20.500.12556/RUL-181095 ​Učinki lantanoidnih ionov na strukturo in lastnosti metaloencima paraoksonaza 1 Effects of lanthanide ions on structure and characteristics of metalloenzyme paraoxonase 1 Paraoksonaza 1 (PON1) je ključen od Ca2+ ionov odvisen hidrolitičen encim, vezan na lipoproteine visoke gostote (HDL), katerega aktivnost in koncentracija sta tesno povezani s številnimi bolezenskimi stanji oksidativnega stresa, vključno s srčno-žilnimi in nevrodegenerativnimi obolenji. Kljub intenzivnim raziskavam še ne poznamo zanesljive metode za neposredno koncentracijsko kvantifikacijo PON1 v bioloških tekočinah. Obstoječi pristopi se zanašajo zgolj na merjenje kinetične aktivnosti z ne-fiziološkimi substrati ali nezanesljivimi encimsko vezanimi imunskimi testi. Namen doktorskega dela je bil postaviti temeljne raziskave o izmenjavi Ca2+ ionov z lantanoidnimi ion ter vezavo aromatskih ligandov v aktivno mesto PON1, ki bi lahko zaradi luminescenčne narave lantanoidov lahko bili uporabni za koncentracijsko kvantifikacijo encima. V doktorskem delu smo sistematično pojasnili, kako vezava Tb3+ ionov vpliva na encimsko aktivnost rekombinantnega encima PON1 (rePON1) in v nadaljevanju njegove fotofizikalne lastnosti ob tvorbi ternarnega kompleksa z 2-hidroksikinolinom (2HQ). Pokazali smo, da Tb3+ ioni reverzibilno inhibirajo laktonazno aktivnost rePON1 po zaporednem dvostopenjskem mehanizmu, ki vključuje tako katalitsko kot strukturno vezavno mesto. S kinetičnim modeliranjem progresivnih krivulj hidrolize substrata dihidrokumarina smo potrdili takojšnjo, nizkoafinitetno zamenjavo katalitskega Ca2+ iona (KI1 = 0,48 μM), ki ji sledi počasna, visokoafinitetna zamenjava na strukturnem mestu (KI2 = 0,91 nM). Presežek Ca2+ ionov inhibiran encim reaktivira, kar nakazuje na ohranitev zgradbe proteina ob zamenjavi ionov. V osrednjem delu smo pokazali, da 2HQ z rePON1 in Tb3+ ioni tvori fotoluminiscenčni ternarni kompleks rePON1:Tb3+:2HQ, ki izkazuje značilno, od Tb3+ ionov odvisno časovno zamaknjeno fosforescenco z emisijski vrhovi pri karakterističnih valovnih dolžinah. Intenziteta fosforescenčnega signala linearno sovpada s koncentracijo rePON1, kar bi lahko omogočalo neodvisno koncentracijsko kvantifikacijo PON1. Kristalna struktura rePON1:Tb3+:2HQ z ločljivostjo 2,35 Å (PDB 9R0Q) je razkrila delno zamenjavo Ca2+ ionov s Tb3+ ioni na katalitskem mestu (47 %), medtem ko je zasedenost na strukturnem mestu pri uporabljenih pogojih nizka (5 %). Inhibitor 2HQ se veže na enak način kot v Ca2+-kompleksu, brez večjih strukturnih odklonov. Z metodo izotermne titracijske kalorimetrije (ITC) smo termodinamsko potrdili, da se 2HQ na kompleks rePON1:Tb3+ veže bistveno šibkeje kot na kompleks rePON1:Ca2+ (približno 30-krat). Do podobnih rezultatov za vezavo 2HQ na kompleks rePON1:Tb3+ smo prišli tudi s titracijo 2HQ ob merjenju fosforescenčnega signala nastalega kompleksa rePON1:Tb3+:2HQ. V doktorski nalogi smo postavili strukturne in mehanistične temelje za uporabo fotoluminiscenčnih lantanoidnih kompleksov za kvantifikacijo encima PON1. Nadaljnje raziskave bi se morale usmeriti v iskanje in razvoj aromatskih ligandov z višjo afiniteto za kompleks rePON1:Tb3+, saj ligandi z nizko afiniteto za popolno zasedenost aktivnih mest PON1 zahtevajo visoke koncentracije le-teh, kar lahko vodi v dušenje lastne fosforescence (angl. self-quenching) ter zmanjšanje intenzitete in zanesljivost signala. Zato smo izvedli tudi netarčne presejalne teste ligandov iz knjižnice 1409 zdravil, ki so bila odobrena na kitajskem trgu s strani nacionalne uprave za medicinske pripomočke (angl. National Medical Products Administration). Pri tem smo odkrili dve farmakološki spojini prulifloksacin in efavirenz, ki sta izkazali za 2- do 3-krat višjo afiniteto do rePON1 kot 2HQ. To je dobro temeljno izhodišče za nadaljnje korake tudi pri vzpostavljanju sistema na detekcijo endogenega PON1 v kompleksnih bioloških vzorcih. Paraoxonase 1 (PON1) is a calcium-dependent hydrolytic enzyme bound to high-density lipoproteins (HDL), whose activity and concentration are tightly linked to various oxidative stress-related pathologies, including cardiovascular and neurodegenerative diseases. Despite intensive research, no reliable method for direct concentration-based quantification of PON1 in biological fluids exists to date. Current approaches rely solely on kinetic activity measurements with non-physiological substrates or on inconsistent enzyme linked immunosorbent assays. The aim of this doctoral work was to establish fundamental research into Ca2+ ion exchange with lanthanide ions and the binding of aromatic ligands at the active site of PON1, which could be exploited for further enzyme quantification. In this thesis, we systematically explained how Tb3+ ion binding affects the enzymatic activity of recombinant PON1 (rePON1) and, subsequently, its photophysical properties upon formation of a ternary complex with 2-hydroxyquinoline (2HQ). We demonstrated that Tb3+ ions reversibly inhibit the lactonase activity of rePON1 through a sequential two-step mechanism involving both the catalytic and structural binding sites. Kinetic modeling of dihydrocoumarin (DHC) hydrolysis progress curves confirmed an immediate, low-affinity exchange of the catalytic Ca2+ ion (KI1 = 0.48 μM), followed by a slower, high-affinity substitution at the structural site (KI2 = 0.91 nM). The inhibited enzyme can be reactivated by an excess of Ca2+ ions, indicating preservation of the overall protein fold during ion replacement. In the central part of the work, we showed that 2HQ forms a photoluminescent ternary complex with rePON1 and Tb3+ ion, which exhibits characteristic Tb3+-dependent time-resolved phosphorescence with emission peaks at the expected wavelengths. The phosphorescence intensity correlated linearly with rePON1 concentration, suggesting the potential for independent concentration-based quantification of PON1. The crystal structure of the rePON1:Tb3+:2HQ complex at 2.35 Å resolution (PDB 9R0Q) revealed partial substitution of catalytic Ca2+ ions with Tb3+ (47%), while the occupancy at the structural site was low (5%) under the tested conditions. The inhibitor 2HQ bound in the same manner as in the Ca2+-complex, without major structural deviations. Isothermal titration calorimetry (ITC) confirmed that 2HQ binds much weaker to the rePON1:Tb3+ complex compared to rePON1:Ca2+ (approximately 30-fold weaker). Similar results for the binding of 2HQ to the rePON1:Tb3+ complex were also obtained by titrating 2HQ while measuring the phosphorescence signal upon the formation of rePON1:Tb3+:2HQ complex. Overall, this doctoral thesis laid structural and mechanistic foundations for the use of photoluminescent lanthanide complexes in PON1 quantification. Future studies should focus on the search and development of aromatic ligands with higher affinity for the rePON1:Tb3+ complex, since low-affinity ligands require high concentrations to fully occupy the PON1 active sites, potentially causing self-quenching and reducing signal intensity and reliability. Therefore, we additionally performed non-targeted screening of ligands from a library of 1,409 drugs approved by the Chinese National Medical Products Administration, leading to the identification of prulifloxacin and efavirenz as promising candidates with 2- do 3-times greater binding affinities for rePON1 compared to 2HQ. These findings form an important starting point for future development of a system capable of detecting endogenous PON1 in complex biological samples. paraoksonaza 1 lantanoidi terbij fosforescenca ternarni kompleks paraoxonase 1 lanthanides terbium fosforescence ternary complex true false false Slovenski jezik Angleški jezik Doktorsko delo/naloga 2026-03-25 08:15:23 2026-03-25 08:15:39 2026-03-26 04:21:47 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 38995 8491.pdf 8491.pdf 1 33C01F70AC475EE7CA0EABB42DF5AB74 636b47ad3c852043ffe636899323ee2cbaf17e11320ca4a3ce2e15a9ec15c1ba 4e917d41-281a-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=231266 Medicinska fakulteta 0 0 0