Annotation of the results obtained in 2019
In the scope of the Project in 2019, a comprehensive in vitro and in vivo analysis of the amyloid properties of the storage protein of pea seeds Pisum sativum L. Vicilin, as well as the RopA and RopB proteins of the nodule bacteria Rhizobium leguminosarum was carried out. Our studies showed that the storage protein of pea seeds, vicilin, as well as both of its domains Cupin-1.1 and Cupin-1.2, which share a beta-barrel structure, form in vitro fibrils visualized by transmission electron microscopy. The Cupin-1.1 and Cupin-1.2 domains form fibrils in the hexafluoroisopropanol solvent, however, for the effective fibrillogenesis of full-length Vicilin, the use of “seeds” from pre-existing fibrils of the same protein is necessary. The fibrils of Vicilin, Cupin-1.1 and Cupin-1.2 domains are resistant to cold and hot ionic detergent sodium dodecyl sulfate (SDS) and are stained with amyloid-specific dye Congo red exhibiting green-yellow birefringence in polarized light, which is considered as the "gold standard" in the amyloid diagnostics. A study of the properties of Vicilin in P. sativum L. seeds in vivo demonstrated that this protein forms detergent-resistant aggregates, the amount of which increases with seed maturation and then significantly decreases with seed germination, which is accompanied by the formation of a proteolytic fragment, the presence of which suggests the participation of protease systems in the degradation of Vicilin aggregates. It was found that the anti-Vicilin antibody signals colocalizes with the amyloid-specific dye Thioflavin-T on seed sections. Protein bodies isolated from seeds, in which vicilin is localized, bind Thioflavin-T and exhibit birefringence in polarized light when are stained with Congo red that proves the amyloid nature of the Vicilin aggregates in vivo. We have shown that Vicilin amyloids resist canning of pea seeds; they can withstand treatment with proteolytic enzymes of digestive tract under near physiological conditions and are toxic to mammalian and fungal cells. Analysis of the amyloid properties of the RopA and RopB proteins of the nodule bacteria R. leguminosarum showed that in vitro recombinant full-length RopA and RopB form fibrils that are resistant to the SDS treatment and exhibit green-yellow birefringence when stained with Congo red dye, which confirms their amyloid nature. Moreover, RopB fibrils are resistant to trypsin and pepsin treatment, while RopA fibrils are unstable in the presence these proteases. An in vivo study of the amyloid properties of RopA and RopB in R. leguminosarum cells showed that these proteins form polymers that are resistant to cold and hot SDS treatment, which are detected by Western blot hybridization with anti-RopA and RopB antibodies using as the separation methods both classical denaturing gel electrophoresis in polyacrylamide gel and semi-denaturing electrophoresis in agarose gel. It was shown that the number of RopA polymers increases when the cells are treated with luteolin flavonoid, which simulates the initial stages of the formation of plant-microbial symbiosis that suggests the functional role of these polymers in plant-microbial interactions. Using immunoelectron microscopy with antibodies against RopA and RopB conjugated to secondary antibodies labeled with colloidal gold, it was shown that these proteins are localized in the extracellular fibrils that comprise specific protein-polysaccharide structures surrounding the R. leguminosarum cells called capsules, which are important at the initial stage of bacteria interaction with the host plant. Staining of the cells of R. leguminosarum with Congo red showed that the capsules bind this dye and exhibit a green-yellow birefringence in polarized light, which confirms the presence of amyloid fibrils in them. Thus, the most important result of the Project in 2019 was the proof of the amyloid properties of the Vicilin seed storage protein of garden pea P. sativum and the RopA and RopB proteins of the root nodule bacteria R. leguminosarum in vitro and in vivo. These data for the first time show the existence of amyloids under native conditions in plants and nodule bacteria, respectively. Our results support the involvement of amyloidogenesis in the storage of proteins in plant seeds and plant-microbial interactions, and also show that beta-barrel domains are important amyloidogenic determinants in prokaryotes and eukaryotes. Publications of the Project in 2019: 1. Kosolapova A.O., Belousov M.V., Sulatskaya A.I., Belousova M.E., Sulatsky M.I., Antonets K.S., Volkov K.V., Lykholay A.N., Shtark O.Y., Vasilieva E.N., Zhukov V.A., Ivanova A.N., Zykin P.A., Kuznetsova I.M., Turoverov K.K., Tikhonovich I.A., Nizhnikov A.A. Two Novel Amyloid Proteins, RopA and RopB, from the Root Nodule Bacterium Rhizobium leguminosarum // Biomolecules, 2019, V.9, e694. 2. Malovichko Y.V., Nizhnikov A.A., Antonets K.S. Repertoire of the Bacillus thuringiensis virulence factors unrelated to major classes of protein toxins and its role in specificity of host-pathogen interactions // Toxins, 2019, V.11, e347. 3. Antonets K.S., Belousov M.V., Sulatskaya A.I., Belousova M.E., Kosolapova A.O., Sulatsky M.I., Andreeva E.A., Zykin P.A., Malovichko Y.V., Shtark O.Y., Lykholay A.N., Volkov K.V., Kuznetsova I.M., Turoverov K.K., Kochetkova E.Y., Demidov O.N., Tikhonovich I.A., Nizhnikov A.A. Accumulation of storage proteins in plant seeds is mediated by amyloid formation // BioRxiv, 2019, 825091 (preprint published 30.10.2019). 4. Nizhnikov A.A., Belousov M., Belousova M., Kosolapova A., Tikhonovich I., Antonets K. The interdependence between amyloid formation and virulence of Proteobacteria // FEBS Open Bio (Suppl.1: Abstracts of the 44th FEBS Congress (Krakow, Poland; 6-11 July 2019)), 2019, V.9, S.1, P. 274. 5. Antonets K., Belousov M., Belousova M., Shtark O., Andreeva E., Zykin P., Kosolapova A., Lykholay A., Volkov K., Malovichko Y., Tikhonovich I., Nizhnikov A.A. Proteomic screening revealed functional amyloid proteins in the seeds of garden pea Pisum sativum L. // FEBS Open Bio (Suppl.1: Abstracts of the 44th FEBS Congress (Krakow, Poland; 6-11 July 2019)), 2019, V.9, S.1, P. 275.

 

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Annotation of the results obtained in 2017
In 2017, we performed a comprehensive bioinformatic screening of amyloidogenic proteins in the proteomes of plants with sequenced and annotated genomes, as well as in bacteria of the order Rhizobiales. The screening was carried out using two different bioinformatic algorithms: Waltz, which searched for short amyloidogenic regions enriched primarily with hydrophobic amino acids, and SARP, which identified potentially amyloidogenic regions enriched with asparagine (N) and glutamine (Q). For each of the analyzed plant and bacterial proteomes, the proportion of proteins with amyloidogenic regions predicted by Waltz and SARP and the fraction of such amyloidogenic regions in the proteome were calculated. The molecular functions and biological processes, as well as the localization of potentially amyloidogenic proteins, were analyzed using the Gene Ontology database. The protein domains enriched with amyloidogenic regions were identified using the PFAM database. The proteomes of 75 species of plants were included in the bioinformatic screening of amyloidogenic proteins using the Waltz and SARP algorithms, and the total number of protein sequences analyzed reached 2.9 million. The obtained data showed that the content of amyloidogenic proteins varies significantly in different plant species even belonging to the same genus. A study of the localization and functions of potentially amyloidogenic proteins predicted by Waltz has shown that many of them are transmembrane proteins that function either as transporters or have motor or kinase activity. In contrast, QN-enriched proteins predicted by SARP are involved in the regulation of transcription and the binding of nucleic acids. Also, QN-enriched proteins are associated with the storage of nutrients (this group includes, mainly, seed storage proteins). Plant proteins with amyloidogenic regions predicted by Waltz have a predominantly membrane localization, while QN-enriched amyloidogenic proteins are localized in the nucleus and cytoplasm. The most important finding is that in the majority of plant species, the evolutionarily conserved Cupin-1 domain, which is characteristic for seed storage proteins belonging to the 7S and 11S globulins, is potentially amyloidogenic. In total, more than 300 proteins containing QN-enriched Cupin-1 domain were detected in 54 of 70 plant species in which this domain is present. It should be noted that a number of other domains of seed storage proteins of plants are also QN-rich. This result agrees with the originally proposed hypothesis on the possible role of amyloidogenesis in the storage of proteins in plant seeds and opens the perspective for further validation of this hypothesis by experimental proteomic approaches. We perfomed bioinformatic screening of amyloidogenic proteins in the proteomes of more than 80 species of bacteria of the order Rhizobiales using the Waltz and SARP algorithms. The work was carried out according to the same scheme as in the previous section. The data obtained showed that the total proportion of proteins containing the regions predicted by Waltz in the Rhizobiales proteomes varied from 11.7 to 22.3%, while the same for QN-rich proteins predicted by SARP varied from 0.2 to 1.4%. Most potentially amyloidogenic proteins predicted by Waltz are localized in the plasma membrane, while QN-rich proteins predicted by SARP are located in the outer membrane or involved in the formation of motor structures, including flagella. The amyloidogenic proteins predicted by Waltz are mainly involved in transport through the plasma membrane, and also participate in secretion. Bacterial proteins predicted by SARP are more diverse in function and involved in transport, biosynthesis, cytokinesis, secretion, as well as stress response. The analysis of bacterial protein domains enriched with amyloidogenic regions predicted by Waltz and SARP showed that flagellin (Flg) and hemolysin (HlyD) domains are potentially amyloidogenic, as well as the domains of proteins involved in the secretion of lipopolysaccharides (LptD). Most of these domains are involved in the virulence of bacteria of the order of Rhizobiales, and for some of them the role in plant-microbial interactions is shown. Overall, the data obtained suggest that amyloidogenic proteins of the root nodule bacteria may potentially participate in the regulation of virulence and molecular mechanisms of symbiosis. The bioinformatic data obtained within the framework of the Project demonstrated promising prospects for further experimental screening of amyloid proteins in plants and root nodule bacteria. For further research, the method of proteomic screening of amyloid proteins PSIA has been optimized for seeds of pea P. sativum, as well as for the free-living form and bacteroids of the root nodule bacterium R. leguminosarum. The main difficulty in proteomic studies in P. sativum was the lack of reference data, since the genome of this species has not been sequenced so far. In this regard, sequencing of the transcriptome of P. sativum seeds was carried out at two stages of the seed development (10 and 20 days) on the Illumina HiSeq4000 platform. Sequencing for each of the steps was carried out in four replicates, one of which was made with a double coverage. A total of about 190,000 transcripts were assembled. Proteins corresponding to the assembled transcripts were predicted using TransDecoder in the SwissProt curated protein database and the Pfam protein domain database. Overall, 126579 proteins and protein fragments longer than 100 amino acids were predicted, more than 78% of which were full-length and more than 85% had orthologs in the plant genomes. Taking together, in 2017 we carried out a comprehensive bioinformatic screening of amyloidogenic proteins in 75 plant species with sequenced genomes and more than 80 species of bacteria of the order of Rhizobiales using the Waltz and SARP algorithms. Also, we performed the optimization of the PSIA approach for further proteomic screening of amyloids in the seeds of P. sativum, as well as in the free-living form and bacteroids of R. leguminosarum. Publications of the Project in 2017: Публикации по результатам выполнения Проекта в 2017 году: 1. Antonets K.S., Nizhnikov A.A. Predicting amyloidogenic proteins in the proteomes of plants // International Journal of Molecular Sciences, 2017, V.18, e2155.. 2. Antonets K.S., Nizhnikov A.A. Amyloids and prions in plants: facts and perspectives // Prion, 2017, V.11, P.300-312. 3. Nizhnikov A.A., Antonets K.S. The study of amyloidogenic properties of plant proteomes // The FEBS Journal (Supplement 1: Abstracts of the 42nd FEBS Congress (Jerusalem, Israel; September 10–14, 2017)), 2017, V.284, S.1, P.211, P.3.1.B001. 4. Antonets K.S., Belousov M.V., Bondarev S.A., Kosolapova A.O., Zhouravleva G.A., Nizhnikov A.A. Identification of novel amyloids in bacterial proteomes // The FEBS Journal (Supplement 1: Abstracts of the 42nd FEBS Congress (Jerusalem, Israel; September 10–14, 2017)), 2017, V.284, S.1, P.211, P.3.1.B002.

 

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Annotation of the results obtained in 2018
In the scope of the Project in 2018, we carried out proteomic screening of proteins forming aggregates resistant to treatment with ionic detergents (one of the properties of amyloids) in the seeds of Pisum sativum L. pea of the Sprint-2 genetic line, as well as in various life forms of the root nodule bacterium Rhizobium leguminosarum bv. viciae strain RCAM1026. Fractions of detergent-resistant proteins were isolated and purified using a series of ultracentrifugation of protein lysates treated with ionic detergents. Detergent-resistant proteins were solubilized and trypsinized. The resulting peptide mixtures were separated by high performance liquid chromatography, after which proteins were identified by mass-spectrometry. As a result, we obtained data on the landscapes of potentially amyloidogenic detergent-resistant proteins of the seeds of the pea Pisum sativum and various life forms of the root nodule bacterium Rhizobium leguminosarum. The result of identification of the detergent-resistant seed proteins agreed with our initial hypothesis of this Project about the role of amyloidogenesis in seed-storage proteins since they, such as vicilins, legumins, and convicilins, were found in the detergent-resistant seed fraction with the highest mass-spectrometry. The dynamics of the accumulation of these proteins in the detergent-resistant fractions changes as the seeds mature: legumins dominate at the initial stages of maturation, then vicilins. It should be noted that pea vicilin contains two evolutionarily conservative domains CUPIN-1, which we identified in a large-scale bioinformatic analysis in 2017 as the most amyloidogenic domains in the proteomes of various species of land plants. Thus, the results of proteomic screening of amyloids in pea seeds agreed with previously performed bioinformatic predictions. Moreover, our analysis of the amyloid properties of vicilin protein in the C-DAG bacterial system showed that this protein forms fibrils upon secretion on the surface of E. coli cells. These fibrils showed birefringence upon Congo red binding, which is the "gold standard" in the diagnosis of amyloids. Thus, in 2018, we obtained important data confirming the role of amyloidogenesis in protein storage in plant seeds. According to result of proteomic screening of detergent-resistant proteins in the free-living culture of the root nodule bacterium Rhizobium leguminosarum, outer membrane porin RopA that forms a beta-barrel type structure similar to that of E.coli OmpA and OmpC porins, was found with the highest mass-spectrometric score. The outer membrane protein RopB was also detected in the detergent-resistant fraction of the free-living culture, but not in the bacteroids of R. leguminosarum. It should be noted, that the level of production of RopB, as well as RopA, increases in the early stages of nodulation, and then almost completely represses. The C-DAG system showed that RopA and RopB form fibrils upon secretion on the surface of E. coli cells, and these fibrils show green birefringence when bind Congo red dye, which confirms their amyloid properties. Virulence factors were revealed previously in our bioinformatic analysis as the most amyloidogenic proteins in bacterial proteomes of the order of Rhizobiales, and in the present work, the amyloid-like properties were shown for RopA and RopB proteins, which, apparently, play an important role in the virulence of the root nodule bacterium R. leguminosarum. Therefore, our findings confirm the idea of the role of amyloidogenesis in the virulence of symbiotic bacteria. It can be assumed that the functional role of amyloidogenesis of virulence factors is to stabilize the structure of these proteins and to protect them from destruction in the external environment. Thus, in 2018 we preformed proteomic screening of amyloid proteins in the seeds of garden pea Pisum sativum and various life forms of the root nodule bacterium Rhizobium leguminosarum. This screening confirmed our previous bioinformatic predictions about relation of amyloidogenesis with the storage of protein in the seeds of plants and virulence of root nodule bacteria, and allowed to identify a number of protein candidates for further confirmation of their amyloid properties in vivo and in vitro. Publications of the Project in 2018: 1. Antonets K.S., Kliver S.F., Nizhnikov A.A. Exploring proteins containing amyloidogenic regions in the proteomes of bacteria of the order Rhizobiales // Evolutionary Bioinformatics, 2018, V.14, P.1-12. 2. Bondarev S.A., Antonets K.S., Kajava A.V., Nizhnikov A.A., Zhouravleva G.A. Protein co-aggregation related to amyloids: methods of investigation, diversity and classification // International Journal of Molecular Sciences, 2018, V.19, e2292. 3. Kosolapova A., Belousov M., Belousova M., Antonets K., Shtark O., Vasilyeva E., Nizhnikov A.A. Identification of amyloid-forming proteins in the proteome of Rhizobium leguminosarum // FEBS Open Bio (Supplement 1: Abstracts of the 43nd FEBS Congress (Prague, Czech Republic; July 7–12, 2018)), 2018, V.8, S.1, P.402. 4. Belousova M., K. Antonets, M. Belousov, O. Shtark, A. Kosolapova, Nizhnikov A.A. Identification of amyloid-forming proteins in higher plants // FEBS Open Bio (Supplement 1: Abstracts of the 43nd FEBS Congress (Prague, Czech Republic; July 7–12, 2018)), 2018, V.8, S.1, P.409.

 

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