Modifikacije proteinov TDP-43 in FUS v stresnih pogojih

Fortuna, Žan (author), Rogelj, Boris (mentor)

Amiotrofična lateralna skleroza (ALS) je redka progresivna bolezen motoričnih nevronov z nizko incidenco in visoko umrljivostjo, pri čemer smrt v večini primerov nastopi v treh do petih letih po diagnozi. Na njen nastanek vplivajo spremembe različnih proteinov, ki vzdržujejo celično homeostazo, znane mutacije pa pojasnijo približno 20 % primerov. Med njima sta TDP-43 in FUS, RNA-vezavna proteina s ključno vlogo pri regulaciji izražanja genov, homeostazi in transportu RNA. Za ALS je značilna izguba jedrnega TDP-43 ter pojav fosforiliranih in ubikvitiniranih citoplazemskih agregatov pri približno 97 % bolnikov, medtem ko so spremembe FUS manj pogoste, vendar bolezen pogosto hitro napreduje. V raziskavah bolezensko stanje modelirajo z različnimi molekulami, ki povzročijo oksidativni, hiperosmotski ali Ca2+ -odvisen stres, saj se mehanizmi degeneracije pri patoloških tkivih pogosto razlikujejo, zato en sam pristop ne pojasni variabilnosti bolezni. V ta namen smo celice HEK293 in SH-SY5Y izpostavili D-sorbitolu, NaAsO2 , H2 O 2 in CaCl 2 v akutnih (1,5 h) in kroničnih (24 h) pogojih ter lizate analizirali z 2D elektroforezo, prenosom western in imunodetekcijo. Pri TDP-43 smo zaznali spremembe v izoelektrični točki in pojav dodatnih oblik z drugačno molekulsko maso, vključno s fosforiliranimi, ubikvitiniranimi, glikoziliranimi, fragmentiranimi in agregiranimi oblikami. D-sorbitol je pI znižal, NaAsO 2 in H2 O 2 sta ga zvišala, CaCl 2 pa izrazitih sprememb ni povzročil. Pri FUS je NaAsO 2 spremenil razporeditev oblik in povečal fosforilacijo Y526, D-sorbitol pI ni bistveno spremenil, H2 O 2 ga je povišal, vpliv CaCl 2 pa je bil najmanjši. Pri SH-SY5Y smo z enakimi metodami določili le fosforilirane, glikozilirane in fragmentirane oblike TDP-43, ki so se razlikovale v pI v primerjavi s HEK293, kar nakazuje na vpliv celičnega tipa na odziv na stres. S HEK293 smo fosforilirane oblike TDP-43 določili s protitelesi proti pS369 in pS409/410 ter z alkalno fosfatazo. Fosforilacija teh mest je bila prisotna tudi v nestresnih pogojih pri različnih oblikah proteina in se ob stresu ni spremenila. N-glikozilacijo smo preverili s PNGazo F in konkanavalinom A, vendar je zaradi dvoumnih rezultatov ne moremo potrditi. Določali smo tudi lokalizacijo inducibilno izražanih fuzijskih proteinov mScarletI-myc-TDP-43 wt in N345K v HEK293. Pri akutnem stresu je TDP-43 ostal pretežno jedrn v obliki zrnatih struktur in ni tvoril izrazitih citoplazemskih agregatov, medtem ko so se ti pri kroničnem NaAsO 2 pri manjšem deležu celic z wt obliko proteina pojavili. Rezultati kažejo, da stresor, trajanje izpostavitve in morda celo celični tip določajo oblike TDP-43 in FUS. Ugotovitve usmerjajo raziskave v diferencirane nevrone in patološka tkiva, kjer bo mogoče neposredno povezati modifikacije, lokalizacijo in odziv na stres.

• Language: Slovenian
• Keywords: ALS, TDP-43, FUS, posttranslacijske modifikacije, 2D elektroforeza
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2026
• PID: 20.500.12556/RUL-178010
• COBISS.SI-ID: 266605827
• Publication date in RUL: 16.01.2026

Secondary language

• Language: English
• Title: Modifications of TDP-43 and FUS proteins under stress conditions

Abstract

Amyotrophic lateral sclerosis (ALS) is a rare, progressive motor neuron disease with low incidence and high mortality, with death occurring in most cases within three to five years after diagnosis. Its onset is influenced by changes in various proteins maintaining cellular homeostasis, while known mutations account for approximately 20 % of cases. Among these are TDP-43 and FUS, RNA-binding proteins with key roles in the regulation of gene expression, RNA homeostasis and transport. ALS is characterized by the loss of nuclear TDP-43 and the appearance of phosphorylated and ubiquitinated cytoplasmic aggregates in approximately 97 % of patients, whereas FUS alterations occur in a smaller subset, but are often associated with rapid disease progression. Experimental studies often model the disease state with molecules inducing oxidative, hyperosmotic, or Ca 2+ stress, because degenerative mechanisms vary across pathological tissues and no single approach explains disease variability. To this end, HEK293 and SH-SY5Y cells were exposed to D-sorbitol, NaAsO2 , H2 O 2 and CaCl 2 under acute (1.5 h) and chronic (24 h) conditions and the lysates were analysed by 2D electrophoresis, western blotting and immunodetection. For TDP-43, we detected changes in the isoelectric point and the appearance of additional forms with different apparent molecular weight, including phosphorylated, ubiquitinated, glycosylated, fragmented and aggregated forms. D-sorbitol decreased pI, NaAsO 2 and H2 O 2 increased it, but CaCl 2 did not cause significant changes. For FUS, NaAsO 2 changed the distribution of forms and increased phosphorylation of Y526, D-sorbitol did not significantly change pI, H2 O 2 increased it, while the effect of CaCl 2 was minimal. In SH-SY5Y, using the same methods, we observed only phosphorylated, glycosylated and fragmented forms of TDP-43, whose pI differed compared to HEK293, indicating a cell-type effect on the stress response. In HEK293, phosphorylated forms of TDP-43 were determined with antibodies against pS369 and pS409/410 and with alkaline phosphatase. Phosphorylation at these sites was also present in non-stress conditions in different forms of the protein and changed upon stress. N-glycosylation was evaluated using PNGase F and concanavalin A, but was unable to be confirmed. We also examined the localization of inducible mScarletI-myc-TDP-43 wt and N345K in HEK293. Under acute stress, TDP-43 remained nuclear in granular structures with no cytoplasmic aggregates observed, whereas under chronic NaAsO 2 they appeared in a small fraction of wt-expressing cells. Overall, stressor, exposure duration and possibly even cell type shape TDP-43 and FUS forms and distribution, motivating future studies in differentiated neurons and patient tissue to directly link modifications, localisation and stress responses.

• Keywords: ALS, TDP-43, FUS, posttranslational modifications, 2D electrophoresis