Title Validating a new cell model for an interactome mapping of ALS-linked optineurin mutation Q398X Title (croatian) Validacija novog staničnog modela za mapiranje interaktoma Q398X mutacije optineurina iz ALS bolesnika Author Antonija Braut Mentor Ivana Munitić (mentor) Committee member Toni Todorovski (predsjednik povjerenstva) Committee member Stribor Marković (član povjerenstva) Granter University of Rijeka Defense date and country 2023-10-26, Croatia Scientific / art field, BIOTECHNICAL SCIENCES Abstract Amyotrophic lateral sclerosis (ALS) is a progressive adult-onset neurodegenerative disease of upper and lower motor neurons that leads to a complete paralysis and death usually in 2-5 years from the disease onset. Although decades of research have been invested in understanding the ALS pathogenesis, the extremely heterogenous genetic background, with more than 20 genes being implicated, makes it difficult to pinpoint the exact mechanisms that lead to development of this disease. Optineurin protein, encoded by the OPTN gene, has been linked to ALS in a small subset of patients. It was suggested that OPTN acts solely through a loss-of-function mechanism in contrast to a toxic gain-of-function mechanism that was suggested for more common genes mutated in ALS, such as C9ORF72, SOD1 and TARDBP. We decided to apply the proximity-dependent labeling method to study the interactome of Q398X optineurin, a mutation that has been found in ALS patients. For that purpose, we generated new HEK293 Flp-In cell models that upon doxycycline treatment express constructs that carry BioID2 enzyme linked to the N-terminus of the wild-type or Q398X optineurin. Our results demonstrated that doxycycline successfully induced the expression of inserted constructs and that BioID2 enzyme was able to conjugate biotin to a wide range of proteins. Additionally, we detected interactions between both wild-type or Q398X optineurin and TBK1 demonstrating that linkage of BioID2 and Myc tag to optineurin N-terminus did not disturb its physiological interactions. Unexpectedly, we found that wild-type optineurin interacted with p-p65, but the Q398X optineurin did not. Although this was likely an indirect interaction of wild-type optineurin, it highlighted the possibility that Q398X mutation loses some relevant interactions in the NF-κB pathway, which contributes to the ALS development. On the other hand, we found no interaction between wild-type or Q398X optineurin with RIPK1, which we attributed to a small labeling radius of BioID2 that was likely insufficient for labeling interaction partners binding to the C-terminus of optineurin. This signified the importance of generating additional cell lines with BioID2 linked to optineurin C-terminus. Combined with our existing cell lines, they should enable the mapping of complete interactomes of wild-type and Q398X optineurin using mass spectrometry and hopefully contribute to the understanding of mechanisms by which Q398X optineurin causes ALS.
Abstract (croatian) Amiotrofična lateralna skleroza (ALS) je progresivna neurodegenerativna bolest odrasle dobi koja zahvaća gornje i donje motoričke neurone. Vodi do potpune paralize i smrti uglavnom u roku 2-5 godine nakon početka bolesti. Iako su već desetljeća istraživanja posvećena razumijevanju patogeneze ALS-a, izuzetna heterogenost genetske pozadine, s više od 20 gena vezanih s njezinim nastankom, stvara teškoće u razumijevanju točnih mehanizama koji vode ka razvoju ove bolesti. Protein optineurin, kojeg kodira gen OPTN, povezan je s ALS-om u maloj skupini bolesnika. Pretpostavlja se da OPTN djeluje isključivo mehanizmom gubitka funkcije (eng. loss-of-function) za razliku od djelovanja putem dobitka novih toksičnih funkcija (eng. gain-of-function) koji je bio predložen za učestalije mutirane gene u ALS-u poput C9ORF72, SOD1 i TARDBP. Odlučili smo koristiti metodu obilježavanja na temelju bliskosti (eng. proximity-dependent labeling) za istraživanje interaktoma Q398X optineurina, mutacije pronađene u pacijentima oboljelih od ALS-a. U tu smo svrhu napravili nove HEK293 Flp-In stanične modele koji po tretmanu s doksiciklinom eksprimiraju proteinske konstrukte koji sadrže optineurin divljeg soja ili Q398X povezan s BioID2 enzimom na N-kraju. Naši su rezultati pokazali da doksiciklin uspješno izaziva ekspresiju umetnutih konstrukata te da BioID2 enzim biotinilira širok raspon proteina. Nadalje, uočili smo interakcije optineurina divljeg soja i Q398X s TBK1, što je pokazalo da vezanje BioID2 enzima i Myc oznake za N-kraj optineurina nije omelo njegove fiziološke interakcije. Neočekivano, pronašli smo da optineurina divljeg soja ulazi u interakciju s p-p65, no ta interakcija nije pronađena u Q398X mutanta. Iako je posrijedi vjerojatno bila indirektna interakcija s optineurinom divljeg soja, ovo saznanje je istaknulo mogućnost da Q398X mutacija gubi neke značajne interakcije u NF-κB signalnom putu i time doprinosi razvoju ALS-a. S druge strane, interakcije s RIPK1 nisu pronađene u optineurina divljeg soja ni u Q398X mutacije, što smo pripisali malenom radijusu obilježavanja BioID2 koji je vjerojatno bio nedovoljan da obuhvati interakcije s partnerima koji se vežu za C-terminalni dio optineurina. To je istaknulo potrebu za stvaranjem dodatnih staničnih linija sa BioID2 enzimom vezanim za C-kraj optineurina. Te bi stanične linije, u kombinaciji s već postojećima, omogućile mapiranje cjelovitog interaktoma optineurina divljeg soja i Q398X mutacije pomoću masene spektrometrije te, nadajmo se, doprinijele razumijevanju mehanizama kojima Q398X optineurin uzrokuje ALS.
Keywords
amyotrophic lateral sclerosis
optineurin
proximity-dependent labeling
BioID2
inflammation
Keywords (croatian)
amiotrofična lateralna skleroza
optineurin
obilježavanje na temelju bliskosti
BioID2
upala
Language english URN:NBN urn:nbn:hr:193:365351 Study programme Title: Medicinal chemistry Type of resource Text File origin Born digital Access conditions Embargoed access Terms of use Created on 2023-10-26 12:15:17
