Proteinske interakcije človeškega matrina 3

Nimac, Jerneja (avtor), Župunski, Vera (mentor)

Matrin 3 (MATR3) je DNA/RNA-vezavni protein jedrnega matriksa, ki je vključen v različne celične procese. Sodeluje pri zgodnjem odzivu na poškodbe DNA, prepisovanju, stabilizaciji mRNA, alternativnem izrezovanju intronov in jedrnem izvozu mRNA. Mutacija S85C v genu MATR3 je povezana z razvojem počasi napredujoče oblike amiotrofične lateralne skleroze (ALS), vendar vloga MATR3 v patogenezi te bolezni ni poznana. Slabo opisane in nepoznane so tudi proteinske interakcije MATR3 in njegovih mutantov. Prav te pa bi bile lahko odgovorne za razvoj ALS. V sklopu magistrskega dela smo želeli z metodo BioID, ki temelji na označevanju bližnjih proteinov z biotinom, identificirati proteine, ki v pogojih in vivo interagirajo s človeškim MATR3 in njegovo mutirano obliko MATR3S85C. V ta namen smo pripravili inducibilne ekspresijske celične linije HEK 293, ki stabilno izražajo fuzijski protein MATR3 oz. MATR3S85C z biotin ligazo BioID2, ali BioID2 brez fuzijskega partnerja. Ugotovili smo, da imata fuzijska proteina MATR3 in MATR3S85C z BioID2 enako celično lokalizacijo kot endogeni MATR3 in da je ligaza BioID2 v teh fuzijah encimsko aktivna. Po preučitvi aktivnosti BioID2 in celične lokalizacije izraženih proteinov smo nadaljevali s testom »pull down«, s katerim smo iz celičnih lizatov uspešno izolirali biotinilirane interakcijske partnerje obeh oblik MATR3. Z barvanjem s srebrom in imunodetekcijo biotiniliranih proteinov po prenosu western smo namreč zaznali proteine, ki so značilni za vzorce z MATR3 in MATR3S85C, ne pa tudi za vzorce z BioID2 brez fuzijskega partnerja. Te interakcijske partnerje bomo identificirali s tehniko tekočinske kromatografije, sklopljene z masno spektrometrijo. Rezultati bodo pripomogli k razumevanju interaktoma MATR3 in MATR3S85C. V eluatih z MATR3 in MATR3S85C smo sicer že zaznali dva znana interakcijska partnerja MATR3, proteina TDP-43 in NONO. Na koncu smo preverili še vpliv MATR3S85C na nastanek stresnih granul in na podlagi rezultatov zaključili, da ta oblika MATR3 prepreči njihovo tvorbo.

• Jezik: Slovenski jezik
• Ključne besede: MATR3, ALS, interakcijski partnerji, BioID
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2021
• PID: 20.500.12556/RUL-133573
• COBISS.SI-ID: 89932291
• Datum objave v RUL: 01.12.2021

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Protein interactions of human matrin 3

Izvleček

Matrin 3 (MATR3) is a DNA/RNA-binding nuclear matrix protein involved in several cellular processes. These include early stages of DNA damage response, transcription, mRNA stability, alternative splicing and mRNA export. The mutation S85C in the MATR3 gene is associated with the development of a slowly progressive form of amyotrophic lateral sclerosis (ALS). However, the role of MATR3 in the pathogenesis of this disease is unknown. Additionally, little is known about the protein-protein interactions of MATR3 and its mutants, even though these interactions could be responsible for ALS development. In the master’s thesis, we aimed to identify proteins that interact with human MATR3 and its mutant form MATR3S85C in vivo with the BioID method, which is based on labelling the nearby proteins with biotin. To this end, we generated cell lines stably expressing fusion protein of MATR3 or MATR3S85C with the biotin ligase BioID2 or BioID2 without a fusion partner. We found that fusion proteins of MATR3 and MATR3S85C with the biotin ligase BioID2 have the same cellular localization as endogenous MATR3 and that BioID2 is enzymatically active. After examining the BioID2 activity and cellular localization of the expressed proteins, we successfully isolated biotinylated interaction partners of MATR3 and MATR3S85C from cell lysates by the pull-down assay. Using silver staining and immunodetection of the biotinylated proteins after Western blotting, we detected proteins specific for samples with MATR3 and MATR3S85C, but not for samples with BioID2 without a fusion partner. These interaction partners will be identified by liquid chromatography – mass spectrometry. Our results will help to understand MATR3 and MATR3S85C interactome. Moreover, we have already detected two known MATR3 interaction partners, TDP-43 and NONO in eluates with MATR3 and MATR3S85C. Finally, we have examined the effect of MATR3S85C on stress granule formation, and based on the results, we have concluded that this form of MATR3 prevents their formation.

• Ključne besede: MATR3, ALS, interaction partners, BioID