Stanislav A. Bondarev

Effect of Charged Residues in the N-domain of Sup35 Protein on Prion [PSI+] Stability and Propagation

Background: The prion domain (PrD) of Sup35p can aggregate to form the [PSI+] prion. Results: Introduction of charged lysine residues (sup35KK) in the Sup35p PrD alters prion properties. Conclusion: Some sup35KK alleles lead to the formation of new prion variants. Significance: Establishment of molecular interactions influencing [PSI+] prion stability and maintenance is a step toward an understanding of prion folding. Recent studies have shown that Sup35p prion fibrils probably have a parallel in-register β-structure. However, the part(s) of the N-domain critical for fibril formation and maintenance of the [PSI+] phenotype remains unclear. Here we designed a set of five SUP35 mutant alleles (sup35KK) with lysine substitutions in each of five N-domain repeats, and investigated their effect on infectivity and ability of corresponding proteins to aggregate and coaggregate with wild type Sup35p in the [PSI+] strain. Alleles sup35-M1 (Y46K/Q47K) and sup35-M2 (Q61K/Q62K) led to prion loss, whereas sup35-M3 (Q70K/Q71K), sup35-M4 (Q80K/Q81K), and sup35-M5 (Q89K/Q90K) were able to maintain the [PSI+] prion. This suggests that the critical part of the parallel in-register β-structure for the studied [PSI+] prion variant lies in the first 63–69 residues. Our study also reveals an unexpected interplay between the wild type Sup35p and proteins expressed from the sup35KK alleles during prionization. Both Sup35-M1p and Sup35-M2p coaggregated with Sup35p, but only sup35-M2 led to prion loss in a dominant manner. We suggest that in the fibrils, Sup35p can bind to Sup35-M1p in the same conformation, whereas Sup35-M2p only allowed the Sup35p conformation that leads to the non-heritable fold. Mutations sup35-M4 and sup35-M5 influence the structure of the prion forming region to a lesser extent, and can lead to the formation of new prion variants.

Prions, amyloids, and RNA: Pieces of a puzzle.

ABSTRACT Amyloids are protein aggregates consisting of fibrils rich in β-sheets. Growth of amyloid fibrils occurs by the addition of protein molecules to the tip of an aggregate with a concurrent change of a conformation. Thus, amyloids are self-propagating protein conformations. In certain cases these conformations are transmissible / infectious; they are known as prions. Initially, amyloids were discovered as pathological extracellular deposits occurring in different tissues and organs. To date, amyloids and prions have been associated with over 30 incurable diseases in humans and animals. However, a number of recent studies demonstrate that amyloids are also functionally involved in a variety of biological processes, from biofilm formation by bacteria, to long-term memory in animals. Interestingly, amyloid-forming proteins are highly overrepresented among cellular factors engaged in all stages of mRNA life cycle: from transcription and translation, to storage and degradation. Here we review rapidly accumulating data on functional and pathogenic amyloids associated with mRNA processing, and discuss possible significance of prion and amyloid networks in the modulation of key cellular functions.

Stoichiometry and Affinity of Thioflavin T Binding to Sup35p Amyloid Fibrils.

In this work two modes of binding of the fluorescent probe thioflavin T to yeast prion protein Sup35p amyloid fibrils were revealed by absorption spectrometry of solutions prepared by equilibrium microdialysis. These binding modes exhibited significant differences in binding affinity and stoichiometry. Moreover, the absorption spectrum and the molar extinction coefficient of the dye bound in each mode were determined. The fluorescence quantum yield of the dye bound in each mode was determined via a spectrofluorimetric study of the same solutions in which the recorded fluorescence intensity was corrected for the primary inner filter effect. As previously predicted, the existence of one of the detected binding modes may be due to the incorporation of the dye into the grooves along the fiber axis perpendicular to the β-sheets of the fibrils. It was assumed that the second type of binding with higher affinity may be due to the existence of ThT binding sites that are localized to areas where amyloid fibrils are clustered.

Structure-based view on [PSI+] prion properties

ABSTRACT Yeast [PSI+] prion is one of the most suitable and well characterized system for the investigation of the prion phenomenon. However, until recently, the lack of data on the 3D arrangement of Sup35p prion fibrils hindered progress in this area. The recent arrival in this field of new experimental techniques led to the parallel and in-register superpleated β-structure as a consensus model for Sup35p fibrils. Here, we analyzed the effect of amino acid substitutions of the Sup35 protein through the prism of this structural model. Application of a newly developed computational approach, called ArchCandy, gives us a better understanding of the effect caused by mutations on the fibril forming potential of Sup35 protein. This bioinformatics tool can be used for the design of new mutations with desired modification of prion properties. Thus, we provide examples of how today, having progress toward elucidation of the structural arrangement of Sup35p fibrils, researchers can advance more efficiently to a better understanding of prion [PSI+] stability and propagation.

FibrilJ: ImageJ plugin for fibrils’ diameter and persistence length determination

Abstract Application of microscopy to evaluate the morphology and size of filamentous proteins and amyloids requires new and creative approaches to simplify and automate the image processing. The estimation of mean values of fibrils diameter, length and bending stiffness on micrographs is a major challenge. For this purpose we developed an open-source FibrilJ plugin for the ImageJ/FiJi program. It automatically recognizes the fibrils on the surface of a mica, silicon, gold or formvar film and further analyzes them to calculate the distribution of fibrils by diameters, lengths and persistence lengths. The plugin has been validated by the processing of TEM images of fibrils formed by Sup35NM yeast protein and artificially created images of rod-shape objects with predefined parameters. Novel data obtained by SEM for Sup35NM protein fibrils immobilized on silicon and gold substrates are also presented and analyzed. Program summary Program title: FibrilJ Program Files doi: http://dx.doi.org/10.17632/ndxb93h4vc.1 Licensing provisions: Apache-2.0 Programming language: ImageJ Macro Language Nature of problem: Amyloids are large protein aggregates that form unbranched fibrils. Formation of amyloids by different proteins leads to the emergence of a number of serious human diseases, including Alzheimer’s, Parkinson’s and Huntington’s diseases. Therefore, various amyloids, in particular their topology, are widely studied in an enormous number of papers. The structural organization of the amyloid aggregates at the molecular level is a key problem in this area. At the moment new methods of amyloids analysis based on electronic and probe microscopy are particularly popular. Usually it is required to process images containing hundreds of amyloids to obtain statistically reliable data on the distribution of the thickness, length and persistence length (a basic mechanical property quantifying the stiffness of a polymer). The lattermost parameter was introduced for the characteristic of bending stiffness of amyloids similarly to that in a model of wormlike chain for polymers. The manual measurements of these parameters take a lot of time and, due to the human factor, are insufficiently representative. ImageJ/FiJi is open-source software and one of the most commonly used program for image processing. The ImageJ/FiJi community provides BoneJ plugin for bones analysis and DiameterJ plugin for nanofibers analysis, which have the function of determining the diameter of the respective objects. Also, approaches that involve calculations of the end-to-end distances and contour lengths of biopolymers were used to determine their persistence lengths using ImageJ/FiJi. These above mentioned plugins are not designed for the study of amyloids and have never been used or validated for them. They do not provide the necessary functionality for the automatic recognition of amyloids on the substrate and filtering objects by a number of parameters. They have unspecified accuracy of these objects measurements and do not calculate the objects distribution on average diameters and persistence lengths. Solution method: For this purpose we developed a FibrilJ plugin for ImageJ/FiJi program with an open source code. It automatically recognizes the fibrils on the surface of a mica, silicon, gold or formvar film and further analyzes them to calculate the distribution of fibrils on average diameters, lengths and persistence lengths. FibrilJ usage may unify the process of diameter and persistence length calculation and it may also contribute to comparison of results obtained by different research groups. Plugin FibrilJ which is presentedin this paper is free of DiameterJ or BoneJ weaknesses. It has been validated by artificially created images of rod-shape objects with predefined parameters and micrographs of Sup35NM protein fibrils.

BetaSerpentine: a bioinformatics tool for reconstruction of amyloid structures

Motivation Numerous experimental studies have suggested that polypeptide chains of large amyloidogenic regions zig-zag in β-serpentine arrangements. These β-serpentines are stacked axially and form the superpleated β-structure. Despite this progress in the understanding of amyloid folds, the determination of their 3D structure at the atomic level is still a problem due to the polymorphism of these fibrils and incompleteness of experimental structural data. Today, the way to get insight into the atomic structure of amyloids is a combination of experimental studies with bioinformatics. Results We developed a computer program BetaSerpentine that reconstructs β-serpentine arrangements from individual β-arches predicted by ArchCandy program and ranks them in order of preference. It was shown that the BetaSerpentine program in combination with the experimental data can be used to gain insight into the detailed 3D structure of amyloids. It opens avenues to the structure-based interpretation and design of the experiments. Availability and implementation BetaSerpentine webserver can be accessed through website: http://bioinfo.montp.cnrs.fr/b-serpentine. Source code is available in git.hub repository (github.com/stanislavspbgu/BetaSerpentine). Contact stanislavspbgu@gmail.com or andrey.kajava@crbm.cnrs.fr. Supplementary information Supplementary data are available at Bioinformatics online.

SFP1‐mediated prion‐dependent lethality is caused by increased Sup35 aggregation and alleviated by Sis1

[PSI+] is the prion form of the translation termination factor Sup35 (eRF3); [PSI+] strains display nonsense suppression. Another prion‐like element, [ISP+], is linked to antisuppression in a specific background. Transcriptional regulator Sfp1 was shown to be responsible for [ISP+] propagation. In this work, we identified SFP1 as a multicopy inducer of [PSI+]‐dependent lethality. Sfp1 is likely to up‐regulate transcription of genes encoding release factors; however, its overproduction increases Sup35, but not Sup45 protein level. Using the synthetic lethality test, we compared the effects of SFP1 and SUP35 over‐expression on the viability of [PSI+] strains. Together with an observation that Sfp1 overproduction leads to an increased accumulation of Sup35 in [PSI+] aggregates, we suggest that excess Sfp1 causes [PSI+] toxicity. Even though SUP45 over‐expression is known to compensate for the [PSI+]‐dependent lethality, it fails to do so when the lethality is caused by SFP1 over‐expression. We discovered that the increased levels of Hsp40 chaperone Sis1 alleviate prion toxicity caused by either SFP1 or SUP35 over‐expression and revert back to normal distribution of Sup35 between monomers and aggregate fractions. Finally, we showed that Sfp1 partially colocalizes with Sup35 aggregates, which may contribute to another mechanism of Sfp1‐derived [PSI+] prion toxicity.

Allelic variants of hereditary prions: The bimodularity principle

ABSTRACT Modern biology requires modern genetic concepts equally valid for all discovered mechanisms of inheritance, either “canonical” (mediated by DNA sequences) or epigenetic. Applying basic genetic terms such as “gene” and “allele” to protein hereditary factors is one of the necessary steps toward these concepts. The basic idea that different variants of the same prion protein can be considered as alleles has been previously proposed by Chernoff and Tuite. In this paper, the notion of prion allele is further developed. We propose the idea that any prion allele is a bimodular hereditary system that depends on a certain DNA sequence (DNA determinant) and a certain epigenetic mark (epigenetic determinant). Alteration of any of these 2 determinants may lead to establishment of a new prion allele. The bimodularity principle is valid not only for hereditary prions; it seems to be universal for any epigenetic hereditary factor.

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.

M60-like metalloprotease domain of the Escherichia coli YghJ protein forms amyloid fibrils

Amyloids are protein fibrils with a characteristic spatial structure. Amyloids were long perceived as the pathogens involved in a set of lethal diseases in humans and animals. In recent decades, it has become clear that amyloids represent a quaternary protein structure that is not only pathological but also functionally important and is widely used by different organisms, ranging from archaea to animals, to implement diverse biological functions. The greatest biological variety of amyloids is found in prokaryotes, where they control the formation of biofilms and cell wall sheaths, facilitate the overcoming of surface tension, and regulate the metabolism of toxins. Several amyloid proteins were identified in the important model, biotechnological and pathogenic bacterium Escherichia coli. In previous studies, using a method for the proteomic screening and identification of amyloids, we identified 61 potentially amyloidogenic proteins in the proteome of E. coli. Among these proteins, YghJ was the most enriched with bioinformatically predicted amyloidogenic regions. YghJ is a lipoprotein with a zinc metalloprotease M60-like domain that is involved in mucin degradation in the intestine as well as in proinflammatory responses. In this study, we analyzed the amyloid properties of the YghJ M60-like domain and demonstrated that it forms amyloid-like fibrils in vitro and in vivo.