Title Utjecaj UV-C zračenja na interakciju KRAS proteina s RAF1 RAS veznom domenom
Title (english) Effect of UV-C radiation on protein interaction between KRAS and RAF1 RAS binding domain
Author Tina Budimir
Mentor Marina Rudan Dimlić (mentor)
Mentor Josip Crnjac (komentor)
Committee member Snježana Štambuk (predsjednik povjerenstva)
Committee member Željana Bašić (član povjerenstva)
Committee member Marina Rudan Dimlić (član povjerenstva)
Granter University of Split University Department of Forensic Sciences Split
Defense date and country 2024-04-19, Croatia
Scientific / art field, discipline and subdiscipline INTERDISCIPLINARY AREAS OF KNOWLEDGE Cognitive Science (Natural, Technical, Biomedical and Healthcare, Social and Humanistic Sciences)
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.
Abstract (english) 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.
Keywords
protein-protein interakcija
ras obitelj
KRAS onkogen
KRAS protein
RAF1
Raf/MEK/ERK signalni put
oksidativni stres
UV-C zračenje
karbonilacija proteina
HEK293 stanice
SIMPL metoda
Language croatian
URN:NBN urn:nbn:hr:227:164938
Study programme Title: Forensic Sciences Study programme type: university Study level: graduate Academic / professional title: sveučilišni magistar/sveučilišna magistra forenzike (sveučilišni magistar/sveučilišna magistra forenzike)
Type of resource Text
File origin Born digital
Access conditions Embargoed access Embargo expiration date: 2026-04-19
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Created on 2024-05-21 06:03:42