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Int J Mol Sci
2022 Jul 20;2314:. doi: 10.3390/ijms23147997.
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Amyloid Properties of the FXR1 Protein Are Conserved in Evolution of Vertebrates.
Velizhanina ME
,
Galkin AP
.
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Functional amyloids are fibrillary proteins with a cross-β structure that play a structural or regulatory role in pro- and eukaryotes. Previously, we have demonstrated that the RNA-binding FXR1 protein functions in an amyloid form in the rat brain. This RNA-binding protein plays an important role in the regulation of long-term memory, emotions, and cancer. Here, we evaluate the amyloid properties of FXR1 in organisms representing various classes of vertebrates. We show the colocalization of FXR1 with amyloid-specific dyes in the neurons of amphibians, reptiles, and birds. Moreover, FXR1, as with other amyloids, forms detergent-resistant insoluble aggregates in all studied animals. The FXR1 protein isolated by immunoprecipitation from the brains of different vertebrate species forms fibrils, which show yellow-green birefringence after staining with Congo red. Our data indicate that in the evolution of vertebrates, FXR1 acquired amyloid properties at least 365 million years ago. Based on the obtained data, we discuss the possible role of FXR1 amyloid fibrils in the regulation of vital processes in the brain of vertebrates.
Figure 1. FXR1 forms SDS-resistant amyloid-like aggregates in the brains of Xenopus laevis, Trachemys scripta, and Gallus gallus domesticus. (A) FXR1 is predominantly found as insoluble aggregates in all vertebrate species studied. Total protein lysate from the brain was treated with 1% SDS and separated into three fractions: of monomers less than 100kDa (M), oligomers larger than 100kDa (O), and insoluble aggregates (A). The fractions were subjected to SDS-PAGE and analyzed by immunoblotting with anti-FXR1 antibodies. (B) Relative intensity of bands corresponding to FXR1 monomers, oligomers, and insoluble aggregates is represented as meanSEM (standard error of the mean) for three independent brain samples for each organism. The green, yellow, and orange columns of the diagram represent the values for monomers, oligomers, and insoluble aggregates, respectively.
Figure 2. Colocalization of FXR1 (red signal) with amyloid-specific dye Thioflavin S (green signal) in neurons of frog (A), turtle (B), and chicken (C) brain sections. Blue signal corresponds to the nuclear dye DAPI; scale bar is 50 m.
Figure 3. Colocalization of FXR1 (green signal) with amyloid-specific dye CR (red signal) in neurons of frog (A), turtle (B), and chicken (C). Blue signal corresponds to the nuclear dye DAPI; scale bar is 50 m.
Figure 4. Analysis of the fibrillar structure and CR staining of the FXR1 protein isolated from the brain of Xenopus laevis (A), Trachemys scripta (B), and Gallus gallus domesticus (C). FXR1 after immunoprecipitation was detected using TEM. CR staining was analyzed in transmitted (BF) and polarized (POL) light.
Figure 5. Anti-amyloid OC antibodies (red signal) recognize cytoplasmic structures in brain neurons of all studied vertebrate species. Blue signal corresponds to the nuclear dye DAPI; scale bar is 50 um.
Figure 6. (A) Schematic representation of the FXR1 protein structure of Rattus norvegicus. The potentially amyloidogenic regions are indicated by yellow boxes. TD—Tudor domains responsible for the recognition of trimethylated peptides; KH1, KH2, and RGG—RNA-binding domains. (B) Comparative analysis of sequences of N-terminal amyloidogenic region of the FXR1 protein in Rattus norvegicus, Gallus gallus domesticus, Trachemys scripta, Xenopus laevis, and Danio rerio. The sequence of Rattus norvegicus was chosen as a reference. Red color indicates amino acid residues that differ from the reference sequence. The yellow frame indicates potentially amyloidogenic sequences identified by the algorithm ArchCandy.
