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.
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