Effect of UV-C radiation on protein interaction between KRAS and RAF1 RAS binding domain

Abstract

Cilj : Svrha ovog rada je opisati i razjasniti kako oksidacija utječe na funkcionalne posljedice interakcije KRAS i RBD (RAF1 RAS-vezna domena) proteina. Metode: Korištenjem HEK293 stanica, proizvedeni su potrebni proteini za analizu. Nakon analize stanica, proteini su izloženi tretmanu UV-C zračenja. Zatim su određene razne varijante pri kojima su proteini stupali u protein-protein interakcija. Zadnji korak je odraditi Western blot analizu koristeći SIMPL metodu gdje su rezultati vidljivi na filmu pomoću metode kemiluminiscencije. Rezultati: Proteini su tretirani raznim dozama UV-C zračenja. Nakon što je uočeno da pri početno postavljenim dozama tretmana, 0.05 J/cm2 i 0.3 J/cm2 , nema vidljivih promjena u interakcijama između proteina, određene su nove doze: 0.4 J/cm2 , 0.6 J/cm2 , 0.8 J/cm2 i 1.0 J/cm2 . Pri dozama od 0.4 J/cm2 i 0.6 J/cm2 nema vidljivih promjena u inerakcijama između proteina. Pri dozi 0.8 J/cm2 , vidljivo je smanjenje intenziteta proteinske vrpce kada su KRAS i RBD proteini tretirani. Krajnja doza zračenja korištena u ovom eksperimentu je 1.0 J/cm2 gdje se uočava potpuni gubitak interakcije između tretiranih KRAS i RBD proteina. Dodatna je provedena mjera karbonilacije proteina pri već postavljenim dozama. Uočava se razlika u izgledu proteinske vrpce između netretiranog proteina i proteina tretiranih različitim dozama UV-C zračenja. Također, karbonilacija je ponovljena za doze 0.8 J/cm2 i 1.0 J/cm2 pri čemu je uočeno da proteinska vrpca RBD protein tretiran dozom 1.0 J/cm2 skoro pa potpuno blijedi. Zaključak: Rezultati dobiveni ovim istraživanjem ukazuju na dozno-ovisni učinak UV-C zračenja na interakciju KRAS-RBD. Slabljenje veze, uočeno pri većoj dozi, implicira da oksidacija proteina može utjecati na stabilnost kompleksa. Važno je istaknuti da su proteinske vrpce kontrolnih uzoraka pojedinačnih proteina (netretiranog proteina) bile jednake onima koje su prošle tretman zračenjem, sugerirajući da samo UV-C zračenje nije uzrok promjena u ekspresiji proteina. Dodatna provjera oksidativne štete je pomoću mjerenja razine proteinske karbonilacije, što je pokazalo povećanje oksidativnog oštećenja u tretiranim uzorcima u usporedbi s netretiranim. Ova opservacija podržava ideju da UV-C zračenje uzrokuje oksidaciju proteina, što dalje može utjecati na njihove interakcije.Aim: The purpose of this thesis is to describe and clarify how oxidation affects the functional consequences of the interaction of KRAS and the RBD (RAF1 RAS-binding domain) protein. Methods: The required proteins for analysis were produced using HEK293 cells. After cell lysis, proteins are exposed to UV-C radiation treatment. Then various variants were determined in which the proteins entered into protein-protein interactions. The last step was to perform a Western blot analysis using the SIMPL method where the results are visible on film using the chemiluminescence method. Results: The proteins were treated with various doses of UV-C radiation. After it was observed that at the initially set treatment doses, 0.05 J/cm2 and 0.3 J/cm2 , there were no visible changes in the interactions between proteins, new doses were determined: 0.4 J/cm2 , 0.6 J/cm2 , 0.8 J/cm2 and 1.0 J/cm2 . At doses of 0.4 J/cm2 and 0.6 J/cm2 there are no visible changes in interactions between proteins. At a dose of 0.8 J/cm2 , a decrease in the intensity of the protein band was visible when KRAS and RBD proteins were treated. The final radiation dose used in this experiment is 1.0 J/cm2 where a complete loss of interaction between the treated KRAS and RBD proteins is observed. An additional measure of protein carbonylation was carried out at the already set doses. There is a difference in the appearance of the protein band between the untreated protein and the protein treated with different doses of UV-C radiation. Also, carbonylation was repeated for doses of 0.8 J/cm2 and 1.0 J/cm2 , where it was observed that the protein band RBD protein treated with a dose of 1.0 J/cm2 almost completely fades. Conclusions: The results obtained in this study indicate a dose-dependent effect of UV-C radiation on the KRAS-RBD interaction. The weakening of the bond, observed at the higher dose, implies that protein oxidation may affect the stability of the complex. It is important to point out that the protein bands of control samples of individual proteins (untreated protein) were the same as those that underwent radiation treatment, suggesting that UV-C radiation alone is not the cause of changes in protein expression. Additionally, oxidative damage was checked by measuring the level of protein carbonylation, which showed an increase in oxidative damage in treated samples compared to untreated ones. This observation supports the idea that UV-C radiation causes oxidation of proteins, which can further affect their interactions