Identification of Amyloidogenic Regions in Pseudomonas aeruginosa Ribosomal S1 Protein.

Bacterial S1 protein is a functionally important ribosomal protein. It is a part of the 30S ribosomal subunit and is also able to interact with mRNA and tmRNA. An important feature of the S1 protein family is a strong tendency towards aggregation. To study the amyloidogenic properties of S1, we isolated and purified the recombinant ribosomal S1 protein of Pseudomonas aeruginosa. Using the FoldAmyloid, Waltz, Pasta 2.0, and AGGRESCAN programs, amyloidogenic regions of the protein were predicted, which play a key role in its aggregation. The method of limited proteolysis in combination with high performance liquid chromatography and mass spectrometric analysis of the products, made it possible to identify regions of the S1 protein from P. aeruginosa that are protected from the action of proteinase K, trypsin, and chymotrypsin. Sequences of theoretically predicted and experimentally identified amyloidogenic regions were used to synthesize four peptides, three of which demonstrated the ability to form amyloid-like fibrils, as shown by electron microscopy and fluorescence spectroscopy. The identified amyloidogenic sites can further serve as a basis for the development of new antibacterial peptides against the pathogenic microorganism P. aeruginosa.

Amyloidogenic Propensities of Ribosomal S1 Proteins: Bioinformatics Screening and Experimental Checking.

Structural S1 domains belong to the superfamily of oligosaccharide/oligonucleotide-binding fold domains, which are highly conserved from prokaryotes to higher eukaryotes and able to function in RNA binding. An important feature of this family is the presence of several copies of the structural domain, the number of which is determined in a strictly limited range from one to six. Despite the strong tendency for the aggregation of several amyloidogenic regions in the family of the ribosomal S1 proteins, their fibril formation process is still poorly understood. Here, we combined computational and experimental approaches for studying some features of the amyloidogenic regions in this protein family. The FoldAmyloid, Waltz, PASTA 2.0 and Aggrescan programs were used to assess the amyloidogenic propensities in the ribosomal S1 proteins and to identify such regions in various structural domains. The thioflavin T fluorescence assay and electron microscopy were used to check the chosen amyloidogenic peptides' ability to form fibrils. The bioinformatics tools were used to study the amyloidogenic propensities in 1331 ribosomal S1 proteins. We found that amyloidogenicity decreases with increasing sizes of proteins. Inside one domain, the amyloidogenicity is higher in the terminal parts. We selected and synthesized 11 amyloidogenic peptides from the Escherichia coli and Thermus thermophilus ribosomal S1 proteins and checked their ability to form amyloids using the thioflavin T fluorescence assay and electron microscopy. All 11 amyloidogenic peptides form amyloid-like fibrils. The described specific amyloidogenic regions are actually responsible for the fibrillogenesis process and may be potential targets for modulating the amyloid properties of bacterial ribosomal S1 proteins.

Should the Treatment of Amyloidosis Be Personified? Molecular Mechanism of Amyloid Formation by Aß Peptide and Its Fragments.

Aß40 and Aß42 peptides are believed to be associated with Alzheimer's disease. Aggregates (plaques) of Aß fibrils are found in the brains of humans affected with this disease. The mechanism of formation of Aß fibrils has not been studied completely, which hinders the development of a correct strategy for therapeutic prevention of this neurodegenerative disorder. It has been found that the most toxic samples upon generation of fibrils are different oligomeric formations. Based on different research methods used for studying amyloidogenesis of Aß40 and Aß42 peptides and its amyloidogenic fragments, we have proposed a new mechanism of formation of amyloid fibrils. In accord with this mechanism, the main building unit for fibril generation is a ring-like oligomer. Association of ring-like oligomers results in the formation of fibrils of different morphologies. Our model implies that to prevent development of Alzheimer's disease a therapeutic intervention is required at the earliest stages of amyloidogenesis-at the stage of formation of ring-like oligomers. Therefore, the possibility of a personified approach for prevention not only of Alzheimer's disease development but also of other neurodegenerative diseases associated with the formation of fibrils is argued.

Rosetta Stone for Amyloid Fibrils: The Key Role of Ring-Like Oligomers in Amyloidogenesis.

Deeper understanding of processes of protein misfolding, aggregation, formation of oligomers, protofibrils, and fibrils is crucial for the development of future medicine in treatment of amyloid-related diseases. While numerous reports illuminate the field, the above processes are extremely complex, as they depend on many varying parameters, such as the peptide concentration, temperature, pH, presence of metal ions, lipids, and organic solvents. Different mechanisms of amyloid fibril formation have been proposed, but the process of the oligomer-to-fibril transition is the least agreed upon. Our studies of a number of amyloidogenic proteins and peptides (insulin, Aß peptides, the Bgl2 protein from the yeast cell wall), as well as their amyloidogenic fragments, have allowed us to propose a model of the fibril structure generation. We have found that the main building block of fibrils of any morphology is a ring-like oligomer. The varying models of interaction of ring oligomers with each other revealed in our studies make it possible to explain their polymorphism. Crucially, the amino acid sequence determines the oligomer structure for the given protein/peptide.

Smooth muscle titin forms in vitro amyloid aggregates.

Amyloids are insoluble fibrous protein aggregates, and their accumulation is associated with amyloidosis and many neurodegenerative diseases, including Alzheimer's disease. In the present study, we report that smooth muscle titin (SMT; 500 kDa) from chicken gizzard forms amyloid aggregates in vitro This conclusion is supported by EM data, fluorescence analysis using thioflavin T (ThT), Congo red (CR) spectroscopy and X-ray diffraction. Our dynamic light scattering (DLS) data show that titin forms in vitro amyloid aggregates with a hydrodynamic radius (Rh) of approximately 700-4500 nm. The initial titin aggregates with Rh approximately 700 nm were observed beyond first 20 min its aggregation that shows a high rate of amyloid formation by this protein. We also showed using confocal microscopy the cytotoxic effect of SMT amyloid aggregates on smooth muscle cells from bovine aorta. This effect involves the disorganization of the actin cytoskeleton and result is cell damage. Cumulatively, our results indicate that titin may be involved in generation of amyloidosis in smooth muscles.