Detection of protein interactions of the MIU domain of SPRTN due to DNA damage

Nesmetano odvijanje osnovnih procesa poput replikacije, transkripcije te popravka DNA esencijalni su za opstanak stanice. Prisustvo specifičnog tipa oštećenja pod nazivom kovalentno vezanje proteina na DNA (engl. DNA-protein crosslink, DPC) onemogućava odvijanje osnovnih staničnih procesa, čime se narušava stanična homeostaza. Zato što akumulacija DPC u stanici doprinosi razvoju tumora te starenju, humana proteaza SPRTN, koja je usko vezana uz proteolitički popravak DPC, predmet je brojnih istraživanja. U ovom radu pozornost je stavljena na nekarakteriziranu domenu MIU proteina SPRTN. Testirali smo interakcijske partnere domene MIU, te time pokušali predvidjeti njenu funkciju koju obavlja za vrijeme proteolitičkog uklanjanja DPC. Tumorske stanične linije HeLa i HepG2 su tretirane formaldehidom i UV-zračenjem u svrhu indukcije DPC. Korištenjem metode pulldown ispitali smo interakciju domene MIU s proteinima XRCC1, CHK1, PMS2, GAPDH, MLH1 te proteinima koji su obilježeni s ubikvitinom ili SUMO-2/3 posttranslacijskom modifikacijom. Interakcija proteina je provjerena metodom Western blot. Domena MIU nije vezala ni jedan testirani protein. Protein XRCC1 te PMS2 su definirani kao novi C-terminalni interaktori proteina SPRTN dok je za CHK1 ustanovljeno da se veže za N-terminalni dio proteina SPRTN. The unobstructed performance of basic processes, such as DNA replication, transcription and repaire, is essential for cell survival. The presence of a specific type of damage called DNAprotein crosslink (DPC) prevents the execution of basic cellular processes, thereby disrupting cellular homeostasis. Due to the fact that an accumulation of DPC in cells contributes to tumor development and aging, a human protease SPRTN, closely related to the proteolytic repair of DPC, is the subject of numerous studies. This study focuses on the MIU domain, an uncharacterized domain of SPRTN protein. We tested interaction partners of the MIU domain, and thereby tried to predict its function during the proteolytic removal of DPC. For the purpose of DPC induction, tumor cell lines HeLa and HepG2 were treated with formaldehyde and UV radiation. Using the pulldown method, we examined interactions of the MIU domain with XRCC1, CHK1, PMS2, GAPDH, MLH1 and proteins labeled with ubiquitin or SUMO-2/3 post-translational modification. Protein interactions were verified by a Western blot method. The MIU domain did not bind any tested protein. The XRCC1 and PMS2 proteins were defined as new C-terminal interactors of SPRTN protein, while CHK1 was found to bind the N-terminal part of SPRTN protein.