Načrtovanje sintezne vnetne poti receptorja STING z uporabo oligomerizacijskih domen

Babnik, Ana (avtor), Hafner Bratkovič, Iva (mentor), Župunski, Vera (komentor)

STING je adaptorski protein signalne poti cGAS-STING, ki je del prirojene imunosti. Receptor cGAS prepozna citosolno DNA, ki izvira iz okužb s patogeni ali poškodb lastnih celic, in preko aktivacije STING sproži sproščanje IFN tipa I in provnetnih citokinov, kar izzove imunski odziv. Signalna pot cGAS-STING ima zaradi aktivacije imunskih celic pomembno vlogo v ustvarjanju protitumorskega imunskega odziva, zaradi česar je privlačna tarča za imunoterapijo raka. Razvili so že več agonistov STING, ki pa lahko ob vnosu v telo sprožijo sistemsko vnetje, zaradi česar so potrebni novi pristopi. Eden izmed pristopov je uporaba umetnih sistemov, ki imajo prednost v tem, da niso odvisni od prisotnosti funkcionalnih poti ter ob primerni dostavi omogočajo lokalno in omejeno aktivacijo. Želeli smo pripraviti t.i. vnetno napravo na osnovi segmenta STING, ki bi izboljšala aktivacijo IRF3 in NFκB. Uporabili smo himerni CTT iz človeka in navadne cebrice, za katerega so de Oliveira Mann in sodelavci pokazali, da vzpodbudi signalizacijo preko obeh transkripcijskih faktorjev. Za izboljšanje signaliziranja smo ubrali različne pristope: pripravili smo konstrukte CTT z različnim številom ponovitev CTT, z dodatkom oligomerizacijskih domen in TDP-43, ki tvori kondenzate preko fazne ločitve, s čimer smo posnemali oligomerizacijo STING na membrani endoplazemskega retikuluma ali Golgijevega aparata. Za analizo vpliva konstruktov na učinkovitost aktivacije IRF3 in NFκB smo konstrukte vnesli v celično linijo HEK293T. Ugotovili smo, da sta za ojačitev aktivacije transkripcijskih faktorjev v konstruktu s CTT potrebna TDP-43 in dodatek oligomerizacijskih domen oziroma TDP-43 in večje število ponovitev CTT. Najučinkovitejši sta bili različici, kjer je bil CTT vezan na protein TDP-43 v kombinaciji s tetramerizacijsko domeno in kjer so bile na TDP-43 vezane tri zaporedne ponovitve CTT. Omenjena konstrukta sta tudi v rakavi celični liniji HeLa aktivirala transkripcijski poti IRF3 in NFκB. V okviru magistrskega dela smo uspešno pripravili konstrukte na osnovi STING, s potencialom za obetaven in inovativen pristop pri imunoterapiji raka, ki ne temelji na izražanju endogenih komponent signalne poti cGAS-STING v rakastih celicah.

• Jezik: Slovenski jezik
• Ključne besede: STING, IRF3, NFκB, sintezna vnetna naprava, imunoterapija raka
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-170200
• COBISS.SI-ID: 242292739
• Datum objave v RUL: 02.07.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Design of a synthetic STING receptor inflammatory pathway using oligomerization domains

Izvleček

STING is an adaptor protein in the cGAS-STING signaling pathway, an important component of innate immunity. The pathway triggers the release of type I interferon (s) and proinflammatory cytokines in response to the recognition of cytosolic dsDNA by receptor cGAS, thereby provoking an immune response. The cGAS-STING signaling pathway plays a crucial role in eliciting an antitumor immune response due to the activation of immune cells, making it an attractive target for cancer immunotherapy. Several STING agonists have already been developed, but they can induce systemic inflammation upon administration, highlighting the need for new approaches. One approach involves the use of synthetic systems, which do not depend on the expression of endogenous molecules. When appropriately introduced, they induce local and contained inflammatory response. We aimed to develop an inflammatory device based on STING that would enhance the activation of IRF3 and NF-κB. We utilized a chimeric CTT from humans and zebrafish, which has been previously shown to stimulate signaling through both transcription factors. To improve signaling, we employed various strategies: we created CTT constructs with different numbers of CTT repeats, added oligomerization domains, and included TDP-43, which forms condensates via phase separation, mimicking the oligomerization of STING on the endoplasmic reticulum or Golgi apparatus membrane. To analyze the constructs' effect on the efficiency of IRF3 and NF-κB activation, we introduced the constructs into the HEK293T cell line. We found that for enhanced transcription factor activation of the CTT, both TDP-43 and the addition of oligomerization domains or TDP-43 and multiple CTT repeats were necessary. The best response was achieved when CTT was linked to the TDP-43 protein in combination with a tetramerization domain and when three consecutive CTT repeats were linked to TDP-43. These constructs also activated IRF3 and NF-κB in the HeLa cervical carcinoma cell line. As part of this master's thesis, we successfully developed a STING variant with the potential to serve as a promising and innovative approach in cancer immunotherapy, which does not rely on the expression of endogenous components of the cGAS-STING signaling pathway in cancer cells.

• Ključne besede: STING, IRF3, NF-κB, synthetic inflammatory device, cancer immunotherapy