The review discusses the influence of various factors (e.g., post-translational modifications and chaperones) on the pathological transformation of amyloidogenic proteins involved in the onset and development of neurodegenerative diseases (Alzheimer’s and Parkinson’s diseases) and spongiform encephalopathies of various origin with special focus on the role of α-synuclein, prion protein, and, to a lesser extent, beta-amyloid peptide. The factors investigated by the authors of this review are discussed in more detail, including posttranslational modifications (glycation and S-nitrosylation), cinnamic acid derivatives and dendrimers, and chaperonins (eukaryotic, bacterial, and phage). A special section is devoted to the role of the gastrointestinal microbiota in the pathogenesis of amyloid neurodegenerative diseases, in particular, its involvement in the transformation of infectious prions and possibly other proteins capable of prion-like transmission of amyloidogenic diseases.

GroEL-like chaperonins encoded by bacterial viruses have unique features compared to other group I and II chaperonins. Here, the properties of the single-ring chaperonin of the bacteriophage OBP Pseudomonas fluorescens were further studied. To understand the role of the chaperonin domains in its function, two deletion variants of the chaperonin OBP were obtained. One represented the isolated apical domain (AD) only and the other was a chaperonin without apical domains (OBPΔAD). It was shown that removal of the apical domains slightly destabilises the chaperonin structure, but does not affect its heptameric assembly. In addition, the chaperonin without the apical domains retains its ATPase activity and exhibits the properties that are characteristic of the full-length chaperonin OBP. In the presence of ATP, the mutant chaperonin OBPΔAD protects the phage endolysin from thermal aggregation and promotes the fibrillation of two amyloidogenic proteins, α-synuclein and the prion protein. In the absence of ATP, OBPΔAD is able to bind α-synuclein monomers in the same way as the full-length chaperonin OBP, thus preventing their spontaneous fibrillation. The recombinant chaperonin apical domain, lacking ATPase activity, binds α-synuclein monomers independently of ATP presence, preventing amyloid formation. The results obtained suggest that, in mutant OBP, which lacks apical domains, substrate proteins, including non-specific ones, are capable of binding to the internal cavity formed by the other two chaperonin domains. The possible role of chaperonin domains in regulating the pathological transformation of amyloidogenic proteins is discussed.

Phage Lydia, a newly isolated siphovirus infecting Pseudomonas aeruginosa, was characterized with respect to its basic kinetic properties and subjected to comparative bioinformatic analysis with related phages. The phage exhibited a restricted host range, with lytic activity observed against 7 of 30 tested isolates. The genome of phage Lydia consists of a 61,986 bp dsDNA molecule and contains 89 predicted genes. Bioinformatic analysis suggests the presence of a DNA modification system, but no apparent genes associated with lysogeny or antibiotic resistance were identified. Taxonomic classification places Lydia within the Mesyanzhinovviridae family, Rabinowitzvirinae subfamily, and Yuavirus genus, with the closest relation to Pseudomonas virus M6. Comprehensive bioinformatic studies, including structural modelling and analysis of phage proteins, as well as comparative taxonomic, phylogenomic, and pangenomic analyses of the Mesyanzhinovviridae family, revealed relationships between proteins of Mesyanzhinovviridae phages, proteins from other phage groups, encapsulins, and a gene transfer agent (GTA) particle from Rhodobacter capsulatus. These analyses uncovered patterns of evolutionary history within the family, characterized by genetic exchange events alongside the maintenance of a common genomic architecture, leading to the emergence of new groups within the family.

Chaperonins are known to be important players in the conversion of amyloidogenic proteins into amyloid precursors in a variety of neurodegenerative diseases. However, the mechanisms of their action is still poorly understood. In this work, we used a single-ring chaperonin of the bacteriophage OBP, which functions in an ATP-dependent manner but has a simpler structure than other chaperonins. The effect of the chaperonin OBP on the conversion of human α-synuclein mutant A53T into amyloid was studied and the cytotoxicity of the formed fibrils was investigated. The phage chaperonin OBP was expressed in HEK293T cells together with the human α-synuclein mutant A53T. Both proteins showed a diffuse distribution within the cell cytoplasm as determined by fluorescence microscopy using specific antibodies. Separate and co-expression of the two proteins did not result in the formation of distinguishable protein aggregates in the cells, nor did it have any effect on cell viability. However, the co-expression of chaperonin and α-synuclein did result in the appearance of some dimeric and oligomeric forms of α-synuclein in the insoluble fraction of the cell lysate. It can therefore be concluded that chaperonin OBP stimulates the amyloid transformation of α-synuclein A53T when both proteins are co-expressed in eukaryotic cells. A comparison of the cytotoxicity of mutant α-synuclein amyloid forms obtained in vitro, both during spontaneous fibrillation and with the participation of the chaperonin OBP, showed that the maximum effect on HEK293T and SH-SY5Y cells, resulting in the death of more than 50 % of the population, was exerted by α-synuclein fibrils formed under chaperonin action in the presence of ATP. In the context of recent data on the spread of amyloid α-synuclein from the gut to the brain, the role of phage chaperonins in the pathological aggregation of amyloidogenic proteins in the human body and the potential use of the OBP chaperonin in cellular models of synucleinopathies are discussed.

The pathogenesis of the various prion diseases is based on the conformational conversion of the prion protein from its physiological cellular form to the insoluble scrapie isoform. Several chaperones, including the Hsp60 family of group I chaperonins, are known to contribute to this transformation, but data on their effects are scarce and conflicting. In this work, two GroEL-like phage chaperonins, the single-ring OBP and the double-ring EL, were found to stimulate monomeric prion protein fibrillation in an ATP-dependent manner. The resulting fibrils were characterised by thioflavin T fluorescence, electron microscopy, proteinase K digestion assay and other methods. In the presence of ATP, chaperonins were found to promote the conversion of prion protein monomers into short amyloid fibrils with their further aggregation into less toxic large clusters. Fibrils generated with the assistance of phage chaperonins differ in morphology and properties from those formed spontaneously from monomeric prion in the presence of denaturants at acidic pH.

Celiac disease and other types of gluten intolerance significantly affect the life quality of patients making them restrict the diet removing all food produced from wheat, rye, oat, and barley flour, and some other products. These disorders arise from protease resistance of poorly soluble proteins prolamins, contained in gluten. Enhanced proteolytic digestion of gliadins might be considered as a prospective approach for the treatment of celiac disease and other types of gluten intolerance. Herein, we tested a range of sulfated polymers (kappacarrageenan, dextran sulfate and different polysaccharides from brown seaweeds, and a synthetic polystyrene sulfonate) for the ability to activate gliadin digestion by human digestive proteases, pepsin and trypsin. Sulfated polysaccharide from Fucus evanescens enhanced proteolytic digestion of gliadins from wheat flour and reduced its cytotoxicity on intestinal epithelial Caco-2 cell culture. Regarding the non-toxic nature of fucoidans, the results provide a basis for polymer-based drugs or additives for the symptomatic treatment of gluten intolerance.

This review discusses a few examples of specific mechanisms mediating the contribution of the GIT microbiota to the development of amyloid neurodegenerative diseases caused by the pathologic transformation of prion protein, or alpha-synuclein. The effect of the bacterial GroE chaperonin system and phage chaperonins (single-ring OBP and double-ring EL) on prion protein transformation has been described. A number of studies have shown that chaperonins stimulate the formation of cytotoxic amyloid forms of prion protein in an ATP-dependent manner. Moreover, it was found that E. coli cell lysates have a similar effect on prion protein, and the efficiency of amyloid transformation correlates with the content of GroE in cells. Data on the influence of some metabolites synthesized by gut microorganisms on the onset of synucleinopathies, such as Parkinson’s disease, is provided. In particular, the induction of amyloid transformation of alpha-synuclein from intestinal epithelial cells with subsequent prion-like formation of its pathologic forms in nervous tissues featuring microbiota metabolites is described. Possible mechanisms of microbiota influence on the occurrence and development of amyloid neurodegenerative diseases are considered.

Chaperonins provide proper folding of proteins in vivo and in vitro and, as was thought until recently, are characteristic of prokaryotes, eukaryotes, and archaea. However, it turned out that some bacteria viruses (bacteriophages) encode their own chaperonins. This review presents results of the investigations of the first representatives of this new chaperonin group: the double-ring EL chaperonin and the single-ring OBP and AR9 chaperonins. Biochemical properties and structure of the phage chaperonins were compared within the group and with other known group I and group II chaperonins.

Chaperonins, a family of molecular chaperones, assist protein folding in all domains of life. They are classified into two groups: bacterial variants and those present in endosymbiotic organelles of eukaryotes belong to group I, while group II includes chaperonins from the cytosol of archaea and eukaryotes. Recently, chaperonins of a prospective new group were discovered in giant bacteriophages; however, structures have been determined for only two of them. Here, using cryo-EM, we resolved a structure of a new chaperonin encoded by gene 228 of phage AR9 B. subtilis. This structure has similarities and differences with members of both groups, as well as with other known phage chaperonins, which further proves their diversity.

Controversial information about the role of chaperonins in the amyloid transformation of proteins and, in particular, α-synuclein, requires a more detailed study of the observed effects due to the structure and functional state of various chaperonins. In this work, two types of phage chaperonins, the double-ring EL and the single-ring OBP, were shown to stimulate α-synuclein fibrillation in an ATP-dependent manner. Chaperonin morphology does not affect the stimulation of α-synuclein amyloid transformation. However, the ATP-dependent effect of single- and double-ring chaperonins on this process differs, which can lead to different morphology of resulting fibrils. Fibril formation seems to proceed without substrate encapsulation in the internal cavity of chaperonin, because of the structural features of phage chaperonins and their ability to function without co-chaperonins. In the absence of ATP, both chaperonins, on the contrary, completely prevent α-synuclein amyloid transformation, which provides the possibility of their use as anti-amyloid agents, in the form of incomplete molecules or mutants with suppressed ATPase activity.

The review highlights various aspects of the influence of chaperones on amyloid proteins associated with the development of neurodegenerative diseases and includes studies conducted in our laboratory. Different sections of the article are devoted to the role of chaperones in the pathological transformation of alpha-synuclein and the prion protein. Information about the interaction of the chaperonins GroE and TRiC as well as polymer-based artificial chaperones with amyloidogenic proteins is summarized. Particular attention is paid to the effect of blocking chaperones by misfolded and amyloidogenic proteins. It was noted that the accumulation of functionally inactive chaperones blocked by misfolded proteins might cause the formation of amyloid aggregates and prevent the disassembly of fibrillar structures. Moreover, the blocking of chaperones by various forms of amyloid proteins might lead to pathological changes in the vital activity of cells due to the impaired folding of newly synthesized proteins and their subsequent processing. The final section of the article discusses both the little data on the role of gut microbiota in the propagation of synucleinopathies and prion diseases and the possible involvement of the bacterial chaperone GroE in these processes.

Background: Chaperonins are a family of molecular chaperones Hsp60 (heat shock proteins 60). GroEL is a bacterial chaperonin. It ensures the correct folding of proteins, using the energy of ATP hydrolysis. Three-dimensional reconstructions of its predicted orthologs were obtained and biochemically characterized in free and nucleotide-bound states for bacteriophages EL Pseudomonas aeruginosa, OBP Pseudomonas fluorescens (Kurochkina, L. P. et al., Journal of virology, 2012; Semenyuk, P. I. et al., Biochemical Journal, 2016; Stanishneva-Konovalova, T. B. et al., Journal of Structural Biology, 2020). Physicochemical studies were carried out for the bacteriophage AR9 Bacillus Subtilis and confirmed that the protein has chaperone activity and does not require co-chaperonin to function (Semenyuk P. I. et al., International Journal of Biological Macromolecules, 2020). Methods: The recombinant chaperonin of the B. subtilis bacterial phage AR9 (gp228) was isolated and purified in a free state and vitrified in Vitrobot Mark IV. Data were collected using a Titan Krios cryo-TEM and processed in Warp, RELION and cryoSPARC software. Results: The final structures of the chaperonin were reconstructed with a C1 and C7 symmetry at the resolution of 4.5 Å and 4.0 Å respectively. Significant heterogeneity of the apical domains was addressed further using 3D classification and symmetry expansion in RELION resulting in a set of classes reflecting the conformational transition of the subunits between different states. At least four different conformational states of the subunit were clearly resolved. Conclusion: Gp228 structure show similarities between bacteriophage chaperonin and also bacterial chaperonin GroEL. It is formed by a single ring consisting of seven identical subunits, each has three domains: equatorial, intermediate, and apical. The subunits of the apo-form chaperonin Gp228 exhibit significant conformational flexibility in the apical and intermediate domains.

The roughly purified extract of E. coli proteins has been studied by cryoelectron microscopy, the class-sums containing 2D projections of two proteins (β-galactosidase and 2-oxoglutarate dehydrogenase complex catalytic domain (ODC-CD)), identified in an extract by tandem mass spectrometry, have been distinguished. The structures of these proteins have been solved at near-atomic resolution. De novo simulation of the ODC-CD structure yielded an atomic model that revealed differences in the positions of some amino acid residues of the active center, in comparison with the known crystal structures.

A prospective technology for reversible enzyme complexation accompanied with its inactivation and protection followed by reactivation after a fast thermocontrolled release has been demonstrated. A thermoresponsive polymer with upper critical solution temperature, poly(N-acryloyl glycinamide) (PNAGA), which is soluble in water at elevated temperatures but phase separates at low temperatures, has been shown to bind lysozyme, chosen as a model enzyme, at a low temperature (10 °C and lower) but not at room temperature (around 25 °C). The cooling of the mixture of PNAGA and lysozyme solutions from room temperature resulted in the capturing of the protein and the formation of stable complexes; heating it back up was accompanied by dissolving the complexes and the release of the bound lysozyme. Captured by the polymer, lysozyme was inactive, but a temperature-mediated release from the complexes was accompanied by its reactivation. Complexation also partially protected lysozyme from proteolytic degradation by proteinase K, which is useful for biotechnological applications. The obtained results are relevant for important medicinal tasks associated with drug delivery such as the delivery and controlled release of enzyme-based drugs.

Journal Article Cryo-EM Analysis and de Novo Modeling of E. coli dihydrolipoamide Succinyltransferase Catalytic Domain Structure Get access Vasily Mikirtumov, Vasily Mikirtumov Moscow Lomonosov University, moscow, Moskva, Russia Search for other works by this author on: Oxford Academic Google Scholar Evgeny Pichkur, Evgeny Pichkur Kurchatov Institute, Moscow, Moskva, Russia Search for other works by this author on: Oxford Academic Google Scholar Olga Tikhonova, Olga Tikhonova Orekhovich Research Institute of Biomedical Chemistry RAS, Moscow, Moskva, Russia Search for other works by this author on: Oxford Academic Google Scholar Lidia Kurochkina, Lidia Kurochkina Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Moscow, Moskva, Russia Search for other works by this author on: Oxford Academic Google Scholar Olga Sokolova Olga Sokolova Lomonosov Moscow State University, Faculty of Biology, Moscow, Moskva, Russia Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 26, Issue S2, 1 August 2020, Pages 1288–1291, https://doi.org/10.1017/S1431927620017596 Published: 01 August 2020

Eukaryotic double-ring chaperonin TRiC is an ATP-dependent protein-folding machine. Most of its substrates are known to form large ordered structures from multiple polypeptide chains. Since these structures are similar to fibrillar and oligomeric forms of amyloidogenic proteins, we hypothesized that TRiC may play a role in the development of neurodegenerative diseases of amyloid nature including prion diseases. Enzyme-linked immunosorbent assay showed that monomeric, oligomeric and fibrillar forms of prion protein (PrP) bind strongly to chaperonin TRiC, whereas glycation reduces the prion protein affinity for chaperonin. Nevertheless, dynamic light scattering, electron microscopy and thioflavin T fluorescence confirmed that all studied forms of PrP undergo an amyloid transformation after interaction with chaperonin, but different forms of prion protein are capable of having different effects on the functional state of TRiC. For example, prion protein monomers completely block its ability to reactivate the chaperonin's natural substrate - sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS). At the same time, PrP oligomers and fibrils only partially prevent the reactivation of GAPDS upon the action of TRiC. The monomeric forms of prion protein glycated by methylglyoxal do not inhibit, but only slow down the chaperone-dependent reactivation of GAPDS. Thus, the interaction of amyloidogenic proteins with chaperonins could cause cell malfunction.

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The effect of a range of synthetic charged polymers on alpha-synuclein aggregation and amyloid formation was tested. Sulfated aromatic polymers, poly(styrene sulfonate) and poly(anethole sulfonate), have been found to suppress the fibril formation. In this case, small soluble complexes, which do not bind with thioflavin T, have been formed in contrast to the large stick-type fibrils of free alpha-synuclein. Sulfated polysaccharide (dextran sulfate), as well as sulfated vinylic polymer (poly(vinyl sulfate)) and polycarboxylate (poly(methacrylic acid)), enhanced amyloid aggregation. Conversely, pyridinium polycation, poly(N-ethylvinylpyridinium), switched the mechanism of alpha-synuclein aggregation from amyloidogenic to amorphous, which resulted in the formation of large amorphous aggregates that do not bind with thioflavin T. The obtained results are relevant as a model of charged macromolecules influence on amyloidosis development in humans. In addition, these results may be helpful in searching for new approaches for synucleinopathies treatment with the use of natural polymers.