Preučevanje vpliva modulatorjev N-acetilglukozaminilacije na proliferacijo nevroblastomske celične linije SH-SY5Y ; A study of N-acetylglucosaminylation modulators on SH-SY5Y neuroblastoma cell line proliferation

N-acetilglukozaminilacija je posttranslacijska modifikacija številnih jedrnih, citoplazemskih in mitohondrijskih proteinov. Preko moduliranja signalnih poti vstopa v interakcijo s potmi fosforilacije in je, kakor ta vpeta v regulacijo mnogih celičnih procesov. Vlogo naj bi imela celo v etiologiji nekaterih kroničnih bolezni. Uravnavata jo dva encima, O-GlcNAc transferaza in O-GlcNAc hidrolaza, ki sta v največji meri prisotna v možganih, imunskih celicah in trebušni slinavki. V magistrski nalogi smo se osredotočili na preučevanje vpliva modulatorjev N-acetilglukozaminilacije na proliferacijo celic SH-SY5Y, z namenom dokaza morebitne vpletenosti N-acetilglukozaminilacije v patologijo nevroblastoma oz. drugih nevroloških obolenj. Uporabljena celična linija SH-SY5Y, je nevroblastomska linija, ki se uporablja kot in vitro model za raziskovanje nevronskih vlog. V prvem delu smo s testom metabolne aktivnosti najprej preverili morebitni vpliv selektivnega zaviralca OGT – OSMI-1 in selektivnega zaviralca OGA - TMG na živost celic SH-SY5Y. Ugotovili smo, da je OSMI-1 (50 µM) za celice toksičen, medtem ko TMG nanje, ni imel vpliva. Nadalje smo uporabili netoksične koncentracije spojin in preverili učinek na proliferacijo celic SH-SY5Y. Pokazal se je trend, vendar razlike niso bile statistično značilne, zato vpliva nismo mogli potrditi. Ravno tako se pod vplivom omenjenih spojin, s pomočjo pretočnega citometra niso pokazale spremembe v celičnem ciklu. V naslednjem koraku smo kljub neuspešnemu dokazu o vplivu na proliferacijo, želeli s pomočjo prenosa western raziskati še vpliv OSMI-1, TMG in vsebnosti glukoze v mediju, na N-acetilglukozaminilacijo proteinov ter izražanje encimov OGT in OGA. OSMI-1 je zavrl N-acetilglukozaminilacijo in izražanje OGA, pri čemer se je izražanje OGT povečalo. TMG je deloval ravno nasprotno in izzval povečanje N-acetilglukozaminilacije in izražanje OGA, izražanje OGT pa se je zmanjšalo. Kjer je bila vsebnost glukoze v mediju višja je prišlo do višje stopnje N-acetilglukozaminilacije in izražanja OGT. V zadnjem delu smo s konfokalnim fluorescenčnim mikroskopom dokazali premik encima OGT iz jedra v citoplazmo celice ob dodatku OSMI-1. To bi lahko nakazovalo na spremembe v znotrajcelični signalizaciji in torej morebitno vpletenost drugih signalnih poti v smrtnost celic, v primeru dodatka spojine OSMI-1. ; O-GlcNAcylation is a post-translational modification known to modify many nuclear, cytoplasmic and mitochondrial proteins. Modulation of cellular pathways by O-GlcNAcylation involves an extensive cross talk with the pathways that are also regulated by protein phosphorylation signaling pathways. Consequently, like phosphorylation O-GlcNAcylation is involved in the regulation of many cellular processes. This protein modification plays a fundamental role also in chronic diseases. O-GlcNAc cycling is modified by two enzymes: O-GlcNAc transferase and O-GlcNAcase, which are highly expressed in brain, immune cells and pancreas. In this master thesis we focused on a study of O-GlcNAcylation modulators on SH-SY5Y cell line proliferation. Our objective was to prove the involvement of O-GlcNAcylation in the pathogenesis of neuroblastoma or some other neurological disease. We used SH-SY5Y neuroblastoma cell line, an in vitro model widely used for the study of neurons. In the first part, a metabolic assay was used to evaluate the effect of the OSMI-1, a selective inhibitor of OGT, and TMG, a selective inhibitor of OGA, on the viability of SH-SY5Y cells. We showed that OSMI-1 (50 µM) is cytotoxic, while TMG had no effect on this parameter. Next, we determined non-toxic concentrations of inhibitors and their potential effect on proliferation of SH-SY5Y cells. The differences were not statistically significant, therefore we could not prove the influence on proliferation. Also, using flow cytometry we were not able to show any differences in cell cycle. In the next step Western blot was used to investigate the effects of OSMI-1, TMG and glucose level on O-GlcNAcylation level and OGT, OGA expression in SH-SY5Y cells. OSMI-1 reduced O-GlcNAcylation of proteins and expression of OGA, but induced the expression of OGT. On the other hand, TMG induced O-GlcNAcylation and expression of OGA, but reduced the expression of OGT. High glucose levels resulted in higher level of protein O-GlcNAcylation and induced expression of OGT, compared to low glucose medium. In the last segment we addressed the effect of OSMI-1 on the intracellular location of OGT in SH-SY5Y cells. Using confocal microscopy we determined that OSMI-1 causes OGT translocalization from the nucleus to the cytoplasm. This indicates changes in the cellular signalization, however further studies are needed to identify which pathways are involved in the observed effect.