magistrsko delo
Iza Oblak (Author), Vera Župunski (Mentor), Marko Dolinar (Thesis defence commission member), Miha Pavšič (Thesis defence commission member)

Abstract

Amiotrofična lateralna skleroza (ALS) je neozdravljiva nevrodegenerativna bolezen, za katero je značilen postopen upad motoričnih funkcij. Najpogostejši genetski vzroki za dedno in sporadično obliko ALS je povečano število ponovitev v genu C9orf72 in drugačnosmiselne mutacije v genih SOD1, TARDBP ter FUS. Identificirali so nove drugačnosmiselne mutacije v genu ANXA11 in jih povezali z ALS. Gen ANXA11 zapisuje protein aneksin A11 (ANXA11), ki spada v večjo proteinsko družino aneksinov. Njegov C-konec je visoko ohranjen in sestavljen iz štirih aneksinskih ponovitev, medtem ko je njegov N-konec dolg, hidrofoben, intrinzično neurejen in sposoben vezave več interakcijskih partnerjev, med katerimi je najbolj poznan kalciklin. Najpogostejša mutacija ANXA11, povezana z ALS, je v evropski populaciji D40G, zaradi katere ANXA11 izgubi sposobnost vezave na kalciklin. Posledično pride do kopičenja ANXA11 v citoplazmi in njegove patološke agregacije. V tem magistrskem delu smo v celicah E. coli seva BL21 [DE3] pLysS izrazili 3 variante proteina ANXA11: ANXA11wt, ki predstavlja celoten zapis proteina, ANXA11D40G, ki predstavlja mutirano obliko proteina, ter ANXA11∆119, ki predstavlja za prvih 118 aminokislin skrajšano obliko proteina. Vse tri oblike smo izolirali z afinitetno kromatografijo preko heksahistidinske oznake in s kromatografijo z ločevanjem po velikosti. Nato smo izvedli agregacijske teste z metodo fluorescenčne spektroskopije, kjer smo kot fluorescenčno barvilo uporabili tioflavin T (ThT). Ugotovili smo, da je hitrost agregacije višja pri mutirani obliki ANXA11D40G glede na ANXA11wt in da je pri mutirani obliki ANXA11D40G potreben krajši čas za nastop agregacije. Dokazali smo pomembnost N-končne domene ANXA11 za njegovo agregacijo.

Keywords

proteini;aneksin A11;agregacija proteinov;agregacijski test;amiotrofična lateralna skleroza;ALS;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [I. Oblak]
UDC: 577.112:616.8-009.5(043.2)
COBISS: 76810243 Link will open in a new window
Views: 405
Downloads: 88
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Other data

Secondary language: English
Secondary title: Aggregation studies of protein annexin A11 and its mutant variants D40G and Δ119
Secondary abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive deterioration of motor function. The most common genetic causes of familial and sporadic ALS are an intronic hexanucleotide repeat expansion in C9orf72 and nonsynonymous mutations in the SOD1, TARDBP, and FUS genes. Novel nonsynonymous mutations in the ANXA11 gene were identified and linked to ALS. The ANXA11 gene encodes annexin A11 (ANXA11) which belongs to the annexin protein family. Its C-terminus is highly conserved and consists of four annexin repeats, while its N-terminus is long, hydrophobic, intrinsically disordered, and has the ability to bind to multiple interaction partners, including calcyclin. Interaction with calcyclin is specifically inhibited by the D40G mutation, which is also the most common ANXA11 mutation in the European population. The altered binding properties lead to accumulation of cytoplasmic ANXA11, promoting its abnormal aggregation. In the E. coli strain BL21 [DE3] pLysS, we expressed three different variants of the ANXA11 protein: ANXA11wt with complete protein sequence, ANXA11D40G with mutated protein sequence and ANXA11∆119 with truncated N-terminal domain up to Ser119. We isolated the proteins by nickel affinity and size exclusion chromatography. We then performed fluorescence spectroscopy using thioflavin T (ThT) as the amyloid dye. The results indicate higher aggregation rates and shorter lag time of the mutant variant ANXA11D40G compared to ANXA11wt. We demonstrated the importance of the N-terminal domain for ANXA11 aggregation.
Secondary keywords: annexin A11;amyotrophic lateral sclerosis;protein aggregation;
Type (COBISS): Master's thesis/paper
Study programme: 1000377
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Pages: 74 str.
ID: 13303773