Several yeast two-hybrid screens for interaction partners identified the proteins glutamate receptor interacting protein, protein inter-acting with C kinase 1 and Golga3 to bind to serine
Trang 1D-Amino acids in the brain: the biochemistry of brain
serine racemase
Florian Baumgart and Ignacio Rodrı´guez-Crespo
Departamento de Bioquı´mica y Biologı´a Molecular, Facultad de Ciencias Quı´micas, Universidad Complutense de Madrid, Spain
The initial purification of brain mammalian serine
racemase was performed by Wolosker, Snyder and
coworkers using 60 brains obtained from rats [1] This
seminal work permitted the isolation of a
homo-geneous protein preparation that displayed the ability
to isomerize l-serine into its enantiomeric d-serine
counterpart In addition, the authors established the
molecular mass of the enzyme, its pH and temperature
dependence, the presence of bound pyridoxal-5¢
phos-phate (PLP) and the exquisite activity regulation
exerted by reagents that react with free SH groups, such as oxidized glutathione The subsequent mole-cular cloning of mouse brain serine racemase, as well
as the comparison with PLP-containing racemases from other organisms, led to the identification of K56
as the lysine residue that formed the Schiff base with the PLP moiety [2] The first recombinant expression and purification experiment was performed by Wolos-ker and coworWolos-kers using HEK293 cells transfected with
a serine racemase–glutathione S-transferase plasmid [3]
Keywords
AMPA receptor; astrocytes; ATP; calcium
activation; D -serine; gliotransmitters; GRIP;
NMDA receptor; PDZ interaction; serine
racemase
Correspondence
I Rodrı´guez-Crespo, Departamento de
Bioquı´mica y Biologı´a Molecular, Facultad de
Ciencias Quı´micas, Universidad
Complutense, Ciudad Universitaria, 28040
Madrid, Spain
Fax: +34 91 394 4159
Tel: +34 91394 4137
E-mail: nacho@bbm1.ucm.es
(Received 30 January 2008, revised 3 April
2008, accepted 4 April 2008)
doi:10.1111/j.1742-4658.2008.06517.x
It has been recently established that in various brain regions d-serine, the product of serine racemase, occupies the so-called ‘glycine site’ within N-methyl d-aspartate receptors Mammalian brain serine racemase is a pyridoxal-5¢ phosphate-containing enzyme that catalyzes the racemization
of l-serine to d-serine It has also been shown to catalyze the a,b-elimina-tion of water from l-serine or d-serine to form pyruvate and ammonia Serine racemase is included within the group of type II-fold pyridoxal-5¢ phosphate enzymes, together with many other racemases and dehydratases Serine racemase was first purified from rat brain homogenates and later recombinantly expressed in mammalian and insect cells as well as in Escherichia coli It has been shown that serine racemase is activated by divalent cations like calcium, magnesium and manganese, as well as by nucleotides like ATP, ADP or GTP In turn, serine racemase is also strongly inhibited by reagents that react with free sulfhydryl groups such
as glutathione Several yeast two-hybrid screens for interaction partners identified the proteins glutamate receptor interacting protein, protein inter-acting with C kinase 1 and Golga3 to bind to serine racemase, having different effects on its catalytic activity or stability In addition, it has also been proposed that serine racemase is regulated by phosphorylation Thus,
d-serine production in the brain is tightly regulated by various factors pointing at its physiologic importance In this minireview, we will focus on the regulation of brain serine racemase and d-serine synthesis by the factors mentioned above
Abbreviations
[Ca 2+ ]cyt, cytosolic calcium concentration; AMPA, a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; AMPAR, AMPA receptor; GluR2, glutamate receptor subunit 2; Golga3, Golgin subfamily A member 3; GRIP, glutamate receptor interacting protein; GSNO, S-nitroso-glutathione; NO, nitric oxide; PDZ, PSD95 ⁄ disc large ⁄ ZO-1; PICK1, protein interacting with C kinase 1; PKC, protein kinase C;
PLP, pyridoxal-5¢ phosphate.
Trang 2The purified enzyme was extremely efficient in terms of
the elimination reaction, using l-serine-O-sulfate as a
substrate and producing pyruvate plus ammonia
However, this purified enzyme failed to catalyze the
elimination when l-serine was used as a substrate A
major breakthrough was the observation that both
divalent cations and nucleotides were actually
cofac-tors of serine racemase [4] Both the racemase and
eliminase reactions of recombinant serine racemase
expressed in mammalian cells when l-serine was used
as a substrate were activated to similar levels in the
presence of divalent cations such as calcium and
mag-nesium [4] This activation by divalent cations was also
observed when serine racemase was recombinantly
expressed and purified from Escherichia coli [5] or
when it was purified from mouse brain [6] When
recombinant serine racemase produced in mammalian
cells was used, in terms of d-serine synthesis (racemase
activity) both magnesium and ATP independently
acti-vated the enzyme and their effect was additive Even
in the presence of the chelating agent EDTA, ATP
was still able to increase serine racemase activity [4] In
the absence of added ATP, mammalian cells expressing
serine racemase became activated, in terms of pyruvate
production, at about 100 lm magnesium However, in
the presence of the nucleotide, the amount of
magne-sium needed for half activation was close to 10 lm [4]
Similar data were obtained when the enzyme purified
from bacteria was used: in the absence of added ATP,
calcium activated the racemase activity of the enzyme
at a half-maximal concentration (EC50) of about
26 lm, although using changes in tryptophan
fluores-cence a binding constant for calcium to serine
race-mase was narrowed down to about 6 lm [5] The
physiological activation of brain serine racemase by
divalent cations is described in detail below
Very recently, no fewer than six PLP-containing
enzymes having broad sequence homology with human
brain serine racemase have been cloned and
recombi-nantly expressed Three recombinant plant serine
race-mases have recently been characterized: those of
Arabidopsis thaliana, Hordeum vulgare (barley) and
Oryza sativa(rice) [7,8] An aspartate racemase that has
a very high homology with brain serine racemase has
recently been cloned and characterized from a bivalve
mollusk [9] The so-called serine racemase from
Saccha-romyces cerevisiaehas been recombinantly expressed in
E coli and characterized, and its properties seem to
indicate that it is a paralog rather than an ortholog of
mammalian serine racemases [10] Likewise, the serine
racemase from the hyperthermophylum
Pyrobacu-lum islandicum was both purified and recombinantly
expressed, and the isolated enzymes were characterized
[11] Finally, the coordinates of the 3D structure of ser-ine racemase from Schizosaccharomyces pombe, another enzyme that displays high homology with mammalian serine racemase, have been recently deposited (Protein Data Bank code 1WTC) Sequence comparison allowed
us to rationalize the dependence of each of these enzymes on divalent cations and nucleotides, and on their binding to other interacting proteins
Regulation of serine racemase by divalent cations and nucleotides
The sequence comparison of human brain serine race-mase with selected homologous proteins is depicted in Fig 1 We recently used the coordinates obtained from the crystal structure of the Mg2+-bound S pombe serine racemase and the Ca2+-bound Thermus thermo-philus threonine deaminase to identify the equivalent positions within mammalian serine racemase that would bind the divalent cation [12] We were able to predict that the metal is hexavalently coordinated and that the cation-binding site is formed by two carboxyl-ate-containing residues, a main-chain carbonyl oxygen and three well-ordered water molecules The positions involved in the interaction with the divalent cation are marked in orange in Fig 1 In human serine racemase, the residues predicted to be directly involved in cal-cium binding are Glu210, Asp216 and Ala214 Consis-tent with this prediction, these three residues, which are conserved in plant and yeast serine racemases, would be responsible for the Ca2+⁄ Mg2+ racemase activation observed for these enzymes [7,8,10] Con-versely, the absence of these residues in the bivalve and Pyrobaculum serine racemases is in agreement with the absence of increased racemization of these enzymes induced by Ca2+or Mg2+[9,11]
The enzyme activation by nucleotides is somehow more puzzling For instance, the activation of the homologous protein E coli Thr dehydratase by AMP was observed as early as 1949 [13] However, in this bacterial enzyme the nucleotide exerted an allosteric role, promoting protein oligomerization and activating the enzyme [14] In the absence of AMP, the Km of
E coli Thr deaminase for Thr was 70 mm and it decreased to 5 mm in the presence of the nucleotide [15] The residues participating in the binding of nucle-otides (shown in red in Fig 1) could also be predicted because the crystal structure of the S pombe serine racemase has the nucleotide AMPpcp bound [12] Interestingly, most of the nucleotide-binding sites are conserved in all the enzymes However, although mam-malian serine racemase is strongly activated by nucleo-tides [4,6,12] this is not the case in their plant
Trang 3Fig 1 Sequence alignment of human serine racemase (gi:11345492), Arabidopsis thaliana serine racemase (gi:84458483), Hordeum vulgare (barley) serine racemase (gi:148356707), Bivalve (Scapharca broughtonii) aspartate racemase (gi:86439930), Saccharomyces cerevisiae homolog of serine racemase (gi:151941446), Schizosaccharomyces pombe serine racemase (gi:71041740) and Pyrobaculum islandicum ser-ine racemase (gi:83582728) Based on the crystal structure of S pombe serser-ine racemase, green arrows depict b-strands and yellow barrels depict a-helices The modelling has previously been described in detail by Baumgart et al [12] The residues involved in calcium binding are shown in orange and those involved in nucleotide binding are shown in red Residues involved in the binding to the PLP moiety are shown
in blue, whereas those involved in protein–protein interaction are shown in green The first four amino acids of the barley serine racemase and the final 88 amino acids of the Pyrobaculum serine racemase are omitted for clarity Alignment was performed using the CLUSTAL
software.
Trang 4orthologs [7,8] In fact, both the bivalve and
Pyrobacu-lum serine racemases are actually inhibited by ATP,
although the former is activated slightly in the
pres-ence of AMP [9,11]
The question hence remains regarding the exact role
of the nucleotide in brain mammalian serine racemase
catalysis because a PLP-dependent racemization does
not require ATP-driven energy An allosteric role may
provide an explanation In fact, homology modeling
indicates that the nucleotide is positioned in the
mono-mer⁄ monomer interface [12 and Fig 2] In contrast to
the case of E coli Thr dehydratase mentioned above,
we were unable to observe changes in the
oligomeriza-tion state of recombinant mouse serine racemase in the
presence and absence of added ATP [12] In fact, all
the recombinant versions of serine racemase mentioned
above are either homodimers [7–9] or homotrimers [11]
in the absence of the nucleotide Consequently, it is
unlikely that ATP might be regulating the quaternary
structure of serine racemase In this regard, as noted by
Wolosker and coworkers [4] ATP is not hydrolyzed
during catalysis, because both ADP and a
nonhydro-lyzable analog of ATP are able to activate the enzyme
to a similar extent Furthermore, in the cytosol the
ATP concentrations are in the 3–6 mm range, an
obser-vation suggesting that serine racemase is always
satu-rated with enough nucleotide to exert its racemase
activity because 100 lm ATP is more than enough to
result in full activity [4,6]
Perhaps it is even more important to know if serine
racemase is activated by Ca2+ or Mg2+ in vivo In
principle, if the Mg2+ concentration in the cellular
cytosol is indeed 600 lm [4,16], the brain serine race-mase would always be ‘on’ However, when type II astrocytes were loaded with radioactive d-serine, its release would be induced by l-glutamate and kainate, agents known to increase intracellular calcium concen-trations [17] Subsequently, we observed the increased release of d-serine by primary astrocytes when gluta-mate, kainate or the calcium ionophore A23187 was added to the cellular medium [5] Likewise, C6 glioma cells increased their secretion of d-serine when incubated with a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) [18] A more direct demon-stration has been recently performed by Mothet and coworkers when they showed that d-serine release is directly related to the increase of cytosolic calcium concentration ([Ca2+]cyt) [19] These authors showed that the removal of extracellular calcium, or the deple-tion of thapsigargin-sensitive intracellular calcium stores, abrogated the release of d-serine [19] It is conceivable that perhaps the increase of [Ca2+]cyt is only involved in the secretion of d-serine previously accumulated in secretion granules [19] although the storage of d-serine in granules in glia has recently been ruled out [18] It is very likely that serine racemase at various intracellular localizations might be challenged with different calcium concentrations, hence regulating its enzymatic activity For instance, direct coupling of serine racemase to the AMPA receptor (AMPAR) via glutamate receptor interacting protein (GRIP) binding might be one way to regulate its d-serine synthesizing activity (see below)
Nitrosylation of serineine racemase
Only scant data are available on possible post-transla-tional modifications of serine racemase in vivo The observation that both oxidized glutathione [1,5] and cys-tamine [5] could inhibit serine racemase provided some evidence that reactive cysteine residues should be pres-ent that are esspres-ential for serine racemase function When
we tested if the nitric oxide (•NO) donor DETA NONOate [(z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium,2-diolate] could alter serine race-mase activity, we obtained a negative result [5] Quite recently, reports of•NO as an inhibitor of serine race-mase in a glioblastoma cell line added a new aspect to
d-serine-dependent modulation of the glutamatergic synapse The authors propose that NMDAR-mediated calcium entry into postsynaptic neurons entails cal-cium⁄ calmodulin-dependent activation of neuronal nitric oxide synthase and the consequent liberation of
•NO Serine racemase is subsequently nitrosylated and inhibited, whereas d-amino oxidase, which is thought to
ACP
PLP
ACP
PLP
Ca 2+
Ca 2+
Fig 2 Molecular model of human serine racemase, as described
by Baumgart et al [12] The calcium ions are depicted as yellow
spheres, the PLP moiety is shown in blue and the nucleotide
analo-gue phosphomethylphosphonic acid adenylate ester (AMP-PCP) is
shown in magenta The molecular modelling was performed using
the crystal structures of the S pombe serine racemase and the
E coli Thr deaminase.
Trang 5counteract serine racemase activity in vivo by
degrada-tion of d-serine, is upregulated by•NO [20,21]
Subse-quent biochemical proof for this model was provided
[22], pinning down the residue that becomes modified
and proposing a structural model for the action of•NO
Apparently, cysteine 113 (out of seven cysteine residues
in the mouse and human serine racemase sequence) can
become nitrosylated, both in the recombinant enzyme
and in transfected cells, using the•NO donor,
S-nitroso-glutathione (GSNO) A molecular model of mouse
ser-ine racemase, based on a yeast homolog, reveals that
residue 113 lies in proximity to the putative
ATP-bind-ing region of the enzyme Nitrosylation would therefore
lead to impaired nucleotide binding and inactivation of
the enzyme It is noteworthy that GSNO is known to
modify cysteines not only with•NO but also with
gluta-thione, leading to protein glutathionylation, another
post-translational modification occurring under
condi-tions of oxidative⁄ nitrosative stress In fact, GSNO is
very frequently used in glutathionylating studies [23] It
is thus conceivable that purified serine racemase
becomes modified by glutathione together with •NO
Experiments with milder nitrosylating reagents that lack
a glutathione moiety would unambiguously
demon-strate if serine racemase is, in fact, modified by•NO
Serine racemase-interacting proteins:
GRIP, PICK1 and Golga3
The carboxy-terminal end of both mouse and human
serine racemase display a -Val-serine-Val-COOH
sequence, a motif reminiscent of the type II consensus
sequence for binding to PSD95⁄ disc large ⁄ ZO-1
(PDZ) domains [24] PDZ domains are among the
most ubiquitous protein–protein interaction motifs in
metazoan genomes and are especially important in the
nervous system for the assembly of synaptic complexes
and scaffolding [25,26] After performing a yeast
two-hybrid screen of serine racemase against a rat
hippo-campus and cortex cDNA library, the hepta-PDZ
protein GRIP was identified as a binding partner of
serine racemase [18] Out of the seven consecutive
PDZ modules found in GRIP, serine racemase was
found to bind specifically to the PDZ6 domain by
means of its C-terminal PDZ-binding motif
Previ-ously, GRIP had been described to interact with
gluta-mate receptors of the AMPA⁄ kainate type [27], where
it is responsible for proper trafficking and assembly of
the receptor and accessory proteins GRIP can bind to
the glutamate receptor subunit 2 (GluR2) subunit of
AMPA receptors via PDZ4⁄ PDZ5, both PDZ domains
working in concert to establish binding [27,28] The
finding of serine racemase interacting with the PDZ6
domain of GRIP and being activated was the first report on cellular interaction partners of serine race-mase and it raised several intriguing questions It was not clear whether GRIP directly activated serine race-mase or if binding led to a translocation to the prox-imity of AMPARs in vivo (Fig 3) Furthermore, the influence of the other PDZ domains of GRIP was not investigated Therefore, other proteins that become associated with GRIP, using some of the other six PDZ domains, might modulate the activity of serine racemase Conversely, d-serine might also change the activity of some GRIP-associated proteins When ser-ine racemase⁄ GRIP interactions were first studied, it was proposed that GRIP was released from AMPARs when they became stimulated and phosphorylated [18], which would lead to GRIP interacting with serine racemase in the cytosol where it would bind to and activate serine racemase With our own results we were able to confirm the interaction of GRIP with serine racemase via PDZ6 [12] However, we observed that binding to PDZ6 alone was not sufficient for activa-tion Rather, the presence of the rest of the C-terminal region of GRIP, that is the PDZ7 module and a link-ing segment between PDZ6 and PDZ7, was required for full activation of serine racemase, both in vitro and
in vivo Although these results do not necessarily pre-clude a translocation process to AMPARs mediated by GRIP, they do show the direct activation of serine racemase by GRIP as a result of the concerted inter-action of several PDZ modules, independent of the subcellular localization This PDZ crosstalk, where an isolated PDZ domain is insufficient to carry out a specific function, has also been observed in other examples, for instance in the requirement of both PDZ4 and PDZ5 for GRIP binding to GluR2 [27,28] Interestingly, the activating effect of GRIP on serine racemase results mainly in a change in Vmax More-over, the response curve to calcium remains unchanged upon binding to GRIP under the experimental condi-tions applied, which indicates that GRIP binding and regulation by calcium must be regarded as independent regulation pathways It has been proposed that serine racemase activation by GRIP takes place in the cytosol after AMPAR phosphorylation and concomitant dis-sociation of GRIP [18] However, because nothing is known about the kinetics of this process, the forma-tion of a ternary complex among the GluR2 subunit
of the AMPA receptor, GRIP and serine racemase cannot be discounted It would be plausible that GRIP brings serine racemase in close proximity to the gluta-mate-activated channel, where serine racemase might
be close to other calcium channels Although the AMPAR is not a calcium channel, it is conceivable
Trang 6that in certain calcium microdomains serine racemase
could become exposed to temporarily high calcium
concentrations To shut the system off, GluR2 could
become phosphorylated, in order to release GRIP and
serine racemase, abolishing the transient activation of
serine racemase by calcium (Fig 3) Because GRIP
can bind to serine racemase, both in the presence and
absence of calcium, it is possible that some other
GRIP-interacting protein that also binds to PDZ6
might disrupt the serine racemase–GRIP interaction,
hence diminishing the activity of the former
In a similar yeast two-hybrid screen using a human
hippocampal cDNA library, a different PDZ
domain-containing protein was found to interact with serine
racemase, also requiring the C-terminal binding motif
[30] Protein interacting with C kinase 1 (PICK1)
con-tains one PDZ domain that is required for interacting
with protein kinase C (PKC) [30,31] or serine racemase
It also contains a Bin⁄ amphiphysin ⁄ Rys domain,
important for the interaction with lipids, and a
coiled-coil domain Furthermore, it has been shown recently
that the PDZ domain of PICK1 is also capable of
inter-acting with lipid membranes, a property crucial for the
clustering of AMPAR and synaptic plasticity [32]
There are no data available regarding the effect of the
binding of PICK1 on serine racemase activity
There-fore, biochemical characterization of the role of the
interaction of serine racemase and PICK1 is needed to
judge the importance of these observations Surely the
interaction of PICK1 with PKC leads to the temptation
to speculate on a possible phosphorylation of serine racemase by PKC [33] As yet, however, there are no data available, either on the details of the interaction of PICK1 with serine racemase, or on the phosphorylation
of serine racemase
Considering that the phosphorylation of Ser880 of the GluR2 subunit of the AMPA receptor, positioned
at the carboxy-terminal end of the polypeptide chain, disrupts its interaction with PDZ4⁄ PDZ5 of GRIP, it
is tempting to speculate that phosphorylation of Ser336 of human serine racemase or of Thr336
of mouse serine racemase might also break their inter-action with PDZ6 of GRIP This putative phosphory-latable residue is located at position -3 of the human (Ser–Val–Ser–Val-COOH) and mouse (Thr–Val–Ser– Val-COOH) sequences, respectively, and both are inserted within amino acid sequences of type II con-sensus PDZ domain-interacting partners [24,26] It has been proposed that PKCa phosphorylates serine race-mase, probably brought into its proximity by PICK1 binding [33] This hypothesis would rationalize a novel mode of regulation of d-serine synthesis through the activation of nonphosphorylated serine racemase by the multi-PDZ domain GRIP We have been unable to identify PKCa as a kinase that modifies purified recombinant serine racemase (unpublished data) although perhaps this might be the case in vivo In addition, both the rat and cow serine racemases are
GluR2
GluR2
L-Ser D-Ser
C C
C
L-Ser D-Ser
C
P
D-Ser
L-Ser D-Ser
Fig 3 Proposed modes of interaction
among serine racemase, GRIP and the
AMPAR (A) A trimeric complex is assumed.
(B) Phosphorylation of the GluR2 subunit of
the AMPAR at Ser880 dissociates GRIP
binding, which remains bound to serine
racemase (C) Serine racemase is active in
the cytoplasm and does not interact with
GRIP, whereas the latter associates with
the GluR2 subunit (D) The simultaneous
phosphorylation of the GluR2 subunit of the
AMPAR together with the phosphorylation
of serine racemase releases GRIP to the
cytoplasm in the absence of any dual
inter-action.
Trang 7truncated in the carboxy-terminal end, hence lacking
GRIP-interacting sequences Further experiments will
demonstrate if the activation of serine racemase by
GRIP and its binding to PICK1 is exclusively present
in certain mammals or if there is sequence splicing at
this region and both rat and cow do have longer
(as-yet unidentified) versions of serine racemase
Consequently, at least four different modes of
inter-action can be envisaged among AMPAR, serine
race-mase and GRIP (Fig 3) Although phosphorylation of
the GluR2 subunit of the AMPAR and the disruption
of its association with GRIP have been unambiguously
demonstrated, the putative carboxy-terminal
phosphor-ylation of serine racemase remains to be established If
GRIP brings serine racemase towards the proximity of
the calcium channel, a theoretical modulation of the
synthesis of d-serine by calcium concentration can be
postulated, in accordance with recent data [18] Using
a mouse brain lysate we observed a trimeric GluR2–
GRIP–serine racemase (data not shown) although we
do not know which mechanisms lead to the
dissocia-tion of serine racemase from GRIP Nevertheless, in
the absence of the association with the AMPAR,
recombinant purified GRIP alone is able to increase
the activity of recombinant purified serine racemase
[12] We have shown that certain GRIP amino acids,
present further down in the sequence than PDZ6, are
responsible for the majority of the observed activation
of serine racemase by GRIP [12]
In another study to identify binding partners of
ser-ine racemase using the yeast two-hybrid technology,
the Golgi-localized protein, Golgin subfamily A
mem-ber 3 (Golga3), was found to interact with serine
race-mase [34] In this case, however, no PDZ interactions
with the C-terminal amino acid triplet of serine were
crucial for binding, but instead, the interaction was
established with its N-terminal 66 residues Binding of
Golga3 increases d-serine synthesis Intriguingly, this is
achieved through a decrease in ubiquitin⁄ proteasomal
degradation of serine racemase, rather than by
modula-tion of the catalytic rate Serine racemase was shown to
have an average half-life of about 4.5 h When Golga3
and serine racemase were cotransfected, both serine
racemase stability and d-serine synthesis increased
con-siderably Thus, it is important to note that in addition
to the modulators mentioned beforehand which directly
influence the catalysis of serine racemase, indirect
effects such as protein stability or subcellular
localiza-tion should be taken into account when investigating
the precise regulation of serine racemase-dependent
d-serine levels at glutamatergic synapses
In conclusion, brain serine racemase, a member of
the type II-fold PLP-dependent racemases⁄
dehydrata-ses, shares several mechanistic properties with other members of the same family, such as activation by nucleotides or divalent cations, although its functional-ity in the brain is also modulated through interaction with specific glial or neuronal proteins
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