Identification of New FG-Repeat Nucleoporins with Amyloid Properties

Abstract

Amyloids are fibrillar protein aggregates with a cross-β structure. More than two hundred different proteins with amyloid or amyloid-like properties are already known. Functional amyloids with conservative amyloidogenic regions were found in different organisms. Protein aggregation appears to be beneficial for the organism in these cases. Therefore, this property might be conservative for orthologous proteins. The amyloid aggregates of the CPEB protein were suggested to play an important role in the long-term memory formation in Aplysia californica, Drosophila melanogaster, and Mus musculus. Moreover, the FXR1 protein demonstrates amyloid properties among the Vertebrates. A few nucleoporins (e.g., yeast Nup49, Nup100, Nup116, and human Nup153 and Nup58), are supposed or proved to form amyloid fibrils. In this study, we performed wide-scale bioinformatic analysis of nucleoporins with FG-repeats (phenylalanine-glycine repeats). We demonstrated that most of the barrier nucleoporins possess potential amyloidogenic properties. Furthermore, the aggregation-prone properties of several Nsp1 and Nup100 orthologs in bacteria and yeast cells were analyzed. Only two new nucleoporins, Drosophila melanogaster Nup98 and Schizosaccharomyces pombe Nup98, aggregated in different experiments. At the same time, Taeniopygia guttata Nup58 only formed amyloids in bacterial cells. These results rather contradict the hypothesis about the functional aggregation of nucleoporins.

Keywords: ArchCandy; C-DAG; FG-repeats; amyloids; evolution; nucleoporins.

Figure A1

Nucleoporins lack conservative regions with amyloidogenic features. Gaps—frequency of gaps in the position of alignment. AAC (Amino Acids Conservatism)—rate of most frequent amino acids. CAP (Conservatism of Amyloidogenic Properties)—frequency of cases when corresponding position is located inside amyloidogenic region.

Figure 1

Many Nups are potential amyloids: (A) Schematic representation of the NPC. (B–D) The distribution of potential amyloids among FG-Nups. The NCBI taxonomy is used to represent trees. The proportion of potential amyloids is shown by the color gradient, where the maximum corresponds to orange and the minimum to dark blue. Each phylum is colored according to the proportion of amyloidogenic proteins in it. Kingdoms are mentioned with icons. A number of analyzed sequences is presented next to the phylum. Trees are grouped according to Nups’ localization in the nuclear pore complex: (B)—Nups of the cytoplasmic ring; (C)—Nups of the inner ring; (D)—Nups of the nuclear ring.

Figure 2

Several nucleoporins possess regions with conservative amyloidogenic properties: Gaps—frequency of gaps in the position of alignment. AAC (Amino Acids Conservatism)—rate of most frequent amino acids. CAP (Conservatism of Amyloidogenic Properties)—frequency of cases when corresponding position is located inside amyloidogenic region. Regions with known structures are highlighted by red frames. Light cyan corresponds to potential conservative amyloidogenic regions.

Figure 3

The Nup58 protein from T. guttata possess amyloid properties: (A) Amyloidogenic regions in nucleoporins. The ArchCandy Cumulative Score presented in plots reflects the ability of the protein to form $\mathsf{\beta }$-arches. (B) Colonies’ color of bacteria, overproducing the amyloidogenic regions of nucleoporins, on the medium containing Congo Red (CR-inducing plate). The control plate, containing only antibiotics, was used to check the amount of plated cells. The construction names are listed in Table 1. (C). Microphotographs of bacterial cells from CR-inducing plate in transmitted (BF) and polarized light (Pol). The scale bar equals 20 μm. (D) TEM microphotographs of the cells from plates were presented on panel (B). Plasmids coding Sup35NM and Sup35M were used as positive and negative controls in C-DAG experiments, respectively. The scale bar equals 1 μm. The conclusion about amyloid properties of a protein based on different experiments is presented below panel (D).

Figure 4

Several Nup100 and Nup145 orthologs from different organisms demonstrate amyloidogenic properties in the C-DAG system: (A) Amyloidogenic regions in nucleoporins. The ArchCandy Cumulative Score presented on plots reflects the ability of the protein to form $\mathsf{\beta }$-arches. (B) Colonies’ color of bacteria, overproducing the amyloidogenic regions of nucleoporins, on the medium containing Congo Red (CR inducing plate). The control plate, containing only antibiotics, was used to check the amount of plated cells. The construction names are listed in Table 1. (C) Microphotographs of bacterial cells from the CR-inducing plate in transmitted (BF) and polarized light (Pol). The scale bar equals 20 μm. (D). TEM microphotographs of the cells from plates were presented on panel (B). The same controls as in Figure 3 (Sup35NM and Sup35M) were presented. The scale bar equals 1 μm. The conclusion about amyloid properties of a protein based on different experiments is presented below panel (D).

Figure 5

Only few Nups aggregate in yeast cells: The microphotographs of orthologs Nup100 (A) and Nsp1 (B) protein fused with GFP overproducing nucleoporins’ fragments fused with GFP. GFP alone was taken as negative control. The scale bar equals 25 μm.