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.

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.

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.

Transcriptional response to the [ISP(+) ] prion of Saccharomyces cerevisiae differs from that induced by the deletion of its structural gene, SFP1.

Currently, several protein-based genetic determinants, or prions, are described in yeast, and several hundred prion candidates have been predicted. Importantly, many known and potential prion proteins regulate transcription; therefore, prion induction should affect gene expression. While it is generally believed that the prion phenotype should mimic the deletion phenotype, this rule has exceptions. Formed by the transcription factor Sfp1p, [ISP(+) ] is one such exception as the [ISP(+) ] and sfp1Δ strains differ in many phenotypic traits. These data suggest that effects of prion formation by a transcription factor and its absence may affect gene expression in a different way. However, studies addressing this issue are practically absent. Here, we explore how [ISP(+) ] affects gene expression and how these changes correspond to the effect of SFP1 deletion. Our data indicate that the [ISP(+) ]-related expression changes cannot be explained by the inactivation of Sfp1p. Remarkably, most Sfp1p targets are not affected in the [ISP(+) ] strain; instead, the genes upregulated in the [ISP(+) ] strain are enriched in Gcn4p and Aft1p targets. We propose that Sfp1p serves as a part of a regulatory complex, and the activity of this complex may be modulated differently by the absence or prionization of Sfp1p.

SUP35 expression is enhanced in yeast containing [ISP+], a prion form of the transcriptional regulator Sfp1.

[ISP+] is a prion form of the global transcriptional regulator Sfp1 in Saccharomyces cerevisiae that manifests phenotypically as an antisuppressor of specific sup35 nonsense suppressor mutations. Although SUP35 is a Sfp1 target, the mechanism of antisuppression is unclear. Here we show that the level of SUP35 transcription in [ISP+] cells containing the sup35 mutation is increased relative to [isp-] cells and cells with a SFP1 deletion. As a result, [ISP+] cells have increased amounts of Sup35 encoded by the mutant allele. Indeed, additional experiments showed that increased amounts of mutant Sup35 may cause antisuppression. Remarkably, [ISP+] effects are not equivalent to those produced by SFP1 deletion, so [ISP+] represents an obvious example of a functionally active prion form of a protein. This feature distinguishes [ISP+] from other yeast prions, where prion switch often has the same effect as inactivation of a prion host gene. We suggest that enhancement of SUP35 expression in [ISP+] cells is caused by specific interaction of Sfp1 in its prion form with some negative SUP35 regulator. We also demonstrate that the advantage of [ISP+] strains over [isp-] strains described in our earlier work is specific for certain genetic background and growth conditions.

Non-Mendelian determinant [ISP+] in yeast is a nuclear-residing prion form of the global transcriptional regulator Sfp1.

Four protein-based genetic determinants or prions-[SWI(+)], [MCA], [OCT(+)], and [MOT3(+)]-are recent additions to the list of well-known Saccharomyces cerevisiae prions, [PSI(+)], [URE3], and [PIN(+)]. A rapid expansion of this list may indicate that many yeast proteins can convert into heritable prion forms and underscores a problem of prion input into cellular physiology. Here, we prove that the global transcriptional regulator Sfp1 can become a prion corresponding to the prion-like determinant [ISP(+)] described earlier. We show that SFP1 deletion causes an irreversible [ISP(+)] loss, whereas increased SFP1 expression induces [ISP(+)] appearance. Cells that display the [ISP(+)] phenotype contain the aggregated form of Sfp1. Indeed, these aggregates demonstrate a nuclear location. We also show that the phenotypic manifestation of Sfp1 prionization differs from the manifestation of SFP1 deletion. These properties and others distinguish [ISP(+)] from yeast prions described to date.