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. Author manuscript; available in PMC: 2022 Sep 1.
Published in final edited form as: Neurotoxicology. 2021 Jul 24;86:85–93. doi: 10.1016/j.neuro.2021.07.005

In vitro inhibition of glutathione-S-transferase by dopamine and its metabolites, 3,4-dihydroxyphenylacetaldehyde and 3,4-dihydroxyphenylacetic acid

Rachel A Crawford 1, Kate R Bowman 1, Brianna S Cagle 1, Jonathan A Doorn 1
PMCID: PMC8440459  NIHMSID: NIHMS1729136  PMID: 34314733

Abstract

Parkinson’s disease is characterized by dopamine dyshomeostasis and oxidative stress. The aldehyde metabolite of dopamine, 3,4-dihydroxyphenylacetaldehyde (DOPAL), has been reported to be cytotoxic and capable of protein modification. Protein modification by DOPAL has been implicated in the pathogenesis of Parkinson’s disease, but the complete pathology is unknown. Our findings show that DOPAL modifies glutathione S-transferase (GST), an important enzyme in the antioxidant defense system. DOPAL, dopamine, and the metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), inhibited the activity of GST isolated from N27 dopaminergic cells at an IC50 of 31.46μM, 82.32μM, and 260.0μM, respectively. DOPAL, dopamine, and DOPAC inhibited commercially available equine liver GST at an IC50 of 23.72μM, 32.17μM, and 73.70μM, respectively. This inhibition was time dependent and irreversible. 1mM ʟ-cysteine or glutathione fully protected GST activity from DOPAL, DA, and DOPAC inhibition. 1mM carnosine partially protected GST activity from DA inhibition. Furthermore, ʟ-cysteine was found to protect GST by forming a putative thiazolidine conjugate with DOPAL. We conclude that GST inactivation may be a part of the broader etiopathology of Parkinson’s disease.

Keywords: Parkinson’s disease, dopamine, DOPAL, glutathione S-transferase, glutathione

1. Introduction

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by motor issues and death of dopaminergic cells in the substantia nigra (Erkkinen et al., 2018). While exact PD etiopathology is unknown, it is thought that oxidative stress and dopamine (DA) dyshomeostasis play important roles (Goldstein et al., 2013). Consequently, the study of DA and its metabolites is paramount. DA is metabolized by monoamine oxidase (MAO) to the biogenic aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL) before detoxification by several aldehyde dehydrogenase enzymes (ALDH) to 3,4-dihydroxyphenylacetic acid (DOPAC) (Figure 1). In 1952, a review by Blaschko hypothesized that biogenic aldehydes produced from catecholamines are toxic to local cells (Blaschko, 1952). This view has evolved into what is now called the “catecholaldehyde hypothesis,” which states that DOPAL is an inherently cytotoxic molecule that can accumulate and lead to cell death (Goldstein et al., 2014). Much evidence supports the catecholaldehyde hypothesis and the concept that DA dyshomeostasis can lead to disease. Interruption of DA sequestration is linked to PD, as demonstrated by the downregulation of vesicular monoamine transporter 2 (VMAT2) in blood samples of PD patients (Molochnikov et al., 2012). VMAT2 sequesters DA in vesicles and its interruption leads to DA leakage to the cytosol where DA can auto-oxidize or undergo metabolism. Furthermore, mice low in VMAT2 present with dopaminergic neuron degeneration and both motor and non-motor PD symptoms (Taylor et al., 2011). The overexpression of MAOB, an enzyme responsible for DOPAL production, results in mice with locomotor issues and dopaminergic cell loss in the substantia nigra (Mallajosyula et al., 2008). Disruption of ALDH activity, and therefore disruption of the detoxification of DOPAL, is highly implicated in PD, evidenced by ALDH1/2 KO mice presenting with PD pathology (Wey et al., 2012) and the epidemiological correlation of PD with the ALDH inhibitor Benomyl, a fungicide (Casida et al., 2014; Fitzmaurice et al., 2013). Furthermore, downregulation of ALDH1A1 and ALDH1A1 mRNA is observed in blood samples of early-stage PD patients (Molochnikov et al., 2012) and dopaminergic neurons of patient brains (Mandel et al., 2005), respectively.

Figure 1.

Figure 1.

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The metabolism of DA.

While it is evident that disruption of the homeostatic pathway of DA can lead to disease, it is not fully known what causes the resulting cytotoxicity. The catecholaldehyde hypothesis conveys the idea that DOPAL, a highly reactive biogenic aldehyde, modifies proteins (Anderson et al., 2016; Anderson et al., 2011; Cagle et al., 2019). DOPAL readily reacts with proteins via its aldehyde constituent, which undergoes a Schiff base reaction with nucleophiles (e.g., lysine) followed by oxidative rearrangement to stable indole-type adducts (Anderson et al., 2016). DA and DOPAC modify proteins via auto-oxidation of the catechol moiety to a semi-quinone radical or ortho-quinone and subsequent Michael addition to thiols (e.g., cysteine) (Anderson et al., 2011; Rabinovic et al., 2000). Likewise, DOPAL can also undergo auto-oxidation of its catechol. This auto-oxidation is thought to increase the reactivity of its aldehyde constituent (Anderson et al., 2016). DOPAL modifies several key proteins that are implicated in PD, supporting the catecholaldehyde hypothesis. For example, DOPAL potently oligomerizes α-synuclein, a hallmark of PD (Follmer et al., 2015; Jinsmaa et al., 2016). DOPAL also inhibits tyrosine hydroxylase, a rate-limiting enzyme in DA synthesis (Mexas et al., 2011; Vermeer et al., 2012). Of specific interest to this report, a 1994 manuscript by Ploemen et al found that the quinone of DA inhibited glutathione S-transferase (GST) (Ploemen et al., 1994). Our laboratory similarly identified GST as a target of DOPAL in a proteomic scan utilizing PC6-3 cells (Jinsmaa et al., 2011). Thus, this study expands upon GST modification by DA and its metabolites.

GST is a ubiquitous enzyme that conjugates the reduced form of glutathione (GSH) to xenobiotics (Dringen and Hirrlinger, 2003; Janáky et al., 2000; Smeyne and Smeyne, 2013). GSH is an abundant and vital antioxidant that protects against oxidative stress. Oxidative stress is thought to contribute to the events that cause dopaminergic neuron degeneration in PD (Jenner, 2003; Jinsmaa et al., 2009), and also makes protein modification by DOPAC, DA, and DOPAL more likely (Anderson et al., 2016).The importance of GSH in oxidative stress models is well documented. For example, GSH depletion potentiates MPTP toxicity (Wullner et al., 1996) and produces dystrophia (Andersen et al., 1996) in dopaminergic neurons, and switches cellular response to nitric oxide from neurotrophic effects to cell death (Canals et al., 2001). The substantia nigra is particularly vulnerable to oxidative stress, partly because it has relatively low GSH levels, potentially a protective measure in itself since precursors to GSH are toxic at high concentrations (Dringen and Hirrlinger, 2003; Janáky et al., 2000). Additionally, the substantia nigra of PD patients has increased iron, a catalyst in the oxidative Fenton reaction (Mounsey and Teismann, 2012). In PD, GST polymorphisms seem to be correlated to the age of onset and environmental response (Ahmadi et al., 2000; Golbe et al., 2007; Wilk et al., 2006). For example, a GST pi G-for-A nucleotide substitution at position 313 is positively associated with age of onset in individuals with the PARK1 mutation (Golbe et al., 2007). Furthermore, exposure to pesticides paired with GST pi mutations is positively associated with PD (McCormack et al., 2002). While there is not yet any research directly linking GST polymorphisms with PD etiology, GST is a highly regulated protein influenced by many different factors, and thus should not be discounted as an important enzyme in PD etiopathology (Smeyne and Smeyne, 2013).

In this study, we have characterized the ability of DA, DOPAC, and DOPAL to modify GST and alter its function. Furthermore, this study investigates a variety of protective compounds (i.e., carnosine, ʟ-cysteine, and GSH) for their efficacy in mitigating GST modification and therefore may have implications for future pharmacotherapeutic work.

2. Materials and Methods

2.1. Chemicals

DA, DOPAC, equine liver GST, and l-cysteine were purchased from Sigma-Aldrich (St. Louis, MO). Carnosine was purchased from Fisher Scientific (Pittsburgh, PA). GSH was purchased from Research Products International (Mt. Prospect, IL). DOPAL was purchased from Cayman Chemicals (Ann Arbor, MI) or synthesized via the method of Fellman (Anderson et al., 2011; Fellman, 1958). All other chemicals were purchased from Sigma-Aldrich or Fisher Scientific at the highest purity available. The N27 cell line was generously provided by Dr. Jau-Shyong at the National Institute of Environmental Health Sciences (Research Triangle Park, NC).

2.2. Isolation of GST from N27 cells

2.2.1. N27 lysate production.

N27 cells, i.e., dopaminergic neuronal cells from rat midbrain, were lysed via lysis buffer (10mM KH2PO4, 0.1% Triton-X, pH 7.4). 1mL of buffer was added per 100 × 20 mm petri. After 15 minutes of incubation with buffer at 37°C, the petri was scraped to disturb cells and the lysate was aliquoted into a tube and frozen at −80° C for one hour. Lysate was then thawed on benchtop to help promote further lysis. Alternatively, lysate was produced via sonication after centrifugation and washing of the pellet. Lysate typically contained ~0.75mg/mL protein, as measured via BCA assay.

2.2.2. GST isolation.

GST was isolated from N27 cells using Glutathione Sepharose™ 4B (GE Healthcare Bio-Sciences, Uppsala, Sweden) following the manufacturer’s recommended protocol. Briefly, lysate was applied to a slurry of Glutathione Sepharose™ 4B and PBS and incubated with agitation for 1 hour at room temperature. After incubation, the mixture was centrifuged, supernatant discarded, and washed twice with PBS. Elution buffer (50mM Tris-HCl, 10mM GSH, pH 8.0) was applied and the mixture was incubated with agitation for 15 minutes at room temperature. After centrifugation, the supernatant was kept, as it contains the isolated GST. The elution process was repeated and the two eluates pooled. Isolation typically yielded ~0.15mg/mL, measured via absorbance at 280nm. Please note that isolation did not yield pure or concentrated protein; consequently, isolation was difficult and samples did not retain enzymatic activity if further purification was attempted. Therefore, samples utilizing N27 GST isolate contain GSH. However, this aids in modeling an in vitro system with intrinsic antioxidants. After the initial evaluation of N27 GST activity modification (described below in 2.4.1), commercially available equine liver GST was used to obtain biochemical results with a relatively pure protein.

2.3. Isoform Evaluation

Commercially available equine liver GST (Sigma Aldrich, St. Louis, MO) and isolated N27 GST were evaluated for their isoform content via LC-MS/MS with an Agilent 1260 Infinity Capillary pump coupled with a Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer from Thermo Scientific. Samples were digested in solution with lysyl endopeptidase and trypsin prior to analysis. The peptides were separated via liquid chromatography and further analyzed via MS/MS fragmentation. The resulting intact and fragmentation patterns were matched to known isoform sequences through MASCOT and Sequest HT software databases.

2.4. Measurement of GST inhibition

2.4.1. N27 GST inhibition.

GST activity was measured via the1-chloro-2,4-dinitrobenzene (CDNB) assay. Prior to the assay, the isolated N27 GST (~0.15mg/mL, 50mM Tris-HCl, 1mM GSH, pH 8.0) was incubated with 5μM, 10μM, 25μM, 50μM, or 100μM DA, DOPAL, or DOPAC for 4 hours at 37°C. N27 GST was incubated without any inhibitor for a positive control. Samples were pipetted into a 96 well assay plate with 20μL sample (i.e., the isolated GST +/− test inhibitors), 20μL of 25mM reduced GSH (0.1mM potassium phosphate, 1mM EDTA, pH 6.5), 5μL of 40mM CDNB (95% ethanol), and 155μL buffer (0.1mM potassium phosphate, 1mM EDTA, pH 6.5). Negative controls contained 175μL buffer, 20μL of 25mM reduced GSH, and 5μL of 40mM CDNB. Kinetic absorbance was measured at 340nm for 15 min at 37°C. The absorbance (y) was plotted vs time (x) and the linear portion of this graph was assessed. The slopes were compared to the positive control.

2.4.2. Equine liver GST inhibition.

GST activity was again measured via CDNB assay. Prior to the assay, 10μL of 0.5mg/mL equine liver GST (diluted in 50mM sodium phosphate buffer, pH 7.4) was incubated with 5μM, 10μM, 25μM, 50μM, or 100μM DA, DOPAC, or DOPAL with a total volume of 100μL (50mM sodium phosphate buffer, pH 7.4) for 4 hours at 37°C. GST was incubated without inhibitor for a positive control. Samples were pipetted into a 96 well assay plate with 5μL of sample (i.e., the diluted equine liver GST +/− test inhibitor), 20μL of 25mM reduced GSH (0.1mM potassium phosphate, 1mM EDTA, pH 6.5), 5μL of 40mM CDNB (95% ethanol), and 170μL of buffer (0.1mM potassium phosphate, 1mM EDTA, pH 6.5). Negative controls contained 175μL buffer, 20μL of 25mM reduced GSH, and 5μL of 40mM CDNB. Kinetic absorbance was measured at 340nm for 15 min at 37°C. The absorbance (y) was plotted vs time (x) and the linear portion of this graph was assessed. The slopes were compared to the positive control.

2.4.3. Determination of time dependency.

To determine time dependency, the experiment described in 2.4.2 was repeated. Prior to the assay, aliquots of the sample mixture were taken after 1, 2, and 4 hours of incubation and frozen at −20° C to stop the reaction. An additional 24-hour time point was taken for inhibitor concentrations at 5μM and 100μM to observe the effect of prolonged exposure. Samples were thawed on benchtop and the CDNB assay was performed as described in 2.4.2.

2.4.4. Determination of reversibility.

To determine the reversibility of GST inhibition, equine liver GST was incubated with or without 100μM DA, DOPAL, or DOPAC as described in 2.4.2. Samples were then applied to Bio-Spin® 6 Tris Columns per manufacturer’s instructions (Bio-Rad Laboratories, Inc., Hercules, CA). GST activity was measured via CDNB assay and compared to GST activity that had not been applied to the Bio-Spin® 6 Tris Columns. These columns allow for dissociation and removal of reversible inhibitors and elution of GST that is unmodified, if modification was indeed reversible. We validated this method by similarly incubating equine liver GST with 500μM caffeic acid and 500μM S-hexylglutathione, independently. Caffeic acid and S-hexylglutathione are known reversible inhibitors of GST (Adang et al., 1991; Kudugunti et al., 2011); GST showed significant inhibition after incubation with caffeic acid or S-hexylglutathione, but after application of samples to the Bio-Spin® 6 Tris Columns, GST regained the full value of its original activity (data not shown). Furthermore, we validated that the columns would indeed remove any free, dissociated DOPAL via HPLC-UV analysis (data not shown); we have previously reported that these columns will remove ~90% of DOPAL (Mexas et al., 2011).

2.5. Nitro blue tetrazolium staining

2.5.1. DOPAL modification of a standard.

The protein bovine serum albumin (BSA) was used as a standard protein to test the protective effects of l-cysteine and carnosine against DOPAL modification. 0.5mg/mL BSA (50mM sodium phosphate buffer, pH 7.4) was incubated with 200μM DOPAL for 4 hours at 37°C; likewise, 0.5mg/mL BSA was incubated with 200μM DOPAL and 100μM, 200μM, 1mM, or 5mM l-cysteine or carnosine for 4 hours at 37°C. These samples were then resolved via SDS-page, transferred to a nitrocellulose membrane, and stained as described in below in 2.5.3.

2.5.2. DOPAL modification of equine liver GST.

Equine liver GST (0.5mg/mL) was incubated with varying concentrations of DOPAL (10μM, 20 μM, 50 μM, 100μM), 100μM DA, or 100μM DOPAC in 50mM sodium phosphate buffer (pH 7.4) for 4 hours at 37°C. Similarly, the 0.5mg/mL GST was also incubated with 100μM DOPAL and either 1mM carnosine, 1mM l-cysteine, or 1mM glutathione. These samples were then resolved via SDS-page, transferred to a nitrocellulose membrane, and stained as described in below in 2.5.3.

2.5.3. Measurement of DOPAL modification.

The above samples were heated with 5μL 6x SDS protein loading buffer at 80°C for 15 minutes. Samples were resolved on a 10% acrylamide SDS PAGE gel, then transferred to nitrocellulose membrane. The membrane was stained with nitro blue tetrazolium chloride (NBT) (0.2mg/mL NBT in 2M potassium glycinate buffer, pH 10) in darkness for approximately 16 hours at 4° C. After NBT staining, protein presence was verified by staining the membrane with Ponceau S (data not shown). Staining was quantified using ImageJ software (National Institutes of Health, Bethesda, MD) and further analyzed using GraphPad Prism (GraphPad Software, San Diego, CA)

2.6. Mass Spectrometry

2.6.1. HPLC-MS Parameters.

Samples were analyzed on an Agilent 6530 Accurate-Mass Q-TOF mass spectrometer interfaced with an Agilent 1260 Series Capillary HPLC (Agilent Technologies, Santa Clara, CA). Samples were injected with a volume of 5μL and chromatographic separation was carried out on a Jupiter C18 column (150 × 1.00mm, 5μ) at 50 μL/min for MS and a Zorbax SB-C18 column (150 × 0.5mm, 5μ) at 15 μL/min for MS/MS. Mobile phase A (UHPLC grade H20, 0.1% FA) and mobile phase B (UHPLC grade ACN, 0.1% FA) were used in the following gradient for MS (A : B): 0 min (97 : 3)→5 min (97:3) →20 min (50 : 50)→30 min (50 : 50)→35 min (97 : 3). The following gradient was used for MSMS (A : B): 0 min (97 : 3)→1 min (97:3) →10 min (40 : 60)→13 min (5 : 95)→17 min (5 : 95)→17.01 min (97 : 3)→30 min (97 : 3). Samples were analyzed in positive mode with dual Agilent jet stream electrospray ionization. Data were collected and analyzed on MassHunter software (Agilent Technologies, Santa Clara, CA.) and visualized with GraphPad Prism (GraphPAD Software, San Diego, CA). Chromatograms presented are representative of at least three trials.

2.6.2. Sample preparation.

1mM l-cysteine was incubated with 100μM DOPAL for 4 hours at 37°C (MS: 10mM ammonium bicarbonate, pH 8.0; MSMS: 50mM sodium phosphate, pH 7.4). The sample was diluted 1:5 in LC/MS grade water with 0.1% formic acid prior to injection.

2.7. Statistical analysis

NBT staining was quantified using ImageJ software (National Institutes of Health, Bethesda, MD). Data were analyzed using the Prism program (GraphPAD Software, San Diego, CA) and, for LC-MS, MassHunter software (Agilent Technologies, Santa Clara, CA). One-way ANOVAs were employed, followed by either the Dunnett test or Tukey test, depending on the experimental conditions.

3. Results

3.1. Both N27-isolated GST and commercial GST are a pi isoform

Commercially available equine liver GST and isolated N27 GST were evaluated for their protein isoform content via LC-MS/MS. The samples’ spectra were matched via Mascot (searched against Sprot-mammalia) and Sequest HT (searched against Sprot-all) databases. The commercially available “equine” liver GST received the highest score from both databases for a pi isoform from Bos taurus (GSTP1; accession P28801). As expected, the N27 GST sample was impure and therefore had top scores for non-GST proteins. The single GST match for the N27 (i.e., rat and dopaminergic) sample was for a pi isoform from Rattus norvegicus (GSTP1; accession P04906), which had a high confidence match.

3.2. GST activity is inhibited by DOPAL, DA, and DOPAC

The GST isolated from dopaminergic N27 cells was incubated with varying concentrations of DOPAL, DA, and DOPAC (2.4.1) prior to measurement of GST activity via CDNB assay. This model contains impurities including other proteins and GSH. N27 GST showed significant inhibition by DOPAL (IC50 = 31.46μM), DA (IC50 = 82.32μM), and DOPAC (IC50 = 260.0μM) (Figure 2).

Figure 2.

Figure 2.

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N27 isolated GST is significantly inhibited by a) DOPAL (IC50 = 31.46μM), b) DA (IC50 = 82.32μM), and c) DOPAC (IC50 = 260.0μM). One-way ANOVA with Dunnett test; controls n=6, 5μM DOPAL n=15, 10μM DOPAL n=15, all other n=3; p<0.05*, p<0.01**, p<0.001***, p<0.0001****

Because of its concentration, purity, and ease of access, commercially available equine liver GST was used for further analysis of activity modification (though it should be noted that commercially available GST also contains GSH because of its purification process). A stepwise reduction in equine liver GST activity is observed with increasing concentrations of DOPAL, DA, and DOPAC (Figure 3). DOPAL is the most potent, with an IC50 of 23.72μM, followed by DA (32.17μM) and DOPAC (73.70μM).

Figure 3.

Figure 3.

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The stepwise inhibition of GST activity with increasing levels of a) DOPAL, b) DA, and c) DOPAC can be observed. One-way ANOVA with Dunnett test; 25μM DA n=7, DA positive control n=5, all other n=3; p<0.05*, p<0.01**, p<0.001***, p<0.0001****

Inhibition of equine liver GST activity seems to be time dependent (Figure 4) and irreversible (Figure 5). Figure 5 demonstrates that following incubation with DOPAL, DA, or DOPAC, GST activity does not return to its full value (as defined by a control with no test inhibitor) after application to Bio-Spin® 6 Tris Columns. In contrast, when known reversible inhibitors (i.e., caffeic acid and S-hexylglutathione) were used in the same procedure, GST activity returned in full (data not shown) compared to control and following application to the column. Further examination of irreversibility was carried out via a 24-hour time point collection for 100μM and 5μM DOPAL, DA, and DOPAC. Figure 6 compares the 24 and 4-hour time point. While the 5μM concentration of all inhibitors showed more variability at 24 hours, there was no significant difference between the 24 and 4-hour time point at this concentration. The 24-hour incubations with 100μM of DOPAL or DA seem to decimate GST activity, though there is only statistical difference between the 24 and 4-hour time points for DA. The 24-hour incubation with DOPAC produces similar GST activity inhibition to the 4-hour incubation.

Figure 4.

Figure 4.

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Inhibition of GST activity by DOPAL, DA, and DOPAC is time dependent. One-way ANOVA with Dunnett test; n=3; p<0.05*, p<0.01**, p<0.001***, p<0.0001****

Figure 5.

Figure 5.

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Inhibition of GST activity by DOPAL, DA, and DOPAC seems to be irreversible. Samples designated as (+) indicate that the sample was applied to a size exclusive column to wash away reversible inhibitors. The (−) samples did not receive this treatment. One-way ANOVA with Tukey’s multiple comparison test; n=3; p>0.05 ns

Figure 6.

Figure 6.

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Inhibition of GST activity after a 24-hour incubation with 5μM or 100μM of DOPAL, DA, or DOPAC. The 24-hour incubation of GST with either 100μM DOPAL or DA almost completely nulls enzyme activity. One-way ANOVA with Tukey’s multiple comparison test; n=3; p<0.05*, p<0.01**, p<0.001***, p<0.0001****

3.3. GST activity is protected by carnosine, glutathione, and ʟ-cysteine

GST activity, as measured via CDNB assay, is protected when 1mM carnosine, glutathione, or l-cysteine are added prior to incubation with 100μM DOPAL, DA, or DOPAC (Figure 7). l-cysteine and glutathione protect GST function from all three inhibitors, with activity retaining 100% of its original function. Carnosine only showed significant protection against inhibition by DA.

Figure 7.

Figure 7.

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GSH or l-cysteine aid in GST retaining 100% of its activity when incubated with a) 100μM DOPAL, b) 100μM DA, and c) 100μM DOPAC. Carnosine aids in partial activity retention when GST is incubated with b) DA. One-way ANOVA with Dunnett test; n=3; p<0.05*, p<0.01**, p<0.001***, p<0.0001****

3.4. Nitro blue tetrazolium staining reveals that GST is modified by DOPAL but is protected by L-cysteine, carnosine, and glutathione

NBT, which turns dark purple in the presence of a redox-cycling moiety such as a catechol, was used to assess modification of GST by DOPAL, DA, and DOPAC. Figure 8 demonstrates staining at the molecular weight marker for GST (~26 kDa) after reaction with 50μM or 100μM DOPAL. This staining, though less apparent, can also be observed for GST that was reacted with 10μM and 20μM DOPAL. Interestingly, no staining is observed for GST that was incubated with DA or DOPAC.

Figure 8.

Figure 8.

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NBT staining reveals that DOPAL creates an adduct with GST.

Figure 9 quantifies NBT staining of BSA that was incubated with 200μM DOPAL +/− varying concentrations of l-cysteine or carnosine. l-cysteine significantly reduced NBT staining at 1mM. Carnosine protection appears to trend towards significance but was not as effective.

Figure 9.

Figure 9.

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l-cysteine significantly reduce protein modification of BSA by 200μM DOPAL, compared to the control of BSA and 200μM DOPAL. Carnosine did not significantly reduce protein modification, though it appears to trend toward significance. One-way ANOVA with Dunnett test; n=3; p<0.05*, p<0.01**, p<0.001***, p<0.0001****

Finally, NBT stain was used to assess equine liver GST modification by 100 μM DOPAL when in the presence of 1mM carnosine, l-cysteine, or glutathione (Figure 10). l-cysteine and glutathione appear to fully protect against protein modification; staining of samples treated with DOPAL and l-cysteine or DOPAL and glutathione appear similar to the untreated control (−) and are significantly different from the DOPAL-treated positive control. Carnosine appears to be moderately protective; it significantly reduces staining compared to the positive control.

Figure 10.

Figure 10.

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l-cysteine, GSH, and carnosine significantly reduce NBT staining of equine liver GST that is treated with DOPAL. Results were compared to the positive control of GST treated with 100μM DOPAL. One-way ANOVA with Dunnett test; n=3; p<0.05*, p<0.01**, p<0.001***, p<0.0001****

3.5. Mass Spectrometry

To investigate the mechanism via which l-cysteine is protective against DOPAL modification, mass spectrometric analysis was employed to structurally characterize the product resulting from the reaction of DOPAL with either scavenger. We identified a DOPA l-cysteine conjugate with a [M+H]+ peak at 256.06 m/z, consistent with a putative thiazolidine conjugate (theoretical [M+H]+ = 256.0638 m/z). This mass is not consistent with the alternative Michael-type addition product (i.e., l-cysteine reaction with the oxidized catechol). Furthermore, MS/MS analysis revealed peaks at 149.00 m/z and 167.01 m/z, consistent with fragmentation for the thiazolidine conjugate but not Schiff base. Therefore, the resultant conjugate we suggest is shown in Figure 11, c. A DOPAL-carnosine conjugate could not be identified. The reaction of DOPAL with GSH was not examined because GSH is thought to act as an antioxidant to mitigate DOPAL toxicity (Nelson et al., 2019) and therefore there is no conjugate product to be observed.

Figure 11.

Figure 11.

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a) The spectrum for the L-cys-DOPAL conjugate, shown at 256.06 amu. b) The spectrum for L-cysteine. c) The putative structure of the L-cys-DOPAL conjugate based on measured mass.

4. Discussion

Neurodegenerative disorders such as PD are characterized by oxidative stress and disruption of DA homeostasis (Dias et al., 2013; Masato et al., 2019). DOPAL, the biogenic aldehyde metabolite of DA, is implicated in PD pathogenesis via its oxidant properties and ability to modify proteins (Anderson et al., 2011). Interrogation of the exact mechanisms of DOPAL neurotoxicity is vital to help define the largely unknown etiopathology of PD. While literature reports affirm DOPAL’s cytotoxicity via protein modification (e.g., modification of alpha-synuclein) (Jinsmaa et al., 2016) there is currently little knowledge detailing the modification of GST by DOPAL. Our lab has previously identified GST as a target of DOPAL modification in PC6-3 cells (Jinsmaa et al., 2011). In addition to expanding our knowledge of DOPAL targets, this particular protein modification is interesting because it demonstrates a potential breach of the antioxidant defense system which is already precariously balanced prior to disease etiology (Islam, 2017). For the first time, this report characterizes the modification of GST by DA, DOPAL, and the acid metabolite DOPAC.

Two forms of GST were used in our characterization of GST modification. First, GST was isolated from rat dopaminergic neurons (i.e., N27 cells) to obtain a biologically relevant form of GST. Because isolation was difficult and further purification or dilution impeded GST activity, these samples were impure and contained undiluted GSH from the purification process. These impurities likely impacted DOPAL modification and the observed results but help aid in representing cellular events. We also used a commercially available GST from Sigma Aldrich to examine the modification of a relatively pure protein. While this commercially available GST also contained GSH, the GST was highly active and therefore further diluted for experimentation. Both GST sources were investigated via LC-MS/MS for their isoform content. Database comparison revealed that both sources are likely a pi isoform, with the Sigma Aldrich GST having a top match of Bos taurus GSTP1 and the rat dopaminergic GST matching with Rattus norvegicus GSTP1. Seven cytosolic GST isoforms are recognized in humans: alpha, pi, mu, sigma, zeta, theta, and omega (Hayes et al., 2005). The brain contains primarily alpha, pi, and mu isoforms and it is thought that mu and pi are most abundant in the substantia nigra (Smeyne et al., 2007; Smeyne and Smeyne, 2013). Furthermore, polymorphisms of the pi isoform are correlated with PD age-of-onset and environmental risk (Delamarre and Meissner, 2017; Golbe et al., 2007; Wilk et al., 2006). Because both forms of GST used in this report are pi isoforms, we propose that the observed results have relevance to PD pathology, though future studies in vivo and with human isoforms are needed.

Modification of GST activity was first evaluated via CDNB assay. Both GST sources showed significant inhibition by DOPAL, DA, and DOPAC. As expected, the N27-isolated GST inhibition was variable, especially at lower concentrations of inhibitor. The results observed are likely impacted by the impurity of the N27-isolated samples; nevertheless, statistically significant inhibition is observed even in a system that mimics intrinsic antioxidant defense and potential concomitant reactions of a cell. Comparatively, the biochemical reaction of the commercially available GST demonstrated a neat, step-wise inhibition by the compounds, with a lower IC50 for all three inhibitors. DOPAL was able to significantly inhibit commercially available GST at 5μM. This is not far off from in vivo concentrations of DOPAL, which is thought to be around 2μM; DOPAL is toxic to dopaminergic cells at 7μM (Schamp and Doorn, 2017). As anticipated because of literature reports of its potent toxicity, DOPAL produced the strongest inhibition of both GST sources, likely due to its aldehyde moiety (Anderson et al., 2011; Jinsmaa et al., 2020). The commercially available GST was further used to demonstrate the time dependence and irreversible nature of GST inhibition by DA, DOPAL, and DOPAC. Of note, a 24-hour incubation with 100 μM DA or DOPAL almost abrogated GST activity, underlining the potential toxicity and the importance of cellular DA homeostasis.

NBT staining was used to further investigate the modification of GST by DA, DOPAL, and DOPAC. NBT can be used to identify quinoproteins (Paz et al., 1991; Rees et al., 2009). If DOPAL, DA, or DOPAC create adducts with GST that structurally allow redox cycling of the catechol moiety or quinone, the NBT will turn dark purple and hence stain the observed GST. Interestingly, reaction of GST with DOPAL results in this purple stain but reaction with DA or DOPAC does not. There are several feasible reasons for this result. First, it should be noted that GST contains a higher percentage of lysine relative to cysteine residues (the specific protein matched via LC-MS/MS and database comparison contains 12 lysine and 4 cysteine residues). Because DOPAL likely reacts with lysine residues, whereas DOPAC and DA favor cysteine (Anderson et al., 2016), it is conceivable this discrepancy in NBT staining is simply because there are more DOPAL modifications. Secondly, it is probable that DOPAL reacts more readily in the described conditions because it does not require prior oxidation to a quinone to modify proteins (Anderson et al., 2016). DA and DC must be oxidatively activated before they can react via Michael addition with cysteine. It is also possible that DA and DC induce an irreversible GST modification (e.g., via ROS production) and then disassociate, which would contrast to the likely covalent modification mechanism of DOPAL based on NBT results. More work is needed to further confirm the mechanisms of inhibition.

After characterization of GST modification by the three inhibitors, we then sought to examine if different protective compounds could prevent this modification. Both CDNB assays and NBT staining were employed to achieve this goal. BSA was used as a standard protein to elucidate the protective properties of carnosine and l-cysteine; while both appeared to reduce NBT staining of BSA treated with DOPAL, only l-cysteine produced a statistically significant reduction. Carnosine, glutathione, and l-cysteine showed protection of GST function and modification, as measured via CDNB assay or NBT staining. Glutathione and l-cysteine were inarguably more efficacious at protecting GST than carnosine. Carnosine appeared to reduce NBT staining of GST treated with DOPAL, but carnosine only showed significant protection of GST activity against DA (as measured via CDNB assay). This is interesting because carnosine is reported to readily scavenge the aldehydes (Gilardoni et al., 2020), and previous work reported its reaction with DOPAL and the aldehyde metabolite of norepinephrine (Nelson et al., 2019). In contrast to the scavenging mechanism of carnosine, previous work showed GSH functions as an antioxidant to mitigate DOPAL toxicity (Anderson et al., 2016; Nelson et al., 2019). More precisely, this means that GSH prevents the oxidative rearrangement of DOPAL after DOPAL first reacts with proteins via reversible Schiff base, thereby preventing production of a more stable product (Anderson et al., 2016). Furthermore, prevention of the auto-oxidation of DOPAL prevents the concomitantly produced ROS (Anderson et al., 2016). Like carnosine, l-cysteine has been reported to be an aldehyde scavenger (Nelson et al., 2019; Wlodek et al., 1993). A scavenger acts as a nucleophile and reacts with electrophilic compounds such as DOPAL, thereby resulting in sequestration of the electrophilic compounds and mitigation of their cytotoxic effects.

Despite reports of l-cysteine’s ability to scavenge aldehydes, we were not certain of the mechanism of action via which l-cysteine protects GST from DOPAL. Therefore, mass spectrometric analysis was performed to structurally characterize the product resulting from the reaction of DOPAL with l-cysteine. These efforts revealed an identifiable DOPAL-l-cysteine conjugate at an [M+H]+ peak of 256.06 m/z, consistent with a putative thiazolidine conjugate. The resultant conjugate we suggest is shown in Figure 11, c. This conclusion is consistent with a report of the biogenic aldehyde of norepinephrine forming a thiazolidine with l-cysteine (Wanner et al., 2020). We were unable to identify a DOPAL-carnosine conjugate, but this conjugate is likely a Schiff base/indole product if these two reactants do indeed produce an adduct (Anderson et al., 2016); if produced, this adduct may be unstable, which would be consistent with our results that show carnosine is not particularly efficacious at protecting GST against modification by DA and its metabolites.

In conclusion, we have characterized GST as a protein target of DA and its metabolites. Furthermore, we have demonstrated that l-cysteine and GSH are protective of GST function which has implications for future pharmacotherapeutic work. Disruption of DA homeostasis in PD pathology can result in cytosolic DA and aberrant DOPAL production, making proteins susceptible to modification. The brain is also susceptible to oxidative stress, especially if cellular levels of antioxidants are low (Sayre et al., 2008). Redox and DA equilibrium are precariously balanced to promote cell health and vitality. GST is an important regulator of oxidative stress; GST is therefore a particularly interesting protein target of DOPAL because this reaction combines the deregulation of both delicate systems and may tip the oxidative balance in a direction that favors disease. Thus, we propose that the modification of GST observed in this report may be part of the broader etiopathology of PD.

Highlights.

  • Dopamine and its metabolites inhibit the activity of glutathione S-transferase.

  • The biogenic aldehyde metabolite of dopamine, 3,4-dihydroxyphenylacetaldehyde (DOPAL), most potently inhibits glutathione S-transferase.

  • Glutathione S-transferase activity is protected from modification by glutathione, carnosine, or ʟ-cysteine.

Acknowledgements

We would like to thank the Protein Facility of the Iowa State University Office of Biotechnology for their helpful protein isoform identification. Thank you to Dr. Jau-Shyong for the generous gift of the N27 cell line. Thank you to Dr. Dave Roman and Dr. Joseph O’Brien for help with Glutathione Sepharose™ 4B troubleshooting. Thank you to Dr. Ettore Gilardoni for mass spectrometry advice. Thank you to Grant Cooling and Dr. Nathan Delvaux for protein purification advice.

Funding Sources

This work was supported by the University of Iowa Pharmacological Sciences Training Program (NIH T32GM067795); the National Institutes of Health (R21 AG057006 & R01 ES029035); the University of Iowa Environmental Health Sciences Center (NIH P30 ES005605) and the Graduate College of the University of Iowa.

Abbreviations

ALDH

aldehyde dehydrogenase

BSA

bovine serum albumin

CDNB

1-chloro-2,4-dinitrobenzene

DA

dopamine

DOPAC

3,4-dihydroxyphenylacetic acid

DOPAL

3,4-dihydroxyphenylacetaldehyde

GSH

glutathione

GST

glutathione S-transferase

MAO

monoamine oxidase

NBT

nitro blue tetrazolium

PD

Parkinson’s disease

Footnotes

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Declaration of Competing Interest

The authors declare no conflicts of interest.

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