Kirill V. Volkov

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.

Genome-wide screen identifies pathways that govern GAA/TTC repeat fragility and expansions in dividing and nondividing yeast cells.

Triplex structure-forming GAA/TTC repeats pose a dual threat to the eukaryotic genome integrity. Their potential to expand can lead to gene inactivation, the cause of Friedreichs ataxia disease in humans. In model systems, long GAA/TTC tracts also act as chromosomal fragile sites that can trigger gross chromosomal rearrangements. The mechanisms that regulate the metabolism of GAA/TTC repeats are poorly understood. We have developed an experimental system in the yeast Saccharomyces cerevisiae that allows us to systematically identify genes crucial for maintaining the repeat stability. Two major groups of mutants defective in DNA replication or transcription initiation are found to be prone to fragility and large-scale expansions. We demonstrate that problems imposed by the repeats during DNA replication in actively dividing cells and during transcription initiation in nondividing cells can culminate in genome instability. We propose that similar mechanisms can mediate detrimental metabolism of GAA/TTC tracts in human cells.

The HAL3-PPZ1 dependent regulation of nonsense suppression efficiency in yeast and its influence on manifestation of the yeast prion-like determinant [ISP(+)].

The efficiency of stop codons read‐through in yeast is controlled by multiple interactions of genetic and epigenetic factors. In this study, we demonstrate the participation of the Hal3‐Ppz1 protein complex in regulation of read‐through efficiency and manifestation of non‐Mendelian anti‐suppressor determinant [ISP+]. Over‐expression of HAL3 in [ISP+] strain causes nonsense suppression, whereas its inactivation displays as anti‐suppression of sup35 mutation in [isp−] strain. [ISP+] strains carrying hal3Δ deletion cannot be cured from [ISP+] in the presence of GuHCl. Since Hal3p is a negative regulatory subunit of Ppz1 protein phosphatase, consequences of PPZ1 over‐expression and deletion are opposite to those of HAL3. The observed effects are mediated by the catalytic function of Ppz1 and are probably related to the participation of Ppz1 in regulation of eEF1Bα elongation factor activity. Importantly, [ISP+] status of yeast strains is determined by fluctuation in Hal3p level, since [ISP+] strains have less Hal3p than their [isp−] derivatives obtained by GuHCl treatment. A model considering epigenetic (possibly prion) regulation of Hal3p amount as a mechanism underlying [ISP+] status of yeast cell is suggested.

Phenotypic expression of epigenetic determinant [ISP+] in Saccharomyces cerevisiae depends on the combination of sup35 and sup45 mutations

Translation fidelity in Saccharomyces yeasts is determined by genetic and epigenetic (prion) factors. A study was made of S. cerevisiae strains containing the nonchromosomal determinant [ISP+], described earlier. Some of its properties suggest that [ISP+] is a prion. [ISP+] is expressed phenotypically as an antisuppressor of two sup35 mutations and can be cured with guanidine chloride (GuHCl). It was shown that sup35 mutants containing [ISP+] carried additional sup45 mutations. These mutations caused amino acid substitutions in different regions of translation termination factor eRF1, encoded by SUP45. Strains bearing the sup35-25 mutation contained the sup45 mutation that caused amino acid substitution at position 400 of eRF1; strains bearing sup35-10 contained the mutation that altered eRF1 at position 75. Thus, the antisuppressor phenotype of the [ISP+] strains proved to depend on the interaction of sup35 and sup45 mutations, as well as on the GuHCl-curable epigenetic determinant.

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.

Expansion of Interstitial Telomeric Sequences in Yeast

Telomeric repeats located within chromosomes are called interstitial telomeric sequences (ITSs). They are polymorphic in length and are likely hotspots for initiation of chromosomal rearrangements that have been linked to human disease. Using our S. cerevisiae system to study repeat-mediated genome instability, we have previously shown that yeast telomeric (Ytel) repeats induce various gross chromosomal rearrangements (GCR) when their G-rich strands serve as the lagging strand template for replication (G orientation). Here, we show that interstitial Ytel repeats in the opposite C orientation prefer to expand rather than cause GCR. A tract of eight Ytel repeats expands at a rate of 4 × 10(-4) per replication, ranking them among the most expansion-prone DNA microsatellites. A candidate-based genetic analysis implicates both post-replication repair and homologous recombination pathways in the expansion process. We propose a model for Ytel repeat expansions and discuss its applications for genome instability and alternative telomere lengthening (ALT).

Screening for amyloid proteins in the yeast proteome

The search for novel pathological and functional amyloids represents one of the most important tasks of contemporary biomedicine. Formation of pathological amyloid fibrils in the aging brain causes incurable neurodegenerative disorders such as Alzheimer’s, Parkinson’s Huntington’s diseases. At the same time, a set of amyloids regulates vital processes in archaea, prokaryotes and eukaryotes. Our knowledge of the prevalence and biological significance of amyloids is limited due to the lack of universal methods for their identification. Here, using our original method of proteomic screening PSIA–LC–MALDI, we identified a number of proteins that form amyloid-like detergent-resistant aggregates in Saccharomyces cerevisiae. We revealed in yeast strains of different origin known yeast prions, prion-associated proteins, and a set of proteins whose amyloid properties were not shown before. A substantial number of the identified proteins are cell wall components, suggesting that amyloids may play important roles in the formation of this extracellular protective sheath. Two proteins identified in our screen, Gas1 and Ygp1, involved in biogenesis of the yeast cell wall, were selected for detailed analysis of amyloid properties. We show that Gas1 and Ygp1 demonstrate amyloid properties both in vivo in yeast cells and using the bacteria-based system C-DAG. Taken together, our data show that this proteomic approach is very useful for identification of novel amyloids.

Search for genes influencing the maintenance of the [ISP+] prion-like antisuppressor determinant in yeast with the use of an insertion gene library

The prion-like determinant [ISP+] manifests itself as an antisuppressor of certain sup35 mutations. To establish that [ISP+] is indeed a new yeast prion, it is necessary to identify the gene that codes for the protein whose prion form is [ISP+]. Analysis of the transformants obtained by transformation of an [ISP+] strain with an insertion gene library revealed three genes controlling the [ISP+] maintenance: UPF1, UPF2, and SFP1. SFP1 codes for a potentially prionogenic protein, which is enriched in Asn and Gln residues, and is thereby the most likely candidate for the [ISP+] structural gene. UPF1 and UPF2 code for components of nonsense-mediated mRNA decay. The [ISP+] elimination caused by UPF1 and UPF2 inactivation was reversible, and Upf1p and Upf2p were not functionally related to phosphatase Ppz1p, which influences the [ISP+] manifestation. Possible mechanisms sustaining the influence of UPF1 and UPF2 on [ISP+] maintenance are discussed.

Co-expression of RelA/p65 and ACTN4 induces apoptosis in non-small lung carcinoma cells

ABSTRACT Alpha-actinin 4 (ACTN4) is an actin-binding protein of the spectrin superfamily. ACTN4 is found both in the cytoplasm and nucleus of eukaryotic cells. The main function of cytoplasmic ACTN4 is stabilization of actin filaments and their binding to focal contacts. Nuclear ACTN4 takes part in the regulation of gene expression following by activation of certain transcription factors, but the mechanisms of regulation are not completely understood. Our previous studies have demonstrated the interaction of ACTN4 with the RelA/p65 subunit of NF-kappaB factor and the effect on its transcriptional activity in A431 and HEK293T cells. In the present work, we investigated changes in the composition of nuclear ACTN4-interacting proteins in non-small cell lung cancer cells H1299 upon stable RELA overexpression. We showed that ACTN4 was present in the nuclei of H1299 cells, regardless of the RELA expression level. The presence of ectopic RelA/p65 in H1299 cells increased the number of proteins interacting with nuclear ACTN4. Stable expression of RELA in these cells suppressed cell proliferation, which was further affected by simultaneous ACTN4 overexpression. We detected no significant effect on cell cycle but the apoptosis rate was increased in cells with a double RELA/ACTN4 overexpression. Interestingly, when expressed individually ACTN4 promoted proliferation of lung cancer cells. Furthermore, the bioinformatics analysis of gene expression in lung cancer patients suggested that overexpression of ACTN4 correlated with poor survival prognosis. We hypothesize that the effect of RELA on proliferation and apoptosis of H1299 cells can be mediated via affecting the interactome of ACTN4.