Title Modelling the schizophrenia-related aggregation of NPAS3 and CRMP1 in neuroblastoma cells Title (croatian) Modeliranje agregacije proteina NPAS3 i CRMP1 povezanih sa šizofrenijom u stanicama neuroblastoma Author Bobana Samardžija Mentor Nicholas Bradshaw (mentor) Committee member Jelena Ban (predsjednik povjerenstva) Committee member Željko Svedružić (član povjerenstva) Granter University of Rijeka Defense date and country 2020-07-31, Croatia Scientific / art field, INTERDISCIPLINARY AREAS OF KNOWLEDGE Abstract Chronic mental illness affects millions of people every year, while its origins and mechanisms remain unclear. Since symptoms of neurodegenerative and mental disorders are similar, our research focuses on protein aggregation. Protein aggregation changes the structure of protein and can cause loss of function or gain of a novel toxic function. There are several proteins characterized as aggregating in mental illnesses, of which we focused on Collapsin Response Mediator Protein 1 (CRMP1) and Neuronal PAS Domain Protein 3 (NPAS3). We expressed variants of mentioned proteins in neuroblastoma cell line and assessed them by immunofluorescence.
CRMP1 has been detected as insoluble in brains of mental disorder patients and seen to aggregate in cell culture. In our research we wanted to confirm aggregation of the long and short CRMP1 variants. The long variant is seen to aggregate alone, while short variant is only known to co-aggregate with other proteins. Since the long variant od CRMP1 has a longer N-terminal section unlike the short variant, we investigated its truncation too. However, we did not see aggregation of long and short CRMP1 variants when expressed alone, nor the effect of N-terminal truncations. Thus, we believe CRMP1 has greater tendency to co-aggregate with other proteins and the following research should explore it in more detail.
Meanwhile, genetic studies have implicated a mutated version of NPAS3 in mental disorders. Also, mutant NPAS3 has been seen as insoluble in cell culture. We examined aggregation of full-length wild type and mutated NPAS3 variants, along with the major NPAS3 structural regions. We observed that mutated NPAS3 does not enter the nucleus and therefore cannot perform its normal function as transcription factor. Interestingly, we have seen that the bHLH1 region of NPAS3 can aggregate by itself and is stabilised by presence of the PAS region. Our results confirmed that NPAS3 aggregation is a consequence of mutation, but there could be other mechanisms which include disruption of bHLH1 region.
Next steps should include further investigation of bHLH1 and PAS regions of NPAS3, with regards to mutation and translocation. Finally, this and similar research could result in model of aggregation for both proteins, which could be translated to other proteins implicated in mental illness.
Abstract (croatian) Milijuni ljudi obolijevaju od kronične mentalne bolesti godišnje, dok su uzorci i mehanizmi još uvijek nepoznati. Simptomi neurodegenerativnih i mentalnih poremećaja su slični, zbog čega se naše istraživanje temelji na agregaciji proteina. Agregacija proteina mjenja strukturu proteina te može dovesti do gubitka funkcije ili stjecanja nove toksične funkcije proteina. Postoji nekoliko proteina čija je agregacija opisana u mentalnim poremećajima, a u ovom radu istražili smo protein posrednik odgovora kolapsina 1 (eng. Collapsin Response Mediator Protein 1 ili CRMP1) i neuronski protein s PAS domenom 3 (eng. Neuronal PAS Domain Protein 3 ili NPAS3). Eksprimirali smo verzije spomenutih proteina u staničnoj liniji neuroblastoma i analizirali ih putem immunofluorescencije.
U mozgovima pacijenata s mentalnim poremećajima, CRMP1 je detektiran u netopljivoj frakciji te je utvrđeno da agregira u staničnoj kulturi. U našem istraživanju željeli smo potvrditi agregaciju duge i kratke verzije CRMP1. Do sada je viđeno kako duga verzija može sama agregirati, dok kratka verzija agregira u prisutnosti drugih proteina. Duga verzija se razlikuje od kratke verzije po svom N-terminalnom dijelu, zbog čega smo dodatno istražili i skraćene verzije. Međutim, nismo vidjeli agregaciju duge ni kratke verzije kao ni utjecaj N-terminalog dijela. Shodno tome, vjerujemo kako CRMP1 ima veću sklonost prema ko-agregaciji s drugim proteinima, što je potrebno detaljnije istražiti u budućnosti.
Međutim, genetska istraživanja su potvrdila prisutnost mutirane verzije proteina NPAS3 u mentalnim poremećajima te je dokazana njegova netopljivost u staničnoj kulturi. Istražili smo agregaciju divlje i mutirane verzije NPAS3 te agregaciju glavnih regija proteina NPAS3. Primijetili smo kako mutirana verzija proteina NPAS3 ne ulazi u jezgru, zbog čega ne može djelovati kao transkripcijski faktor. Zanimljivo je kako smo vidjeli da bHLH1 regija može samostalno agregirati te je stabilizirana prisustvom PAS regije. Naši rezultati su potvrdili kako je agregacija NPAS3 posljedica mutacije, no moguće je da postoje i drugi mehanizmi agregacije koji uključuju ometanje bHLH1 regije. Slijedeći koraci bi trebali uključivati daljnje istraživanje bHLH1 i PAS regija, uzimajući u obzir mutaciju i translokaciju povezanu s proteinom NPAS3. U konačnici, slična istraživanja mogu dovesti do modela agregacije, koji bi se mogao primijeniti i na druge proteine implicirane u mentalnim poremećajima.
Keywords
protein aggregation
NPAS3
CRMP1
mental illness
mental disorders
Keywords (croatian)
agregacija proteina
NPAS3
CRMP1
mentalne bolesti
mentalni poremećaji
Language english URN:NBN urn:nbn:hr:193:398147 Project Number: IP-2018-01-9424 Study programme Title: Drug research and development Type of resource Text File origin Born digital Access conditions Open access Terms of use Created on 2020-07-30 11:33:19
