Vrednotenje lastnosti vezave fluorescenčnih sond na fibrile amiloida β in inzulina

Petrovčič, Veronika (avtor), Knez, Damijan (mentor)

Izguba nativne strukture proteinov oziroma peptidov ob spremembi različnih fizikalno-kemijskih parametrov in nepravilno zvijanje lahko vodita v nastanek netopnih amiloidnih fibrilov, katerih kopičenje v organih in tkivih lahko povzroči nastanek in/ali napredovanje različnih nevrodegenerativnih, presnovnih in funkcionalnih motenj, kot je na primer Alzheimerjeva bolezen. Razumevanje mehanizma agregacije, občutljivo zaznavanje ter poznavanje strukture in stabilnosti amiloidnih fibrilov, so bistveni za zgodnje zaznavanje agregacije proteinov, postavitev diagnoze ter odkrivanje novih terapevtskih pristopov. Zaradi številnih prednosti je ena najpogosteje uporabljanih metod pri raziskavah agregacije amiloidogenih proteinov ali peptidov in zaznavanju amiloidnih fibrilov fluorescenčna spektroskopija, pri kateri uporabljamo različne fluorescenčne sonde. V eksperimentalnem delu magistrske naloge smo in vitro spremljali potek agregacije in potrdili nastanek fibrilov inzulina z merjenjem relativne intenzitete emisije fluorescence tioflavina T, nastanek fibrilov amiloida β1–42 pa smo potrdili z mikroskopskim pregledom in z merjenjem intenzitete emisije fluorescence tioflavina T. Sledilo je merjenje emisijskih spektrov preučevanih fluorescenčnih sond, iz katerih smo določili emisijski maksimum in povečanje intenzitete emisije fluorescence preučevane sonde po vezavi na amiloidne fibrile. Za nadaljnje raziskave in razvoj so najprimernejše sonde z emisijskim maksimumom nad 600 nm, ki izkazujejo veliko razliko oziroma precej večjo relativno intenziteto emisije fluorescence po vezavi na amiloidne fibrile v primerjavi z emisijo fluorescence proste, nevezane sonde. Za posamezne fluorescenčne sonde smo določili tudi konstanto disociacije, ki opredeljuje jakost vezave sonde na amiloidne fibrile. Konstante disociacije za preučevane fluorescenčne sonde so v nizko mikromolarnem oziroma nanomolarnem razredu velikosti. Za preučevanje specifičnosti in selektivnosti vezave sond na amiloidne fibrile smo merili emisijske spektre sond v prisotnosti govejega in človeškega serumskega albumina. Rezultati kažejo, da je razlika med relativno intenziteto emisije fluorescence sond v prisotnosti in odsotnosti govejega ali človeškega serumskega albumina zelo majhna, kar zagotavlja specifičnost in selektivnost preučevanih fluorescenčnih sond za amiloidne fibrile.

• Jezik: Slovenski jezik
• Ključne besede: agregacija, amiloidni fibrili, inzulin, amiloid β, fluorescenčna spektroskopija, fluorescenčne sonde
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2024
• PID: 20.500.12556/RUL-159322
• Datum objave v RUL: 06.07.2024

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Characterization of the binding properties of fluorescent probes to amyloid β and insulin fibrils

Izvleček

The loss of the native structure of proteins or peptides due to changes in various physical and chemical factors as well as misfolding can lead to the formation of insoluble amyloid fibrils. The accumulation of these fibrils in organs and tissues can lead to the development and progression of various neurodegenerative, metabolic, and functional disorders, such as Alzheimer's disease. Understanding the aggregation mechanism, sensitive detection and knowledge of the structure and stability of amyloid fibrils are essential for early detection of protein aggregation, diagnosis, and the discovery of new therapeutic approaches. Due to its numerous advantages, fluorescence spectroscopy using fluorescent probes is one of the most commonly used methods in the study of amyloidogenic protein or peptide aggregation and the detection of amyloid fibrils. In the experimental part of the master's thesis, we first followed the course of aggregation in vitro and confirmed the formation of insulin fibrils by measuring the relative emission intensity of thioflavin T fluorescence. The formation of amyloid β1–42 fibrils was confirmed by microscopic examination and by measuring the emission intensity of thioflavin T fluorescence. Next, we measured the emission spectra of the fluorescent probes, from which we determined the maximum emission wavelength and the increase in fluorescence emission intensity of the fluorescent probe studied upon binding to amyloid fibrils. Probes with a maximum emission wavelength above 600 nm, which show a significant difference or a much higher relative fluorescence emission when bound to amyloid fibrils compared to the fluorescence emission of the free, unbound probe, are the most suitable for further research. For individual probes, we also determined the binding dissociation constant, which shows the binding affinity of the probe for amyloid fibrils. The values of the binding dissociation constants are in the low micromolar or nanomolar range. To address the binding specificity and selectivity of the probes for amyloid fibrils, we also measured the emission spectra of the fluorescent probes in the presence and absence of bovine and human serum albumin. The results show that the difference in the relative fluorescence emission intensity of the fluorescent probes in the presence and absence of bovine or human serum albumin is very small, demonstrating specificity and selectivity of the investigated probes for amyloid fibrils.

• Ključne besede: aggregation, amyloid fibrils, insulin, amyloid β, fluorescence spectroscopy, fluorescent probes