Detekcija interakcij proteinov TTF2, PRPF40A in CRACD z matrinom 3 s protitelesi

Čarman, Jasna (avtor), Župunski, Vera (mentor)

Amiotrofična lateralna skleroza je hitro napredujoča nevrodegenerativna bolezen motoričnega živčevja, za katero je značilna mišična atrofija in postopen upad motoričnih funkcij. Učinkovitih terapij za upočasnitev napredovanja bolezni še ni, zato večina pacientov umre zaradi odpovedi dihal in zadušitve. Eden od vzrokov za ALS so mutacije v genih za RNA-vezavne proteine, kot je jedrni protein matrin 3. Ta se pri ALS kopiči v jedru, pri čemer pride do nepravilnosti v organizaciji kromatina, transkripciji in izrezovanju intronov, ali pa agregira v citoplazmi in poruši transport in stabilnost RNA. Ena od točkovnih mutacij tega proteina, povezana z ALS, je S85C. Interakcije matrina 3 in njegovih mutantov z drugimi proteini so slabo opisane. Zato smo v okviru diplomskega dela želeli potrditi interakcije matrina 3 in njegovega mutanta S85C s proteini TTF2, PRPF40A in CRACD. Uporabili smo vzorce lizatov celic HEK 293 TR-Flp-In, pripravljene z metodo BioID. Pri tej metodi preiskovani matrin 3 pripravimo v fuziji z biotin ligazo BioID2, ki ob dodatku biotina biotinilira proteine v bližini, ki so potencialni interaktorji preiskovanega proteina. Nato biotinilirane proteine izoliramo s tehniko "pull down". Predhodno so potencialne interaktorje določili z masno spektroskopijo, mi pa smo jih želeli potrditi s prenosom western. Uspešno smo kot inetraktor detektirali protein TTF2, ki ga je bilo nekoliko več v vzorcu interaktorjev matrina 3 z mutacijo S85C. Proteinov PRPF40A in CRACD pa smo v vzorcih z matrinom 3 detektirali manj kot v vzorcu lizata neinduciranih celic, zato interakcij matrina 3 s PRPF40A in s CRACD nismo potrdili. Poleg tega smo analizirali kolokalizacijo matrina 3 in proteina TTF2 z imunocitokemijsko tehniko. Ugotovili smo, da je protein TTF2 pretežno prisoten v citoplazmi, medtem ko smo matrin 3, ki je sicer jedrni protein, detektirali tako v jedru kot v citoplazmi. V nekaterih primerih smo opazili kolokalizacijo obeh proteinov, vendar matrina 3, ki je endogeni protein, nismo detektirali v vseh celicah, zato bi morali poskuse ponoviti in optimizirati.

• Jezik: Slovenski jezik
• Ključne besede: ALS, matrin 3, TTF2, PRPF40A, CRACD
• Vrsta gradiva: Diplomsko delo/naloga
• Tipologija: 2.11 - Diplomsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-171862
• COBISS.SI-ID: 253408771
• Datum objave v RUL: 03.09.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Detection of TTF2, PRPF40A, and CRACD interactions with matrin-3 by antibodies

Izvleček

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease of the motor nervous system, characterized by muscle atrophy and a gradual decline in motor function. Effective therapies to slow disease progression are currently lacking, and most patients die from respiratory failure and asphyxiation. One of the causes of ALS involves mutations in genes encoding RNA-binding proteins, such as the nuclear protein matrin 3. In ALS, matrin 3 accumulates in the nucleus, leading to abnormalities in chromatin organization, transcription, and intron splicing, or it aggregates in the cytoplasm, disrupting RNA transport and stability. One of the point mutations in this protein associated with ALS is S85C. The interactions between matrin 3 and its mutant forms with other proteins are poorly described. Therefore, in this thesis, we aimed to confirm the interactions of wild type matrin 3 and its S85C mutant with the proteins TTF2, PRPF40A, and CRACD. We used HEK 293 TR-Flp-In cell lysate samples prepared with the BioID method. In this approach, the protein of interest (matrin 3) is fused with the biotin ligase BioID2, which, upon the addition of biotin, biotinylates nearby proteins that are potential interactors. These biotinylated proteins are then isolated using a pull-down technique. Potential interactors had previously been identified by mass spectrometry, and our goal was to confirm them using western blotting. We successfully detected the TTF2 protein as an interactor, with slightly higher levels observed in the sample containing the S85C matrin 3 mutant. However, PRPF40A and CRACD were detected at lower levels in the matrin 3 samples compared to the lysate of non-induced cells, and therefore, interactions between matrin 3 and PRPF40A or CRACD could not be confirmed. In addition, we analyzed the colocalization of matrin 3 and TTF2 using immunocytochemical technique. We found that TTF2 is predominantly present in the cytoplasm, whereas matrin 3, which is normally a nuclear protein, was detected both in the nucleus and the cytoplasm. In some cases, colocalization of the two proteins was observed. However, endogenous matrin 3 was not detected in all cells, so these experiments would need to be repeated and optimized.

• Ključne besede: ALS, matrin 3, TTF2, PRPF40A, CRACD