Dopamiini retseptoritele ligandi sidumise uurimist võimaldavate katsesüsteemide arendamine

Väitekirja elektrooniline versioon ei sisalda publikatsioonePolüsahhariidid, nukleiinhapped ja valgud on eluks vajalikud biopolümeerid. Inimese organismis on neist kõige enim valke, mis on ühtlasi ka kõige mitmekülgsemad biomolekulid, seda nii oma struktuuri kui ka funktsiooni poolest. Valgud vastutavad mitmete eluks vajalike funktsioonide läbiviimise eest. Näiteks ensüümvalgud kiirendavad organismis toimuvaid keemilisi reaktsioone. Lisaks on valgud olulised ainete transportijad, pakuvad mehaanilist tuge ja immuunkaitset ning osalevad närvisignaali ülekandes. Käesolevas töös keskenduti retseptorvalkude, täpsemalt G-valguga seotud retseptorite uurimisele. Need valgud paiknevad raku membraanis, kus need vahendavad närvisignaali ülekannet raku väliskeskkonnast raku sisemusse. Signaaliülekande toimumiseks peab retseptoriga seostuma teatud tüüpi keemiline ühend, mida nimetatakse ligandiks. Ligandi seostumine retseptorile põhjustab retseptori struktuuri muutumise, mis omakorda mõjutab raku sees olevaid signaalmolekule. Dopamiini retseptorid on G-valguga seotud retseptorite perekonda kuuluvad valgud, mis vahendavad inimese organismis mitmeid olulisi funktsioone. Häired dopamiinergilises signaaliülekandes võivad põhjustada mitmesuguseid haigusi, millest tuntuimad on skisofreenia ja Parkinsoni tõbi. Seetõttu on dopamiini retseptorid olulised ravimite sihtmärgid ning detailsed teadmised dopamiini retseptorite toimimise kohta on äärmiselt vajalikud. Antud töö käigus keskenduti katsesüsteemide arendamisele, eesmärgiga iseloomustada ligandide seostumist erinevatele dopamiini retseptoritele. Katseid viidi läbi nii natiivsete retseptoritega (koeproovid) kui ka erinevate rekombinantsete dopamiini retseptoritega, mille saamiseks kasutati erinevaid ekspressioonisüsteeme (imetajarakud, putukarakud, pungunud bakuloviirused). Retseptorite ja ligandide vaheliste interaktsioonide iseloomustamiseks rakendati mitmesuguseid, põhiliselt fluorestsentsil põhinevaid meetodeid.Biopolymers, like polysaccharides, nucleic acids and proteins are essential for all living organisms. Besides water, proteins are the most abundant type of molecules in the human body. Proteins are also the most versatile of all biomolecules, performing many functions required for life. Some proteins have catalytic activity and function as enzymes, others serve as structural elements, signal receptors, or transporters that carry specific substances into or out of cells. This thesis focuses on the receptor proteins, specifically to G protein-coupled receptors that are located in the cell’s plasma membrane. These proteins are responsible for detecting signals outside the cell by binding various chemical compounds, called ligands. Ligand binding to a receptor can initiate a conformational change in the structure of the protein, which in turn leads to a chain of biochemical events inside the cell. Dopamine receptors, belonging to the family of G protein-coupled receptors, mediate several important functions in the human body. Abnormal dopaminergic signaling can lead to several neurological and psychiatric disorders, such as schizophrenia and Parkinson’s disease. Therefore, dopamine receptors are relevant drug targets and acquiring further information about the signal transduction process mediated by dopamine receptors is essential. This thesis focused on the development of novel assay systems to characterize ligand binding to different subtypes of dopamine receptors. Experiments were carried out with native receptors (tissue homogenates) and with various recombinant protein expression systems (mammalian and insect cells, budded baculovirus particles). Additionally, several methods were implemented to study receptor-ligand interactions with the emphasis on fluorescence-based methods

Determination of biological activity of gonadotropins hCG and FSH by Forster resonance energy transfer based biosensors

Determination of biological activity of gonadotropin hormones is essential in reproductive medicine and pharmaceutical manufacturing of the hormonal preparations. The aim of the study was to adopt a G-protein coupled receptor (GPCR)-mediated signal transduction pathway based assay for quantification of biological activity of gonadotropins. We focussed on studying human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH), as these hormones are widely used in clinical practice. Receptor-specific changes in cellular cyclic adenosine monophosphate (cAMP, second messenger in GPCR signalling) were monitored by a Forster resonance energy transfer (FRET) biosensor protein (T)Epac(VV) in living cells upon activation of the relevant gonadotropin receptor. The BacMam gene delivery system was used for biosensor protein expression in target cells. In the developed assay only biologically active hormones initiated GPCR-mediated cellular signalling. High assay sensitivities were achieved for detection of hCG (limit of detection, LOD: 5 pM) and FSH (LOD: 100 pM). Even the smallscale conformational changes caused by thermal inactivation and reducing the biological activity of the hormones were registered. In conclusion, the proposed assay is suitable for quantification of biological activity of gonadotropins and is a good alternative to antibody- and animal-testing-based assays used in pharmaceutical industry and clinical research.Peer reviewe

Optimizing the Expression of Human Dopamine Receptors in Escherichia coli

The human dopamine receptors D2S and D3 belong to the group of G protein-coupled receptors (GPCRs) and are important drug targets. Structural analyses and development of new receptor subtype specific drugs have been impeded by low expression yields or receptor instability. Fusing the T4 lysozyme into the intracellular loop 3 improves crystallization but complicates conformational studies. To circumvent these problems, we expressed the human D2S and D3 receptors in Escherichia coli using different N- and C-terminal fusion proteins and thermostabilizing mutations. We optimized expression times and used radioligand binding assays with whole cells and membrane homogenates to evaluate KD-values and the number of receptors in the cell membrane. We show that the presence but not the type of a C-terminal fusion protein is important. Bacteria expressing receptors capable of ligand binding can be selected using FACS analysis and a fluorescently labeled ligand. Improved receptor variants can thus be generated using error-prone PCR. Subsequent analysis of clones showed the distribution of mutations over the whole gene. Repeated cycles of PCR and FACS can be applied for selecting highly expressing receptor variants with high affinity ligand binding, which in the future can be used for analytical studies

Lunasin-induced behavioural effects in mice: Focus on the dopaminergic system

The present study for the first time is devoted to identify central effects of synthetic lunasin, a 43 amino acid peptide. A markedly expressed neuroleptic/cataleptic effect was observed at low (0.1–10 nmol/mouse) centrally administered doses in male C57Bl/6 mice. Lunasin considerably reduced the amphetamine hyperlocomotion but weakly apomorphine climbing behaviour. No influence on ketamine and bicuculline effects was observed. Binding assay studies demonstrated modest affinity of lunasin for the dopamine D1 receptor (Ki = 60±15 M). In a functional assay of cAMP accumulation on live cells lunasin antagonised apomorphine effect on D1 receptor activation (pEC50 = 6.1±0.3), but had no effect in cells expressing D2 receptors. The obtained data suggest that lunasin’s action at least in part is provided via dopaminergic D1 receptor pathways. However, other non-identified mechanisms (probably intracellular) may play an important role in lunasin’s central action. Nevertheless further studies of lunasin are promising, particularly taking into account a necessity for novel type of antipsychotic drug

BRET- and fluorescence anisotropy-based assays for real-time monitoring of ligand binding to M2 muscarinic acetylcholine receptors

BRET and fluorescence anisotropy (FA) are two fluorescence-based techniques used for the characterization of ligand binding to G protein-coupled receptors (GPCRs) and both allow monitoring of ligand binding in real time. In this study, we present the first direct comparison of BRET-based and FA-based binding assays using the human M-2 muscarinic acetylcholine receptor (M2R) and two TAMRA (5-carboxytetramethylrhodamine)-labeled fluorescent ligands as a model system. The determined fluorescent ligand affinities from both assays were in good agreement with results obtained from radioligand competition binding experiments. The assays yielded real-time kinetic binding data revealing differences in the mechanism of binding for the investigated fluorescent probes. Furthermore, the investigation of various unlabeled M2R ligands yielded pharmacological profiles in accordance with earlier reported data. Taken together, this study showed that BRET- and FA-based binding assays represent valuable alternatives to radioactivity-based methods for screening purposes and for a precise characterization of binding kinetics supporting the exploration of binding mechanisms

The expression of <i>Wfs1</i> and <i>Drd1a</i> in the adult chick brain.

<p>The expression of <i>Wfs1</i> and <i>Drd1a</i> in the adult chick brain, shown by mRNA <i>in situ</i> hybridization on coronal brain sections. The section plane is shown on image L. The probes are indicated on the left side of the figure. Both, <i>Wfs1</i> and <i>Drd1a</i> show strong expression in rostral to medial MSt (A-F). In Acb and StPalAcb, the expression of <i>Wfs1</i> is substantially weaker than in the surrounding striatal structures (A-B). The expression of <i>Drd1a</i> is weak in Acb and StPalAcb, and is missing in SPO (D-F). In LSt, both <i>Wfs1</i> and <i>Drd1a</i> expression show strengthening gradient in rostrocaudal direction (A-G,J). <i>Wfs1</i>-expressing cells in PHi are shown in higher magnification (I). In the adult brain, ADo and APir are delineated with <i>Wfs1</i> expression, but remain hardly distinguishable by <i>Drd1a</i> expression (H,K). Note that GP is devoid of both <i>Drd1a</i> and <i>Wfs1</i> (B,E,G,J). For abbreviations, see list. Scale bar is 1 mm in A-H and J-K and 500 μm in I.</p

Binding of D₁/D₅ specific ligand [³H]SCH23390 to hippocampal membranes of wt and <i>Wfs1</i> knockout mice.

<p>Comparison of specific binding of radioligand [³H]SCH23390 to hippocampal membranes of wt and <i>Wfs1</i> knockout mice. (A) Binding curve of [³H]SCH23390 binding to pooled samples of wt (triangle) and <i>Wfs1</i> knockout (circle) mice. The membrane suspensions (3 mg/well) were incubated with different concentrations of [³H]SCH23390 for 60 min and bound radioactivity was measured. Data are presented as mean ± SEM from experiments (n = 3) performed in duplicates. (B) The level of [³H]SCH23390 binding sites of individual wt and <i>Wfs1</i> knockout mice determined in hippocampal membrane suspensions (6.7 mg/ml.) incubated with 4 nM radioligand. Data presented as mean ± SEM of all the mice tested. *P < 0.05. Data of individual mice are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172825#pone.0172825.s006" target="_blank">S1 Table</a>.</p

The expression of <i>Wfs1</i> and <i>Drd1a</i> in the mouse intercalated amygdala and in its putative avian homologue, StC, in chick.

<p>The expression of <i>Wfs1</i> and <i>Drd1a</i> in the mouse intercalated amygdala and in its putative avian homologue, StC, in chick. A, B–in situ hybridization on coronal sections of the mouse brain. C, D–immunohistochemistry on coronal sections of the mouse brain. E, F–in situ hybridization on the coronal sections of the chick brain. The intercalated nuclei of the amygdala (arrowheads) are expressing both Wfs1 and Drd1a in mouse brain (A, B). Wfs1 and D1 proteins are both strongly expressed in the intercalated nuclei (C, D). The insets in C and D show closer view on the intercalated nucleus between the BL and claustrum-endopiriform formation. In chick brain, the StC is expressing both <i>Wfs1</i> and <i>Drd1a</i>. For abbreviations, see list. Scale bar is 100 μm in A-D and 1 mm in E-F.</p