In Silico and in Vitro Study of Trace Amines (TA) and Dopamine (DOP) Interaction with Human Alpha 1-Adrenergic Receptor and the Bacterial Adrenergic Receptor QseC
Arif Luqmana,b,c Viol Dhea Kharismac Ruben Amian Ruiza Friedrich Götza
aMicrobial Genetics, Interfaculty Institute of Microbiology and Infection Medicine Tübingen (IMIT), University of Tübingen, Tübingen, Germany, bBiology Department, Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia, cGenerasi Biologi Indonesia (Genbinesia) Foundation, Gresik, Indonesia
Key Words
Trace amines • Dopamine • QseC • α1-adrenergic receptor
Abstract
Background/Aims: Trace amines (TA) are small organic compounds that have neuromodulator activity due to their interaction with some neuron-related receptors, such as trace amine associated receptors (TAARs), α2-adrenergic receptor (α2-AR) and ß-adrenergic receptor (ß-AR). However, there is little information on whether TA and dopamine (DOP) can interact with other adrenergic receptors (ARs) such as the mammalian α1-AR and the bacterial counterpart QseC, which is involved in quorum sensing of some Gram-negative pathogens. The aim of this study was to investigate the interaction of TA and DOP with α1-AR and QseC. Methods: We performed an in silico study using 3D structure from SWISS MODEL and analyzed the protein interaction via molecular docking using PyMol, PoseView and PyRX 8.0. For the in vitro study, we investigated the QseC kinase activity by measuring the remaining ATP in a reaction containing QseC-enriched membrane incubated together with purified QseB and EPI, TA, DOP, or PTL respectively. We also measured the intracellular Ca++ levels, which represents the α1-AR activation, in LNCAP (pancreatic cell line) cells treated with EPI, TA, DOP and PTL respectively using a fluorescence-based assay. The LNCAP cell proliferation was measured using an MTT-based assay. Results: Our in silico analysis revealed that TAs and DOP have high binding affinity to the human α1-AR and the bacterial adrenergic receptor (QseC), comparable to epinephrine (EPI). Both are membrane-bound kinases. Experimental studies with pancreatic cell line (LNCAP) showed that the TAs and DOP act as α1-AR antagonist by counteracting the effect of EPI. In the presence of EPI, TA and DOP trigger an increase of the intracellular Ca++ levels in the LNCAP cells leading to an inhibition of cell proliferation. Although in silico data suggest an interaction of TA and DOP with QseC, they do not inhibit the kinase activity of QseC, a histidine kinase receptor involved in quorum sensing which is also sensitive to EPI. Conclusion: Our study showed that the TAs and DOP act as α1-AR antagonist but no effect was observed for QseC.
Trace amines (TA) are monoamines which are regarded as undesirable by-products of food and beverage fermentation [1-4]. However, they also play a role as a neurotransmitter in invertebrates such as insects [5-7], or as neuromodulators in vertebrates where they are ligands of the trace amine associated receptors (TAARs) [8-10]. The term ‘trace’ is based on their low concentrations in the central nervous system (CNS) in comparison to the endogenous catecholamines, such as serotonin, epinephrine (EPI) or dopamine (DOP) [11]. According to some reports, activation of TAAR1 in the brain by TA might contribute to the generation of schizophrenia, depression and other neurodegenerative and neuropsychiatric diseases [12, 13]. The psychotic syndromes might be triggered by TAAR1 activation via TA that inhibits the expression of transporters for neurotransmitters and consequently their re-uptake is affected [14].
TA, DOP and EPI behave differently for certain ARs. On α2-AR, TA and DOP act as agonist like EPI, whereas on α2-AR, TA and DOP act as partial allosteric antagonists in contrast to EPI [15-17]. Since a number of commensal skin bacteria produce TA, we asked the question whether bacteria communicate with their host via TA. Indeed, it has been shown that the TA and DOP increase bacterial internalization in the intestinal epithelia via an agonistic effect on α2-AR [18, 19]. Furthermore, skin commensals producing TAs such as Staphylococcus epidermidis, accelerates wound healing by antagonizing the effect of EPI which inhibits cell motility by β2-adrenergic receptor (β2-AR) activation [20]. As β2-AR antagonists, TA and dopamine (DOP) abrogate the effect of EPI, thus accelerating wound healing both in vitro and in a mouse model.
To our knowledge, there are no reports on how TA and DOP act on α1-AR. Such an investigation of the interaction of TA and DOP with α1-AR might provide more insight on the significance of TA, if any, in vertebrates. Since ARs are expressed in many parts of the human body, they are important receptors that can affect the body's metabolism. Recently, some Gram-negative bacteria have also been reported to have an AR-like receptor involved in quorum sensing (QS) regulation. This receptor, named QseC, is one of the quorum sensing receptors first reported in Escherichia coli. QseC is a sensor kinase that can be activated by either the autoinducer-3, but also by EPI and norepinephrine [21]. The activation of this QseC receptor leads to an upregulation of various virulence factors in pathogenic E. coli, such as flagella, shiga toxins, intimin, and locus of enterocyte effacement (LEE) island [21, 22]. Therefore, it would be interesting to investigate the interaction of other potential compounds such as TA and DOP on QseC. Here we performed in silico and in vitro interaction studies of TA and DOP with α1-AR and QseC. TA and DOP showed high binding affinity towards α1-AR and QseC. TA and DOP share some interaction sites with the known agonist (EPI) in silico. In the in vitro studies, however, TA and DOP differed in their effect on α1-AR and QseC.
Materials and Methods
In silico study
Ligand and protein interaction. We modeled the α1-AR and QseC using SWISS MODEL software [23] to obtain the 3D structure of protein receptors. We then analyzed the ligand and resulted 3D structure of the protein interactions using PyMol. We further analyzed the ligand-receptor interaction for more information about the type of chemical interaction and its interaction sites namely the position and type of chemical bond interactions such as hydrophobic, hydrogen, or phi-alkyl using PoseView [24, 25]. The blind docking method was used in this study because the active site of the target protein was unknown, therefore the grid was directed at the entire surface of the target protein [26].
Molecular docking. We used PyRx 0.8 software to perform molecular docking between the ligands (TRY, PEA, TYM, DOP, PTL, EPI and Autoinducer-3) and the receptors, α1-AR and QseC. The molecular docking study was conducted to obtain the binding affinity value between the ligand and the receptor. For this analysis, a blind docking was carried out to reveal the possible affinity of the ligands to all sites of the receptor [26].
In vitro study
Preparation of QseB and QseC-enriched membrane. Plasmids pET28a-qseC and pET28a-qseB were constructed for the expression and purification of QseC and QseB in E: coli BL21. qseC and qseB genes were amplified from E. coli K12 using specific primers (Supplementary Table S1) and inserted into HindIII-linearized pET28a plasmid using Hi-Fi DNA Assembly Master Mix (New England Biolabs), and the construct was transformed into E. coli DC10B (for all supplementary material see www.cellphysiolbiochem.com). Clones were selected on 30 μg/mL kanamycin-containing Luria-Bertani (LB) agar. The constructed plasmid from selected clones were isolated and transformed into E. coli BL21; clones were selected on 30 μg/mL kanamycin containing LB agar. Selected clones containing pET28a-qseC and pET28a-qseB were grown in 30 μg/mL kanamycin-containing LB broth overnight at 37oC with 150 rpm shaking. The grown cultures were reinoculated into fresh 30 μg/mL kanamycin-containing LB medium and the protein expression was induced using a final concentration of 0.5 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The cells were pelleted and resuspended in PBS-containing protease inhibitor cocktail. Cells were disintegrated with glass beads (Roth, Karlsruhe, Germany) using FastPrep instrument (MP Biomedicals). Cell lysate from E. coli BL21 pET28a-qseB was subjected to QseB purification using Ni-NTA resin (Qiagen). Cell lysate from E. coli BL21 pET28a-qseC was subjected to be pelleted using ultracentrifuge at 235,000 x g for 30 min. The pellet was collected and resuspended in 20 mM Tris pH 7.5 with 1 mM MgCl2. This QseC-enriched membrane was subjected to in vitro kinase assay. Membrane fractions from non-induced culture of E. coli BL21 pET28a-qseC was used as negative control.
In vitro kinase activity assay. The in vitro assays were carried out using the prepared QseC-enriched membrane fraction and purified QseB in the presence of ATP (5 mM), MgCl2 (25 mM) in HNG buffer (HEPES 100 mM, NaCl 300 mM, Glycerol 20%). To induce the kinase reaction, we added EPI (1 mM). We also added TAs, DOP, PTL (1 mM) or water (as negative control) to the reaction to investigate the kinase inhibition. The in vitro assays were perfomed in room temperature for 30 min. After 30 min, it was centrifuged at 5000 g for 10 min and the supernatant was taken to measure the remaining ATP using Kinase Glo-Max (Promega). The luminescent intensity is inversely correlated with the kinase activity. The values from negative control were considered as baseline for zero kinase activity and was used to calculate the kinase activity for the reactions with the addition of TAs, DOP, and PTL.
Intracellular Ca++ measurement assay. We seeded the LNCaP cells in 96-well microtiter black flat bottom plate with 1x105 cells/well and incubated for 24 h at 37°C in 5% CO2. The LNCAP cells were treated with the neurochemicals (TRY, PEA, TYM and DOP) and PTL (50 µg/ml) with and without EPI (50 µg/ml). EPI was added 5 min after neurochemicals, incubated further for 12 min and the Ca++ levels were measured every 3 min. The intracellular calcium measurement assays were performed using Fluo-8 Calcium Flux Assay Kit – No Wash (Abcam) according to the protocol provided by the company.
Cell proliferation assay. Prior the cell proliferation assay, LNCaP cells were seeded in a 96-well microtiter flat bottom plate with 1x105 cells/well and incubated for 24 h at 37°C in 5% CO2. The LNCAP cells were treated with the neurochemicals (TRY, PEA, TYM and DOP) and PTL with final concentration 50 µg/ml with and without EPI (50 µg/ml). The cytotoxicity assay was performed using the Cell Proliferation Kit I (MTT; Roche, Germany) according to the protocol provided by the company.
Statistical significance
Multiple comparisons were analyzed using one-way ANOVA with Dunnett post-test. Normal distributions were analyzed by Student’s t test, with the significance defined as p < 0.05 where n represents independent biological replicates.
Results
TA and DOP have similar binding affinity as EPI on α1-AR and QseC in silico
It has been reported that TA and DOP act as agonists on α2-AR and as partial antagonists on ß2-AR [15-17]. For this reason, we wondered whether TA and DOP can also affect other types of AR, such as α1-AR and QseC. In order to answer this question, we first performed in silico analyses of the binding affinity of TA and DOP on α1-AR and QseC. We used EPI and PTL as controls because they were reported to act as agonists (EPI) and antagonist (PTL) on both α1-AR and QseC. We also used autoinducer-3 (AI-3), a bacterial endogenous QseC agonist, as an additional positive control since the structure has been characterized recently [27]. Indeed, we could show that TA and DOP have similar binding affinity to α1-AR and QseC as EPI. The binding affinity of TA and DOP are even stronger than AI-3 to QseC (Table 1). This results lead to the suggestion that TA and DOP are probably able to interact with α1-AR and QseC.
TA and DOP have similar binding sites as EPI and PTL on α1-AR in silico
Next, we analyzed the binding sites of TA and DOP on α1-AR and QseC. EPI and PTL were included in these analyses for comparison. In particular, we investigated the chemical interactions, such as hydrophobic, hydrogen and π interaction between the ligands and the receptors. Our in silico studies showed that TRY, TYM, EPI and PTL have an overlapping binding region at α1-AR, while PEA and DOP interacted with another site (Fig. 1). Although TA and DOP have a similar binding affinity to α1-AR, they bind to different sites of α1-AR (Table 1). The binding studies suggest that TRY and TYM interact with α1-AR at the same orthosteric site, while PEA and DOP interact with α1-AR at an allosteric site (Table 2 and Fig. 1).
TA and DOP have similar binding sites as EPI and PTL on QseC in silico
TA and DOP, like EPI and PTL, have similar interactions sites on QseC (Fig. 2). They share at least three similar chemical interaction sites on QseC (Table 2), suggesting that TA and DOP bind to a common orthosteric motif. The recently elucidated endogenous agonist, AI-3, is also interacting with QseC at orthostheric site at position Tyr367 and Arg366, similar to EPI, PTL, TA and DOP (Supplementary Fig. S1 and Table 2). Due to the high binding affinity of TA and DOP to QseC and the high similarity of their interaction sites in QseC compared to the known agonists (EPI) and antagonists (PTL), we postulate that TA and DOP might have an effect on the activity of QseC.
TA and DOP act as α1-AR antagonist in vitro
Since TAs and DOP have a high affinity to α1-AR, we performed in vitro experiments to confirm the interaction and to study the effect of TAs and DOP on α1-AR. In this experiment, we used LNCaP as a model cell line that expresses α1a-AR [28]. To address this, we measured the intracellular Ca++ level of the treated LNCaP cells, since activation of α1-AR leads to an increase of the intracellular Ca++ level due to the activation of Ca++ channel. We could show that the addition of EPI (50 μg/mL) alone significantly increased intracellular Ca++ levels compared to the control after 3 min of incubation. If EPI (50 μg/mL) is added together with DOP (50 μg/mL), the Ca++ level decreased to a level similar to the control. Treatment with TAs and DOP without EPI did not show a significant effect on Ca++ levels (Fig. 3A). Cell viability experiments using LNCaP treated with TAs and DOP (each 50 μg/mL) with and without EPI (50 μg/mL) showed no difference compared to the control, with one exception; LNCaP treated only with EPI showed higher cell viability (Fig. 3B). The higher cell viability was probably due to the increased cell growth induced by the oscillation of intracellular Ca++ level [29-31].
TAs and DOP do not inhibit the QseBC kinase activity
In order to confirm the in silico prediction of TA interaction with QseC, we carried out in vitro kinase assays of QseC and QseB with additions of TAs and DOP respectively in the presence and absence of EPI. We also included PTL as negative control because PTL has antagonistic effects against QseC [21]. Surprisingly, the in vitro experiments showed that the TA and DOP do not have a significant effect on the kinase activity of QseBC, irrespective of the presence and absence of EPI (Fig. 4).
Discussion
Trace amines (TA) are reported to be present endogenously in the human body at very low concentrations [32]. However, TA are also produced by microorganisms and many of them are part of the human microbiota as either gut [19] or skin microbiota [20]. TA can interact not only with TA associated receptors (TAARs) but also with some adrenergic receptors (ARs), such as α2-AR and ß-AR [15-17]. These receptors are also reported to be able to interact with dopamine (DOP), an endogenous biogenic neurotransmitter [33, 34].
On α2-AR, TA and DOP act as agonist similar to EPI and NEPI [15, 17, 19]. TA and DOP interact with α2-AR at the orthosteric site similar to EPI and NEPI. As for α2-AR, TA and DOP have been reported to interact with the allosteric site as partial antagonist [16]. Our in silico experiments showed that TA and DOP have similar binding affinity towards α1-AR compared to EPI (Table 1). This suggests that as competitive (TRY and TYM) and allosteric (PEA and DOP) ligands, both have a similar high probability to bind to the receptor as EPI (Table 2 and Fig. 1). This could be the reason why TA and DOP antagonize the effect of EPI at the Ca++ level as well as LNCAP cell proliferation (Fig. 3).
The activation of α1-AR is followed by an activation of Ca++ transporter and an increase of cytoplasmic Ca++. The presence of TA and DOP inhibit the activation of α1-AR by EPI either via competitive or allosteric antagonist interaction and subsequently the increase of Ca++ is also abrogated as shown in Fig. 3A. TA and DOP inhibit cell proliferation as effective as PTL (Fig. 3B). This is due to the antagonistic effect of TA and DOP on α1-AR, which causes the inhibition of the activity of the Ca++ transporter. As a result, Ca++ levels became lower and consequently inhibiting the cell proliferation, which is regulated by the Ca++ level [29-31]. Another effect related to the Ca++ level is smooth muscle contraction [35]. As α1-AR is expressed in many organs and tissues, such as in urinary bladder [36], prostate [37], urethra [36], heart [38] and adipocytes [39], the presence of TA and DOP will have an effect to the function of these organs or tissues due to the decrease of smooth muscle contraction or proliferation.
As TA and DOP showed an interaction with mammalian AR, we wondered whether TA and DOP might also interact with bacterial AR known as QseC. QseC plays an important role in the regulation of many virulence factors of pathogenic Gram-negative bacteria such as E. coli [40, 41], Salmonella enterica [42, 43], Aggregatibacter actinomycetemcomitans [44], or Actinobacillus pleuropneumoniae [45]. Our in silico prediction studies showed that TA and DOP have a similar high binding affinity towards QseC as with the known agonist (EPI) and antagonist (PTL); all of these compounds also interact at orthosteric sites. On the basis of these results, we postulate that TA and DOP should have an effect on the activity of QseC. However, TA and DOP showed no significant effect on QseC kinase activity (Fig. 4). The discrepancy between in silico and in vitro experiments is not uncommon, since a certain inaccuracy is inherent in the in silico prediction. Computational predictions, however, are not always accurate [22]; hence one has to verify the in silico results experimentally. Furthermore, TA, DOP, EPI and PTL share only one interaction site with AI-3, namely the orthosteric Tyr367 of QseC (Table 2). A comparison of the binding affinities shows that the binding affinity of EPI and PTL to QseC is stronger than that of AI-3 (Table 1). This suggests that the mammalian ligands are more efficient than the bacteria's own ligand AI-3, which is formed by threonine dehydrogenase and "abortive" tRNA synthetase [27].
The importance of this work is that we show that TA interact not only with α2-AR and ß-AR but also with α1-AR. This is a new finding demonstrating the versatility of TA in its interaction with mammalian AR. When studying the interaction of the human α1-AR with the ligands, our in silico and experimental data agree very well (Table 3). As shown in Fig. 5A, EPI acts on α1-AR as an agonist. It triggers a cascade of reactions starting with Gq (G alpha q) signaling, the activation of phospholipase C followed by activation of Ca++ channel. In LNCAP cells, diacylglycerol (DAG)-directed TRPC1 and TRPC3 Ca-channels are present. It is assumed that in prostate cancer epithelial cells, α1-ARs is functionally coupled to the Ca++ permeable TRPC1 and TRPC3 Ca-channels [46].
Accumulation of intracellular Ca++ eventually leads to an increase in cell proliferation. However, in the presence of PTL, TRY, TYM, PEA and DOP, the activation of α1-AR is antagonized (Fig. 5B). Interestingly, PTL, TRY and TYM are binding to the same binding site as EPI (orthosteric) thus acting as competitive inhibitors. On the other hand, PEA and DOP are binding to different binding site thus acting as allosteric inhibitors. It is noteworthy to mention that the three TA differ with respect to the binding site. While TRY and TYM bind to orthosteric sites, PEA is binding allosterically. This observation indicates that in future, we will have to analyze each of the TA molecules individually. The finding that TA act as α1-AR antagonists is important because they may have a negative effect on cardioprotection, as it has recently reported that Gq coupling is required for cardioprotection by an alpha-1A-AR agonist [47].
While the in silico binding studies of TA and DOP with the human α1-AR correspond very well with their activity, this was not the case with the bacterial QseC. Our in silico study demonstrated that the binding of TA and DOP with QseC was similar to that for EPI and PTL. However, TA and DOP had no effect on the phosphorylation activity. A summary of these results is shown in Fig. 6.
We thank Dr Sook-Ha Fan for proofreading the final version of the manuscript.
Author contributions
FG and AL designed the study. AL, VDK and RAR designed and performed the experiments. FG, AL and VDK wrote the manuscript.
Funding
This work was supported by infrastructural funding from the Deutsche Forschungsgemeinschaft (DFG), Cluster of Excellence EXC 2124 Controlling Microbes to Fight Infections. AL was supported by the Ministry for Science, Research and the Arts of Baden-Württemberg (MWK) “AntibioPPAP”.
Statement of Ethics
Disclosure Statement
The authors have no conflicts of interest to declare.
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