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The Journal of Neuroscience, September 15, 1998, 18(18):7111-7117
Dopamine Inactivates Tryptophan Hydroxylase and Forms a
Redox-Cycling Quinoprotein: Possible Endogenous Toxin to Serotonin
Neurons
Donald M.
Kuhn1, 2 and
Robert
Arthur Jr.1
1 Cellular and Clinical Neurobiology Program,
Department of Psychiatry and Behavioral Neurosciences, and
2 Center for Molecular Medicine and Genetics, Wayne State
University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
Exposure of tryptophan hydroxylase (TPH), the initial and
rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, to dopamine under mild oxidizing conditions (iron + H2O2) or in the presence of tyrosinase
results in a concentration-dependent inactivation of the enzyme.
Dopamine, iron, H2O2, or tyrosinase alone does not alter TPH activity. Similarly,
N-acetyldopamine oxidized with one equivalent of sodium
periodate causes a concentration-dependent inactivation of TPH as well.
TPH is protected from dopamine-induced inactivation by reduced
glutathione, ascorbic acid, and dithiothreitol but not by the radical
scavengers DMSO, mannitol, or superoxide dismutase. Parallel studies
with [3H]dopamine reveal a high negative
correlation between inhibition of catalysis and incorporation of
tritium into the enzyme. Those reducing agents and antioxidants that
protect TPH from inactivation are effective in preventing the labeling
of TPH by [3H]dopamine. Acid hydrolysis and HPLC
with electrochemical detection (HPLC-EC) analysis of inactivated
TPH revealed the formation of cysteinyl-dopamine residues within the
enzyme. Exposure of dopamine-modified TPH to redox-cycling staining
after SDS-PAGE confirmed the formation of a quinoprotein. These results
indicate that dopamine-quinones covalently modify cysteinyl residues in
TPH, leading directly to the loss of catalytic activity, and establish
that TPH could be a target for dopamine-quinones in vivo
after drugs (e.g., neurotoxic amphetamines) that cause
dopamine-dependent inactivation of TPH. Redox cycling of a
TPH-quinoprotein could also participate in the serotonin neuronal
toxicity caused by these same drugs.
Key words:
tryptophan hydroxylase; dopamine; quinones; neurotoxic
amphetamines; quinoproteins; serotonin
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INTRODUCTION |
The neurotransmitter dopamine (DA)
can have quite divergent effects on the brain, especially when neurons
are placed under the influence of certain pharmacological agents. On
one hand, the reinforcing effects of many addictive drugs such as
morphine, cocaine, and nicotine are thought to be mediated by
drug-induced release of DA in the limbic forebrain (Pontieri et al.,
1995 ; Koob and Nestler, 1997). On the other hand, DA plays an important role in the neurotoxic actions of methamphetamine and
3,4-methylenedioxymethamphetamine (MDMA) on DA and serotonin
(5-HT) neurons alike (Seiden and Sabol, 1996; Gibb et al.,
1997). A common effect among the neurotoxic amphetamines is their
ability to cause the inhibition of tryptophan hydroxylase (TPH)
(Schmidt et al., 1986; Stone et al., 1986), the initial and
rate-limiting enzyme in the biosynthesis of 5-HT (Jequier et al., 1967)
and a phenotypic marker for 5-HT neurons. This property alone could
have numerous secondary effects considering the wide variety of
physiological functions that are mediated by 5-HT neurotransmission
(Whitaker-Azmitia and Peroutka, 1990). Altered 5-HT neurotransmission
is also thought to play a role in several psychiatric conditions, and
evidence of diminished 5-HT function in human MDMA and methamphetamine
users has been presented (Steele et al., 1994; Green et al., 1995).
Certain aspects of DA chemistry make it a strong candidate for an
endogenous neurotoxicant. DA readily oxidizes nonenzymatically to
corresponding quinones (Graham, 1978; Graham et al., 1978), and it is
well known that quinones can modify proteins (Graham et al., 1978;
Gieseg et al., 1993; Hastings and Zigmond, 1994; Hastings et al.,
1996b; Terland et al., 1997) and DNA (Stokes et al., 1996). The
metabolism of DA by monoamine oxidase also produces
H2O2 (Maker et al., 1981; Cohen et al., 1997),
and the generation of reactive oxygen species downstream of the
peroxide, such as superoxide or hydroxyl radicals (Halliwell, 1992),
could also underlie the damaging effects of DA in neural tissue. High concentrations of DA can inhibit DA (Berman et al., 1996; Berman and
Hastings, 1997) and glutamate transporter function in vitro (Berman and Hastings, 1997) and cause toxic effects in brain or cultured neurons (Rosenberg, 1988; Filloux and Townsend, 1993; Hastings
et al., 1996a; Hoyt et al., 1997; Lai and Yu, 1997). Studies of DA
cellular toxicity have not yet identified a specific target protein or
nucleotide in brain tissue. In view of the effect of the neurotoxic
amphetamines on TPH, and considering that DA has been implicated in the
actions of these drugs in vivo, we hypothesized that TPH
could be a target for DA-induced modification. Direct tests of this
hypothesis are now possible via the use of a recombinant TPH that can
be purified to homogeneity and used for in vitro mechanistic
studies relevant to the neurotoxic amphetamines (Kuhn and Arthur,
1997b). We demonstrate here that DA-quinones inactivate TPH, suggesting
that this enzyme could be a target for DA-quinones in vivo
under conditions that elevate DA release (neurotoxic amphetamines)
and/or synthesis [L-3,4-dihydroxyphenylalanine (L-DOPA)] and that cause DA-dependent inhibition of TPH
(Gibb et al., 1997).
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MATERIALS AND METHODS |
Materials. The following materials were obtained from
Sigma (St. Louis, MO): DA, dithiothreitol (DTT), ferrous ammonium
sulfate, glutathione (GSH), N-acetyldopamine
(N-acetyl-DA), GSH-agarose beads, tryptophan,
H2O2, nitroblue tetrazolium (NBT),
sodium periodate, mushroom tyrosinase, and prestained molecular weight
standards. The glutathione S-transferase (GST)-fusion
protein vector pGEX4T-2 and thrombin protease (7500 U/mg) were
purchased from Pharmacia (Piscataway, NJ). T4 DNA ligase and
restriction endonucleases were purchased from New England Biolabs
(Beverly, MA). Catalase was a product of Boehringer Mannheim
(Indianapolis, IN). Tetrahydrobiopterin was purchased from Dr. B. Schircks Laboratories (Jona, Switzerland). Isopropyl
 -D-thiogalactopyranoside (IPTG) was obtained from Gold Biotechnologies (St. Louis, MO). BL-21 (Escherichia coli)
cells were purchased from Invitrogen (Carlsbad, CA).
[3H]DA
(3,4-[7-3H]dihydroxyphenylethylamine; 24.1 Ci/mmol) was obtained from NEN Life Science Products (Boston,
MA). BioSpin 6 chromatography columns were obtained from Bio-Rad
(Hercules, CA).
Cloning and expression of TPH. TPH was cloned and expressed
as a GST-fusion protein as described previously (D'Sa et al., 1996b; Kuhn and Arthur, 1997a). A deletional mutant constituting the catalytic domain of TPH (amino acids 99-444) was expressed in
BL-21 (E. coli) cells. This form of TPH retains the
essential catalytic properties of the wild-type enzyme and has proved
useful in mechanistic studies of the catalytic properties of the enzyme (D'Sa et al., 1996a; Kuhn and Arthur, 1997b; Kuhn et al., 1997). Bacteria transformed with the plasmid bearing the TPH cDNA were grown
overnight at 37°C and induced with 0.1 mM IPTG for 2 hr at 30°C. Bacteria were washed with 10% glycerol and resuspended in
0.1 vol of 50 mM Tris-HCl, pH 7.5. After sonication and
centrifugation (40,000 × g) to sediment insoluble
material, the supernatants were adsorbed on GSH-agarose for 30 min at
4°C. Affinity beads with immobilized TPH were washed three times with
50 vol of 50 mM Tris-HCl, pH 7.5, at 4°C and were used
immediately as previously described (D'Sa et al., 1996b; Kuhn and
Arthur, 1997b). In some experiments (specified below), TPH was removed
from the GST-fusion tag by digestion with thrombin protease (10 U of
protease per mg of protein) at room temperature for 1 hr. The cleaved
TPH protein was separated from the GSH-affinity beads by filtration
through glass wool.
Treatment of TPH with dopamine. The TPH-fusion protein
(~15 µg of protein per tube) was treated with varying
concentrations of DA (5-200 µM) at 30°C for 15 min in
a volume of 1 ml while still bound to GSH-affinity beads. After removal
of DA by three washes of 100 vol of 0.05 M Tris-HCl, pH
7.5, residual enzyme activity was determined. In some experiments,
enzyme preparations were also incubated with iron (ferrous ammonium
sulfate, 100 µM) and/or H2O2 (1 mM) in all possible combinations with DA under the same
incubation conditions. DA, as a representative catecholamine [dihydroquinone (QH2)], can be oxidized to
semiquinones (·QHs) and quinones (Qs) by the following
series of reactions (Graham, 1978; Klegaris et al., 1995):
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(1)
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(2)
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(3)
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(4)
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The oxidation of DA at neutral pH (Eqs. 1-3) was facilitated by
the Fenton reaction (Eq. 4). Because the oxidation of DA can produce a
variety of quinones, two additional methods were used to study the
effects of DA-derived quinones on TPH. First, N-acetyl-DA was oxidized with one equivalent of NaIO4 to form a more
stable o-quinone (Graham, 1978; Graham et al., 1978), and
this solution was added to TPH and incubated as described above.
Second, DA was converted directly to its quinone with tyrosinase (50 U/ml). Agents being tested for the ability to protect against the DA effect on TPH were added before DA and remained present throughout the
15 min incubation period. When attempts were made to reverse the
effects of DA on TPH (e.g., with GSH or DTT), the enzyme was first
treated with DA (and other agents) and washed free of all reagents. GSH
or DTT was added to the enzyme, and samples were incubated at 4°C for
30 min. After three additional buffer washes, residual TPH activity was
determined.
Assay of TPH catalytic activity. TPH activity was assayed by
measuring the formation of 5-hydroxytryptophan from tryptophan as
described previously (D'Sa et al., 1996a,b; Kuhn and Arthur, 1997b;
Kuhn et al., 1997). TPH activity was assayed while the protein remained
bound to the GSH-affinity beads because adsorption of the TPH-fusion
protein to the beads does not interfere with the catalytic function of
the enzyme. The levels of protein in all enzyme samples were measured
with bead-bound preparations using the method of Bradford (1976).
Controls contained the appropriate buffers, solvents, and beads
unexposed to protein. The GSH-agarose beads did not cause the bovine
serum albumin standard curve to depart from linearity.
Treatment of TPH with [3H]dopamine. To
test whether DA was binding to TPH, we incubated the purified enzyme
with varying concentrations of [3H]DA alone or
under conditions that convert DA to a quinone (above). These studies
used TPH that was cleaved from the GST-fusion tag by thrombin. Protein
(~20 µg per tube) was incubated with [3H]DA at
30°C for 15 min in the presence of 50 mM Tris-HCl, pH 7.5, in a volume of 200 µl, and reactions were terminated by the addition of 1 ml of ice-cold 10% trichloroacetic acid (TCA). Samples were then incubated on ice for 10 min, and acid-insoluble material was
trapped on Whatman GF/B filter disks by vacuum filtration. The disks
were washed with an additional 5 ml of 10% TCA, and after drying, the
amount of acid-precipitable tritium was assayed by liquid scintillation
spectrometry.
Acid hydrolysis and HPLC with electrochemical detection
analysis for cysteinyl-dopamine. TPH (~3.5 mg) cleaved from the
GST-fusion tag with thrombin was exposed to L-DOPA (200 µM) ± tyrosinase (50 U/ml) as described above.
Hydrolysis and HPLC analysis for cysteinyl-DA was then performed as
described previously (Kato et al., 1986; Hastings and Zigmond, 1994).
Reactions were arrested with an equivalent volume of 10% TCA
followed by incubation at 4°C for 2 hr. The resulting precipitates
were collected by centrifugation and washed three times with 10 ml of
ice-cold 5% TCA. The final pellet was suspended in 0.75 ml of 6 M HCl containing 5% thioglycolic acid, transferred to a
hydrolysis vessel, and purged extensively with nitrogen before sealing.
Protein samples were heated to 110°C for 16 hr in a sand bath.
Hydrolyzed samples were then mixed with 50 mg of acid-washed alumina in
the presence of 2.7 M Tris-HCL, pH 8.6, sodium bisulfite
(0.2 mM), and EDTA (1% w/v) to extract catechol-modified
amino acid residues. Samples were mixed with alumina for 10 min at room
temperature and washed extensively with water. Catechols were eluted
with 0.4 ml of 0.4 M perchloric acid. The eluate was
analyzed for cysteinyl-DA by HPLC with electrochemical detection
(HPLC-EC). The HPLC mobile phase (0.05 M sodium citrate, 0.05 M NaH2PO4, 200 µM EDTA, 1.5 mM heptanesulfonic acid, and 14% methanol, pH 3.3) was pumped at a flow rate of 0.7 ml/min through
a Beckman 5 µm-C18 column, and samples were oxidized at a
glassy carbon electrode set to a potential of +0.75 V versus Ag/AgCl.
The presence of cysteinyl-DA was confirmed by comparison of samples
with cysteinyl-DA standards prepared as described by Rosengren et al.
(1985). Just before injection onto the HPLC column, an internal
standard containing 3-4 ng of free DA was added to samples and
cysteinyl-DA standards.
SDS-PAGE and redox-cycling staining. TPH was treated with DA
alone or under conditions that convert DA to a quinone (above) and then
was exposed to SDS-PAGE (Laemmli, 1970). Gels were electroblotted to
nitrocellulose membranes at 100 V for 2 hr at 4°C in a buffer of 25 mM Tris-HCl, pH 8.3, containing 192 mM glycine
and 20% (v/v) methanol. Quinoproteins were detected by staining blots
with NBT (0.24 mM in 2 M potassium glycinate
buffer, pH 10) as described by Paz et al. (1991). The
blue-purple-stained quinoproteins were photographed, blots were then
stained for total protein with Ponceau S (0.1% in 5% acetic acid),
and red-stained protein bands were rephotographed.
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RESULTS |
Effects of DA on TPH
It is known that catechols can inhibit TPH enzyme activity via
competitive interactions with protein-bound iron (Johansen et al.,
1991; D'Sa et al., 1996a,b). However, preincubation of TPH with DA for
15 min at 30°C, followed by removal of free DA, did not influence
enzyme activity. If TPH was exposed to DA under mild oxidizing
conditions (iron + H2O2), DA exerted
quite significant effects on the enzyme. The results in Figure
1 show that DA caused a
concentration-dependent inactivation of TPH when incubated in the
presence of 100 µM iron and 1 mM
H2O2. Concentrations of DA as low as 10 µM resulted in partial inactivation of TPH (to 54% of
the control level), and the enzyme was totally inhibited at concentrations of 50-100 µM DA in the presence of iron
and H2O2. Exposure of TPH to iron or
H2O2 individually was without effect. Similarly, coincubation of TPH and DA with either iron or
H2O2 had no effect on the enzyme. The
combination of iron + H2O2 caused a 10-15%
reduction in TPH activity (data not shown). When tyrosinase was used to
convert DA to its quinone in place of the Fenton reaction (Eq. 4), the
results were similar, a concentration-dependent inactivation of TPH
that was less potent than iron + H2O2. Finally,
a more stable o-quinone [N-acetyl-DA oxidized
with NaIO4 (Graham, 1978)] was a very potent inhibitor of
TPH. Concentrations of the N-acetyl-DA-derived o-quinone as low as 5 µM caused a 75%
reduction in TPH activity. Neither N-acetyl-DA nor
NaIO4 alone modified TPH activity (data not shown). The
concentration effects of all DA-quinones were statistically significant
(p < 0.001, ANOVA for each).
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Figure 1.
Effect of DA on TPH activity. TPH was exposed to
the indicated concentrations of DA at 30°C in the presence of either
100 µM ferrous ammonium sulfate + 1 mM
H2O2 or tyrosinase at 50 U/ml. When
N-acetyl-DA was used, it was first treated with a molar
equivalent of NaIO4 and added immediately to the enzyme
sample. Controls contained DA without other additions. Incubations with
tyrosinase were performed for 30 min, and all others were for 15 min.
After incubations, enzyme samples were washed free of all test reagents
by washing the enzyme in 50 vol of 50 mM Tris-HCl, pH 7.5, at 4°C, and the amount of TPH activity remaining was
determined. Results represent the mean ± SEM for four independent
experiments performed in duplicate. The main effect of DA (or
N-acetyl-DA) concentration was statistically significant
(p < 0.001, ANOVA) for each
condition.
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Protection of TPH from DA-mediated inactivation
Several reducing agents, antioxidants, and radical scavengers were
tested for the ability to protect TPH from DA-induced inactivation. Figure 2 shows that GSH (50 µM), ascorbic acid (200 µM), and DTT (500 µM) protected TPH from inactivation caused by any one of the DA-quinones. These same concentrations of GSH, ascorbic acid, and
DTT were without effects on TPH in the absence of DA-quinones (data not
shown). The overall effect of protectant was significant (p < 0.001, ANOVA), and individual comparisons
of GSH, ascorbic acid, and DTT with their respective controls (for each
quinone) were also significant (p < 0.01, Bonferroni post hoc comparison). Superoxide dismutase (250 U/ml), mannitol (10 mM), and DMSO (50 mM) were
ineffective in protecting the enzyme from inactivation caused by the
DA-quinones. Catalase (0.13 U/ml) did not protect against tyrosinase or
N-acetyl-DA-induced inactivation (data not shown). Attempts
were also made to recover TPH activity after inhibition had occurred.
DTT (10-20 mM) or GSH (5-10 mM) was
ineffective in reversing the inactivation caused by DA (data not
shown).
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Figure 2.
Protection of TPH from inactivation by DA. TPH was
exposed to 20 µM DA at 30°C for 15 min in the presence
of 100 µM ferrous ammonium sulfate and 1 mM
H2O2 or for 30 min in the presence of
tyrosinase at 50 U/ml as described for Figure 1.
N-acetyl-DA was oxidized with a molar equivalent of
NaIO4, and the resulting solution was added to
enzyme preparations at a concentration of 5 µM. GSH (50 µM), ascorbic acid (200 µM), or DTT (500 µM) was added just before DA or
N-acetyl-DA. The protectants were without effect on TPH
in the absence of DA. Results represent the mean ± SEM for four
independent experiments performed in duplicate for each reagent. The
overall effect of protectant was significant
(p < 0.001, ANOVA), and the individual
effects of GSH, ascorbic acid, and DTT were statistically different
from their respective controls under all conditions (at least
p < 0.01, Bonferroni post hoc
comparison).
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Binding of [3H]DA to TPH
TPH was treated with iron + H2O2,
and [3H]DA was included to determine whether
protein-DA adducts were being formed. The results from these
experiments are shown in Figure 3. It can
be seen that incubation of TPH in the presence of
[3H]DA and iron plus H2O2
resulted in a concentration-dependent increase in the incorporation of
tritium into acid-precipitated protein. This effect was statistically
significant (p < 0.001, ANOVA). Results were
the same if [3H]DA was treated with tyrosinase in
place of iron plus H2O2 (data not shown). At
saturating concentrations of DA, the labeling of TPH with tritium was
substoichiometric, resulting in the incorporation of ~0.8 moles of
[3H]DA per mole of TPH subunit.
[3H]DA did not label TPH in the absence of iron
and H2O2. GSH (50 µM) completely
prevented tritium incorporation into TPH (Fig. 3), as did ascorbic acid
and DTT at concentrations that protect TPH from inactivation by DA
(data not shown). A plot of TPH inactivation and tritium incorporation
into the enzyme versus the log concentration of DA is presented in
Figure 4 and reveals a close relationship between these two dependent measures. Each separate interaction of DA
with TPH was linear, and the two measures (inhibition of activity and
tritium labeling) exhibited a high negative correlation (r =  0.983).
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Figure 3.
Binding of [3H]DA to TPH. TPH
was cleaved from the GST-fusion tag by incubation with thrombin
protease as described in Materials and Methods. The purified, cleaved
enzyme was incubated with the indicated concentrations of
[3H]DA at 30°C for 15 min in the presence of 100 µM ferrous ammonium sulfate and 1 mM
H2O2. Controls contained the same amount of
isotope but omitted iron + H2O2. Reactions were
stopped by the addition of 1 ml of ice-cold 10% trichloroacetic acid.
Precipitated protein was trapped on a Whatman GF/B filter by vacuum
filtration. Filters were washed with 5 ml of acid and dried, and
tritium was quantified by liquid scintillation counting. Nonspecific
binding of [3H]DA was defined as the amount of
label present in samples containing 2 mM DA (unlabeled) and
was subtracted from all tubes. In some experiments, GSH (50 µM), ascorbic acid (200 µM), or DTT (500 µM) was added to enzyme preparations just before
[3H]DA. Only the results of GSH are plotted for
the sake of clarity. The results represent the mean ± SEM for
four experiments run in duplicate. The effect of
[3H]DA binding to TPH was statistically
significant (p < 0.001, ANOVA).
[3H]DA binding in the presence of GSH was
significantly different from the [3H]DA-binding
curve (p < 0.001, ANOVA).
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Figure 4.
Relationship between 3H incorporation
and TPH activity. The results from [3H]DA binding
to TPH and the inactivation of enzyme activity by DA-quinones were
plotted versus the log concentration of DA. Each relationship was
linear, and the two measures exhibited a high negative correlation
(r = 0.983).
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Acid hydrolysis of TPH
TPH (3.5 mg) was treated with DA (200 µM) + tyrosinase (50 U/ml) and then exposed to acid hydrolysis and HPLC-EC in
an attempt to establish the identity of the amino acid residue modified
by DA-quinones. The results are presented in Figure
5. Standards of enzymatically generated
cysteinyl-DA in Figure 5A contain four major peaks and
represent cysteinyl-DOPAC at 7.6 min, an internal standard of free DA
at 8.95 min, and cysteinyl-DA eluting with a retention time (RT)
of 11.05 min. An injection spike (present in all standard and sample
injections) also eluted at 6.15 min. These chromatographic results are
consistent with previous studies investigating cysteinyl-DA (Rosengren
et al., 1985; Kato et al., 1986; Hammond and Zigmond, 1994). Figure
5B shows that the chromatographic profile of TPH treated
with DA + tyrosinase contained the same peaks seen in the standard,
with identical retention times. Peak 4, with the same
retention time as cysteinyl-DA (11.05 min), establishes that TPH had
been modified to contain cysteinyl-DA. The cysteinyl-DA peak is also
clearly resolved from the DA internal standard, as shown in both
chromatographic traces. If TPH was treated with DA or
tyrosinase alone, the cysteinyl-DA peak was not observed (data not
shown).
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Figure 5.
HPLC-EC analysis of DA-modified TPH. TPH (3.5 mg)
was treated with DA (200 µM) + tyrosinase (50 U/ml) and
exposed to acid hydrolysis and HPLC-EC analysis as described.
Chromatographs represent standards of enzymatically generated
cysteinyl-catechols (A) and samples of
DA-quinone-modified TPH (B). The peaks in
A and B are identified as follows:
peak 1, injection spike, RT = 6.15 min; peak
2, cysteinyl-DOPAC, RT = 7.6 min; peak 3,
internal standard of free DA added to each sample just before
injection, RT 8.95 min; and peak 4, cysteinyl-DA,
RT = 11.05 min.
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Redox-cycling staining of TPH
TPH was treated with DA (100 µM) or under conditions
that convert DA to a quinone and then was exposed to redox-cycling
staining. The results in Figure
6A demonstrate that DA
alone did not convert TPH to a quinoprotein (lane 1).
However, incubation of TPH with DA + iron and
H2O2 (lane 2),
N-acetyl-DA (10 µM) + NaIO4
(lane 3), or DA + tyrosinase (50 U/ml; lane
4) resulted in the formation of a quinoprotein. The
quinoprotein has the same Mr as native TPH
(i.e., ~40,000), consistent with the predicted
Mr of the TPH catalytic core. We did not observe
evidence that TPH cross-linked to higher Mr
species after treatment with DA-quinones. Replacement of glycinate
buffer with valinate abolished redox-cycling staining of TPH (data not
shown). Figure 6B is a Ponceau S stain of the same blot shown in Figure 6A and confirms that all
lanes contained approximately the same amount of
protein.
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Figure 6.
Redox-cycling staining of DA-modified TPH. TPH was
purified and cleaved free of the GST-fusion tag as described in
Materials and Methods. Enzyme (8-10 µg) was then treated with 100 µM DA alone (lane 1), with 100 µM DA in the presence of 100 µM ferrous
ammonium sulfate + 1 mM H2O2
(lane 2), with 10 µM
N-acetyl-DA oxidized with NaIO4 (lane
3), or with 100 µM DA + 50 U/ml tyrosinase
(lane 4) for 15 min at 30°C. SDS-stop solution
was added to each tube, and samples were subjected to SDS-PAGE on 10%
acrylamide gels. Proteins were transferred to nitrocellulose sheets,
and blots were subjected to redox-cycling staining with 0.24 mM NBT in 2 M potassium glycinate, pH 10.0 (A). After redox-cycling staining, blots were
washed with water and restained for total protein with Ponceau S
(B). The molecular weight standards indicated to
the left of blots are prestained ovalbumin (49.5 kDa)
and prestained carbonic anhydride (32.5 kDa). This experiment was
repeated on five separate occasions with the same results.
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DISCUSSION |
TPH is presently shown to be inactivated after exposure to DA
under specified conditions. All aspects of the data support the
conclusion that DA-derived quinones are the agents mediating the
inactivation. First, preincubation with DA alone had no effect on TPH
activity. Second, conditions that prompt the conversion of DA to one of
its quinones lead to significant reductions in TPH catalytic function.
Because DA can be the source of numerous, different quinones (Graham,
1978; Graham et al., 1978; Kelgaris et al., 1995), we tested three
representative ones in an attempt to establish the generality of their
effects on TPH. DA was oxidized by iron + H2O2
to semiquinones and quinones as described in Equations 1-3. DA was
also converted enzymatically to the aminochrome by tyrosinase, and
finally, N-acetyl-DA was oxidized by NaIO4 to
the more stable o-quinone (Graham, 1978; Graham et al.,
1978). Each quinone caused significant inactivations of TPH. Third,
GSH, DTT, and ascorbic acid protected TPH from inactivation by each of
the DA-derived quinones. Antioxidants and reducing agents are well
known to prevent quinone formation from DA or to reduce the DA-quinone
back to DA (Graham, 1978; Graham et al., 1978; Hastings and Zigmond,
1994; Hastings et al., 1996b; Berman and Hastings, 1997). Quinones are electron-deficient compounds with high reactivity toward low molecular weight thiols, and the protectants could also exert their effects, in
essence, by competing with protein sulfhydryls for the quinones (van
Iwaarden et al., 1992). These same antioxidants and reductants also
prevent DA-quinone-induced cellular (Hastings et al., 1996a,b; Hoyt et
al., 1997; Terland et al., 1997) and DNA toxicity (Stokes et al.,
1996).
The chemical properties of DA allow other possible avenues of influence
on TPH in addition to quinones. The enzymatic metabolism of DA by
monoamine oxidase yields H2O2 that could alter
TPH (Kuhn and Arthur, 1996, 1997a,b). This possibility can be
eliminated by the results that showed that scavengers of
H2O2 (catalase), the hydroxyl radical (DMSO),
or superoxide dismutase did not prevent the DA-mediated
inactivation of TPH. Finally, catechol compounds such as DA and
L-DOPA chelate ferric iron and can inhibit TPH by
preventing iron redox during catalysis (Johansen et al., 1991; D'Sa et
al., 1996a,b). The possibility that DA is inactivating TPH via
chelation of iron also seems unlikely. Free DA was removed from the
enzyme by extensive washing before assays for catalytic activity, and
DA alone had no effect under the incubation conditions used here. DA
also caused a covalent modification of TPH (see below) that is
inconsistent with the competitive and reversible mechanism by which DA
chelates TPH-bound iron (Johansen et al., 1991). The recombinant TPH
used here is isolated as an apoenzyme, dependent on exogenous iron for
expression of catalytic activity (D'Sa et al., 1996a,b; Kuhn and
Arthur, 1996). Consequently, the enzyme contains little iron to
serve as a target for DA binding.
TPH that had been cleaved from its GST-fusion tag was incubated with
[3H]DA under conditions similar to those used for
catalytic studies to determine whether DA-TPH adducts were being
formed. A concentration-dependent and saturable incorporation of
tritium into the enzyme was observed under conditions that convert DA
to a quinone. [3H]DA alone did not label the
enzyme. The labeling of TPH by [3H]DA-quinone was
acid-stable, suggesting a covalent modification of the protein. GSH,
ascorbic acid, and DTT, at the same concentrations that protect TPH
from DA-mediated inactivation, almost completely blocked labeling of
TPH by [3H]DA. A plot of TPH inactivation and
tritium incorporation into the enzyme against the logarithm of the DA
concentration revealed a high negative correlation between these
measures (r = 0.983). Therefore, increasing
incorporation of tritium into TPH is associated with decreasing
catalytic function of the enzyme.
Quinones are highly reactive with nucleophilic groups including protein
sulfhydryls. TPH is a very unstable enzyme, and alterations in its
oxidation status can dramatically alter its catalytic function (Kuhn et
al., 1980). In view of the covalent modification of the enzyme by
DA-quinones and the close relationship between loss of activity and
[3H]DA-quinone incorporation into TPH, it was
hypothesized that the quinone-induced inactivation was mediated by the
alteration of cysteinyl residues. This hypothesis was tested by
exposing DA-quinone-inactivated TPH to acid hydrolysis and HPLC-EC
analysis. The results established that the inactivated enzyme contained cysteinyl-DA. Our results agree well with previous demonstrations that
DA can lead to the formation of cysteinyl-catechols via reactive quinones (Hastings and Zigmond, 1994). They also strengthen the possibility that the toxic effects of intrastriatal DA injections (Hastings et al., 1996b) and the inhibition of DA (Berman et
al., 1996) and glutamate transporter function (Berman and Hastings, 1997) by DA are mediated by DA-quinone attack on critical cysteinyl residues associated with these proteins.
TPH was exposed to redox-cycling staining after SDS-PAGE and was indeed
converted to a quinoprotein by each DA-quinone under the same
conditions that inactivate catalytic function. DA alone or conditions
that do not diminish enzyme activity (e.g., iron, H2O2, N-acetyl-DA,
NaIO4, or tyrosinase) did not result in
redox-cycling staining. These results represent the first demonstration
of this interesting post-translational modification in TPH. We do not yet know whether quino-TPH behaves as a "true" quinoprotein, an enzyme with a quinone-containing prosthetic group in its active site
(Anthony, 1996; Klinman, 1996). This possibility is under investigation, but until evidence supporting a role for a
quinone-prosthetic group function in modified TPH is gathered,
quino-TPH is most conservatively referred to as an "artificial"
quinoprotein (see Velez-Pardo et al., 1996).
The covalent modification of TPH by DA-quinones to form a redox-cycling
quinoprotein represents a novel finding for this important brain enzyme
and provokes interesting speculation with regard to 5-HT neurotoxicity.
As a quinoprotein, TPH would undergo continuous redox cycling that
could contribute to depletion of cellular energy stores and endogenous
reductants. Indeed, redox-cycling proteins have been implicated in
several forms of neurotoxicity (Brunmark and Cadenas, 1989; Velez-Pardo
et al., 1996), and redox cycling is likely to be more pronounced with
protein-bound quinones than with free quinones in solution (Paz et al.,
1991). Cellular defense mechanisms that might be expected to protect
against oxidative stress (e.g., GSH or ascorbate) could actually
contribute to redox cycling of a quinoprotein (Paz et al., 1991; Gieseg
et al., 1993; Velez-Pardo et al., 1996). Quinoproteins could also have
effects that are longer-lived than those of reactive oxygen species,
DA, or DA-quinones in solution (Hastings et al., 1996a,b; Shen
and Dryhurst, 1996; Shen et al., 1997). TPH represents the first
identified target for DA-quinone attack, and as a phenotypic marker for
5-HT neurons, it would be in a position to have detrimental cellular effects in vivo via its redox-cycling properties.
The results from the present in vitro studies may be
relevant for in vivo conditions known to cause deficits in
5-HT neurons. Substituted amphetamine drugs such as MDMA and
methamphetamine inactivate TPH (Stone et al., 1989a,b; Fleckenstein et
al., 1997) and lead to losses in 5-HT nerve endings (Seiden and Sabol,
1996; Gibb et al., 1997). The pattern of the effects of these drugs on
the 5-HT neuronal system is consistent with the possibility that a
DA-quinone could play a important role. First, the drug-induced 5-HT
alterations are clearly dependent on DA (Gibb et al., 1997). Second,
the in vivo inhibition of TPH by MDMA seems to involve the
modification of critical cysteinyls in the enzyme (Stone et al.,
1989a,b). Third, the 5-HT deficits caused by MDMA are prevented with
injections of ascorbate or cysteine (Gudelsky, 1996).
A role for a TPH-quinoprotein in amphetamine-induced neurotoxicity is
certainly plausible, as discussed above, but the specificity of
DA-quinone modification of proteins is an important issue to address to
define the scope of any role played by quinoproteins. TPH has 10 cysteinyl residues in its primary structure (Darmon et al., 1988) and
would seem to be a good substrate for cysteinyl-based quinone attack.
Very few proteins have been identified heretofore as catechol-induced
quinoproteins. Exposure of crude brain extracts to high concentrations
of DA results in the formation of just two redox-cycling quinoproteins
(Liu et al., 1985; Velez-Pardo et al., 1996). The identity of these
proteins has not been established beyond the characterization of their
molecular weights of 45 and 56 kDa. These proteins were originally
referred to as serotonin-binding proteins (Tamir and Huang, 1974), and
the 56 kDa protein is the same molecular weight as TPH. Studies of a
very limited set of other catechol-quinone-labeled proteins indicate
that they vary widely in the extent to which they are labeled (Kato et
al., 1996). Therefore, it does not seem that catechol-quinones
nonspecifically label large numbers of proteins. The rather selective
modification of proteins by DA-quinones would be consistent with the
limited neuronal deficits caused by MDMA and methamphetamine.
GSH is probably the major determinant of cellular redox potential, and
its intracellular concentration ranges from 1 to 10 mM
(Hwang et al., 1992). Because GSH is so effective at protecting TPH
from DA-quinone-induced inactivation in vitro, one must
also question whether TPH could ever be modified by DA-quinones
in vivo. Reaction-rate kinetics provide a plausible basis
for quinone modification of proteins, even in the presence of GSH. For
example, Denu and Tanner (1998) have shown that protein phosphatases
are inactivated by H2O2. Because the rate of
inactivation is 100-fold faster than the rate of reduction (and
reactivation) of the enzyme by GSH, inactivation takes place in the
presence of physiological concentrations of reductants (Denu and
Tanner, 1998). DA- and amphetamine-induced toxicity certainly occurs
in vivo, despite the presence of GSH. It certainly seems
possible that TPH could be at least one of the neuronal proteins
targeted by drug-induced toxic mechanisms.
| |
FOOTNOTES |
Received March 5, 1998; revised June 8, 1998; accepted June 25, 1998.
This research was supported by the National Institute on Drug Abuse
Grant DA10756 and by the Joe Young, Sr., Psychiatric Research Fund of
the Department of Psychiatry and Behavioral Neurosciences. We thank Dr.
Doyle Graham for his comments and advice on dopamine-based quinones.
Correspondence should be addressed to Dr. Donald M. Kuhn, Department of
Psychiatry and Behavioral Neurosciences, Gordon H. Scott Hall, Room
2125, 540 East Canfield, Detroit, MI 48201.
| |
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Abstract
Introduction
Compound via MeSH
