Role of the Gut Microbiome and Bacterial Amyloids in the Development of Synucleinopathies.

Less than ten years ago, evidence began to accumulate about association between the changes in the composition of gut microbiota and development of human synucleinopathies, in particular sporadic form of Parkinson's disease. We collected data from more than one hundred and thirty experimental studies that reported similar results and summarized the frequencies of detection of different groups of bacteria in these studies. It is important to note that it is extremely rare that a unidirectional change in the population of one or another group of microorganisms (only an elevation or only a reduction) was detected in the patients with Parkinson's disease. However, we were able to identify several groups of bacteria that were overrepresented in the patients with Parkinson's disease in the analyzed studies. There are various hypotheses about the molecular mechanisms that explain such relationships. Usually, α-synuclein aggregation is associated with the development of inflammatory processes that occur in response to the changes in the microbiome. However, experimental evidence is accumulating on the influence of bacterial proteins, including amyloids (curli), as well as various metabolites, on the α-synuclein aggregation. In the review, we provided up-to-date information about such examples.

AmyloComp: A Bioinformatic Tool for Prediction of Amyloid Co-aggregation.

Typically, amyloid fibrils consist of multiple copies of the same protein. In these fibrils, each polypeptide chain adopts the same ß-arc-containing conformation and these chains are stacked in a parallel and in-register manner. In the last few years, however, a considerable body of data has been accumulated about co-aggregation of different amyloid-forming proteins. Among known examples of the co-aggregation are heteroaggregates of different yeast prions and human proteins Rip1 and Rip3. Since the co-aggregation is linked to such important phenomena as infectivity of amyloids and molecular mechanisms of functional amyloids, we analyzed its structural aspects in more details. An axial stacking of different proteins within the same amyloid fibril is one of the most common type of co-aggregation. By using an approach based on structural similarity of the growing tips of amyloids, we developed a computational method to predict amyloidogenic ß-arch structures that are able to interact with each other by the axial stacking. Furthermore, we compiled a dataset consisting of 26 experimentally known pairs of proteins capable or incapable to co-aggregate. We utilized this dataset to test and refine our algorithm. The developed method opens a way for a number of applications, including the identification of microbial proteins capable triggering amyloidosis in humans. AmyloComp is available on the website: https://bioinfo.crbm.cnrs.fr/index.php?route=tools&tool=30.

How Big Is the Yeast Prion Universe?

The number of yeast prions and prion-like proteins described since 1994 has grown from two to nearly twenty. If in the early years most scientists working with the classic mammalian prion, PrPSc, were skeptical about the possibility of using the term prion to refer to yeast cytoplasmic elements with unusual properties, it is now clear that prion-like phenomena are widespread and that yeast can serve as a convenient model for studying them. Here we give a brief overview of the yeast prions discovered so far and focus our attention to the various approaches used to identify them. The prospects for the discovery of new yeast prions are also discussed.

Identification of New FG-Repeat Nucleoporins with Amyloid Properties.

Amyloids are fibrillar protein aggregates with a cross-ß 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.

The [PSI+] prion modulates cytochrome c oxidase deficiency caused by deletion of COX12.

Cytochrome c oxidase (CcO) is a pivotal enzyme of the mitochondrial respiratory chain, which sustains bioenergetics of eukaryotic cells. Cox12, a peripheral subunit of CcO oxidase, is required for full activity of the enzyme, but its exact function is unknown. Here experimental evolution of a Saccharomyces cerevisiae Δcox12 strain for ∼300 generations allowed to restore the activity of CcO oxidase. In one population, the enhanced bioenergetics was caused by a A375V mutation in the cytosolic AAA+ disaggregase Hsp104. Deletion or overexpression of HSP104 also increased respiration of the Δcox12 ancestor strain. This beneficial effect of Hsp104 was related to the loss of the [PSI+] prion, which forms cytosolic amyloid aggregates of the Sup35 protein. Overall, our data demonstrate that cytosolic aggregation of a prion impairs the mitochondrial metabolism of cells defective for Cox12. These findings identify a new functional connection between cytosolic proteostasis and biogenesis of the mitochondrial respiratory chain.

NOS1AP Interacts with α-Synuclein and Aggregates in Yeast and Mammalian Cells.

The NOS1AP gene encodes a cytosolic protein that binds to the signaling cascade component neuronal nitric oxide synthase (nNOS). It is associated with many different disorders, such as schizophrenia, post-traumatic stress disorder, autism, cardiovascular disorders, and breast cancer. The NOS1AP (also known as CAPON) protein mediates signaling within a complex which includes the NMDA receptor, PSD-95, and nNOS. This adapter protein is involved in neuronal nitric oxide (NO) synthesis regulation via its association with nNOS (NOS1). Our bioinformatics analysis revealed NOS1AP as an aggregation-prone protein, interacting with α-synuclein. Further investigation showed that NOS1AP forms detergent-resistant non-amyloid aggregates when overproduced. Overexpression of NOS1AP was found in rat models for nervous system injury as well as in schizophrenia patients. Thus, we can assume for the first time that the molecular mechanisms underlying these disorders include misfolding and aggregation of NOS1AP. We show that NOS1AP interacts with α-synuclein, allowing us to suggest that this protein may be implicated in the development of synucleinopathies and that its aggregation may explain the relationship between Parkinson's disease and schizophrenia.

Structure and Polymorphism of Amyloid and Amyloid-Like Aggregates.

Amyloids are protein aggregates with the cross-ß structure. The interest in amyloids is explained, on the one hand, by their role in the development of socially significant human neurodegenerative diseases, and on the other hand, by the discovery of functional amyloids, whose formation is an integral part of cellular processes. To date, more than a hundred proteins with the amyloid or amyloid-like properties have been identified. Studying the structure of amyloid aggregates has revealed a wide variety of protein conformations. In the review, we discuss the diversity of protein folds in the amyloid-like aggregates and the characteristic features of amyloid aggregates that determine their unusual properties, including stability and interaction with amyloid-specific dyes. The review also describes the diversity of amyloid aggregates and its significance for living organisms.

Amyloid fibril length distribution from dynamic light scattering data.

The study of the aggregation of amyloid proteins is challenging. A new approach to processing dynamic light scattering data was developed and tested using aggregates of the well-known model Sup35NM amyloid. After filtering and calculating the moving averages of autocorrelation functions to reduce impacts of noise, each averaged autocorrelation function is converted to the fibril length distribution via numerical modeling. The processing results were verified using atomic force and scanning electron microscopy data. Analysis of fibril length distribution changes over time gives valuable information about the aggregation process.

The Human NUP58 Nucleoporin Can Form Amyloids In Vitro and In Vivo.

Amyloids are fibrillar protein aggregates with a cross-ß structure and unusual features, including high resistance to detergent or protease treatment. More than two hundred different proteins with amyloid or amyloid-like properties are already known. Several examples of nucleoporins (e.g., yeast Nup49, Nup100, Nup116, and human NUP153) are supposed to form amyloid fibrils. In this study, we demonstrated an ability of the human NUP58 nucleoporin to form amyloid aggregates in vivo and in vitro. Moreover, we found two forms of NUP58 aggregates: oligomers and polymers stabilized by disulfide bonds. Bioinformatic analysis revealed that all known orthologs of this protein are potential amyloids which possess several regions with conserved ability to aggregation. The biological role of nucleoporin amyloid formation is debatable. We suggest that it is a rather abnormal process, which is characteristic for many proteins implicated in phase separation.

Direct proof of the amyloid nature of yeast prions [PSI+] and [PIN+] by the method of immunoprecipitation of native fibrils.

Prions are proteins that can exist in several structurally and functionally distinct states, one or more of which is transmissible. Yeast proteins Sup35 and Rnq1 in prion state ([PSI+] and [PIN+], respectively) form oligomers and aggregates, which are transmitted from parents to offspring in a series of generations. Several pieces of indirect evidence indicate that these aggregates also possess amyloid properties, but their binding to amyloid-specific dyes has not been shown in vivo. Meanwhile, it is the specific binding to the Congo Red dye and birefringence in polarized light after such staining that is considered the gold standard for proving the amyloid properties of a protein. Here, we used immunoprecipitation to extract native fibrils of the Sup35 and Rnq1 proteins from yeast strains with different prion status. These fibrils are detected by electron microscopy, stained with Congo Red and exhibit yellow-green birefringence after such staining. All these data show that the Sup35 and Rnq1 proteins in prion state form amyloid fibrils in vivo. The technology of fibrils extraction in combination with standard cytological methods can be used to identify new pathological and functional amyloids in any organism and to analyze the structural features of native amyloid fibrils.

Symmetry Breaking in an Excited Quadrupolar Acridine-Dione Derivative Driven by Hydrogen Bonding.

An acridine-dione derivative (3,3,11,11-tetramethyl-8,16-diphenyl-3,4,8,10,11,12,13,16-octahydroacridino[4,3-c]acridine-1.9(2H,5H)dion) with quadrupolar motif has been synthesized and its stationary and transient spectra have been measured. Stationary absorption and fluorescence spectra as well as nonstationary spectra show no signs of symmetry breaking (SB) in aprotic solvents, even of high polarity. The specific features of SB are revealed in alcohol solvents through a considerable red shift of stationary fluorescence spectra and the appearance of a new excited state absorption band in transient absorption spectra. SB is due to the formation of asymmetric strong hydrogen bonds, mainly on one side of the molecule. An unexpected regularity of symmetry breaking is found in mixtures of aprotic dimethylformamide and protic methanol, where methanol acts as a fluorescence quencher. It is revealed that there is no quenching as long as the methanol concentration is less than the critical value of 9 M. This leads to the conclusion that SB in such mixtures is possible only if the concentration of the protic solvent exceeds a certain threshold value.

The Role of Rotational Motion in Diffusion NMR Experiments on Supramolecular Assemblies: Application to Sup35NM Fibrils.

Pulsed-field gradient (PFG) NMR is an important tool for characterization of biomolecules and supramolecular assemblies. However, for micrometer-sized objects, such as amyloid fibrils, these experiments become difficult to interpret because in addition to translational diffusion they are also sensitive to rotational diffusion. We have constructed a mathematical theory describing the outcome of PFG NMR experiments on rod-like fibrils. To test its validity, we have studied the fibrils formed by Sup35NM segment of the prion protein Sup35. The interpretation of the PFG NMR data in this system is fully consistent with the evidence from electron microscopy. Contrary to some previously expressed views, the signals originating from disordered regions in the fibrils can be readily differentiated from the similar signals representing small soluble species (e.g. proteolytic fragments). This paves the way for diffusion-sorted NMR experiments on complex amyloidogenic samples.

Quantitative assessment of chaperone binding to amyloid aggregates identifies specificity of Hsp40 interaction with yeast prion fibrils.

Yeast self-perpetuating protein aggregates (yeast prions) provide a framework to investigate the interaction of misfolded proteins with the protein quality control machinery. The major component of this system that facilitates propagation of all known yeast amyloid prions is the Hsp104 chaperone that catalyzes fibril fragmentation. Overproduction of Hsp104 cures some yeast prions via a fragmentation-independent mechanism. Importantly, major cytosolic chaperones of the Hsp40 group, Sis1 and Ydj1, oppositely affect yeast prion propagation, and are capable of stimulating different activities of Hsp104. In this work, we developed a quantitative method to investigate the Hsp40 binding to amyloid aggregates. We demonstrate that Sis1 binds fibrils formed by the Sup35NM protein with higher affinity compared to Ydj1. Moreover, the interaction of Sis1 with the fibrils formed by the other yeast prion protein, Rnq1, is orders of magnitude weaker. We show that the deletion of the dimerization domain of Sis1 (crucial for the curing of [PSI+] by excess Hsp104) decreases its affinity to both Sup35NM and Rnq1 fibrils. Taken together, these results suggest that tight binding of Hsp40 to the amyloid fibrils is likely to enhance aggregate malpartition instead of fibril fragmentation.

Estimation of amyloid aggregate sizes with semi-denaturing detergent agarose gel electrophoresis and its limitations.

Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) was proposed by Vitaly V. Kushnirov in the Michael D. Ter-Avanesyan's laboratory as a method to compare sizes of amyloid aggregates. Currently, this method is widely used for amyloid investigation, but mostly as a qualitative approach. In this work, we assessed the possibilities and limitations of the quantitative analysis of amyloid aggregate size distribution using SDD-AGE results. For this purpose, we used aggregates of two well-characterized yeast amyloid-forming proteins, Sup35 and Rnq1, and developed a protocol to standardize image analysis and process the result. A detailed investigation of factors that may affect the results of SDD-AGE revealed that both the cell lysis method and electrophoresis conditions can substantially affect the estimation of aggregate size. Despite this, quantitative analysis of SDD-AGE results is possible when one needs to estimate and compare the size of aggregates on the same gel, or even in different experiments, if the experimental conditions are tightly controlled and additional standards are used.

Nonsense Mutations in the Yeast SUP35 Gene Affect the [PSI+] Prion Propagation.

The essential SUP35 gene encodes yeast translation termination factor eRF3. Previously, we isolated nonsense mutations sup35-n and proposed that the viability of such mutants can be explained by readthrough of the premature stop codon. Such mutations, as well as the prion [PSI+], can appear in natural yeast populations, and their combinations may have different effects on the cells. Here, we analyze the effects of the compatibility of sup35-n mutations with the [PSI+] prion in haploid and diploid cells. We demonstrated that sup35-n mutations are incompatible with the [PSI+] prion, leading to lethality of sup35-n [PSI+] haploid cells. In diploid cells the compatibility of [PSI+] with sup35-n depends on how the corresponding diploid was obtained. Nonsense mutations sup35-21, sup35-74, and sup35-218 are compatible with the [PSI+] prion in diploid strains, but affect [PSI+] properties and lead to the formation of new prion variant. The only mutation that could replace the SUP35 wild-type allele in both haploid and diploid [PSI+] strains, sup35-240, led to the prion loss. Possibly, short Sup351-55 protein, produced from the sup35-240 allele, is included in Sup35 aggregates and destabilize them. Alternatively, single molecules of Sup351-55 can stick to aggregate ends, and thus interrupt the fibril growth. Thus, we can conclude that sup35-240 mutation prevents [PSI+] propagation and can be considered as a new pnm mutation.

Design of a New [PSI +]-No-More Mutation in SUP35 With Strong Inhibitory Effect on the [PSI +] Prion Propagation.

A number of [PSI +]-no-more (PNM) mutations, eliminating [PSI +] prion, were previously described in SUP35. In this study, we designed and analyzed a new PNM mutation based on the parallel in-register ß-structure of Sup35 prion fibrils suggested by the known experimental data. In such an arrangement, substitution of non-charged residues by charged ones may destabilize the fibril structure. We introduced Q33K/A34K amino acid substitutions into the Sup35 protein, corresponding allele was called sup35-M0. The mutagenized residues were chosen based on ArchCandy in silico prediction of high inhibitory effect on the amyloidogenic potential of Sup35. The experiments confirmed that Sup35-M0 leads to the elimination of [PSI +] with high efficiency. Our data suggested that the elimination of the [PSI +] prion is associated with the decreased aggregation properties of the protein. The new mutation can induce the prion with very low efficiency and is able to propagate only weak [PSI +] prion variants. We also showed that Sup35-M0 protein co-aggregates with the wild-type Sup35 in vivo. Moreover, our data confirmed the utility of the strategy of substitution of non-charged residues by charged ones to design new mutations to inhibit a prion formation.

Spectral Dynamics of Nitro Derivatives of Xanthione in Solutions.

Nitro derivatives of xanthione, 2,7-dinitro-9 H-xanthene-9-thione and 2,4,7-trinitro-9 H-xanthene-9-thione, have been first synthesized and their stationary and transient spectra have been measured. The stationary spectra show that the attachment of the nitro groups to the xanthione scaffold leads to strong quenching of S2 → S0 fluorescence and the decrease of the oscillator strength of the S2 ← S0 electronic transition. Analysis of the transient absorption spectra uncovers the ultrafast stimulated emission quenching from the second excited state, S2, in the both derivatives. A kinetic scheme has been suggested to rationalize the complex spectral dynamics of the transient absorption signal. The kinetic scheme is deduced from the analysis of the transient spectra and supported by the quantum-chemical calculations, which predict the existence of a dark state and S2 state splitting into two close levels. The ultrafast transitions between S2 state sublevels and the transition into the dark state play a crucial role in spectral dynamics. These new features discovered in the nitro derivatives of xanthione distinguish essentially their spectral dynamics from that observed in xanthione.

Low-Temperature Effect on the Electronic Structure and Spectral-Fluorescent Properties of Highly Dipolar Merocyanines.

Absorption and fluorescence spectra of a vinylogous series of reversely solvatochromic merocyanines based on benzimidazole and malononitrile have been studied in frozen ethanol solutions at 77 K. It is found that they possess negative thermochromism-in contrast to both positively solvatochromic merocyanines and negatively solvatochromic symmetrical ionic polymethines-and even stronger negative thermofluorochromism. It has been deduced from the spectral data that at low temperature their electronic structure becomes more dipolar, deviating substantially from the virtual ideal polymethine in both the ground and the excited states. At that, owing probably to the high polarity and ordering of frozen ethanol, the dipolarity of the studied merocyanines increases with the polymethine chain lengthening-the tendency not observed for them in common solvents. The conclusions, based on the spectral data analysis, have been verified by the (TD)DFT-PCM simulations of the dyes within the four-level scheme of electronic transitions.

Protein Co-Aggregation Related to Amyloids: Methods of Investigation, Diversity, and Classification.

Amyloids are unbranched protein fibrils with a characteristic spatial structure. Although the amyloids were first described as protein deposits that are associated with the diseases, today it is becoming clear that these protein fibrils play multiple biological roles that are essential for different organisms, from archaea and bacteria to humans. The appearance of amyloid, first of all, causes changes in the intracellular quantity of the corresponding soluble protein(s), and at the same time the aggregate can include other proteins due to different molecular mechanisms. The co-aggregation may have different consequences even though usually this process leads to the depletion of a functional protein that may be associated with different diseases. The protein co-aggregation that is related to functional amyloids may mediate important biological processes and change of protein functions. In this review, we survey the known examples of the amyloid-related co-aggregation of proteins, discuss their pathogenic and functional roles, and analyze methods of their studies from bacteria and yeast to mammals. Such analysis allow for us to propose the following co-aggregation classes: (i) titration: deposition of soluble proteins on the amyloids formed by their functional partners, with such interactions mediated by a specific binding site; (ii) sequestration: interaction of amyloids with certain proteins lacking a specific binding site; (iii) axial co-aggregation of different proteins within the same amyloid fibril; and, (iv) lateral co-aggregation of amyloid fibrils, each formed by different proteins.

Overproduction of Sch9 leads to its aggregation and cell elongation in Saccharomyces cerevisiae.

The Sch9 kinase of Saccharomyces cerevisiae is one of the major TOR pathway effectors and regulates diverse processes in the cell. Sch9 belongs to the AGC kinase family. In human, amplification of AGC kinase genes is connected with cancer. However, not much is known about the effects of Sch9 overproduction in yeast cells. To fill this gap, we developed a model system to monitor subcellular location and aggregation state of overproduced Sch9 or its regions fused to a fluorescent protein. With this system, we showed that Sch9-YFP forms detergent-resistant aggregates, and multiple protein regions are responsible for this. This finding corroborated the fact that Sch9-YFP is visualized as various fluorescent foci. In addition, we found that Sch9 overproduction caused cell elongation, and this effect was determined by its C-terminal region containing kinase domains. The constructs we present can be exploited to create superior yeast-based model systems to study processes behind kinase overproduction in cancers.