Intracellular monoADP-ribosylation modifies proteins that have roles in cell signalling and metabolism, such as the chaperone GRP78⁄ BiP, the b-subunit of heterotrimeric G-proteins and gl
Trang 1Physiological relevance of the endogenous
mono(ADP-ribosyl)ation of cellular proteins
Maria Di Girolamo, Nadia Dani, Annalisa Stilla and Daniela Corda
Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti, Italy
Enzyme-modulated mono(ADP-ribosyl)ation was
ori-ginally identified as the mechanism of action of several
of the bacterial toxins [1] The diphtheria, cholera,
per-tussis and clostridia toxins are
mono(ADP-ribo-syl)transferases (ARTs; EC 2.4.2.31), and they are
known to cause various pathologies after their
translo-cation into mammalian host cells Once inside the cell,
they act by modifying specific host cell proteins, such
as elongation factor 2, the a-subunit of the
hetero-trimeric GTP-binding (G) proteins, the small GTPases
Rho and Rac, and monomeric actin ([2–4], and
refer-ences therein)
More recently, a series of enzymes that are related to
these toxins have been identified in cells, and their
potential physiological roles have been explored ([5,6] and references therein; Table 1) The best known of these ARTs are ectoenzymes that are either glycosyl-phosphatidylinositol (GPI)-anchored or secretory Both the toxins and these toxin-related eukaryotic ARTs function through the transfer of an ADP-ribose residue from bNAD+to a specific amino acid of the acceptor protein, with the creation of an N- or S-glycosidic link-age and the release of nicotinamide The free amino acid arginine (the most frequently modified residue of the protein substrates) and its analogue agmatine have been widely used as substrates to characterize the enzymatic activities of these mono(ADP-ribosyl)transferases [7] The mono(ADP-ribosyl)ation reaction is distinct from
Keywords
mono(ribosyl)ation;
ADP-ribosyltransferase; ART; G-protein; defensin;
apoptosis; P 2 X 7
Correspondence
D Corda or M Di Girolamo, Consorzio
Mario Negri Sud, Department of Cell
Biology and Oncology, 66030 Santa Maria
Imbaro (Chieti), Italy
Fax: +39 0872 570 412
Tel: +39 0872 570 338
E-mail: corda@negrisud.it,
mdigirolamo@negrisud.it
Website: http://www.negrisud.it/en/dcbo
(Received 18 April 2005, accepted 18 July
2005)
doi:10.1111/j.1742-4658.2005.04876.x
The mono(ADP-ribosyl)ation reaction is a post-translational modification that is catalysed by both bacterial toxins and eukaryotic enzymes, and that results in the transfer of ADP-ribose from bNAD+ to various acceptor proteins In mammals, both intracellular and extracellular reactions have been described; the latter are due to glycosylphosphatidylinositol-anchored
or secreted enzymes that are able to modify their targets, which include the purinergic receptor P2X7, the defensins and the integrins Intracellular mono(ADP-ribosyl)ation modifies proteins that have roles in cell signalling and metabolism, such as the chaperone GRP78⁄ BiP, the b-subunit of heterotrimeric G-proteins and glutamate dehydrogenase The molecular identification of the intracellular enzymes, however, is still missing A better molecular understanding of this reaction will help in the full definition of its role in cell physiology and pathology
Abbreviations
ART, mono(ADP-ribosyl)transferase; ARTT, ADP-ribosylating turn-turn; DRAG, dinitrogenase reductase activating glycohydrolase; DRAT, dinitrogenase reductase ADP-ribosyltransferase; FGF-2, basic fibroblast growth factor; G-protein, GTP binding protein; GPI, glycosyl-phosphatidylinositol; IDDM, insulin-dependent diabetes mellitus; MIBG, meta-iodobenzylguanidine; NADase, NAD glycohydrolase; PARP, poly(ADP-ribose) polymerases; PDGF, platelet-derived growth factor.
Trang 2that catalysed by the poly(ADP-ribose) polymerases
(PARPs; EC 2.4.2.30), which instead transfer branched
polymers of ADP-ribose to their target proteins, via
an O-glycosidic bond (reviewed in [7a])
In mammalian cells, the mono(ADP-ribosyl)ation
reaction is also regulated by enzymes that are able
to reverse these post-translational modifications: the
cytosolic ADP-ribosyl hydrolases and the cytosolic and
extracellular pyrophosphatases [8,9] With the former,
the protein-ADP-ribose linkage is hydrolysed to release
the ADP-ribose moiety, while with the
pyrophospha-tases, it is the pyrophosphate linkage that is
hydro-lysed, to release AMP and thus to leave a
ribosylphosphate attached to the protein
The mammalian
ecto-ADP-ribosyl-transferases (ARTs)
The mammalian ARTs are coded for by a family of
structurally and functionally related genes To date,
five mammalian enzymes (ART1–5) have been cloned,
although only four of these are expressed in humans,
due to a defective art2 gene that has a stop signal in
the coding region Conversely, there are six ARTs
expressed in mouse, due to the duplication of the art2
gene ([5,6,10], and references therein)
This enzyme family shares very limited amino acid
sequence identity, with 20–30% seen among the ART
paralogue members within any species; the exception
here is mouse ART2.1 and ART2.2, where their
sequence identity (85%) indicates the recent
evolution-ary duplication of the mouse art2 gene [11] The
ART2 enzymes have also been cloned from rat, and in
this case the two isoforms are known as ART2.a and
ART2.b and they are coded for by two alleles of a
single-copy gene [12] These allelic differences between
the rat art2a and art2b genes result in a sequence
variation of only 10 amino acids between the ART2.a
and ART2.b proteins, although this alters their
enzymatic properties: while both can catalyze the hydrolysis of NAD to ADP-ribose and nicotinamide, only ART2.b is capable of auto-ADP-ribosylation [13]
As the human and mouse genome sequences have been completely determined, all of the recognizable mem-bers of these toxin-related ARTs have now been identi-fied for these two species, with the identity among orthologues ranging from 75% to 85% As an exam-ple, the deduced amino-acid sequence of mouse ART1, which was the first cloned and characterized mamma-lian arginine-specific ART, is 77% and 73% identical
to human and rabbit ART1, respectively [11,14] Despite the low similarity at the level of their amino-acid sequences, there are common structural features that characterize this family of mammalian ARTs [15,16] The catalytic domain of these enzymes
is completely coded for by a single exon in all of the ARTs, and it contains a conserved glutamate residue that has been demonstrated to be crucial for the cata-lytic activity of the bacterial toxins and of ART1 and ART2 (mouse and rat) by site-specific mutagenesis In ART1 from rabbit, even the conservative glutamate
240 to aspartate (E240D) substitution abolishes the transfer of ADP-ribose to the arginine used as an acceptor; the neighbouring E238 has also been shown
to be important for ADP-ribose transfer [17,18] In several of the ARTs, the replacement of this second glutamate abolishes the ability of these transferases to use arginine as acceptors, thus further supporting the hypothesis that this region is involved in substrate recognition [19] According to the structural model proposed by Rappuoli and colleagues [16], this cata-lytic domain is composed of 70–100 amino acids and consists of three regions Region 1, which is near the N-terminal portion of the protein and is characterized either by a conserved histidine (as in diphtheria toxin, ART3 and the PARPs) or by a conserved arginine (as in pertussis toxin, cholera toxin, the heat-labile enterotoxins and the other ARTs); region 2, which is
Table 1 Mammalian ARTs See text for details and relevant references.
Ectoenzymes
ART1 Human, rat, mouse Integrin, defensin, FGF-2, PDGFBB ⁄ Arg Inhibits substrate activity
Endoenzymes
Trang 3characterized either by hydrophobic amino acids that
are involved in NAD+binding or by the
serine-x-ser-ine motif (where x represents threonserine-x-ser-ine, serserine-x-ser-ine or
alan-ine); and region 3, which is highly acidic and is
characterized by the conserved glutamate residue The
arginine-serine-glutamate-x-glutamate motif (R-S-EQE;
which spans regions 1–2–3, respectively) is present in
cholera toxin and in ART1, 2 and 5, and it is typical
of the arginine-specific ARTs This motif is missing in
ART3 and ART4 [20,21] Through a comparative
ana-lysis of crystallographic structures, Han and Tainer
[19] have more recently extended the significance of the
region 3 sequences by identifying an ADP-ribosylating
turn-turn (ARTT) motif that they have implicated in
substrate recognition Consistent with the relevance of
the ARTT motif, it has been shown that the
auto-ADP-ribosylation of ART2.b is abolished by
muta-tions of its R204, which is part of the ARTT motif
Similarly, if the Y204 of ART2.a is mutated to an
arginine (Y204R), it is possible to promote ART2.a
auto-ADP-ribosylation [22]
The other common structural features of the ARTs
include an a-helix-rich N-terminal region, which
repre-sents a signal sequence for extracellular proteins, and a
C-terminal region folded into b-sheets, which is
char-acteristic of GPI-anchored membrane proteins [23] As
mammalian (human and mouse) ART5 does not
con-tain this hydrophobic C-terminal signal sequence, it is
a secreted protein [24] Finally, there are four cysteines
involved in disulfide bridge formation that are
con-served among all of the ART isoforms Thus,
accord-ing to the rat ART2.2 crystal structure, C21 (C53 in
mART1) forms a disulfide bond with another
con-served cysteine at 223 (C272 in mART1) at the
C-ter-minus of the molecule, which stabilizes the folding of
the a-helix-rich domain [11] C121 and C173 form the
second disulfide bond, which is also located at the
pro-tein surface and is also important for propro-tein
stabiliza-tion [25]
The possibility that the N- and C-terminal domains
of the ARTs are involved in the regulation of ART
activity has been recently investigated by measuring
both the transferase and NAD glycohydrolase
(NADase) activities of truncated mutants of ART1
[26] In mouse ART1, the amino acids at 24–38 (an
ART1-specific extension) modulate both the
trans-ferase and NADase activities, and amino acids 39–45
(a common ART coil) are essential for both activities
The removal of the C-terminal basic domain decreases
the transferase, but enhances the NADase activity The
N- and C-terminal regions of ART1 are therefore
required for its transferase activity, while the enhanced
NADase activity of the shorter mutants indicates that
there are sequences outside of the catalytic site that exert structural constraints, and that modulate the sub-strate specificity and catalytic activity [26]
The ecto-ARTs: expression and function
ART1 is predominantly expressed in skeletal muscle, heart and lung, and in neutrophils and T-cell lym-phoma cells [27,28] Its substrates include integrin a7
in mouse skeletal muscle cells, where ADP-ribosylation has been proposed to have a role in myogenesis, as an increase in arginine-specific ADP-ribosylation has been observed during their differentiation into myotubes [29]
Of note, the human defensin HNP-1 is among the most recently identified substrates of ART1 [30] The defensins are 2–6 kDa cationic peptides that are con-sidered to be the major components of innate anti-microbial immunity, and that are thought to act by disrupting the microbial membrane [31] They can also be considered to be components of adaptive immunity, because cytokine stimulation of human natural killer cells and T- and B-lymphocytes leads to the production of the defensins Interestingly, a-defen-sins 1–3 are secreted by CD8 T-cells from immuno-logically stable HIV-1-infected individuals (long-term nonprogressors) and they are able to suppress HIV-1 replication [32]
Thus ART1 has been shown to modify HNP-1 on R14 in an in vitro assay [30] This ADP-ribosylated HNP-1 loses its antimicrobial and cytotoxic activity, although it significantly increases the release of IL-8 from A549 cells, as compared to unmodified HNP-1 Conversely, the two peptides (unmodified and ADP-ribosylated) have similar chemotactic activities when evaluated for their ability to recruit T-lymphocytes [30] These data are consistent with the concept that, once modified, HNP-1 acquires specific biological activities that can result in the recruitment of neutro-phils (by the release of IL-8 from epithelial cells) and
in the modulation of its own antimicrobial and cyto-toxic activities These latter aspects are particularly relevant, as this study also identified ADP-ribosylated HNP-1 in the bronchoalveolar lavage fluid from smok-ers (but not from nonsmoksmok-ers); this would indicate that ADP-ribosylated HNP-1 is produced during the inflammatory response (and loses its antimicrobial activity) The relevance of these data also resides in the fact that this was the first demonstration of endo-genous ADP-ribosylation in humans [30,33]
Additional substrates of ART1 have been identified
in various different cell lines overexpressing this
Trang 4ecto-enzyme, and these include growth factors and
mem-brane receptors ART1-transfected rat adenocarcinoma
(NMU) cells were used to demonstrate
ADP-ribosyla-tion of basic fibroblast growth factor (FGF-2), which
had been detected initially on the surface of adult
bovine aortic endothelial and human hepatoma cells
[34,35] As FGF-2 has a high affinity for heparin, it is
localized and possibly sequestered by the heparin
sul-fates on the cell surface and in the extracellular matrix
Heparin also inhibits the ADP-ribosylation reaction,
which would imply that the heparin binding of FGF-2
and its ADP-ribosylation are mutually exclusive
Fur-thermore, the ADP-ribosylated site of FGF-2 is in its
receptor-binding domain, and so it is possible that
ADP-ribosylation modulates the binding of FGF-2 to
its receptor and to heparin, thus regulating its
avail-ability to the cell [34,35]
In ART1-transfected V79 Chinese hamster lung
fibroblasts, platelet-derived growth factor-BB
(PDGF-BB) is the best substrate for ART1, whereas its
struc-tural homologue PDGF-AA is not a substrate [36]
ADP-ribosylated PDGF-BB loses its ability to
stimu-late mitogenic and chemotactic responses in human
pulmonary smooth muscle cells, and it shows a
reduced capacity for binding to PDGF receptors in
competition-binding experiments, as compared to
unmodified BB [36] This indicates that
PDGF-BB-dependent signalling can be regulated by ART1
activity at the cell surface
When the EL-4 mouse T-cell lymphoma cell line was
stably transfected with ART1, the T-cell receptor
sig-nalling was inhibited in the presence of NAD via the
ADP-ribosylation of integrin LFA-1 and other
co-receptor proteins [37,38] These effects have been
pro-posed to result from a failure of the T-cell receptors
and coreceptors to associate into a functional receptor
cluster Thus these T-cell responses would be
modula-ted by mono(ADP-ribosyl)ation of cell surface proteins
[37,38]
In general, the role of the ARTs in T-cell signalling is
not clear, although the expression of ART2 on T-cells
has been well characterized Thus ART2 is known to be
expressed in resting T-cells and in natural killer cells,
and it appears to be specific to the immune system This
presence of ART2 on the surface of immune cells would
thus suggest an immunomodulatory activity, and
indeed, a significant disposition to develop autoimmune
diabetes has been shown to depend on the absence of
ART2 expression on rat T-cells [39–42]
What can perhaps be defined as the most intriguing
function of ART2 was recently uncovered by
Koch-Nolte and coworkers, namely that
ADP-ribosy-lation activates the P2X7 purinoceptor ([43], Fig 1A)
P2X7 is a member of the P2X family of ATP-gated ion channels, and it is widely expressed on several types of blood cells [44] This specific purinoceptor has attrac-ted interest because of its particular ability to induce the formation of large membrane pores Thus the acti-vation of P2X7 with millimolar concentrations of ATP triggers calcium fluxes, phosphatidylserine exposure and apoptosis [44] These same effects are trig-gered by NAD at micromolar concentrations via the ADP-ribosylation of P2X7 However, these effects are not seen in ART2-deficient T-cells, demonstrating that the activation of P2X7 by NAD is ART2-dependent [43] These data provide an explanation for the previ-ous demonstrations that extracellular NAD induces rapid apoptosis in naive T-cells by a mechanism invol-ving ADP-ribosylation of cell surface molecules [45] Altogether, these data show that not only are ART1 and ART2 expressed in cells of the immune system, but also that these two arginine-specific ARTs have a clear role in the regulation of the immune response However, it is somewhat disappointing that one of the most interesting physiological roles has been defined for an ART that is not expressed in human cells Thus,
it is important to understand what the human counter-part of mouse ART2 might be, and whether these mouse T-cell effects can be extended to human cells The biological functions of ART3, ART4 and ART5 remain poorly defined [20,24] ART3 and ART5 are strongly expressed in human testis, whereas ART4 is preferentially expressed in human lymphatic tissue In human monocytes, the cell-surface ADP-ribosylated proteins are modified on their cys-teine residues, suggesting that ART3 and ART4 are cysteine-specific ARTs [21] This is consistent with the observation that in in vitro assays neither of these two ARTs displays arginine-specific enzymatic activity when expressed in and purified from Sf9 insect cells In the same study, human ART5 was seen to be an argin-ine-specific ART, unlike mouse ART5, which shows a potent NADase activity [5]
A point that still needs to be clarified is the occur-rence of the extracellular NAD+ that is required to sustain the ADP-ribosylation reaction The steady-state concentration of NAD+in the serum of healthy individuals is around 0.1 lm, and it can be kept low
by the extracellular NAD-glycohydrolase CD38 (both soluble and membrane-associated) [46]; thus, to be util-ized by ecto-ARTs, extracellular NAD+ should reach the concentration of 1–10 lm that is required for ADP-ribosylation of P2X7 [43], or higher if the Km of the ARTs (from in vitro assays) is considered [47] The probable mechanism is that NAD+ is released from cells, where its concentration is in the range of
Trang 50.5–1.0 mm, as a consequence either of cell lysis during
inflammatory immune reactions and apoptosis, or of
nonlytic release, for example through the connexin 43
channels [48]
Intracellular
mono(ADP-ribosyl)tran-ferases and endogenous substrates
Although the ARTs that are able to modify
extracellu-lar proteins are the only well characterized family,
mono(ADP-ribosyl)ation has also been demonstrated
for intracellular proteins involved in cell signalling and
metabolism (Table 1; [6] and references therein) The
enzymatic activities involved here have been shown to
be both cytosolic and membrane associated, although
there is very little further information available
concerning their identities The first example of a
well defined intracellular ADP-ribosylation cycle was
reported in prokaryotes An intracellular ART activ-ity (dinitrogenase reductase ADP-ribosyltransferase; DRAT) was characterized in the photosynthetic bac-terium Rodospirillum rubrum [49], where it regulates nitrogen fixation through mono(ADP-ribosyl)ation on R101 of the dinitrogenase reductase [50] This reaction
is reversible and the dinitrogenase reductase is fully reactivated by an ADP-ribosylarginine-hydrolase known as dinitrogenase reductase activating glyco-hydrolase (DRAG) Surprisingly, there is no significant amino acid sequence similarity between DRAT and the bacterial toxins that have ADP-ribosyltransferase activity; only a few key residues are conserved across the two families [23]
The same scenario could occur for the two families
of mammalian ADP-ribosyltransferases: the ecto-ARTs and the endo-ARTs These endo-ARTs appear to be part of a completely different family of proteins that
A
B
Fig 1 (A) Schematic representation of the
mammalian mono(ADP-ribosyl)ation
reac-tions The figure shows both the extracellular
mono(ADP-ribosyl)ation that is catalysed by
the ARTs and the intracellular reaction that is
catalysed by the yet undefined
mono(ADP-ribosyl)transferases The upper section
(extracellular space, OUT) shows the
ART2-dependent ADP-ribosylation of the P 2 X 7
purinergic receptor The ADP-ribosylated
receptor is activated and leads to T-cell
apop-tosis The lower section (intracellular space,
IN) shows the ADP-ribosylation ⁄
deribosyla-tion cycle of the heterotrimeric G-protein b
subunit that is catalysed by a
membrane-associated, intracellular
ADP-ribosyltransf-erase (iART) and by a cytosolic
ADP-ribo-sylhydrolase (ARH) The dashed arrow
indicates possible hormonal regulation of this
iART The effectors that are uncoupled from
the bc dimer by ADP-ribosylation are
indica-ted by the red line, while the red arrow
indi-cates coupling (see text for details) (B)
Schematic representation of the product of a
mono(ADP-ribosyl)ation reaction The
N-gly-cosidic linkage between the ADP-ribose
residue and Arg129 on the heterotrimeric
G-protein b-subunit is illustrated.
Trang 6shows no structural relationship to the ecto-ARTs
des-cribed above An example consistent with this is seen
in the sirtuin family It has recently been shown that
yeast silent information regulator 2 protein (Sir2p, a
NAD+-dependent histone⁄ protein deacetylase) has
ADP-ribosyltransferase activity, and while it
deacety-lates histones, it also catalyses the
mono(ADP-ribo-syl)ation of the removed acetyl group [51,52] Clearly,
this is not a ‘classical’ reaction that involves the
modi-fication of a target protein, but it involves small
mole-cule substrates It is therefore similar to that seen for a
bacterial ribosyltransferase that is able to
ADP-ribosylate and inactivate the antibiotic rifampicin [53],
and a yeast enzyme that is able to transfer ADP-ribose
from NAD+ to a phosphate group in tRNA [54] To
date, seven human homologues of Sir2p have been
des-cribed (sirtuins 1–7), and they are characterized by
ART activity [51,52], although they share no obvious
sequence homologies with the ARTs themselves
Obvi-ously, the sirtuins could represent the prototypes of a
novel intracellular ART family The alternative
possi-bility that the ectocellular ARTs can modify
intracellu-lar substrates can also be considered In this situation,
either there needs to be a search for new isoforms that
do not contain the signal peptide, or it needs to be
shown that one or more of the ecto-ARTs can be shed
from the membrane and can translocate into the
cyto-plasm This could be achieved in a way similar to that
of the bacterial toxins, which have their own specific
receptors on the plasma membrane [55] We are now
actively working to identify and define potential new
intracellular ART isoforms
The intracellular mono(ADP-ribosyl)ation reactions
have been associated with cell signalling and metabolism
in intact cells They modify three substrate proteins: the
endoplasmic reticulum-resident chaperone GRP78⁄ BiP,
the b-subunit of heterotrimeric G-proteins, and the
mitochondrial glutamate dehydrogenase GDH
The mono(ADP-ribosyl)ation of GRP78⁄ BiP leads
to its inactivation The modified GRP78⁄ BiP has been
detected in response to conditions that deplete the
endoplasmic reticulum of processible proteins or that
result in nutritional stress (such as lowered
tempera-ture, amino acid and glucose starvation), and has been
related to the rate of protein synthesis and processing
in intact Swiss 3T3 and GH3 pituitary cells [56–58]
According to the model proposed by Laitusis and
col-leagues [56], in cells with high rates of protein
synthe-sis, unmodified GRP78⁄ BiP is complexed with protein
folding intermediates; a slowing of protein synthesis
results in accumulation of the free, active form of
GRP78⁄ BiP, which is subjected to subsequent
inactiva-tion by ADP-ribosylainactiva-tion The ADP-ribosylated form
of the chaperone thus provides a buffering system that allows the rates of protein processing to be balanced with those of protein synthesis It should be noted that while this mono(ADP-ribosyl)ation occurs intracellu-larly, from a topological point of view the catalytic domain of the enzyme involved (that has not yet been characterized) needs to be located in the lumen of the endoplasmic reticulum to modify its substrate, GRP78⁄ BiP Thus, this intracellular reaction occurs out of the cytosolic compartment
Direct evidence of functional, intracellular mono-(ADP-ribosyl)ation has been reported for the G-protein b-subunit ([59], Fig 1) This reaction modifies R129 of the b-subunit (Fig 1B) and is catalysed by a plasma-membrane-associated, but not GPI-anchored, intracellular ART that has not yet been molecularly characterized The mono(ADP-ribosyl)ated b-subunit becomes the substrate of a cytosolic, ribosylhydro-lase ([59], Fig 1), which completes a cellular ADP-ribosylation⁄ de-ADP-ribosylation cycle that controls the activation⁄ inactivation of the bc-dimer Import-antly, b-subunit mono(ADP-ribosyl)ation has also been detected in intact cells, under both resting [59] and sti-mulated conditions, thus indicating the physiological potential of this reaction In intact cells under resting conditions, approximately 0.2% of the total bc-hetero-dimer is modified; this could correspond to a cellular pool of free bc-heterodimer that remains inactive This hypothesis is supported by the demonstration that the b-subunit is modified only as a free heterodimer, and that mono(ADP-ribosyl)ation inactivates the b-subunit by impairing its interactions with its effector enzymes This has been shown directly in the case of type 1 adenylyl cyclase, phosphoinositide 3-kinase and phospholipase
C [59,60] Thus, the ADP-ribosylation⁄ deribosylation cycle modulates the function of the b-subunit It is of particular interest here that the ADP-ribosylation of the b-subunit has also been shown to be under hormonal control: it can be increased upon activation of specific G-protein-coupled receptors (e.g thrombin, serotonin and cholecystokinin receptors), indicating that the active bc-heterodimer released from different classes of G-pro-teins can be a substrate for the endogenous mono(ADP-ribosyl)transferase [60] Thus, while activation of these receptors will lead to the activation and dissociation of the G-protein a- and the bc-subunits, this can initiate a parallel inactivation of bc-subunit function that would potentially regulate the duration of bc and a signalling, through the selective termination of the bc function
An ADP-ribosylation⁄ deribosylation cycle has also been proposed to occur in mitochondria, and involves the cysteine-specific ADP-ribosylation of mitochondrial GDH in intact Hep-G2 cells [61] The modified
Trang 7cysteine has been recently identified as Cys119 [61a].
As for GRP78⁄ BiP and for the G-protein b-subunit,
however, the nature of this ADP-ribosyltransferase
activity remains uncharacterized This cycle appears to
be completed by an ADP-ribosylcysteine hydrolase
that is also present in mitochondria
Further substrates of mono(ADP-ribosyl)ation have
also been identified, including the membrane-fissioning
protein CtBP3⁄ BARS [62,63], and the cytoskeletal
pro-teins actin, tubulin and desmin [64–66] However, to
date there has been no direct evidence for their in vivo
modification Overall, a better understanding of the
various and diverse biological roles of
ADP-ribosyla-tion of cellular proteins and peptides will be essential
to fully define the role of this modification in normal
and disease states
The ADP-ribosylation reaction as a potential
new drug target
A lack of ART2 expression has been correlated with
an enhanced sensitivity to autoimmune disease in
several animal models [40,67] For example, in
dia-betes-prone BioBreeding (DP-BB) rats, a model for
autoimmune insulin-dependent diabetes mellitus
(IDDM), a defective expression of ART2 in their
T-cells is associated with an increased susceptibility to
the disease [39–42] Conversely, the prevention of
IDDM has been described in the same DP-BB rats
fol-lowing a transfusion with ART2-positive T-cells The
development of IDDM has also been observed in
dia-betes-resistant BioBreeding (DR-BB) rats when they
are treated with a monoclonal antibody against ART2
[42] Thus, ART2 expression appears to confer
protec-tion to IDDM in this animal model of the disease [42]
In the ART2.2 natural knock-out NZW mouse, the
development of a lupus-like glomerulonephritis has
been shown, again supporting the hypothesis that
ART2-positive T-cells confer protection against
auto-immune disease [68] Polymorphisms have been
repor-ted for ART2.1 in the C57B1⁄ 6 mouse, where a stop
codon at position 481 leads to an ART enzyme that
lacks the GPI-anchor site and that has a reduced
transferase activity [69] However, it is important to
stress that in these mouse models, disease development
is under the control of several genetic factors, and thus
a reduction or absence of ART2 expression is
neces-sary but not sufficient for the onset of autoimmune
pathologies In line with this, both the NZW mouse
and other mouse models that are natural knock-outs
for ART2.1 (e.g C57B1⁄ 6, BXSB), and the
experi-mentally induced ART2 double knock-out do not
show any evident immunological defects [70]
All of the data discussed above are consistent with the hypothesis that NAD-induced cell death via the acti-vation of the P2X7 receptor has a role in immune responses However, as ART2 knock-out mice show normal numbers and a normal distribution of T-cells [70], this NAD-dependent cell death cannot be crucial in the generation and maintenance of the T-cell Rather, it
is possible that ART2-induced T-cell death has a role during mechanical tissue injury or microbial inflamma-tory processes with severe cytolysis Under these circum-stances, the massive release of intracellular antigens is combined with high local concentrations of inflamma-tory cytokines, raising the danger of activation of auto-reactive T-cells Thus ART2-induced T-cell death could provide a safeguard mechanism against the undesirable activation of irrelevant and potentially autoreactive T-cells during an inflammatory response [43] When extended to the identification of the counterpart in human cells, these findings open the exciting prospect of using NAD and its metabolites to modify the function
of the P2X7receptor and other purinoceptors [43] Other mechanisms that can benefit from ADP-ribosylation-related drugs have emerged from a number
of recent reports ADP-ribosylation has been coupled
to intracellular events that are associated with smooth muscle cell vasoreactivity, cytoskeletal integrity and free radical damage [71,72] Additionally, there is evidence that ADP-ribosylation is required for smooth muscle cell proliferation [71,72] Recent data have provided a direct link between mono(ADP-ribosyl)ation and smooth muscle cell proliferation and migration: meta-iodobenzylguanidine (MIBG), a selective inhibitor of arginine-dependent mono(ADP-ribosyl)ation, blocks the stimulation of DNA and RNA synthesis, prevents smooth muscle cell migration, and suppresses the induc-tion of c-fos and c-myc gene expression MIBG pro-motes the phosphorylation of the Rho effector PRK1⁄ 2, suggesting that mono(ADP-ribosyl)ation participates in
a Rho-dependent signalling pathway that is required for immediate early gene expression Furthermore, expres-sion of the c-fos gene is the earliest proliferative event that has shown sensitivity to MIBG treatment, and it represents a novel mechanism by which mono(ADP-ribosyl)ation can influence cellular processes [71,72]
As the heterotrimeric G-proteins have key roles in cell regulation and the bc complex is essential in a wide range of G-protein functions, including apoptosis, chemotaxis, secretion and cell proliferation and differentiation, we believe that our finding of the mono(ADP-ribosyl)ation of the endogenous b-subunit identifies a potential target for drug development Indeed, recent data have shown that cellular invasion induced by src, met and leptin can be abrogated by
Trang 8constitutively activated forms of the Gao⁄ i subunits,
and can be induced by the coexpression of Gb1c2 [73]
Moreover, depletion of free Gbc heterodimers by
the C-terminus of the b adrenergic receptor kinase
(ct-bARK) results in a remarkable decrease in cellular
adhesion and spreading on a collagen matrix [74]
Thus Gbc dimers can be seen to be positive effectors
of invasion pathways that are induced by oncogenes
and epigenetic factors
In line with the proposal that the ADP-ribosylated
defensins represent tools for the treatment of pulmonary
inflammation and other lung diseases [30,33], other
ADP-ribosylated peptides that mimic the modified
por-tions of the various ADP-ribosylation substrates and
inhibitors of the ADP-ribosylation reaction itself show
potential for the treatment of various pathologies,
inclu-ding autoimmune syndromes and proliferative diseases
Acknowledgements
We wish to thank Dr C.P Berrie for editorial
assist-ance, Ms E Fontana for preparation of the Figures
and the Italian Association for Cancer Research
(AIRC, Milano, Italy), Telethon, Italy (project
n GGP030295) and the MIUR for financial support
N.D is supported by a fellowship from AIRC
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