Ligand binding, allosteric modulation and constitutive activity of melanocortin-4 receptors
Failid
Kuupäev
2020-07-14
Autorid
Ajakirja pealkiri
Ajakirja ISSN
Köite pealkiri
Kirjastaja
Abstrakt
Biokeemilised analüüsid on sageli suunatud valkudega seotud reaktsioonide uurimisele, kuna neil on elutähtis roll organismide biokeemiliste protsesside reguleerimisel. Membraanivalgud, mida nimetatakse retseptoriteks, reguleerivad füsioloogilisi ja ainevahetusprotsesse, toimides rakuväliste signaalide vahendajatena. Spetsiifilised keemilised signaalmolekulid, mida nimetatakse ligandideks, võivad signaali ülekande algatada retseptoritega seostumisel. Imetajate suurim retseptoriperekond, G-valguga seotud retseptorid (GPCR-id), vahendab rakuvälist signaali enamasti rakusiseste G-valkude kaudu, mis aktiveerivad edasisi signaaliülekande radasid.
Melanokortiin-4 (MC4) retseptorid on peamiselt kesknärvisüsteemis leiduvad GPCR-id, mis reguleerivad kehas mitmeid olulisi funktsioone, sealhulgas energia homeostaasi, toitumist ja seksuaalseid funktsioone. Seetõttu pakuvad need retseptorid ravimiarendusfirmadele suurt huvi. MC4 retseptorite poolt vahendatav signaaliülekanne rakku on keeruline dünaamiline protsess, mille kõiki iseärasusi senini veel täpselt ei mõisteta.
Käesoleva doktoritöö üldiseks eesmärgiks oli saada lisateavet MC4 retseptorite signaaliülekande kohta, täpsemalt nende ligandi seostumise, allosteerilise modulatsiooni ja konstitutiivse aktiivsuse kohta. Töö üldise eesmärgi saavutamiseks püstitati mitu konkreetset ülesannet, mille hulka kuulus fluorestsentsil põhinevate ligandi seostumise ja funktsionaalse meetodite arendus MC4 retseptoritele; uute fluorestsentsligandide väljatöötamine ja iseloomustamine MC4 retseptorite jaoks; ning metalliioonide mõju uurimine MC4 retseptorite signaaliülekandele.
Ligandi seostumise ja MC4 retseptori aktivatsiooni uurimiseks kasutasime fluorestsentsanisotroopial (FA) põhinevat ligandi seostumise meetodit ja Försteri resonantsi energiaülekandel (FRET) põhinevaid biosensoreid. Saadud tulemuste alusel leiti, et suure afiinsusega ligandi seostumiseks MC4 retseptoritele on vaja Ca2+ ioonide juuresolekut millimolaarses kontsentratsioonis. Zn2+ ja Cu2+ ioonid toimisid aga kui negatiivsed allosteerilised modulaatorid, mis inhibeerivad ligandi seostumist MC4 retseptoritele mikromolaarsetes kontsentratsioonides. Samas Zn2+ suurendas MC4 retseptorite aktivatsiooni, kuid Cu2+ inhibeeris retseptorite aktivatsiooni isegi alla konstitutiivset ehk baastaset. Kuna need ioonid esinevad nii kudedes kui ka rakukultuuri kasvulahustes, siis nende mõju retseptorsüsteemidele tuleb hoolikalt jälgida.
Biochemical studies are often targeted at protein-related interactions as they play a key role in the regulation of biochemical processes in living organisms. Membrane proteins called receptors regulate physiologic and metabolic processes by acting as mediators of extracellular signals. Specific signaling molecules, called ligands, can initiate the signal transduction by binding to the receptors. The largest receptor family in mammals, G protein-coupled receptors (GPCRs), mediate the extracellular signal into the cell mainly through intracellular G proteins that activate further signaling pathways. Melanocortin-4 (MC4) receptors are GPCRs expressed mainly in the central nervous system, which signaling pathways regulate several important functions in the body, including energy homeostasis, eating behavior and sexual functions. Therefore, these receptors are of great interest for drug development companies. The signal transduction of MC4 receptors is a complex dynamic process, where much information remains yet elusive. The general aim of this dissertation was to obtain additional information about the signal transduction of MC4 receptors, in particular their ligand binding, allosteric modulation and constitutive activity. To achieve the general goal of the work, several specific tasks were set, which included the development of fluorescence-based ligand binding and functional methods for MC4 receptors; development and characterization of new MC4 receptor fluorescent ligands; and characterization of the modulation of MC4 receptor signaling by different metal ions. Fluorescence anisotropy (FA) based ligand binding assay and Förster resonance energy transfer (FRET) based functional assays were used for the MC4 receptors studies in this work. Based on the obtained results, it was concluded that the presence of Ca2+ ions at millimolar concentrations is required for the high affinity ligand binding to MC4 receptors. However, Zn2+ and Cu2+ ions acted as negative allosteric modulators that inhibit ligand binding to MC4 receptors at micromolar concentrations. At the same time, Zn2+ increased the activation of MC4 receptors, but Cu2+ inhibited the activation of receptors even below the basal (constitutive) level. As these ions are present in both tissues and cell culture media, their effects on receptor systems should be closely monitored.
Biochemical studies are often targeted at protein-related interactions as they play a key role in the regulation of biochemical processes in living organisms. Membrane proteins called receptors regulate physiologic and metabolic processes by acting as mediators of extracellular signals. Specific signaling molecules, called ligands, can initiate the signal transduction by binding to the receptors. The largest receptor family in mammals, G protein-coupled receptors (GPCRs), mediate the extracellular signal into the cell mainly through intracellular G proteins that activate further signaling pathways. Melanocortin-4 (MC4) receptors are GPCRs expressed mainly in the central nervous system, which signaling pathways regulate several important functions in the body, including energy homeostasis, eating behavior and sexual functions. Therefore, these receptors are of great interest for drug development companies. The signal transduction of MC4 receptors is a complex dynamic process, where much information remains yet elusive. The general aim of this dissertation was to obtain additional information about the signal transduction of MC4 receptors, in particular their ligand binding, allosteric modulation and constitutive activity. To achieve the general goal of the work, several specific tasks were set, which included the development of fluorescence-based ligand binding and functional methods for MC4 receptors; development and characterization of new MC4 receptor fluorescent ligands; and characterization of the modulation of MC4 receptor signaling by different metal ions. Fluorescence anisotropy (FA) based ligand binding assay and Förster resonance energy transfer (FRET) based functional assays were used for the MC4 receptors studies in this work. Based on the obtained results, it was concluded that the presence of Ca2+ ions at millimolar concentrations is required for the high affinity ligand binding to MC4 receptors. However, Zn2+ and Cu2+ ions acted as negative allosteric modulators that inhibit ligand binding to MC4 receptors at micromolar concentrations. At the same time, Zn2+ increased the activation of MC4 receptors, but Cu2+ inhibited the activation of receptors even below the basal (constitutive) level. As these ions are present in both tissues and cell culture media, their effects on receptor systems should be closely monitored.
Kirjeldus
Väitekirja elektrooniline versioon ei sisalda publikatsioone
Märksõnad
ligands, G-proteins, melanocortin receptors