Ovrednotenje načrtovanih signalosomov na osnovi adapterskega proteina TRIF

Maučec, Ana (author), Hafner Bratkovič, Iva (mentor), Pavšič, Miha (comentor)

Adapter, ki vsebuje domeno receptorjev Toll/interlevkin-1 (TIR) in sproži interferon β (TRIF), je protein prirojene imunosti, ki sodeluje pri aktivaciji transkripcijskih dejavnikov jedrnega dejavnika κB (NF-κB), interferonskega regulatornega dejavnika 3 (IRF3) in aktivacijskega proteina 1 (AP-1) navzdol od Toll-u podobnega receptorja 3 (TLR3) in TLR4 ter posreduje pri sprožitvi različnih vrst celične smrti. Sinteza provnetnih citokinov in interferonov tipa I omogoča aktivacijo obrambnih mehanizmov proti bakterijam in virusom ter spodbuja pridobljeni imunski odziv, vključno s protitumorskim. Zaradi stimulacije protitumorskega odziva je TRIF zanimiva tarča za imunoterapijo raka. Imunski odziv lahko sprožimo z uporabo agonistov TLR, vendar je njihova terapevtska uporabnost omejena. V zadnjem času se vse več pristopov osredotoča na pripravo sinteznih konstruktov, ki temeljijo na delih proteinov prirojene imunosti in lahko omogočajo tudi časovni ter prostorski nadzor nad aktivacijo imunskega odziva. Da bi posnemali delovanje naravnih signalnih kompleksov TRIF v celici, signalosomov, smo pripravili sintezne konstrukte na osnovi adapterja TRIF. Oligomerizacijo TRIF, ki je potrebna za aktivacijo IRF3 in NF-κB, smo dosegli z dodatkom domene z nizko kompleksnostjo (LCD) DNA-vezavnega proteina 43, ki se veže na transaktivacijski odzivni element (TDP43), saj le-ta omogoča spontan nastanek proteinskih kondenzatov. Da bi bil naš sistem inducibilen, smo pripravljenim sinteznih signalosomom dodali tudi dve ponovitvi homooligomerizacijske domene B (dDmrB), ki ob dodatku majhne molekule oligomerizira. V modelnem sistemu, celični liniji HEK293, smo pokazali, da sintezni signalosomi na osnovi TRIF omogočajo inducibilno tvorbo pretežno citosolnih celičnih skupkov ob dodatku dimerizatorja AP20187, neodvisno od zaporedja proteinskih domen v sinteznem signalosomu. Za njihovo tvorbo ni bila zadostna le oligomerizacija dDmrB, temveč tudi prisotnost LCD. Pokazali smo tudi, da vnos konstruktov v celice omogoča inducibilno aktivacijo transkripcijskih dejavnikov NF-κB in IRF3. Pripraviti smo tudi signalosome, ki bi pretežno aktivirali IRF3, zato smo jim odstranili motiv za homotipske interakcije proteinov, povezanih z receptorjem (RHIM). Skrajšani signalosomi so inducibilno aktivirali IRF3, vendar je bila raven aktivacije precej nižja kot pri signalosomih z RHIM, predvidoma zaradi izgube stabilnega ogrodja, ki ga tvori RHIM. Sintezne signalosome smo izrazili še v tumorski celični liniji HeLa, kjer smo zaznali aktivacijo IRF3. V okviru magistrskega dela smo uspešno pripravili funkcionalne inducibilne sintezne signalosome na osnovi TRIF s terapevtskim potencialom za imunoterapijo raka.

• Language: Slovenian
• Keywords: TRIF, sintezni signalosomi, NF-κB, IRF3, LCD
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2026
• PID: 20.500.12556/RUL-186149
• COBISS.SI-ID: 290825475
• Publication date in RUL: 27.08.2026

Secondary language

• Language: English
• Title: Validation of designed signalosomes based on TRIF adapter protein

Abstract

Toll/interleukin-1 receptor/resistance protein (TIR) domain containing adapter inducing interferon-β (TRIF) is an innate immunity adaptor protein that activates the transcription factors nuclear factor κB (NF-κB), interferon regulatory factor 3 (IRF3), and activator protein 1 (AP-1) downstream of Toll-like receptor 3 (TLR3) and TLR4, and induces various types of cell death. The production of proinflammatory cytokines and type I interferons enables effective host antibacterial and antiviral defence, as well as stimulation of adaptive immune system, including antitumour immunity. Given its ability to stimulate antitumour immunity, TRIF is a compelling target for cancer immunotherapy. The use of TLR agonists to elicit the immune response is limited, so new synthetic biology-based approaches that utilise components of innate immune proteins and offer spatiotemporal control of signalling pathways are gaining traction. We designed TRIF-based synthetic constructs that would mimic natural signalling complexes of TRIF in cells, i.e. signalosomes. We intended to achieve TRIF oligomerization, essential for IRF3 and NF-κB activation, by utilising low complexity domain (LCD) of TAR DNA- binding protein 43 (TDP43), capable of constitutively forming protein condensates. To make our system inducible, our signalosomes contained two repeats of homooligomerization domain B (dDmrB) that undergoes oligomerization in the presence of a small molecule. In our model system, HEK293 cell line, we demonstrated inducible formation of mainly cytosolic TRIF puncta upon addition of AP20187 dimeriser, regardless of the order of protein domains in synthetic signalosomes. Oligomerisation of dDmrB alone was insufficient to enable the formation of protein condensates without the presence of LCD. We also showed inducible activation of NF-κB and IRF3 after introduction of synthetic signalosomes into the cells. Additionally, to prepare signalosomes capable of predominantly activating IRF3, the receptor-interacting serine/threonine-protein kinases (RIP) homotypic interaction motif (RHIM) was removed. Shorter constructs inducibly activated IRF3, but to a much lower extent than those containing RHIM, presumably due to the loss of a stable signalling framework formed by RHIM. We expressed synthetic signalosomes in the cancer cell line, HeLa, as well and detected IRF3 activation. In the scope of this master thesis, we successfully prepared functional inducible TRIF-based synthetic signalosomes with potential for use in cancer immunotherapy.

• Keywords: TRIF, synthetic signalosomes, NF-κB, IRF3, LCD