Alexey P. Galkin

Correlation of somatic hypermutation specificity and A-T base pair substitution errors by DNA polymerase η during copying of a mouse immunoglobulin κ light chain transgene

To test the hypothesis that inaccurate DNA synthesis by mammalian DNA polymerase η (pol η) contributes to somatic hypermutation (SHM) of Ig genes, we measured the error specificity of mouse pol η during synthesis of each strand of a mouse Ig κ light chain transgene. We then compared the results to the base substitution specificity of SHM of this same gene in the mouse. The in vitro and in vivo base substitution spectra shared a number of common features. A highly significant correlation was observed for overall substitutions at A-T pairs but not for substitutions at G-C pairs. Sixteen mutational hotspots at A-T pairs observed in vivo were also found in spectra generated by mouse pol η in vitro. The correlation was strongest for errors made by pol η during synthesis of the non-transcribed strand, but it was also observed for synthesis of the transcribed strand. These facts, and the distribution of substitutions generated in vivo, support the hypothesis that pol η contributes to SHM of Ig genes at A-T pairs via short patches of low fidelity DNA synthesis of both strands, but with a preference for the non-transcribed strand.

[NSI+]: a novel non-Mendelian nonsense suppressor determinant in Saccharomyces cerevisiae

Non-Mendelian determinants that control heritable traits in yeast are subdivided into two major groups—one that includes DNA- or RNA-based elements and another that comprises protein-based factors that are analogous to mammalian prion. All yeast non-Mendelian determinants show dominant inheritance, and some of them demonstrate cytoplasmic infectivity. Only prions, however, harbor-specific features, such as high frequency of induction following overproduction of prion-encoding protein, loss of the protein’s normal function, and reversible curability. Here, we describe a novel nonchromosomal determinant that, in addition to [PSI+] and [ISP+], is involved in epigenetic control of nonsense suppression. This determinant, which we have designated [NSI+], causes nonsense suppression in the strains bearing the N-terminal-deleted or -modified SUP35 gene, but has no manifestation in the strains with the intact copy of SUP35. [NSI+] shows dominant non-Mendelian inheritance, reversible curability and may be transmitted by cytoduction, albeit with low frequency. Similar to yeast prions, this determinant can be cured by deletion or mutational inactivation of Hsp104. We have shown that [NSI+] does not correspond to the already identified yeast prions. Based on the data obtained, we hypothesize that [NSI+] is a novel prion factor involved in epigenetic control of nonsense suppression.

Proteomic Screening for Amyloid Proteins

Despite extensive study, progress in elucidation of biological functions of amyloids and their role in pathology is largely restrained due to the lack of universal and reliable biochemical methods for their discovery. All biochemical methods developed so far allowed only identification of glutamine/asparagine-rich amyloid-forming proteins or proteins comprising amyloids that form large deposits. In this article we present a proteomic approach which may enable identification of a broad range of amyloid-forming proteins independently of specific features of their sequences or levels of expression. This approach is based on the isolation of protein fractions enriched with amyloid aggregates via sedimentation by ultracentrifugation in the presence of strong ionic detergents, such as sarkosyl or SDS. Sedimented proteins are then separated either by 2D difference gel electrophoresis or by SDS-PAGE, if they are insoluble in the buffer used for 2D difference gel electrophoresis, after which they are identified by mass-spectrometry. We validated this approach by detection of known yeast prions and mammalian proteins with established capacity for amyloid formation and also revealed yeast proteins forming detergent-insoluble aggregates in the presence of human huntingtin with expanded polyglutamine domain. Notably, with one exception, all these proteins contained glutamine/asparagine-rich stretches suggesting that their aggregates arose due to polymerization cross-seeding by human huntingtin. Importantly, though the approach was developed in a yeast model, it can easily be applied to any organism thus representing an efficient and universal tool for screening for amyloid proteins.

[NSI+] determinant has a pleiotropic phenotypic manifestation that is modulated by SUP35, SUP45, and VTS1 genes

We recently discovered the novel non-chromosomal determinant in Saccharomyces cerevisiae [NSI+] (nonsense suppression inducer), which causes omnipotent nonsense suppression in strains where the Sup35 N-terminal domain is deleted. [NSI+] possesses yeast prion features and does not correspond to previously identified yeast prion determinants. Here, we show that [NSI+] enhances nonsense codon read-through and inhibits vegetative growth in S. cerevisiae. Using a large-scale overexpression screen to identify genes that impact the phenotypic effects of [NSI+], we found that the SUP35 and SUP45 genes encoding the translation termination factors eRF3 and eRF1, respectively, modulate nonsense suppression in [NSI+] strains. The VTS1 gene encodes an NQ-enriched RNA-binding protein that enhances nonsense suppression in [NSI+] and [nsi−] strains. We demonstrate that VTS1 overexpression, like [NSI+] induction, causes translational read-through and growth defects in S. cerevisiae.

Identification of genes encoding potentially amyloidogenic proteins that take part in the regulation of nonsense suppression in yeast Saccharomyces cerevisiae

Previously, we demonstrated that SUP35 N-terminal deletion creates a specific genetic back-ground permitting the identification of novel genes and epigenetic determinants controlling nonsense suppression. In the present study, using a genomic screen, we found three genes encoding potentially amyloidogenic proteins, whose overexpression affects nonsense suppression in the strain producing chimeric Aβ-Sup35MC protein on the background of SUP35 deletion encoding releasing factor eRF3. These genes, NAB2, NAB3, and VTS1, were found to participate in the regulation of nonsense suppression in yeast S. cerevisiae.

Yeast chaperone Hsp104 controls gene expression at the posttranscriptional level

Yeast chaperone Hsp104 is known as a protein responsible for dissociation of aggregates of heat-damaged proteins and prion aggregates into smaller pieces or monomers. The effects of Hsp104 on PrP-GFP and GFP were analyzed. PrP-GFP forms high-molecular-weight aggregates, whereas GFP is unable to aggregate in yeast cells. Hsp104 proved to regulate the amount of PrP-GFP and GFP in yeast cells, and the direction of chaperone action depended on the promoters controlling the production of these proteins. Overproduction of Hsp104 increased the levels of PrP-GFP and GFP when their genes were controlled by the CUP1 promoter. In contrast, overproduction of Hsp104 decreased the levels of PrP-GFP and GFP in the case of their expression under the control of the GPD promoter. The effects of Hsp104 were not related to any changes in the contents of mRNAs of the genes under investigation nor to the ability of the proteins to form aggregates. Thus, the Hsp104 functions were not confined to dissociation of protein aggregates. Hsp104 was assumed to regulate gene expression at the posttranscriptional level.

Interactions of [ NSI + ] prion-like determinant with SUP35 and VTS1 genes in Saccharomyces cerevisiae

Previously we characterized [NSI+], determinant, that possesses the features of a yeast prion. This determinant causes the nonsense suppression in strains that bear different N-substituted variants of Sup35p, which is a translation release factor eRF3. As a result of the genomic screen, we identified VTS1, the overexpression of which is a phenotypic copy of [NSI+]. Here, we analyzed the influence of SUP35 and VTS1 on [NSI+]. We demonstrated nonsense suppression in the [NSI+] strains, which appears when SUP35 expression was decreased or against a background of general defects in the fidelity of translation termination. [NSI+] has also been shown to increase VTS1 mRNA amounts. These findings facilitate the insight into the mechanisms of nonsense suppression in the [NSI+] strains and narrow the range of candidates for [NSI+] determinant.

[Yeast prions, mammalian amyloidoses, and the problem of proteomic networks].

Prion proteins are infective amyloids and cause several neurodegenerative diseases in humans and animals. In yeasts, prions are detected as the cytoplasmic heritable determinants of a protein nature. Yeast prion [PSI], which results from a conformational rearrangement and oligomerization of translation termination factor eRF3, is used as an example to consider the structural-functional relationships in a potentially prion molecule, specifics of its evolution, and interactions with other prions, which form so-called prion networks. In addition, the review considers the results of modeling mammalian prion diseases and other amyloidoses in yeast cells. A hypothesis of proteomic networks is proposed by analogy with prion networks, involving interactions of different amyloids in mammals.

Proteomic Analysis of Escherichia coli Protein Fractions Resistant to Solubilization by Ionic Detergents

Amyloids are protein fibrils adopting structure of cross-beta spine exhibiting either pathogenic or functionally significant properties. In prokaryotes, there are several groups of functional amyloids; however, all of them were identified by specialized approaches that do not reveal all cellular amyloids. Here, using our previously developed PSIA (Proteomic Screening and Identification of Amyloids) approach, we have conducted a proteomic screening for candidates for novel amyloid-forming proteins in Escherichia coli as one of the most important model organisms and biotechnological objects. As a result, we identified 61 proteins in fractions resistant to treatment with ionic detergents. We found that a fraction of proteins bearing potentially amyloidogenic regions predicted by bioinformatics algorithms was 3-5-fold more abundant among the identified proteins compared to those observed in the entire E. coli proteome. Almost all identified proteins contained potentially amyloidogenic regions, and four of them (BcsC, MukB, YfbK, and YghJ) have asparagineand glutamine-rich regions underlying a crucial feature of many known amyloids. In this study, we demonstrate for the first time that at the proteome level there is a correlation between experimentally demonstrated detergent-resistance of proteins and potentially amyloidogenic regions predicted by bioinformatics approaches. The data obtained enable further comprehensive characterization of entirety of amyloids (or amyloidome) in bacterial cells.

Overexpression of genes encoding asparagine-glutamine-rich transcriptional factors causes nonsense suppression in Saccharomyces cerevisiae

We previously conducted a search for genes whose overexpression causes nonsense suppression in Saccharomyces cerevisiae with a background of modified expression of Sup35 variants. In this study, we analyzed the influence of genes encoding asparagine-glutamine-rich transcriptional factors on this process. We demonstrated that the overexpression of ABF1, GLN3, FKH2, MCM1, MOT3, and REB1 affects nonsense suppression in S. cerevisiae. The data obtained highlight the interrelation between the fundamental processes of transcription and translation in the living cell.