Biology subjects

Brazda, V.

Publications and source records attributed to Brazda, V..

7 recordsLinked to original sources

bioRxiv2026

Unraveling Viral peptide-G4 Interactions: the NS3 Protease Domain of Yellow Fever Virus Binds G-Quadruplexes with High Specificity and Affinity

G-quadruplexes (G4s) are critical nucleic acid secondary structures that play pivotal roles in regulating gene expression. In this study, we conducted a proteome-wide in silico analysis across multiple viruses causing hemorrhagic fevers to identify candidate proteins containing a conserved G4-binding motif. Four peptides belonging to Marburg, Ebola, Hantaan and Yellow fever viruses were shown to bind to G4 in vitro. We selected the NS3 protease domain of Yellow Fever virus for further validation. Biochemical assays demonstrated that the NS3 protease domain binds G4 structures with high specificity and affinity, particularly favoring the parallel conformation. Molecular docking and simulations further revealed that the NS3 protease domain interacts with the terminal G-tetrads and loop regions of G4 via key residues, including PHE40, adopting an insertion and stacking composite binding mode. These findings expand our understanding of virus - G4 interactions and offer novel potential targets for G4-based antiviral strategies. Bullet points- We screened viruses causing hemorrhagic fevers for potential G4-binding peptides. - Four peptides belonging to Marburg, Ebola, Hantaan and Yellow fever viruses were shown to bind to G4 in vitro. - Biochemical assays demonstrated that the NS3 protease domain of YFV binds G4 structures with high specificity and affinity.

biophysics↗

bioRxiv2024

G-quadruplexes in Haloferax volcanii

Archaea, a domain of microorganisms found in diverse environments including the human microbiome, represent the closest known prokaryotic relatives of eukaryotes. This phylogenetic proximity positions them as a relevant model for investigating the evolutionary origins of nucleic acid secondary structures such as G-quadruplexes (G4s), which play regulatory roles in transcription and replication. Although G4s have been extensively studied in eukaryotes, their presence and function in archaea remain poorly characterized. In this study, a genome-wide analysis of the halophilic archaeon Haloferax volcanii identified over 5, 800 potential G4-forming sequences. Biophysical validation confirmed that many of these sequences adopt stable G4 conformations in vitro. Using G4-specific detection tools and super-resolution microscopy, G4 structures were visualized in vivo in both DNA and RNA across multiple growth phases. Comparable findings were observed in the thermophilic archaeon Thermococcus barophilus. Functional analysis using helicase-deficient H. volcanii strains further identified candidate enzymes involved in G4 resolution. These results establish H. volcanii as a tractable archaeal model for G4 biology.

molecular biology↗

bioRxiv2023

The presence of a G4 prone sequence upstream of a minimal promoter increases transcriptional activity in the yeast S. cerevisiae

Non-canonical secondary structures in DNA are increasingly being revealed as critical players in DNA metabolism, including modulating the accessibility and activity of promoters. These structures comprise the so-called G-quadruplexes (G4s) that are formed from sequences rich in guanine bases. Using a well-defined transcriptional reporter system, we sought to systematically investigate the impact of the presence of G4 structures on transcription in yeast S. cerevisiae. To this aim, different G4 prone sequences were modeled to vary the chance of intramolecular G4 formation, analyzed in vitro by Thioflavin T binding test and circular dichroism and then placed at the yeast ADE2 locus on chromosome XV, downstream and adjacent to a P53 response element (RE) and upstream from a minimal CYC1 promoter and Luciferase 1 (LUC1) reporter gene in isogenic strains. While the minimal CYC1 promoter provides for basal reporter activity, the P53 RE enables LUC1 transactivation under the control of the human P53 family proteins expressed under the inducible GAL1 promoter. Thus, the impact of the different G4 prone sequences on both basal and P53 family proteins dependent expression was measured after shifting the yeast cells onto galactose containing medium. The results showed that the presence of G4 prone sequences upstream of a yeast minimal promoter can increase its basal activity proportionally to their potential to form intramolecular G4 structures; consequently, this improved accessibility, when present near the target binding site of P53 family transcription factors can be exploited in order to regulate the transcriptional activity of P53, P63 and P73 proteins.

cancer biology↗

bioRxiv2020

Unheeded SARS-CoV-2 protein? Look deep into negative-sense RNA

SARS-CoV-2 is a novel positive-sense single-stranded RNA virus from the Coronaviridae family (genus Betacoronavirus), which has been established as causing the COVID-19 pandemic. The genome of SARS-CoV-2 is one of the largest among known RNA viruses, comprising of at least 26 known protein-coding loci. Studies thus far have outlined the coding capacity of the positive-sense strand of the SARS-CoV-2 genome, which can be used directly for protein translation. However, it has been recently shown that transcribed negative-sense viral RNA intermediates that arise during viral genome replication from positive-sense viruses can also code for proteins. No studies have yet explored the potential for negative-sense SARS-CoV-2 RNA intermediates to contain protein coding-loci. Thus, using sequence and structure-based bioinformatics methodologies, we have investigated the presence and validity of putative negative-sense ORFs (nsORFs) in the SARS-CoV-2 genome. Nine nsORFs were discovered to contain strong eukaryotic translation initiation signals and high codon adaptability scores, and several of the nsORFs were predicted to interact with RNA-binding proteins. Evolutionary conservation analyses indicated that some of the nsORFs are deeply conserved among related coronaviruses. Three-dimensional protein modelling revealed the presence of higher order folding among all putative SARS-CoV-2 nsORFs, and subsequent structural mimicry analyses suggest similarity of the nsORFs to DNA/RNA-binding proteins and proteins involved in immune signaling pathways. Altogether, these results suggest the potential existence of still undescribed SARS-CoV-2 proteins, which may play an important role in the viral lifecycle and COVID-19 pathogenesis. Contactpetr.pecinka@osu.cz; tlb20@cam.ac.uk

bioinformatics↗

bioRxiv2020

Cross kingdom analysis of putative quadruplex-forming sequences in fungal genomes: novel antifungal targets to ameliorate fungal pathogenicity?

Fungi contribute to upwards of 1.5 million human deaths annually, are involved in the spoilage of up to a third of food crops, and have a devastating effect on plant and animal biodiversity. Moreover, this already significant issue is exacerbated by a rise in antifungal resistance and a critical requirement for novel drug targets. Quadruplexes are four-stranded secondary structures in nucleic acids which can regulate processes such as transcription, translation, replication, and recombination. They are also found in genes linked to virulence in microbes, and quadruplex-binding ligands have been demonstrated to eliminate drug resistant pathogens. Using a computational approach, we identified putative quadruplex-forming sequences (PQS) in 1362 genomes across the fungal kingdom and explored their potential involvement in virulence, drug resistance, and pathogenicity. Here we present the largest analysis of PQS in fungi and identified significant heterogeneity of these sequences throughout phyla, genera, and species. Moreover, PQS were genetically conserved. Notably, loss of PQS in cryptococci and aspergilli was associated with pathogenicity. PQS in the clinically important pathogens Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans were located within genes (particularly coding regions), mRNA, repeat regions, mobile elements, tRNA, ncRNA, rRNA, and the centromere. Genes containing PQS in these organisms were found to be primarily associated with metabolism, nucleic acid binding, transporter activity, and protein modification. Finally, PQS were found in over 100 genes associated with virulence, drug resistance, or key biological processes in these pathogenic fungi and were found in genes which were highly upregulated during germination, hypoxia, oxidative stress, iron limitation, and in biofilms. Taken together, quadruplexes in fungi could present interesting novel targets to ameliorate fungal virulence and overcome drug resistance.

microbiology↗

bioRxiv2020

The changes in the p53 protein across the animal kingdom pointing to its involvement in longevity

Recently, the quest for the mythical fountain of youth has turned into extensive research programs aiming to extend the healthy lifespan in humans. Despite advances in our understanding of the aging process, the surprisingly extended lifespan and cancer resistance of some animal species remains unexplained. The p53 protein plays a crucial role in tumor suppression and in tissue homeostasis and aging. Long-lived, cancer-free African elephants, have 20 copies of TP53 gene including 19 retrogenes (38 alleles) which are partially active, whereas humans possess only one copy of TP53 and have an estimated cancer mortality of 11-25%. The mechanism through which p53 contributes to the resolution of the Petos paradox in the Animalia remains vague. Thus, in this work, we took advantage of the available datasets and inspected the p53 amino acid sequence of phylogenetically related organisms that show variations in the lifespan. We discovered new correlations between specific amino acid deviations in p53 and the lifespans across different animal species. We found that species with extended lifespan have certain characteristic amino acid substitutions in the p53 DNA binding domain that alter its function as depicted from the Phenotypic Annotation of p53 Mutations, using PROVEAN tool or SWISS-MODEL workflow. Our findings show a direct association between specific amino acid residues in p53 protein, changes in p53 functionality and the extended animal lifespan, and further highlight the importance of p53 protein in aging.

bioinformatics↗

bioRxiv2020

In-depth Bioinformatic Analyses of Human SARS-CoV-2, SARS-CoV, MERS-CoV, and Other Nidovirales Suggest Important Roles of Noncanonical Nucleic Acid Structures in Their Lifecycles

Noncanonical nucleic acid structures play important roles in the regulation of molecular processes. Considering the importance of the ongoing coronavirus crisis, we decided to evaluate genomes of all coronaviruses sequenced to date (stated more broadly, the order Nidovirales) to determine if they contain noncanonical nucleic acid structures. We discovered much evidence of putative G-quadruplex sites and even much more of inverted repeats (IRs) loci, which in fact are ubiquitous along the whole genomic sequence and indicate a possible mechanism for genomic RNA packaging. The most notable enrichment of IRs was found inside 5'UTR for IRs of size 12+ nucleotides, and the most notable enrichment of putative quadruplex sites (PQSs) was located before 3'UTR, inside 5'UTR, and before mRNA. This indicates crucial regulatory roles for both IRs and PQSs. Moreover, we found multiple G-quadruplex binding motifs in human proteins having potential for binding of SARS-CoV-2 RNA. Noncanonical nucleic acids structures in Nidovirales and in novel SARS-CoV-2 are therefore promising druggable structures that can be targeted and utilized in the future.

bioinformatics↗