Vpliv SigmaR1 na agregacijo TDP-43 pri nevrodegenerativnih boleznih

Maklin, Ana (author), Župunski, Vera (mentor)

Nevrodegenerativne bolezni so v današnjem času vedno večji problem zdravstva in družbe. Simptomi nevrodegenerativnih bolezni so posledica progresivne izgube nevronov centralnega ali perifernega živčnega sistema. Ena glavnih značilnosti nevrodegeneracije je agregacija endogenih proteinov v netopna inkluzijska telesca. Primer takšnega proteina je TAR (angl. transactivation response) DNA-vezavni protein 43 (TDP-43). V normalnih celicah se TDP-43 nahaja predvsem v jedru in ima pomembno vlogo pri regulaciji genov, procesiranju in prerazporejanju RNA. V patoloških pogojih pride do cepitve, hiperfosforilacije in ubikvitinacije TDP-43, kar vodi v akumulacijo in agregacijo TDP-43 v citoplazmi. Še eden izmed kritičnih proteinov pri nevrodegeneraciji je šaperon endoplazemskega retikuluma (ER) sigma receptor 1 (Sig-1R). Sig-1R se nahaja na s holesterolom bogatih predelih membrane ER, lipidnih raftih. Disregulacijo TDP-43 se lahko neposredno poveže z agregacijo Sig-1R. Šaperon Sig-1R se v preživelih motoričnih nevronih pacientov s sporadično amiotrofično lateralno sklerozo akumulira in bi lahko imel nevroprotektivno vlogo. V magistrskem delu smo se osredotočili na in vitro agregacijo TDP-43 in vpliv proteina Sig-1R na potek agregacije TDP-43. V celicah E. coli BL21[DE3] smo izrazili proteina TDP-43-MBP in Sig-1R brez ΔN-končnega transmembranskega dela. S pomočjo afinitetne kromatografije in kromatografije z ločevanjem po velikosti smo izolirali zadostno količino obeh proteinov v topni obliki. V nadaljevanju smo izvedli agregacijske in interakcijske teste. Zanimala nas je agregacija TDP-43 in vpliv Sig-1R na agregacijo TDP-43. Kot inhibitor agregacije TDP-43 smo uporabili tRNA, za katere je znano, da prispevajo k topnosti TDP-43, za ΔNSig-1R pa smo hipotezo o inhibiciji agregacije testirali. Agregacijske teste smo izvedli z merjenjem motnosti raztopine pri valovni dolžini absorbance 395 nm. Dodatek tRNA je zmanjšal oziroma preprečil agregacijo TDP-43. Za ΔNSig-1R nismo uspeli dokazati, da preprečuje agregacijo TDP-43. V nadaljevanju smo izvedli interakcijske teste z metodo interferometrije z biološkimi plastmi (BLI). Tako med TDP-43 in tRNA kot tudi ΔNSig-1R je prišlo do šibke interakcije, vendar bi morali zaradi tehničnih napak v delu eksperiment ponoviti.

• Language: Slovenian
• Keywords: nevrodegeneracije, agregacija, protein, TDP-43
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-141274
• COBISS.SI-ID: 132055043
• Publication date in RUL: 27.09.2022

Secondary language

• Language: English
• Title: The impact of SigmaR1 on TDP-43 aggregation in neurodegeneration

Abstract

Neurodegenerative diseases affect millions of people worldwide. The symptoms of neurodegeneration are caused by the progressive loss of neurons in the central or peripheral nervous system. Protein aggregation into insoluble inclusion bodies is one of the main hallmarks of neurodegeneration. TAR (Trans Activation Response) DNA-binding protein 43 (TDP-43) is one of the critical proteins in neurodegeneration. In normal cells, TDP-43 is mainly present in the nucleus and plays an important role in RNA regulation. Under pathological conditions, cleavage, hyperphosphorylation and ubiquitination of TDP-43 can occur. These posttranslational modifications lead to cytoplasmic accumulation and aggregation of TDP-43. Another critical protein in neurodegeneration is the chaperone at the endoplasmic reticulum (ER) Sigma receptor 1 (Sig-1R). Dysregulation of TDP-43 can be directly caused by Sig-1R aggregation. ER chaperone Sig-1R accumulates in motor neurons of ALS patients and may have neuroprotective functions. In our work we focused on the in vitro aggregation of TDP-43 and the influence of the Sig-1R protein on the course of TDP-43 aggregation. We expressed TDP-43_MBP and Sig-1R protein lacking N-terminal transmembrane region (ΔNSig-1R) in E.coli BL21[DE3] cells. In the process of protein purification with affinity chromatography and size-exclusion chromatography we isolated enough soluble TDP-43-MBP and ΔNSig-1R for further aggregation and interaction experiments. To analyze TDP-43 aggregation, we utilized spectrofotometric turbidity measurements (optical density at 395 nm). Aggregation was followed in the presence of a previously known aggregation inhibitor tRNA and a hypothetical inhibitor ΔNSig-1R. Addition of tRNA decreased or prevented aggregation of TDP-43. Addition of ΔNSig-1R did not prevent TDP-43 aggregation. We utilized bio-layer interferometry (BLI) for interaction analysis of TDP-43 with tRNA and ΔNSig-1R. There was a weak interaction between TDP-43 and tRNA as well as ΔNSig-1R, but due to technical errors during our work, the experiment should be repeated.

• Keywords: neurodegeneration, aggregation, protein, TDP-43