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Vpliv fosforilacije na znotrajcelično razporejanje proteina FUS v diferenciranih celicah SH-SY5Y FlpIn
ID Gartner, Eva (Author), ID Motaln, Helena (Mentor) More about this mentor... This link opens in a new window, ID Rogelj, Boris (Comentor)

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Abstract
Amiotrofična lateralna skleroza (ALS) in frontotemporalna demenca (FTD) sta hitro napredujoči nevrodegenerativni bolezni, za kateri je značilno postopno odmiranje nevronov. Razlog za razvoj obolenj so mutacije v različnih genih, k razvoju pa naj bi prispevale tudi posttranslacijske modifikacije RNA-vezavnih proteinov. Eden izmed takih genov je gen FUS, ki nosi zapis za istoimenski protein. Protein FUS (ang. »fused in sarcoma«) je primarno jedrni RNA/DNA vezavni protein, ki sodeluje pri številnih procesih kot so prepisovanje RNA, metabolizem RNA, odziv na poškodbe DNA in odziv na celični stres. Različne funkcije protein FUS opravlja tako v jedru kot v citoplazmi, zato mora za normalno delovanje krožiti med njima. Slednje bi mu lahko omogočala posttranslacijska modifikacija in sicer fosforilacija zadnjega tirozinskega ostanka s strani kinaz iz družine Src. V magistrskem delu smo opredelili vpliv fosforilacije C-končnega tirozinskega ostanka na mestu 526 (Y526) v proteinu FUS na znotrajcelično razporejanje proteina FUS in proteina FUS z delecijo C-končne domene, ki nosi zapis za jedrni signalizacijski signal (NLS), v diferenciranih celicah SH-SY5Y FlpIn. Uporabili smo predhodno razvite celične linije SH- SY5Y FlpIn in sicer SH-SY5Y-mSc, SH-SY5Y-mScFUS in SH-SY5Y-mScFUSdNLS. Vse tri linije celic inducibilno izražajo rdeči fluorescenčni protein mScarlet (mSc), z mScarlet označen protein FUS (mScFUS) ali z mScarlet označen protein FUS brez C-končne regije (mScFUSdNLS). Uspešnost izražanja proteinov mSc, mScFUS in mScFUSdNLS v celicah smo potrdili s fluorescenčno konfokalno mikroskopijo in detekcijo teh proteinov po prenosu western. Najprej smo s pomočjo literature in eksperimentalnega dela prilagodili diferenciacijski protokol, s katerim smo celice diferencirali v nevronom podobne celice, z dodatkom retinojske kisline in dejavnika BDNF. Izražanje proteinov, značilnih za nevronske celice, smo potrdili s fluorescenčno konfokalno mikroskopijo in detekcijo teh proteinov po prenosu western. Diferencirane celice SH-SY5Y FlpIn vseh treh celičih linij smo nato transficirali s plazmidi z zapisom za kinaze c-Src, c-Fyn in c-Abl. Za primerjavo smo transficirali tudi nediferencirane celice SH-SY5Y FlpIn. Tik pred transfekcijo smo celice inducirali z doksiciklinom, kar je spodbudilo izražanje vstavljenih mSc, mScFUS in mScFUSdNLS. Vpliv fosforilacije na znotrajcelično razporejanje proteina FUS smo ovrednotili z uporabo specifičnega protitelesa proti-FUSp-526Y. Ugotovili smo, da se fosforiliran protein FUSp-526Y v primeru izražanja aktivnih kinaz c-Src in c-Fyn nahaja predvsem v celičnem jedru, medtem ko ga v primeru izražanja aktivne kinaze c-Abl najdemo v citoplazmi. Fosforilacija proteina FUS na zadnjem tirozinskem ostanku moti običajen prenos proteina FUS med jedrom in citoplazmo. S tem potencialno vpliva na kopičenje proteina FUS v citoplazmi in nastanek toksičnih agregatov.

Language:Slovenian
Keywords:fosforilacija, protein FUSp-526Y, diferenciacija, SH-SY5Y FlpIn, kinaze družine Src
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2023
PID:20.500.12556/RUL-153122 This link opens in a new window
COBISS.SI-ID:178508035 This link opens in a new window
Publication date in RUL:18.12.2023
Views:585
Downloads:46
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Secondary language

Language:English
Title:An effect of phosphorylation on intracellular localization of FUS protein in differentiated SH-SY5Y cells
Abstract:
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are both rapidly progressive neurodegenerative diseases. They are both characterized by progressing neuron death. Mutations in different genes can contribute to development of these diseases, as well as post translational modification of RNA-binding proteins. One of genes, where mutation can occur, is FUS gene, encoding FUS protein (FUsed in Sarcoma). It is an RNA/DNA-binding protein, which is primarily located in the cell nucleus and has roles in many processes like gene transcription, RNA metabolism, DNA damage response and cell stress response. Because it functions in both cell nucleus and cytoplasm, it must shuttle between them to execute normal functions. A post translational modification like phosphorylation of FUS last C-terminal tyrosine residue by Src family kinases could be involved in that. We wanted to evaluate the effect of phosphorylation of FUS at its last tyrosine (52) on intracellular distribution of normal FUS and FUS without C-terminal domain (NLS) in differentiated SH-SY5Y FlpIn cells. We used previously generated SH-SY5Y FlpIn cell lines, SH-SY5Y-mSc, SH-SY5Y-mScFUS and SH-SY5Y-mScFUSdNLS. Upon induction they all express red fluorescent proteins mScarlet (mSc), FUS protein tagged with mScarlet (mScFUS) or FUS protein with deleted C-terminal end, tagged with mSarlet (mScFUSdNLS). We confirmed successful expression of mSc, mScFUS and mScFUSdNLS in cells with fluorescent confocal microscopy and western blot analyses. First we adapted a differentiation protocol based on literature and our empircal work. We differentiated the cells of all three FlpIn cell lines to neuron-like cells by adding retinoic acid and neurotrophic factor BDNF. We confirmed differentiated phenotype of the cells and their expression of neuron-specific proteins with fluorescent confocal microscopy and western blot analyses. We transfected differentiated SH-SY5Y FlpIn cells of all three cell lines with plasmids encoding active c-Src, c-Fyn and c-Abl kinases. For comparison we also transfected undifferentiated SH-SY5Y FlpIn cells with them. Prior transfection we induced the cells with doxycycline to enable expression of mSc, mScFUS and mScFUSdNLS. We evaluated the effect of kinase mediated phosphorylation of FUS on its intracellular distribution by using specific anti-FUSp-526Y antibodies respectively. We demonstrated that in cells expressing active c-Src and c-Fyn kinase, phosphorylated FUS p-526Y is mostly found in the nucleus, whereas in cells expressing active c-Abl kinase, FUSp-526Y is mostly found in the cytoplasm. Phosphorylation of the FUS protein last tyrosine residue inhibits its import into the nucleus and disrupts normal shuttling of FUS between nucleus and cytoplasm. This way it potentially contributes to aggregation of FUS protein in the cytoplasm and formation of toxic FUS aggregates.

Keywords:phosphorylation, FUSp-526Y protein, differentiation, SH-SY5Y FlpIn, Src family kinases

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