Validacija proteinskih interaktorjev proteina TDP-43

Razboršek Fink, Klara (author), Rogelj, Boris (mentor)

Protein z molekulsko maso 43 kDa, ki se veže na transaktivacijski odzivni element molekule DNA (TDP-43), je DNA/RNA-vezavni protein iz družine heterogenih jedrnih ribonukleoproteinov. Sodeluje pri uravnavanju metabolizma RNA, transporta mRNA in uravnava izrezovanje več genov. Nahaja se lahko v jedru ali citoplazmi, pri čemer je njegova prisotnost v citoplazmi močno povezana s patološkimi procesi. Citoplazemski agregati proteina TDP-43 so najpogostejša nevropatološka značilnost amiotrofične lateralne skleroze (ALS). Proteinske interakcijske partnerje proteina TDP-43 tako v fizioloških kot tudi patoloških procesih so preučevali že v številnih raziskavah, vendar veliko število odkritih interaktorjev še ni validiranih. V sklopu magistrskega dela smo želeli ovrednotiti potencialne interaktorje ATXN2L, HNRNPK, NUP93 in SFPQ proteina TDP-43wt in TDP-43dNLS, oblike TDP-43 brez jedrnega lokalizacijskega signala, ki delno posnema obnašanje TDP-43 v patologiji. Validirati smo želeli interakcije s TDP-43wt oziroma TDP-43dNLS. V ta namen smo z metodo APEX2 preverjali prisotnost teh interaktorjev v eluatih po metodi »pull down«. Prav tako smo ovrednotili inducibilne ekspresijske celične linije HEK 293, ki stabilno izražajo proteina TDP-43wt in TDP-43dNLS v fuziji s fluorescenčnim proteinom mScarlet-myc. Ugotovili smo, da ima fuzijski protein TDP-43wt z mScarlet pričakovano jedrno lokalizacijo, TDP-43dNLS v fuziji z mScarlet pa citoplazemsko. Z imunodetekcijo smo po prenosu western zaznali interakcijske partnerje TDP-43wt in njegove mutante TDP-43dNLS. Po celični lizi smo izvedli celično frakcionacijo, s katero smo ločili citoplazemsko in jedrno frakcijo in z imunodetekcijo po prenosu western detektirali prisotnost interakcijskih partnerjev v posamezni frakciji. Te celice pa smo uporabili tudi za preparate in imunocitokemijo za natančnejšo lokalizacijo oblik TDP-43 in potencialnih interakcijskih partnerjev. Naši rezultati nakazujejo, da NUP93, HNRNPK in SFPQ interagirajo s TDP-43wt v jedru in v pogojih, ki posnemajo ALS, kolokalizirajo s TDP-43 v citoplazmo. ATXN2L pa je pridobljen interaktor TDP-43dNLS. Pridobljeni rezultati bodo pripomogli k razumevanju interaktoma TDP-43 in TDP-43dNLS.

• Language: Slovenian
• Keywords: TDP-43, validacija, interakcijski partnerji, ALS
• Work type: Master's thesis/paper
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2026
• PID: 20.500.12556/RUL-187023
• Publication date in RUL: 08.09.2026

Secondary language

• Language: English
• Title: Validation of TDP-43 protein interactors

Abstract

Transactive response DNA binding protein 43 kDa (TDP-43) is a DNA/RNA-binding protein that belongs to the family of heterogeneous nuclear ribonucleoproteins. It is involved in the regulation of RNA metabolism and mRNA transport and regulates the splicing of several genes. It can be found in the nucleus or the cytoplasm, and its presence in the cytoplasm is strongly associated with pathological processes. Cytoplasmic aggregates of the TDP-43 protein are the most common neuropathological features of amyotrophic lateral sclerosis (ALS). Protein interaction partners of TDP-43 in both physiological and pathological processes have been studied in a large number of research studies; however, many of the identified interactors have not yet been validated. As part of this master’s thesis, we sought to evaluate potential interactors of ATXN2L, HNRNPK, NUP93, and SFPQ for both TDP-43wt and TDP-43dNLS - a form of TDP-43 lacking a nuclear localization signal that mimics the behavior of TDP-43 in pathological conditions. We sought to validate interactions with TDP-43wt and TDP-43dNLS. To this end, we used the APEX2 method to detect the presence of these interactors in pull down eluates. We also evaluated inducible HEK 293 cell lines that stably express the TDP-43wt and TDP-43dNLS fusion proteins fused to the fluorescent protein mScarlet-myc. We found that the TDP-43wt fusion protein with mScarlet exhibited the expected nuclear localization, while TDP-43dNLS fused with mScarlet exhibited cytoplasmic localization. Using immunodetection following Western blotting, we identified the interaction partners of TDP-43wt and its mutant, TDP-43dNLS. Following cell lysis, we performed cell fractionation to separate the cytoplasmic and nuclear fractions and used Western blot analysis to detect the presence of interaction partners in each fraction. We also used these cells for immunocytochemistry to more precisely localize TDP-43 forms and interaction partners. We found that NUP93, HNRNPK, and SFPQ interact with TDP-43wt in the nucleus and, under conditions that mimic ALS, colocalize with TDP-43 in the cytoplasm. ATXN2L, on the other hand, is an acquired TDP-43dNLS interactor. The results obtained will contribute to our understanding of the interactome of TDP-43 and TDP-43dNLS.

• Keywords: TDP-43, validation, interaction partners, ALS