Amyloids are fibrillar proteins with a cross-β structure. Pathological amyloids are associated with the development of a number of incurable diseases, while functional amyloids regulate vital processes. The detection of unknown amyloids in living objects is a difficult task, and therefore the question of the prevalence and biological significance of amyloids remains open. We present a description of two methods, the combination of which makes it possible to find and identify amyloid proteins in the proteome of various organisms. The method of proteomic screening for amyloids allows the detection of the proteins that form SDS-resistant aggregates. SDS resistance is a general feature of amyloid fibrils. Protein aggregates resistant to SDS treatment can be collected by ultracentrifugation and further identified by mass spectrometry. However, in addition to amyloids, SDS-resistant aggregates contain some non-amyloid proteins. To test the amyloid properties of proteins identified by proteomic screening, we developed the method of fibril immunoprecipitation followed by Congo red staining and birefringence analysis. The methods of proteomic screening and immunoprecipitation of fibrillar proteins have been successfully tested and applied for the identification of amyloid proteins in yeast and vertebrates.

The FXR1 protein regulates the stability and translation of a number of RNA molecules and plays an important role in the regulation of cellular processes under normal conditions and stress. In particular, this protein is known to be a negative regulator of the key proinflammatory cytokine TNF alpha. We had previously shown that FXR1 functioned in the amyloid form in neurons of the brain of jawed vertebrates. Under stress conditions, FXR1 is incorporated into stress granules in some cell lines, but such studies have not been conducted for neuronal cells. Here, we showed the ability of the FXR1 protein to form cytoplasmic granules in a neuroblastoma cell line under various types of stress. This protein colocalizes with core proteins of neuronal stress granules upon heat shock and sodium arsenite treatment. We also showed that FXR1 colocalizes with anti-amyloid antibodies OC under both normal and stress conditions. Given that stress granules are dynamic structures, we propose that amyloid FXR1-containing RNP particles interact with other stress granule proteins through weak intermolecular hydrogen bonds. Using a yeast model system, we found that FXR1 colocalizes and physically interacts with stress granule proteins such as TIA-1, FMRP, FXR2, and SFPQ. Overall, our results provide new insights into the role of the RNA-binding protein FXR1 in neuronal stress response. We believe that FXR1 inactivation in neuronal stress granules can contribute to an increase in the level of the proinflammatory cytokine TNF alpha in neurodegenerative diseases.

Drosophila melanogaster is the oldest classic model object in developmental genetics. It may seem that various structures of the fruit fly at all developmental stages have been well studied and described. However, recently we have shown that some specialized structures of the D. melanogaster eggshell contain an amyloid fibril network. Here, we demonstrate that this amyloid network is formed by the chorionic protein s36. The s36 protein colocalizes with the amyloid-specific dyes Congo Red and Thioflavin S in the micropyle, dorsal appendages, and pillars. The fibrils of s36 obtained from the eggs demonstrate amyloid properties. In the context of the CG33223 gene deletion, the s36 protein is produced but is not detected in the eggshell. The absence of amyloid fibrils of s36 in the eggshell disrupts the endochorion morphology and blocks the development of the micropyle, dorsal appendages, and pillars, leading to sterility. Our data show for the first time that amyloid fibrils are essential for morphogenesis modulation. We suggest that attachment of follicle cells to the s36 extracellular fibrils triggers signaling to enable subsequent cellular divisions needed for building the specialized eggshell structures.