possible role of chromatin to be acetylated. Noticing a form of
coveries, including: the discovery of histone in the regulation of gene expression. Some tubulin that migrated differently dur-
acetyltransferases (HATs), histone dea- laboratories had previously suspected that ing two-dimensional gel electrophoresis,
cetylases (HDACs) and acetyl-Lys-binding histones might be repressing RNA synthesis, L’Hernault and Rosenbaum showed that

258 | APRIL 2015 | VOLUME 16 www.nature.com/reviews/molcellbio

© 2015 Macmillan Publishers Limited. All rights reserved

 PERSPECTIVES

16,000 1,600 encoding this protein revealed that it was the

Phosphorylation orthologue of Gcn5, a yeast transcription reg-
14,000 Acetylation 1,400
ulator 24. Second, in a chemical biology tour
(PubMed citations per year)

(PubMed citations per year)

12,000 1,200 de force, Schreiber and colleagues used an
affinity matrix based on the HDAC inhibitor
Phosphorylation

10,000 1,000

Acetylation
trapoxin to purify a 46-kDa bovine thymus
8,000 800 protein25. Trapoxin had previously been iden-
tified by Yoshida and colleagues in screens
6,000 600
for small molecules that induce the differen-
4,000 400 tiation of tumour cells (a common function
of HDAC inhibitors)26,27. Microsequencing of
2,000 200
the trapoxin-bound protein revealed a
0 0 bovine orthologue of the yeast transcription
1964 1974 1984 1994 2004 2014
Year regulator Rpd3 (REF. 25). Rpd3 had previ-
ously been identified in several independent
Figure 1 | Publications on protein acetylation. The graphsNature show the growth in the number of papers
Reviews | Molecular Cell Biology
published on the topic of protein acetylation during the past 50 years, in comparison to the growth in mutant suppressor screens for transcription
the number of papers published on protein phosphorylation (searched in PubMed). Note that the scale repressors28. In perfect but opposing sym-
of the y axis for protein phosphorylation (on the left) is tenfold larger than for protein acetylation (on metry to the identification of Gcn5 as a HAT,
the right). A clear inflexion point in the rate of publication is visible in the field of protein acetylation histone deacetylase 1 (HDAC1) was cloned
following the two papers by Allis24 and by Schreiber25 in 1996. as the human orthologue of Rpd3 and shown
to exhibit HDAC activity in vitro.
These discoveries were followed by the
tubulin incorporated radiolabelled acetate11. cloned the first histone acetyltransferase, identification of additional HATs associ-
Piperno et al. developed a monoclonal HAT1, by screening a collection of yeast ated with transcription regulation, such as
antibody specific for acetylated α‑tubulin12 temperature-sensitive mutants with an CREB-binding protein (CBP), E1a‑binding
and mapped its binding to a unique residue, enzymatic assay for histone H4 acetylation22. protein p300 (EP300; also known as p300),
Lys40 (REFS 13,14). These important experi- Disappointingly, hat1 mutants had no obvi- the TAF(II)250 subunit of transcription
ments demonstrated the power of using ous growth defects or phenotypes other than factor IID (TFIID) and several members of
modification-specific antibodies, which lack of enzymatic activity. the MYST family of proteins (named for its
would prove to be key tools in the study of founding members MOZ, YBF2/SAS3, SAS2
protein acetylation. Paradoxically, although The epigenetic control of gene expression. and TIP60)29–37 (see also REF. 38 for a review).
α‑tubulin acetylation has been studied for Two independent discoveries electrified the In parallel to this work, detailed biochemical
the past 30 years and is a highly conserved field in 1996 and finally established a clear work led to the identification and the first
modification, it remains poorly understood. causal link between histone acetylation and characterization of the large multiprotein
In the late 1980s, pioneering studies by transcription regulation. First, using an complexes in which many of these proteins
several groups established a link between elegant in gel assay to purify a type A HAT function: the SAGA complex 39, the Sin3
acetylated core histones and transcription- (thought to be transcription-associated), complex 40–43 and the NURD complex 44,45.
ally active genes15, and identified the key Allis and colleagues identified a catalytically Rapidly thereafter, the first EP300
part played by specific Lys residues on active, 55-kDa protein from macronuclei inhibitor, Lys-CoA, was created by
histone tails in the silencing of transcrip- of the protozoan Tetrahymena thermoph- Cole and colleague­s by covalently linkin­g
tion at telomeres and in the control of the ila23. Purification and cloning of the gene CoA and a Lys residue, producing a
mating locus in yeast 16–19. In parallel, Turner
and colleagues used site-specific antibodies
for different histone acetylation marks and Figure 2 | Vincent Allfrey (1921–2002): acety-
indirect immunofluorescence on polytene lation pioneer. Vincent Allfrey, a native New
Yorker, started his career as a young laboratory
chromosomes to show that distinct acetyla-
helper in the illustrious company of Oswald
tion modifications were localized on differ- Avery’s laboratory at the Rockefeller Institute,
ent chromosomal domains. These findings New York, USA. There, he purified pneumococcal
further supported the hypothesis that his- DNA using an experimental system that would be
tones acetylated at particular sites mediated later used by Avery and colleagues to prove that
unique and specific effects on gene expres- genetic information is linked to DNA. After
sion by modifying the degree of chromatin obtaining his Ph.D. from Columbia University,
condensation20,21 (FIG. 4). New York, USA, Allfrey joined the laboratory of
Alfred Mirsky, who was the first to isolate chroma-
Acetylation modifiers and readers tin. He rapidly became interested in the link
between chromatin and gene regulation, and is
The stage was now set for the flurry of dis-
credited with first proposing the model that his-
coveries, made between 1995 and 2000, of tone modifications, including histone acetylation
acetylation-modifying enzymes, their role and methylation, control gene expression by
in transcription regulation, their structures, regulating access to the DNA. Image (dated
multiprotein complexes and novel targets. 1992) is by Robert Reichert, courtesy of The
Sternglanz and colleagues identified and Rockefeller University Public Affairs, USA.

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 16 | APRIL 2015 | 259

© 2015 Macmillan Publishers Limited. All rights reserved

PERSPECTIVES
pseudo­substrate mimicking an enzymatic
intermediate of the acetylation reaction46.
A similar rapid flow of discoveries of
HDACs occurred, first with the identifica-
tion of other Rpd3‑related proteins, HDAC2
and HDAC3 (REFS 47–50), which, together
with HDAC1 and HDAC8, comprise the
class I family of HDACs. A separate family of
HDACs was identified based on their close
homology to yeast Hda1, a protein related
to, but distinct from, Rpd3. The members of
this family (HDAC4–HDAC7 and HDAC9)
are called class II HDACs51–54. HDAC10
and HDAC11 were discovered later and
completed the class II and IV families,
respectively 55–57.
Following these discoveries, the first
protein crystal structures were solved for
an HDAC (a homologue of Rpd3 from
the hyperthermophilic bacterium Aquifex
aeolicu­s)58, for type A HATs (the acetyl-
transferase domains of PCAF (also known
as KAT2B) and Gcn5 (REFS 59–61)) and for
a class III HDAC (discussed below) — a
Sir2 (silent information regulator subunit 2)
orthologue from Archaeoglobus fulgidus, in
complex with NAD+ (REF. 62). During the
same period, a growing number of non-
histone proteins, mostly involved in tran-
scription regulation, were rapidly revealed to
undergo acetylation, thereby progressively
expanding the scope of cellular protein
acetylation. The first such proteins were the
tumour suppressor p53 (REF. 63), the HIV
transcriptional activator Tat 64,65, the tran-
scription factor nuclear factor-κB (NF-κB)66
and others (reviewed in REF. 67).
Reading protein acetylation. Analogous to
what had been observed for protein phos-
phorylation, whereby SRC-homology 2
(SH2) domains specifically recognize
peptides containing phosphorylated Tyr
residues, Zhou and colleagues reported that
the bromodomain specifically recognizes
acetylated Lys residues68. This introduced the
concept of ‘reader’ proteins to the acetylation
field in addition to the previously identi-
fied ‘writers’ (HATs) and ‘erasers’ (HDACs)
(FIG. 4). Bromodomains were found to be
present in diverse types of nuclear proteins,
including HATs, ATP-dependent chromatin-
remodelling proteins, helicases, methyltrans-
ferases, transcription co-activators, nuclear
scaffolding proteins, and in the bromo­
domain and extraterminal (BET) protein
family (reviewed in REF. 38). The potential
recruitment of these transcription mediators
to an acetylated binding partner explained
in part how acetylation could be linked to a
plethora of functional outcomes.
260 | APRIL 2015 | VOLUME 16
Soon after the publication of the land-
mark paper by Zhou and colleagues68,
Owens et al. crystallized an acetylated histone
H4 peptide in complex with the yeast Gcn5
bromo­domain, and identified a canonical
Asn residue that is present in most bromo­
domains and is the critical interacting residue
in the acetyl-Lys binding pocket69. This core
structure is conserved across all eight bro-
modomain families identified so far, despite
little sequence homology. In a remarkable,
more recent effort, several groups crystalized
29 of the 61 bromodomains encoded in the
human genome and annotated the shared and
unique structural characteristics, as well as
the ligand preferences, of individual bromo-
domains within different families70. Recently,
the YEATS domain (named after the five
founding domain-containing proteins, Yaf9,
ENL, AF9, Taf14 and Sas5) was also identified
1964
n-butyrate is identified as
1978
an inhibitor of HDACs9,10
1983
Mutation of Lys residues in
histone tails highlight their role 1990
in gene regulation in yeast16–19
Allis and colleagues
1993
identify Gcn5 as a HAT24
HDAC1 is identified as an orthologue
1996
of Rpd3 by Schreiber and colleagues25
TSA First structures of
HATs and HDACs 1999
determined58–61
Zn
2000
C
Sinclair and colleagues show
2003
that resveratrol is a SIRT1
OH
activator88,89
HO
2006
OH
JQ1 and I-BET are identified as the
2010
first bromodomain inhibitors91,92
Figure 3 | Milestones in protein acetylation. Timeline of the history of protein acetylation research.
Nature Reviews
FDA, Food and Drug Administration; HATs, histone acetyltransferases;
Sir2, silent information regulator 2; SIRT1, sirtuin 1.
© 2015 Macmillan Publishers Limited. All rights reserved
as an acetyl reader, with some selectivity for
acetylated Lys9 from histone H3 (H3K9Ac).
This suggested that additional acetyl readers
might be identified in the future71.
Sensing the metabolic state of the cell. In the
span of a few years, the field of acetylation
had gone from having no known acetylation-
modifying enzymes, to the elucidation of the
structure and mechanism of action of more
than 20 HATs and HDACs. Furthermore, a
surprise was in store: the Sir proteins, known
as transcription repressors at telomeres and
ribosomal DNA, were long suspected to be
regulators of protein acetylation because
yeast Sir mutants exhibited increased histone
acetylation72. Frye identified five human
cDNAs with homology to the yeast SIR2 gene
(he called them sirtuins) and showed that
they metabolized NAD+ (REF. 74).
Allfrey and colleagues propose
that histone acetylation
regulates gene expression7
Discovery of tubulin
acetylation11
O O
OH
N
H
H3C
N
Yoshida and colleagues
CH3 identify the natural
products trapoxin and
trichostatin A as HDAC
inhibitors26,27
Zhou and colleagues identify
the bromodomain as an
acetyl-Lys-binding domain68
Guarente and colleagues
demonstrate that Sir2 is an
NAD+-dependent deacetylase75
Cole and colleagues synthesize
Lys–CoA as a pseudosubstrate
HAT inhibitor46
Zhao and colleagues perform
the first proteomic screen for
acetylation sites81
Vorinostat receives FDA approval
for the treatment of advanced
primary cutaneous T cell lymphoma
| Molecular
HDACs, Cell Biology
histone deacetylases;
www.nature.com/reviews/molcellbio

Ac
Acetylated chromatin
Open and transcriptionally active
Writer
Deacetylated chromatin
Compact and transcriptionally repressed
Figure 4 | Histone acetylation, chromatin condensation and gene expression. Acetylation targets
Lys residues in the amino-terminal tails of core histone proteins. A string of nucleosomes is shown with
the tails protruding when acetylated. Acetylation of the tail domains inhibits the folding of nucleo-
some arrays into secondary and tertiary chromatin structures, with acetylation of histones H2B and H4
having the greatest effect on tertiary structure formation103–105. Thus, histone tail acetylation results in
chromatin decondensation, thereby allowing access to transcription factors and other transcription
co‑activators.
Soon after, Guarente and colleagues
elegantly demonstrated that yeast Sir2 and
mouse SIRT2 proteins are HDACs, the
activity of which is uniquely dependent
on NAD+ (REF. 74). During deacetylation,
NAD+ is cleaved, releasing nicotinamide
and the ADP–ribose covalently linked to the
removed acetyl group (acetyl–ADP–ribose)
(BOX 1). Based on previous work showing
that a gain‑of‑functio­n mutation of Sir2 was
associated with an increased lifespan in yeast,
Guarente further proposed that the depend-
ency of Sir2 on NAD+ for its enzymatic activ-
ity enabled it to sense the energy status of
the cell. Indeed, NAD+ levels increase when
cellu­lar nutrient levels are restrictively low,
and this activates Sir2 and other sirtuins,
thereby transducing a metabolic signal to
various proteins, including histones, by dea-
cetylating them (BOX 1). At the time of its pro-
posal, this model that chromatin-modifying
proteins sense changes in the environment
through their effect on intermediary metabo-
lites was a bold idea, one which is currently
gaining much experimental support for
acetyl­ation and other chromati­n PTMs
(reviewed in REF. 75).
Acetylation goes global
With the exception of tubulin, most acetylated
proteins appeared to be nuclear and associ-
ated with transcription regulation. In 2000,
Kouzarides wondered in a review whether
“acetylation was a regulatory modification to
rival phosphorylation” (REF. 76). Soon after,
however, the observations that several sirtuins
were localized in the cytoplasm (for exam-
ple, SIRT2 (REF. 77)) or in mitochondria (for
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
PERSPECTIVES
were mitochondrial proteins, and the pre-
Reader
diction was made that mitochondria might
Ac
Ac Ac represent a unique environment where acety-
lation is abundant. In the same year, the first
Gene On mitochondrial acetylated protein targeted
by SIRT3 was identified — acetyl-CoA syn-
thetase81,82 — demonstrating the regulatory
role of protein acetylation in mitochondrial
Chromatin metabolism.
remodelling Eraser
Three years later, Mann and colleagues
used high-resolution mass spectrometry
to look deeper into the cellular acetylome.
They identified 3,600 Lys acetylation sites in
Gene Off
1,750 proteins83, and observed that acetyla-
tion was particularly prominent in large
macromolecular complexes involved in a
Nature Reviews | Molecular Cell Biology range of cellular activities, such as chromatin
remodelling, the cell cycle, splicing, nuclear
transport and actin nucleation. With such a
large number of acetylated proteins involved
in numerous cellular processes, protein
acetyl­ation was finally established as a
globall­y important PTM.
example, SIRT3 (REFS 78,79)), suggested that
acetylation might be more broadly distributed Targeting acetylation with drugs
than had been anticipated. This prediction The discovery of protein acetylation and of
became partly validated in 2006 by the first the proteins that write, erase or read acetyl
reported proteomic survey of protein acetyla- groups within proteins has led to the identifi-
tion. Using a new enrichment approach based cation of many novel epigenetic drug targets.
on acetylatio­n-specific antibodies, Zhao and The first FDA-approved acetylation-modify-
colleagues identified 388 acetylation sites in ing agent was the HDAC inhibitor vorinostat
195 proteins80. Thus, in one single experi- (also known as suberanilohydroxamic acid
ment, more acetylated proteins had been (SAHA)), initially identified as an agent
identified than in the preceding 40 years. inducing the differentiation of tumour
Remarkably and unexpectedly, many of these cells in vitro and subsequently as an HDAC
Box 1 | The interface between protein acetylation and metabolism
Metabolism can influence protein acetylation by changes in the cellular concentration of NAD+ and
acetyl-coenzyme A (acetyl-CoA). For example, during fasting the relative concentration of NAD+
increases, leading to an increase in the enzymatic activity of sirtuins and the deacetylation of their
targets (see the figure). In contrast to kinases, the enzymatic activity of which is largely
independent of fluctuations in ATP concentrations, the activity of acetyltransferases varies as a
function of acetyl-CoA concentrations. When nutrient abundance increases, the cellular
concentration of acetyl-CoA increases, leading to an increase in acetyltransferase activity and
target protein acetylation.
↓ Nutrients
↑ NAD+ Ac CoA
Lys
Protein
Sirtuin Acetyltransferase
Nicotinamide Lys ↑ Acetyl-CoA
+ acetyl–ADP–ribose
Protein
↑ Nutrients
Nature Reviews | Molecular Cell Biology
VOLUME 16 | APRIL 2015 | 261
© 2015 Macmillan Publishers Limited. All rights reserved
PERSPECTIVES
inhibitor 84 (as was the case for another HDAC
inhibitor, n-butyrate). Vorinostat, which
is approved for the treatment of cutaneous
T cell lymphoma, and two other subsequently
approved HDAC inhibitors — romidepsin
(Istodax, FK228, FR901228; Celgene)85 and
panabinostat — are currently being tested
in a large number of cancer clinical trials.
Interestingly, the three drugs are also being
tested in HIV-infected individuals to reac-
tivate latent HIV that persists in patients
treated with antiretroviral therapy (ART). It is
anticipated that treatment with these HDAC
inhibitors will allow the patients’ immune
system, in combination with ART, to destroy
the latently infected cells86 and could lead to
curing HIV infections.
With regard to sirtuins, the focus of
drug development has instead been on
identifying enzymatic activators, with the
hope that they would increase lifespan by
mimicking the gain-of-function mutation
in Sir2. Sinclair and colleagues reported the
identification of the polyphenol resvera-
trol (which is abundant in red wine) as the
first Sir2 and SIRT1 activator, and further
showed that it increased lifespan in yeast and
meta­zoans87,88. Whereas subsequent observa-
tions by other groups have questioned these
results and the selectivity of resveratrol, a
recent comprehensive study, also carried
out by Sinclair and colleagues, has brought
renewed support to a mechanistic model in
which sirtuin-activating compounds allos-
terically activate SIRT1 (REF. 89). Thus, such
compounds could be therapeutically relevant
for many age-related diseases.
The discovery of small-molecule inhibi-
tors that disrupt the interaction between the
bromodomain and acetyl-Lys was recently
reported. The first two such compounds,
JQ1 and I‑BET, although structurally dis-
tinct, harbour a triazole ring that forms
a hydrogen bond with the canonical Asn
residue present within the acetyl-Lys pocket
of most bromodomains90,91. The Knapp and
Bradner laboratories showed that JQ1 binds
the first bromodomain of the BET protein
BRD4, which is an important tethering
factor of positive transcription elongation
factor b (P‑TEFb). Studies with JQ1 have
initially focused on a rare type of squamous
cell carcinoma, which is driven by oncogenic
BRD4 protein fusions, and more recently
were expanded to cancers with aberrant Myc
expression92. Interestingly, Tarakovsky and
colleagues showed that the bromodomain
inhibitor I‑BET functions as an immuno-
suppressant, suggesting possible clinical
applications beyond cancer for this new class
of drugs91.
262 | APRIL 2015 | VOLUME 16
The development of HAT inhibitors has
proved more difficult, but recent successes
by Cole and colleagues93 in developing
highly selective EP300 inhibitors indicate
that the development of epigenetic drugs
against HATs is likely to become fruitful
as well.
The future of protein acetylation
A key direction for future research will be
the identification of all proteins the acety-
lation of which is regulated by different
acetyltransferases and deacetylases. As a
first step in this direction, three recently
published studies described the identifica-
tion of the target sites of the mitochondrial
SIRT3 in mouse tissues94,95 and in cell lines94.
It is important to note that although
many acetylation sites have been identified,
there is little information on the stoichiom-
etry of individual sites (that is, the percent-
age of each site that is acetylated in cells).
In fact, two recent studies addressing this
important question found that different
acetylation sites have markedly different
stoichiometries96,97. Many sites in mito-
chondrial and cytoplasmic proteins showed
low stoichiometry and a strong correlation
between stoichiometry and acetyl-CoA
level­s, consistent with protein acetyla-
tion also being a non-enzymatic process.
By contrast, high abundance of acetylation
was found mostly in nuclear proteins such
as histones, HDAC and HAT complexes
and transcription factors, but also in a sub-
set of mitochondrial proteins that either
use or generate acetyl-CoA. Non-enzymatic
acetylation was first observed in the 1970s
on histones98 and might be particularly
relevant in mitochondria, where the local
higher pH (7.9) and high concentration
of acetyl-CoA favour the reaction. Given
the slow kinetics of non-enzymatic protein
acetylation99, it is not likely to be relevant
for organisms such as Escherichia coli and
Saccharomyces cerevisiae, which divide rap-
idly and dilute their pool of acetylated pro-
teins. Instead, non-enzymatic acetylation
might be more biologically relevant in post-
mitotic tissues of multicellular organisms,
in which acetylation of individual proteins
can progressively accumulate over extended
periods of time.
Recent exciting developments indicate
that acetylation was only the first repre-
sentative of a broad class of Lys modifica-
tions, known as Lys acylations. Interestingly,
some sirtuins seem to target these newly
described modifications, as SIRT5 was
recently shown to be a Lys desuccinylase
and demalonylase100,101.
© 2015 Macmillan Publishers Limited. All rights reserved
Finally, our understanding of the nexus
between intermediary metabolites and
protein acetylation is still rudimentary and
mostly based on NAD+ and acetyl-CoA con-
centrations. Recent results indicate that the
ketone body β-hydroxybutyrate, an impor-
tant metabolite during fasting, functions as a
class I HDAC inhibitor and induces histone
hyperacetylation during fasting102.
Expanding on these observations and
carrying out further molecular studies of the
role of acetylation in regulating cellular pro-
cesses represent the main challenges for the
future. In addition, exploiting the identified
regulators of acetylation as potential thera-
peutic targets will fulfil the full potential of
the field of protein acetylation.
Conclusion
Allfrey’s daring hypothesis laid the founda-
tion for the field of histone PTMs and epi-
genetic regulation. During the past 50 years,
protein acetylation has emerged not only
as an important epigenetic regulator but
also as a PTM, uniquely coupled to cellular
metabolism (BOX 1). Although acetylation
still has a lot of catching up to do on protein
phosphoryl­ation (FIG. 1), a solid foundation
has been established and much exciting
work remains to be done.
Eric Verdin and Melanie Ott are at the
Gladstone Institutes, University of California,
San Francisco, 1650 Owens Street, San Francisco,
California 94158, USA.
Correspondence to E.V.
e‑mail: everdin@gladstone.ucsf.edu
doi:10.1038/nrm3931
Published online 30 December 2014
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Acknowledgements
We thank D. Allis, L. Pilus and P. Cole for discussions during the
preparation of this manuscript. Work in the laboratories of E.V.
and M.O. is supported by the National Institutes of Health (NIH)
and by the Gladstone Institutes. We apologize to colleagues
whose work we could not cite owing to space limitations.
Competing interests statement
The authors declare competing interests: see Web version for
details.
FURTHER INFORMATION
Eric Verdin’s homepage:
http://gladstoneinstitutes.org/scientist/verdin
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http://gladstoneinstitutes.org/scientist/ott
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