Mol Cell. 2015 Jul 16;59(2):309-20. doi: 10.1016/j.molcel.2015.06.013. Epub 2015 Jul 9. Identification of a Class of Protein ADP-Ribosylating Sirtuins in Microbial Pathogens.
SIRUINS are an ancient family of NAD(+)-dependent deacylases connected with the
regulation of fundamental cellular processes including metabolic
homeostasis and genome integrity.
We show the existence of a hitherto
unrecognized class of sirtuins, found predominantly in microbial
pathogens. In contrast to earlier described classes, these sirtuins
exhibit robust protein ADP-ribosylation activity.
In our model
organisms, Staphylococcus aureus and Streptococcus pyogenes, the
activity is dependent on prior lipoylation of the target protein and can be reversed by a sirtuin-associated macrodomain protein. Together, our data describe a sirtuin-dependent reversible protein ADP-ribosylation system and establish a crosstalk between lipoylation
and mono-ADP-ribosylation. We propose that these posttranslational
modifications modulate microbial virulence by regulating the response to
host-derived reactive oxygen species.
• A class of sirtuins (SirTMs) is identified in microbial pathogens
• SirTMs are linked to macrodomains and act as protein ADP-ribosyltransferases
• Protein ADP-ribosylation by SirTMs is strictly lipoylation dependent and reversible
• SirTMs modulate the response to oxidative stress
..
Introduction
Sirtuins are a diverse enzyme family of NAD+-dependent
protein deacylases that control a variety of cellular processes
including cell cycle progression, maintenance of genome integrity, and
metabolic homeostasis (Asher and Schibler, 2011; Choi and Mostoslavsky, 2014).
The overall structure of sirtuins is comprised of a highly conserved
Rossmann fold and a more diverse zinc coordinatingdomain (Yuan and Marmorstein, 2012).
The reaction mechanism is initialized by activation of NAD+,
followed by a nucleophilic attack and release of nicotinamide.
In the
case of deacylation, a reactive imidate intermediate is formed that can
undergo base-exchange with nicotinamide, thereby inhibiting reaction
progression (reviewed in Sauve, 2010).
Phylogenetically, the sirtuin family can be divided into five classes (I–IV and U, see Figure 1A) (Frye, 2000; Greiss and Gartner, 2009), and a correlation between sirtuin class and substrate preference was recently suggested (Dölle et al., 2013; He et al., 2012).
For example, human SIRT1, a class I sirtuin, is most efficient at
deacetylation, whereas SIRT5, belonging to class III, has highest
activity toward succinylation (Du et al., 2011; Feldman et al., 2013).
Although sirtuins appear to be primarily deacylases, several studies
have suggested that some also possess protein ADP-ribosyltransferase
activity (Haigis et al., 2006; Kowieski et al., 2008).
Posttranslational ADP-ribosylation influences various cellular
processes, such as transcription, chromatin organization, nitrogen
fixation, and DNA repair, via modification of different acceptor
proteins (Barkauskaite et al., 2013; Feijs et al., 2013; Nordlund and Högbom, 2013)
Figure 1Macrodomain-Associated Sirtuins Form a Distinct Class within the Sirtuin Family
In
this study, we report on the identification of a distinct class of
sirtuins (SirTMs) found primarily in pathogenic microorganisms and show
that these function as protein ADP-ribosyl transferases. Members of this
sirtuin class are genetically linked to a specific subclass of
macrodomain proteins, which reverse the sirtuin catalyzed
ADP-ribosylation. Our structural and biochemical analysis suggest that
SirTMs possess class-specific features that may explain the preference
for protein ADP-ribosylation. Moreover, we show that in Staphylococcus aureus and Streptococcus pyogenes
the sirtuin-mediated ADP-ribosylation is dependent on another
posttranslational modification—lipoylation. We propose that a crosstalk
between these two types of protein modifications is important for the
response of microbial pathogens to oxidative stress, a potent host
defense mechanism.
Results
Identification of a Distinct Class of Sirtuins
SirTMs Lack Protein Deacylase Activity
Taken together, these results suggest that the pathogenic SirTMs lack deacylation activity.
Class M Sirtuins Are Lipoylation-Dependent ADP-Ribosyltransferases
As some macrodomain-containing proteins are known to hydrolyze protein ADP-ribosylation (Jankevicius et al., 2013,Slade et al., 2011, Sharifi et al., 2013),
we assessed the ability of the operon-encoded macrodomains to remove
the SirTM-mediated MARylation of the GcvH-L. Strikingly, the operon
macrodomains (SpyMacro and SauMacro) catalyzed
efficiently the modification reversal, as indicated by the loss of
radiolabel from GcvH-L, whereas the homologous protein de-MARylating
macrodomains from human and E. coli (MacroD1 and YmdB, respectively) did not exhibit this activity (Figures 4F and S4H).
We further characterized the reaction product of the Macro reaction by
TLC. While protein bound ADPr is immobile, the Macro cleavage product
co-migrates with ADPr released in the human PARP1/PARG control reaction (Figure 4G) (Slade et al., 2011
We propose that the pathogenic SirTM/Macro pair coevolved as an ON/OFF switch for the GcvH-L ADP-ribosylation.
SpySirTM Structure Reveals an Unexpected Catalytic Residue
To gain further insight into the SirTM catalyzed ADP-ribosylation, we determined the crystal structure of SpySirTM, both in the ligand-free form as well as bound to ADPr or NAD+.
The overall fold for these three structures is identical with a low
root-mean-square deviation (RMSD) (0.29–0.31 Å). The models contain
residues 3–292 (residues 7–10 are not visible in the electron density
maps) and follow a typical sirtuin fold comprised of a large Rossmann
fold and a small bipartite zinc coordinating domain (Figure 5A; Table 2). SpySirTM is closest in structure to sirtuins from Thermotoga maritima (TmSir2, PDB: 2H4F) and Saccharomyces cerevisiae (HST2, PDB: 1SZC) (Figures S5A and S5B)
The ligand-SpySirTM complexes reveal that the general mode of NAD+ binding to SpySirTM is similar to that observed in other sirtuins (Yuan and Marmorstein, 2012) (Figures S5A and S5B). No major conformational changes are observed upon binding of either ADPr or NAD+, with the exception of slight readjustment of the loop region between β6 and α14 as well as following β7 (Figure 5B).
This movement allows the highly conserved Asn258 to interact with the
2′- and 3′-OH groups of the adenine ribose, while the imidazole group of
the relatively poorly conserved His259 rotates by ∼110° to allow
stacking against the adenine moiety. In addition, the pyrophosphate is
coordinated by the class-specificGVGx[NT]TP motif (residues 229–235),
found in the β6–α14 loop.
The amide
moiety of nicotinamide is bound by direct and water-mediated polar
contacts with Ala34, Phe42, Ala119, and Asp120, thus positioning the
nicotinamide and the distal ribose in a conformation similar to that
observed for TmSir2 (Figure S5B).
The conserved Asn118 coordinates a highly ordered water molecule, which
was proposed to facilitate the initial hydrolysis of nicotinamide from
NAD+ (Zhao et al., 2004).
On the α-face of the ribose the 3′-OH group interacts with the
absolutely conserved residue Gln137, which replaces the catalytic
histidine found in all other sirtuin classes. Gln137 makes further
contact with the Nδ atom of the similarly conserved residue Arg192 (Figure 5B).
Together, these interactions appear to be necessary for the correct
positioning of the nicotinamide ribose, and by extension, the GcvH-L
substrate. To assess the importance of these residues, we performed
MARylation assays with WT and mutant SpySirTM. While WT SpySirTM modified SpyGcvH-L, a mutation of Asn118, Gln137, or Arg192 dramatically decreased the catalytic activity (Figure 5C).
Interestingly, substitution of Gln137 with the general base histidine,
found in other sirtuins, also leads to a complete loss of activity,
suggestive of a distinct catalytic mechanism for class M sirtuins.
SirTM Function Is Linked to Redox Response
Discussion
In
the present study, we have identified a distinct class of sirtuins
(SirTMs). This class is highly conserved and occurs either as part of a
bacterial operon or, in fungi, as a macrodomain-SirTM fusion enzyme.
SirTMs: Not Deacylases, but ADP-Ribosyl Transferases
Our
results show that SirTMs do not possess any appreciable deacylation
activity against a number of different endogenous and host-derived
substrates (Figures 3 and S3).
In accordance with the functional analysis, our sequence and structural
analysis revealed the absence of a catalytic histidine residue, crucial
for the proposed deacylase mechanism and found in all other hitherto
described sirtuins (Sauve, 2010,
Yuan and Marmorstein, 2012)
and that may explain the apparent absence of deacylation activity in
SirTMs. On the other hand, our data show a robust MARylation activity by
SirTMs that is highly specific for the GcvH-L, a protein encoded by the
same operon. Furthermore, the SirTM-dependent ADP-ribosylation is
specifically and efficiently reversed by the pathogenic macrodomain
proteins. Collectively, our data argue for protein ADP-ribosyl
transferase as the primary activity of SirTMs.
Operon Proteins in the Response to Oxidative Stress
The
data presented here strongly support the notion that ADP-ribosylation
of GcvH-L is dependent on its prior lipoylation. The operon-encoded
LplA2/GcvH-L pair is only distantly related to the canonical GCS, but
nonetheless, the catalytic and the lipoate attachment residues are
conserved in the proteins from pathogenic species. While S. pyogenesnaturally lacks a canonical GCS,S. aureuspossesses a complete GCS as well as the operon-encoded LplA2/GcvH-L pair (Spalding and Prigge, 2010 and this study). Interestingly, our analysis showed that the canonical SauLplA1 can modify both GcvH proteins, while SauLplA2 appears to have selectivity toward GcvH-L (Figure S2C).
This may indicate that lipoylation of GcvH-L is the preferred target
for LA attachment under conditions of operon activation.
Surmann et al., 2014),
consistent with an involvement in the oxidative stress response. This
is further supported by several observations: (1) LA was shown to
possess antioxidant properties in models of oxidative stress (Packer et al., 1995
), (2) it was reported that Mycobacterium tuberculosis
utilizes two components of the α-ketoacid dehydrogenase complex in a
lipoyl-dependent defense against the oxidative immune response of the
host (Bryk et al., 2002),
and (3) the operon is genetically associated with the putative
oxidoreductases LLM and OYE. Both enzymes are as yet uncharacterized.
However, their families have been implicated in detoxification of ROS (
Chaiyen et al., 2012,Odat et al., 2007),
and we show that the oxidoreductase OYE directly interacts with
lipoylated GcvH-L. These findings suggest that GcvH-L could act as
carrier protein for the ROS scavenging lipoyl moiety and/or as a
substrate for oxidoreductases. Evidence obtained from the fungal
pathogenC. albicans in this study points toward an involvement of SirTMs in the oxidative stress response. Accordingly, in C. albicans the expression of mfs1
is very low under standard growth conditions, and the gene is only
expressed to a considerable degree upon oxidative stress either
chemically induced (Enjalbert et al., 2009) or by exposure to human blood (Fradin et al., 2003
).
Collectively, these results are consistent with a primary cellular
function of Mfs1 when cells encounter oxidative stress, such as upon
host interactions. In line with a specific function in host-pathogen
interactions, engulfment of S. aureusby host (human) cells led to an induction of the macrodomain encoded in the SirTM operon (Surmann et al., 2014).
Combining
these observations together with the high prevalence of the SirTM
operon in known pathogenic microorganisms and the importance of lipoate
scavenging for microbial pathogenesis (Spalding and Prigge, 2010),
we propose that the SirTM operon or SirTM-macrodomain fusion proteins
in eukaryotic microbes modulate the response of microbial pathogens to
oxidative stress and host-pathogen interactions.
Crosstalk between Lipoylation and ADP-Ribosylation
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