From the Department of Neurochemistry, Institute of
Neurology, 1 Wakefield Street, London WC1N 1PJ, Great Britain, the
§ Glaxo-IMCB Group, Institute of Molecular and Cell Biology,
10 Kent Ridge Crescent, Singapore 119076, and the
Department of
Medicine, University College London, University Street, London WC1E
6JJ, Great Britain
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ABSTRACT |
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Rac1 is a member of the Rho family of small molecular mass GTPases that act as molecular switches to control actin-based cell morphology as well as cell growth and differentiation. Rac1 and Rac2 are specifically required for superoxide formation by components of the NADPH oxidase. In binding assays, Rac1 interacts directly with p67phox, but not with the other oxidase components: cytochrome b, p40phox, or p47phox (Prigmore, E., Ahmed, S., Best, A., Kozma, R., Manser, E., Segal, A. W., and Lim, L. (1995) J. Biol. Chem. 270, 10717-10722). Here, the Rac1/2 interaction with p67phox has been characterized further. Rac1 and Rac2 can bind to p67phox amino acid residues 170-199, and the N terminus (amino acids 1-192) of p67phox can be used as a specific inhibitor of Rac signaling. Deletion of p67phox C-terminal sequences (amino acids 193-526), the C-terminal SH3 domain (amino acids 470-526), or the polyproline-rich motif (amino acids 226-236) stimulates Rac1 binding by ~8-fold. p21Cdc42Hs/Rac-activated kinase (PAK) phosphorylates p67phox amino acid residues adjacent to the Rac1/2-binding site, and this phosphorylation is stimulated by deletion of the C-terminal SH3 domain or the polyproline-rich motif. These data suggest a role for cryptic Rac-binding and PAK phosphorylation sites of p67phox in control of the NADPH oxidase.
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INTRODUCTION |
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Members of the Rho family, Rac1, Cdc42Hs, and RhoA, play essential roles in growth factor-mediated changes in cell morphology associated with the formation of actin microfilaments and "focal complexes" (1-4). They also act downstream of Ras in distinct parts of the transformation process (5-8) and activate Jun N-terminal protein kinase (9, 10) and entry into the G1 phase of the cell cycle (11). Rho family interacting proteins that may play a role as effectors in these signaling pathways include kinases (12-15), GTPase-activating proteins (e.g. n-chimaerin) (16), and adaptors (e.g. p67phox) (17, 18).
Rac1, cytochrome b (gp91phox and p22phox), p67phox, and p47phox comprise the minimal components necessary for superoxide formation by the NADPH oxidase in vitro (19). Another oxidase component, p40phox (20), interacts with both p67phox and p47phox (21, 22), but its function is not clear. Rac1 binds p67phox (17, 18), and p67phox can bind p47phox, which in turn can bind p22phox through an SH3 domain-polyproline interaction (23-25). Reconstitution of the oxidase components in vitro does not accurately reflect complex formation in vivo. For example, the p67phox deletion mutant (amino acids 1-246) can replace p67phox in vitro, but not in vivo (26). Both p47phox and p67phox are phosphorylated during oxidase activation (27-30), and the isolation and identification of the kinases involved are essential to gain a better understanding of the mechanism by which complex formation and oxidase activity are controlled in vivo. PAK has been shown to be activated by fMet-Leu-Phe in neutrophils and to phosphorylate p47phox in vitro (31). Hitherto, kinases that can phosphorylate p67phox have not been identified.
We have been using the NADPH oxidase as a model protein complex to investigate the mechanism by which Rho family GTPases activate cellular pathways. In this study, the Rac1-p67phox interaction has been investigated in more detail. Using binding assays, we show that the Rac1-binding site of p67phox is cryptic, located between amino acids 170 and 199, and that the N-terminal fragment (amino acids 1-192) can be used as an inhibitor of the Rac signaling pathway. The binding sites in p67phox for Rac1 and p40phox are distinct. Recombinant PAK purified from Escherichia coli can phosphorylate p67phox; the phosphorylation site(s) are cryptic and located adjacent to the Rac1-binding site. Deletion of either the polyproline-rich sequence (aa1 226-236) or the C-terminal SH3 domain (aa 460-526) led to increases in Rac1 binding and PAK phosphorylation, suggesting that there is an intramolecular interaction between these two domains of p67phox that gives rise to the crypticity. Taken together, these data suggest that unfolding of p67phox, via disruption of a potential intramolecular "SH3 domain-polyproline" interaction, may play a role in control of the NADPH oxidase.
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MATERIALS AND METHODS |
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Cell Culture and Microinjection-- Swiss 3T3 fibroblast cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum and antibiotic/antimycotic (Life Technologies, Inc.) at 37 °C and 5% CO2. Swiss 3T3 cells were serum-starved for 24-48 h before being microinjected with p67phox protein (0.1-1 mg/ml) and observed by phase-contrast microscopy as described previously (4, 16).
Purification of p67phox from Insect
Cells--
Sf9 cells in IPL-41 medium (Life Technologies, Inc.)
containing 10% fetal calf serum were grown in suspension in an orbital shaker. 800 ml of cell culture were infected to saturation with p67phox cDNA containing baculovirus (or control, not
infected) and were harvested after 72 h. The cells were washed
with 50 mM Tris, pH 7.5, 50 mM NaCl, and 1 mM EDTA supplemented with 1 mM dithiothreitol, 1 mM diisopropyl fluorophosphate, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml
N-p-tosyl-L-lysine
chloromethyl ketone. The cells were disrupted by sonication (MSE
Soniprep), and cell debris was removed by centrifugation (30,000 × g, 15 min). At this stage, cell extracts were used either for experiments or for purification of p67phox by column
chromatography; a Hi-load Q-Sepharose column was used with a 50-500
mM NaCl gradient, followed by a phenyl-Sepharose column
eluted with a 1 to 0 M NH4SO4
gradient, resulting in a p67phox preparation of ~95% purity
as judged by SDS-PAGE.
Expression and Purification of Recombinant Proteins in E. coli--
Cdc42Hs, Rac1, p40phox, p67phox, PAK, and
p67phox deletion mutants were expressed in pGEX plasmids or
derivatives, and protein was purified using glutathione-Sepharose as
described previously (4, 32, 33) or as detailed below. p67phox
cDNA was cloned into pGEX-2T as described previously (19). The
C-terminal truncated forms of p67phox (aa 1-58, 1-192, and
1-238) were made by digestion of full-length p67phox in
pGEX-2T with two enzymes: EcoRI plus StuI,
Bpu11021, or BglII. The DNA was then purified,
blunt-ended with Klenow fragment, and religated with T4 DNA ligase. The
resulting plasmids contained the first 174, 376, and 714 nucleotides,
respectively, of the p67phox coding sequence. C-terminal
p67phox was made by digestion of full-length cDNA in
pGEX-2T with BamHI and EcoRI, followed by
purification of the DNA fragment starting from the internal
BamHI site to the EcoRI site (nucleotide 967 to
the end) and ligation into BamHI/EcoRI-digested
pGEX-3X. The other constructs were made by polymerase chain reaction
amplification using full-length p67phox cDNA in pGEX as a
template. In all cases, the sense primer contained a BamHI
site, and the antisense primer contained an EcoRI site. DNA
encoding aa 170-238 was digested with AvrII and
EcoRI, blunt-ended with Klenow fragment, and religated. This
new plasmid encoded aa 170-199. Mutations were made with the USE kit
(Amersham Pharmacia Biotech). In all cases, the correct sequence was
confirmed by nucleotide sequencing using an Applied Biosystems
automated sequencer. p67phox fragment 1-199 was a kind gift
from A. Hall (University College London, London). p67phox
deletion mutant protein fragment 1-192 was unstable and had to be used
immediately after purification. Protein was quantified by the method of
Bradford (34). p67phox deletion proteins were used as GST
fusions unless otherwise stated.
Cdc42Hs and Rac1 Binding Assays--
p21 probes were prepared by
incubating Cdc42Hs or Rac1 (5 µg) with 1 µl of
[-32P]GTP (6000 Ci/mmol, 10 mCi/ml, 1.6 µM; NEN Life Science Products) in 50 µl of exchange
buffer (50 mM NaCl, 25 mM Mes, pH 6.5, 25 mM Tris-HCl, pH 7.5, 1.25 mM EDTA, 1.25 mg/ml
bovine serum albumin, and 1.25 mM dithiothreitol) for 10 min at room temperature. Protein was directly applied to nitrocellulose
filters (5-20 µg) and then incubated with p21 probes in Petri dishes
containing 4 ml of binding buffer (50 mM NaCl, 25 mM Mes, pH 6.5, 25 mM Tris-HCl, pH 7.5, 1.25 mM MgCl2, 1.25 mg/ml bovine serum albumin, 1.25 mM dithiothreitol, and 0.5 mM GTP). Filters
were analyzed as described previously (18). Background binding was
variable, and therefore, it was essential to make comparisons of
p21-target interactions within the same experiment. p21 probes were
used uncleaved and cleaved.
Kinase Assays--
PAK was expressed as a GST fusion protein
and purified as described previously (35).
PAK-GST (96 kDa) was not
cleaved to allow visualization of potential p67phox
phosphorylation (67 kDa). In vitro kinase assays were
carried out as follows.
PAK protein (0.125-0.25 µg) was incubated
with 5 µg of either full-length p67phox or fragments in
kinase buffer (50 mM Hepes, pH 7.0, 5 mM
MgCl2, 5 mM MnCl2, 1 mM
dithiothreitol, and 0.5% Triton X-100) with 20 µM ATP/2
µCi of [
-32P]ATP (5000 Ci/mmol, 10 mCi/ml; Amersham
Pharmacia Biotech) for 15 min at 30 °C. The reaction was stopped by
the addition of 5× sample buffer, followed by boiling for 5 min. The
proteins were then separated by SDS-PAGE. The dye front was removed,
and the gels were stained with Coomassie Blue to visualize proteins.
Gels were then destained, fixed, and dried before being exposed to Kodak film for between 10 min and 5 h.
Immunoblot Analysis-- Proteins were separated on SDS-polyacrylamide gel, transferred to nitrocellulose by semidry blotting, and incubated with blocking buffer, followed by incubation with either anti-p40phox (20) or anti-Ste20 (Upstate Biotechnology, Inc.) antibodies. The blots were developed using ECL reagents (Amersham Pharmacia Biotech) to visualize the antibodies as described previously (18).
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RESULTS |
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Localization of the Rac1/2-binding Site of
p67phox--
To localize the Rac1-binding site of
p67phox, different fragments of this protein were expressed as
GST fusions and purified on glutathione affinity columns (Fig.
1A, arrowheads mark
full-length protein). p67phox proteins (~5 µg, based on the
percentage of full-length protein present in the sample) were then
dot-blotted onto nitrocellulose and probed with Rac1
Q61L-[-32P]GTP. Rac1 binding to full-length
p67phox was weaker than to aa 1-238 (
239-526) (Fig.
1B). Rac1 binding to N-terminal fragments of p67phox
(aa 1-238, 1-199, and 1-192) was similar. However, Rac1 did not bind
p67phox fragment 1-58 or 1-131 (Fig. 1B,
dots 9 and 10). This suggests that aa
131-192 of p67phox are essential for Rac1 binding. Next,
deletions of p67phox fragment 1-238 were examined. Rac1 bound
aa 170-238 and 170-199 of p67phox, although binding was
significantly weaker then that obtained with aa 1-192 (see below).
Rac1 did not bind to p67phox fragment 192-238 or the
C-terminal part of the protein (aa 300-526) (Fig. 1B). To
confirm that the Rac1-binding site of p67phox was localized to
aa 170-199, a deletion of 6 aa of full-length p67phox was made
(
178-184). This protein did not bind Rac1 (data not shown). Taken
together, these data suggest that the Rac1-binding site of
p67phox is located between aa 170-192 and that sequence 1-170
may stabilize the Rac1-p67phox interaction, possibly through
the presence of tetratricopeptide repeats (TPRs) (see
"Discussion"), but is not essential for binding.
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p67phox Fragment 1-192 Can Be Used as an Inhibitor of Rac Signaling-- The RBD of p67phox is highly specific for Rac1 in binding assays; it did not interact at all with Cdc42Hs or RhoA (see below). Thus, the RBD could serve as a specific inhibitor of signaling by preventing Rac1 from interacting with targets. To examine this possibility, the effect of p67phox fragment 1-192 on Rac1-dependent phorbol 12-myristate 13-acetate (PMA)-induced ruffling was investigated. Fig. 4 shows a time-lapse experiment of two cells, under phase-contrast microscopy, with the cell in panel A injected with p67phox protein fragment 1-192, showing that p67phox fragment 1-192 does indeed inhibit PMA-induced membrane ruffling. Furthermore, p67phox fragment 1-192 is specific in its effect on ruffling, as it did not inhibit the formation of either filopodia/retraction fibers (Cdc42Hs-mediated) or stress fibers (RhoA-mediated) (data not shown). We also examined the effects of the smaller fragments of p67phox, 170-199 and 170-238, which bind to Rac1, but much more weakly than fragment 1-192 (Fig. 3). These proteins gave variable results, although a degree of inhibition of PMA-induced ruffling did occur. A quantitative assay may be required to determine the effect of these smaller peptides on Rac1 signaling.
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Crypticity of the Rac1-binding Site-- Deletion of the C terminus of p67phox (aa 193-526) resulted in an 8-9-fold increase in Rac1 binding (Fig. 5). One possible explanation for this enhancement is that the C-terminal SH3 domain may bind the polyproline-rich sequence (at aa 226-236) and hinder Rac1 binding to aa 170-199. To examine this possibility, p67phox proteins with either the polyproline-rich sequence or the C-terminal SH3 domain deleted were tested for their ability to bind Rac1. Fig. 5 shows that, in both cases, there was an ~8-fold increase in Rac1 binding to the p67phox deletion mutants. These results are consistent with the idea of a cryptic binding site in p67phox for Rac1 due to the presence of an intramolecular SH3-polyproline interaction.
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Distinct Rac1- and p40phox-binding Sites on p67phox-- p40phox has been proposed to interact with two parts of p67phox: in the N-terminal region, where Rac1 also binds (22, 40), and in the C-terminal region between the two SH3 domains (21). Therefore, the possibility exists that Rac1 binding may be affected by p40phox. To investigate this, Rac1 binding to full-length p67phox or aa 1-192 was measured in the presence of increasing amounts of p40phox. The presence of p40phox at up to 10 times the concentration of p67phox did not affect Rac1 binding (Fig. 6A), thus making it unlikely that the binding sites of Rac1 and p40phox overlap.
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A PAK-like Kinase Copurifies with Insect Cell
p67phox--
We have previously reported that Cdc42Hs can
apparently bind p67phox purified from insect cells (18). To
localize the Cdc42Hs-binding site of p67phox, deletion mutants
were used in dot-blot assays as described for Rac1. Cdc42Hs did not
bind any of the deletion mutants or the full-length p67phox
protein produced in E. coli (data not shown). Nevertheless,
we observed good binding of Cdc42Hs to p67phox purified from
insect cells. A possible reason for this difference between E. coli-expressed and insect cell-expressed p67phox could be
that a Cdc42Hs-binding protein copurifies with the latter p67phox protein. To examine this, extracts from insect cells
infected with control virus were compared with extracts infected with
p67phox virus and with purified p67phox protein from
insect cells. Interestingly, a 68-kDa Cdc42Hs-binding band was present
in all three protein preparations (Fig.
7, lanes 1-3).
This band was found to migrate slightly above p67phox when a
Coomassie Blue-stained gel was overlaid with the x-ray film from the
Cdc42Hs binding experiment. The level of this protein was reduced in
the insect cell extracts expressing p67phox. The 68-kDa
Cdc42Hs-binding protein did not bind wild-type Rac1 under the
conditions of the assay (18). To determine whether this 68-kDa protein
could be a Ste20/PAK-like serine/threonine protein kinase present in
insects cells, an immunoblotting analysis was carried out using
anti-Ste20 antibody. This antibody is raised against the kinase domain
of Ste20 and does cross-react with mammalian neutrophil PAK proteins
(,
, and
isoforms).4 The anti-Ste20
antibody did indeed react against the 68-kDa protein present in insect
cell purified p67phox (Fig. 7, lane 9).
Furthermore, the purified p67phox preparations from insect
cells possessed a 68-kDa kinase activity (data not shown). Thus, the
Cdc42Hs binding to p67phox that we initially observed (18) is
an artifact due to the presence of a 68-kDa Cdc42Hs-binding protein in
insect cell p67phox preparations.
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PAK Phosphorylation Sites of p67phox Are
Cryptic--
Since a 68-kDa PAK-like protein copurified with
p67phox in insect cells, we examined whether PAK could
phosphorylate p67phox in vitro. Recombinant PAK
purified from E. coli was fully active and nonresponsive to
the presence of Rac1 or Cdc42Hs in either autophosphorylation (Fig.
8A) or myelin basic protein
phosphorylation (data not shown) assays. However, in some experiments,
we did observe the appearance of a novel phosphorylated band at around 68 kDa when Cdc42Hs or Rac1 was added (Fig. 8A,
lanes 2'-3'). This 68-kDa band is probably a
breakdown product of PAK-GST. Interestingly, if the recombinant PAK
protein was treated with alkaline phosphatase, its ability to
phosphorylate substrate was reduced significantly (data not shown),
suggesting that an E. coli kinase can activate PAK. The
recombinant PAK protein was able to phosphorylate p67phox (Fig.
8B). In attempts to localize the PAK phosphorylation sites of p67phox, we observed, as for the Rac1 binding, that deletion
of the C-terminal part (aa 239-526) stimulated PAK phosphorylation
(Fig. 8C). p67phox fragments 1-58, 1-131, and
300-526 were not phosphorylated by PAK. The best p67phox
protein fragment substrate for PAK was aa 170-238 (Fig. 8C,
lane 6), which has four potential phosphorylation
sites located between aa 203 and 233 (Fig.
9). Since aa 192-238 were not
phosphorylated by PAK, the peptide sequence between aa 170 and 191 must
be essential for PAK interaction. PAK phosphorylation of
p67phox was also stimulated by deletion of either the
C-terminal SH3 domain (aa 1-460) or the polyproline-rich sequence
(
226-236), suggesting that an SH3-polyproline interaction gives
rise to cryptic PAK phosphorylation sites (Fig. 8D, compare
lanes 2 and 6).
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DISCUSSION |
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Rac1/2-binding Site of p67phox--
Rac1 interacts
directly with p67phox in a GTP-dependent manner
(17, 18). Amino acid residues 1-199 are sufficient for Rac1 binding
(17). In this study, we have localized the Rac1/2-binding site to aa
170-199. Rac1 did not interact with the C-terminal portion (aa
300-526), excluding the possible presence of more than one
Rac1-binding site. However, sequences N-terminal to the binding site
are required for full activity, possibly for stabilizing the binding
site (similar observations were made for Cdc42Hs binding to fragments
of PAK and a novel Cdc42Hs interacting protein, CBP5). The
p67phox deletion mutants 58K (41) and
178-184 no longer
bind Rac1. Since the Rac-binding site is located between aa 170 and
199, we believe that the former mutation destabilizes the protein
possibly by disrupting the second TPR located between aa 37 and 70 of
p67phox. TPR domains are thought to form amphipathic helices
that self-associate and stabilize protein structures (42). The
Rac1-binding sequence of p67phox does not align with the
Rac1-binding regions of POR1, tubulin, or p140Sra-1 (37-39). Isolation
of more Rac1-specific targets will be required before a Rac1-specific
binding motif can be generated.
PAK Phosphorylation--
p47phox phosphorylation has been
shown to be important for oxidase activity (46). In particular, the
observation that mutation of residue 379 destroys activity suggests
that isolation of the kinase(s) involved would be an important step in
understanding control of NADPH oxidase activity. Recent work has shown
that PAK1 and
PAK2 immunoprecipitated from fMet-Leu-Phe-activated neutrophils can phosphorylate full-length p47phox and a peptide
covering aa 324-331 (31). Constitutively active recombinant PAK also
phosphorylates p47phox, but not p67phox (31).
p67phox is phosphorylated in vivo (29), and in this
study, we found that recombinant
PAK was able to phosphorylate
p67phox in vitro. These results suggest different
substrate specificity for PAK isoforms.
Role of Cryptic Rac1-binding and PAK Phosphorylation Sites in p67phox-- In resting neutrophils, the components of the oxidase are present as one membrane and two cytosolic complexes. Membrane-bound cytochrome b has two subunits, p22phox and gp91phox, both transmembrane proteins. p67phox and p40phox are in tight association in the cytosol, whereas p47phox associates with the p67phox-p40phox complex more loosely (20, 22). The small molecular mass GTP-binding protein Rac1 (or Rac2 in human neutrophils) associates with the Rho GDP dissociation inhibitor also in the cytosol (47). We propose that p67phox, like p47phox (24, 25), exists in a closed conformation via SH3 domain-polyproline interactions (Fig. 10). Activation of the oxidase is induced by the unfolding of p47phox and p67phox, leading to new SH3-polyproline interactions coupled with dissociation of Rac1 from its complex with the Rho GDP dissociation inhibitor (Fig. 10, step 1). These initial events (priming) could be induced, for example, by receptor activation of phospholipase A2, and this is supported by the observation that arachidonic acid disrupts an SH3 domain-polyproline interaction in p47phox (24). This does not exclude other potential mechanisms for unfolding of p67phox and p47phox, for instance, by phosphorylation. Indeed, there is evidence that phosphorylation of p47phox is required for its translocation to the membrane (27). Protein unfolding would allow interaction between the p67phox C-terminal SH3 domain and the p47phox polyproline-rich region (aa 360-380), thus exposing both SH3 domain(s) of p47phox. Translocation of the p67phox-p40phox-p47phox complex to the membrane would follow due to interaction of the exposed N-terminal SH3 domain of p47phox with the polyproline-rich region of p22phox.
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FOOTNOTES |
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* This work was supported in part by the Glaxo-Singapore Research Fund and the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 171-278-1552; Fax: 171-278-7045.
1 The abbreviations used are: aa, amino acid(s); PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; Mes, 4-morpholineethanesulfonic acid; CRIB, Cdc42Hs/Rac1 interacting domain; TPR, tetratricopeptide repeat; RBD, Rac1-binding domain; PAK, p21Cdc42Hs/Rac-activated kinase; PMA, phorbol 12-myristate 13-acetate.
2 The CRIB motif, a sequence of 16 aa, first identified in proteins such as PAK, ACK, and WASP, has been suggested as a consensus sequence for Cdc42Hs/Rac1 binding.
3 S. Govind and S. Ahmed, unpublished data.
4 K. Marler and S. Ahmed, unpublished data.
5 L. V. Forbes, O. Truong, S. J. Moss, and A. W. Segal, unpublished data.
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REFERENCES |
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