Mehano-zaznavanje na osnovi INSRTR-ja

magistrsko delo

Gregor Strniša (Avtor), Roman Jerala (Mentor), Marko Novinec (Član komisije za zagovor), Gregor Gunčar (Član komisije za zagovor), Ajda Taler-Verčič (Komentor)

Povzetek

Mehanoreceptorji igrajo ključno vlogo v mnogih celičnih procesih, kot je na primer izražanje proteinov, hkrati pa omogočajo celicam, da se odzovejo na spremembe v svoji okolici. Nedavne raziskave so pokazale, da lahko kot mehanoreceptorji delujejo tudi T in B celični receptorji. V okviru magistrske naloge smo načrtovali nov mehanoreceptor, ki smo ga osnovali na proteinu CAR (himerni antigenski receptor) in konstruktu INSRTR ngGFP. Sistem INSRTR temelji na vstavitvi peptida, ki tvori obvito vijačnico v proteinu na način, da slednji ne spremeni delovanja proteina. Dodatek peptida, ki z vstavljenim tvori obvito vijačnico (nastanek dimera), vodi v spremembo strukture in s tem vpliva na funkcijo proteina. Pripravili smo fuzijske proteine tako, da smo v zapis za CAR vstavili zapis za avtoinhibiran fluorescenčni protein ngGFP. Fuzijske proteine smo izražali v celicah HEK293T, uspešnost izražanja pa smo dokazali s prenosom western. Funkcijo umetnega mehanoreceptorja smo preverjali z dodatkom celic Raji k transfeciranim celicam. Celice Raji izražajo antigen CD19, protein, proti kateremu je bil usmerjen scFv našega fuzijskega proteina. Interakcija med receptorjem CAR in antigenom, izraženim na celicah Raji, bi povzročila aktivacijo mehanosenzorja (povišanje fluorescence), kar smo spremljali s konfokalno mikroskopijo. Pred dodatkom celic Raji smo zaznali fluorescenco na membrani celic, transfeciranih z avtoinhibiranimi in konstitutivno aktivnimi konstrukti. Dodatek celic Raji ni imel učinka na spremembo fluorescence iz česar smo sklepali, da mehanosenzor ne deluje pod temi pogoji. Pripravili smo nove konstrukte, kjer smo odstranili variabilni del receptorja. Avtoinhibirane konstrukte smo aktivirali z dodatkom peptida, ki z vstavljenim peptidom tvori obvito vijačnico, ter zaznali fluorescenco, lokalizirano na celični membrani. Preverili smo vpliv različne dolžine povezovalnega zaporedja med vijačnicama. Poleg dolžine povezovalnega zaporedja smo želeli ugotoviti tudi vpliv odstranitve avtoinhibitorne zanke. V ta namen smo v povezovalno zaporedje dodali cepitveno mesto za proteazo TEV. Ob dodatku proteaze TEV k celicam smo opazili spremembo fluorescence. Pripravili smo več različnih fuzijskih proteinov in z njimi dokazali novo možnost uporabe obvitih vijačnic in načina za pripravo novih mehanosenzorjev.

Ključne besede

mehanozaznavanje;mehanoreceptorji;himerni antigenski receptor;CAR;alosterija;motiv obvite vijačnice;INSRTR;magistrska dela;

Podatki

Jezik:	Slovenski jezik
Leto izida:	2025
Tipologija:	2.09 - Magistrsko delo
Organizacija:	UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik:	[G. Strniša]
UDK:	577.2:577.112(043.2)
COBISS:	239257603
Št. ogledov:	185
Št. prenosov:	93
Ocena:	0 (0 glasov)
Metapodatki:

Ostali podatki

Sekundarni jezik:	Angleški jezik
Sekundarni naslov:	INSRTR based mechanosensing
Sekundarni povzetek:	Mechanoreceptors play a key role in many cellular processes, such as protein expression, and at the same time allow cells to respond to changes in their environment. Recent research has shown that T and B cell receptors can also act as mechanoreceptors. We designed a new mechanoreceptor, based on the CAR protein (chimeric antigen receptor) and the INSRTR ngGFP construct. The INSRTR system is based on inserting a peptide which forms a coiled coil in the protein in a way that does not change the function of the protein. The addition of a peptide which forms a coiled coil with the insert (formation of a dimer) leads to a change in structure and thus affects the function of the protein. We prepared fusion proteins by inserting the sequence for autoinhibited ngGFP into the sequence for CAR. The fusion proteins were expressed in HEK293T cells, and the success of the expression was demonstrated by western blotting. The function of the mechanoreceptor was tested by addition of Raji cells to the transfected cells. Raji cells express the CD19 antigen, a protein against which the scFv of our fusion protein was directed. The interaction between the CAR receptor and the antigen expressed on Raji cells would result in activation of the mechanosensor (increased fluorescence), which was monitored by confocal microscopy. Before the addition of Raji cells, we observed fluorescence on the membrane of cells transfected with the autoinhibited and constitutively active constructs. The addition of Raji cells had no effect on the change in fluorescence, which led us to conclude that the mechanosensor was not functional. We prepared new constructs based on the previous ones, in which we removed the variable part of the receptor. The autoinhibited constructs were activated by adding a solution of a peptide that forms a coiled helix with the inserted peptide, and observed the fluorescence localized on the cell membrane. We tested the effect of different lengths of the connecting sequence between the helices. In addition to the length of the linker sequence, we also wanted to determine the effect of removing the autoinhibitory loop. For this purpose, we added a cleavage site for the TEV protease to the linker sequence. Upon addition of TEV protease to the cells, we observed a change in fluorescence. We prepared several different fusion proteins and used them to demonstrate a new possibility of using coiled helices and a way to modify mechanosensors.
Sekundarne ključne besede:	mechanosensing;CAR;allostery;INSRTR;Beljakovine;Univerzitetna in visokošolska dela;
Vrsta dela (COBISS):	Magistrsko delo/naloga
Študijski program:	1000377
Komentar na gradivo:	Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Strani:	1 spletni vir (1 datoteka PDF (60 str.))
ID:	26525966