Development and application of the biosensor assay for measurements of cyclic adenosine monophosphate in studies of G protein-coupled receptor signaling
Date
2015-07-09
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Abstract
Maailma rahvastik on pidevas kasvus ning vananemises ning sellest on tingitud mõnd haigust või sündroomi põdevate patsientide üha suurenev hulk. Enamik haigustest on põhjustatud häiretest organismi metabolismi või rakkude signaaliülekande regulatsioonis. G valguga seotud retseptorid (GPCR) moodustavad suure rakkudevahelist suhtlust reguleeriva valkude perekonna. Keemilise signaaliülekande moduleerimine on levinud ravistrateegia, mida kinnitab ka tõsiasi, et ligi kolmandik kõigist tänapäeva retseptiravimitest on suunatud just GPCR-tele. Ravimid ehk bioaktiivsed ühendid on seega tänapäeva maailma asendamatu osa.
Uute sünteetiliste ühendite otsinguil on väga olulisteks uute molekulide testimine ning saadud katsetulemuste võrdlus struktuurselt sarnaste või kehaomaste signaalimolekulide omadustega. See aitab iseloomustada ühendite erinevaid omadusi ning annab väärtuslikku informatsiooni uute ühendite disainiks. Käesolevas doktoritöös arendati välja katsesüsteem erinevate bioloogiliselt aktiivsete molekulide iseloomustamiseks elusates rakkudes. Selleks kasutati fluorestsentsil põhinevat biosensorit.
Biosensori kasutamiseks on vajalik selle olemasolu uuritavas objektis (rakud toodavad sensorvalku ise, lähtudes neisse eelnevalt viidud sensori geenijärjestusest). Saavutamaks biosensori ühtlast taset erinevates uuritavates rakkudes kasutati käesolevas töös putukaviiruseid. Inimesele ohutud viirused transpordivad sensorit kodeeriva geenijärjestuse uuritavasse rakku, kus sellest sünteesitakse uus valk, tundlik biosensor. Süsteem võimaldas kasutatava viiruse kogusega reguleerida biosensori taset, mille tulemuseks oli ühtlane ja hea korratavusega valkude tootmine ning usaldusväärne katsesüsteem.
Töö käigus demonstreeriti biosensori rakendatavust mitmetes erinevates rakkudes väga erinevate retseptorite aktivatsiooni uurimiseks. Tänu katsesüsteemi tundlikkusele sobib see näiteks ka kehaomaste hormoonide kontsentratsiooni määramiseks erinevates proovides, mis on olulise tähtsusega nii meditsiini kui ka uute tehnoloogiate arendamise seisukohalt. Töö tulemusena on loodud tööriist, mille abil on võimalik määrata erinevate struktuursete omadustega ühendite bioloogilist aktiivsust vastavate retseptorite aktivatsiooni kaudu.
World’s population is constantly growing and aging. As a result, the number of patients suffering from some sort of illness or disease is rising as well. Most of the illnesses are caused by disturbances in cellular signaling or metabolism of the organism. G protein-coupled receptors (GPCR) form a large family of cell surface proteins responsible for the regulation of signal transduction. Over one third of all todays’ prescription drugs are targeted to GPCRs. In search for new synthetic compounds targeting these receptors functional testing is of great importance. Obtained experimental data need to be evaluated and compared with data from structurally similar or endogenous ligands. This yields information about the properties of the new ligand and for molecular design of future compounds. In the current thesis an assay system for characterization of different biologically active molecules on living cells was developed based on the use of fluorescent biosensor proteins. To use a biosensor, it needs to be present inside the cells of interest. To achieve a reliable and uniform cellular level of the biosensor protein, a gene delivery system based on the use of insect viruses was employed. These viruses, harmless to humans, transport the biosensor gene sequence in to the cells, where a new functional protein is synthesized and is ready to be used in functional experiments. The level of protein expression can be regulated by the amount of the applied virus, which results in homogeneous expression and a reliable assay with high reproducibility. During the studies the applicability of the new biosensor assay was demonstrated by monitoring the activation of very different GPCRs in various cells. Due to high sensitivity of the assay it is also suitable for quantification of endogenous hormones in different probes and preparations. This is of importance for the field of medicine as well as for the development of novel technologies. The result of this work is the developed biological tool that enables us to monitor GPCR activation for measurement of biological activity of compounds with various structural properties.
World’s population is constantly growing and aging. As a result, the number of patients suffering from some sort of illness or disease is rising as well. Most of the illnesses are caused by disturbances in cellular signaling or metabolism of the organism. G protein-coupled receptors (GPCR) form a large family of cell surface proteins responsible for the regulation of signal transduction. Over one third of all todays’ prescription drugs are targeted to GPCRs. In search for new synthetic compounds targeting these receptors functional testing is of great importance. Obtained experimental data need to be evaluated and compared with data from structurally similar or endogenous ligands. This yields information about the properties of the new ligand and for molecular design of future compounds. In the current thesis an assay system for characterization of different biologically active molecules on living cells was developed based on the use of fluorescent biosensor proteins. To use a biosensor, it needs to be present inside the cells of interest. To achieve a reliable and uniform cellular level of the biosensor protein, a gene delivery system based on the use of insect viruses was employed. These viruses, harmless to humans, transport the biosensor gene sequence in to the cells, where a new functional protein is synthesized and is ready to be used in functional experiments. The level of protein expression can be regulated by the amount of the applied virus, which results in homogeneous expression and a reliable assay with high reproducibility. During the studies the applicability of the new biosensor assay was demonstrated by monitoring the activation of very different GPCRs in various cells. Due to high sensitivity of the assay it is also suitable for quantification of endogenous hormones in different probes and preparations. This is of importance for the field of medicine as well as for the development of novel technologies. The result of this work is the developed biological tool that enables us to monitor GPCR activation for measurement of biological activity of compounds with various structural properties.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Keywords
G-valgud, retseptorid, biosensorid, adenosiinmonofosfaat, G-proteins, receptors, biosensors, adenosine monophosphate