Biology subjects

Petkov, P.

Publications and source records attributed to Petkov, P..

3 recordsLinked to original sources

bioRxiv2024

Interferon-γ as a Potential Inhibitor of SARS-CoV-2 ORF6 Accessory Protein

ORF6 protein of the SARS-CoV-2 virus plays a crucial role in blocking the innate immune response of the infected cells by inhibiting interferon pathways. Additionally, it binds and immobilises the RAE1 protein onto the cytoplasmic membranes, thereby blocking the transport of mRNA from the nucleus to the cytoplasm. In all these cases the host cell proteins are tethered by the flexible C-terminus of ORF6. A possible strategy to inhibit the biological activity of ORF6 is to bind its C-terminus with suitable ligands. Our in silico experiments suggest that hIFN{gamma} binds the ORF6 protein with high affinity, thus impairing its interactions with RAE1 and, consequently, its activity in viral invasion. The here reported in vitro studies reveal a shift of the localization of RAE1 in ORF6 overexpressing cells upon treatment with hIFN{gamma} from predominantly cytoplasmic to mainly nuclear, resulting in restoration of the export of mRNA from the nucleus. We also explored the expression of GFP in transfected with ORF6 cells by means of fluorescence microscopy and qRT-PCR, finding that treatment with hIFN{gamma} unblocks the mRNA trafficking and reinstates the GFP expression level. The ability of the cytokine to block ORF6 is also reflected in minimising its negative effects on DNA replication by reducing accumulated RNA-DNA hybrids. Our results, therefore, suggest hIFN{gamma} as a promising inhibitor of the most toxic SARS-CoV-2 protein.

molecular biology↗

bioRxiv2020

Heparin as an Anti-Inflammatory Agent

Timely control of the cytokine release syndrome (CRS) at the severe stage of COVID-19 is key to improving the treatment success and reducing the mortality rate. The inhibition of the activity of the two key cytokines, IFN{gamma} and IL-6, can significantly reduce or even reverse the development of the cytokine storm. The objective of our investigations is to reveal the anti-inflammatory potential of heparin for prevention and suppression of the development of CRS in acute COVID-19 patients. The effect of low-molecular-weight heparin (LMWH) on IFN{gamma} signalling inside the stimulated WISH cells was investigated by measuring its antiproliferative activity and the translocation of phosphorylated STAT1 in the nucleus. The mechanism of heparin binding to IFN{gamma} and IL-6 and therefore inhibition of their activity was studied by means of extensive molecular-dynamics simulations. We find that LMWH binds with high affinity to IFN{gamma} and is able to inhibit fully the interaction with its cellular receptor. It also influences the biological activity of IL-6 by binding to either IL-6 or IL-6/IL-6R thus preventing the formation of the IL-6/IL-6R/gp130 signaling complex. Our conclusion is that heparin is a potent anti-inflammatory agent that can be used in acute inflammatory conditions, due to its potential to inhibit both IFN {gamma} and IL-6 signalling pathways. Based on our results and available clinical observations, we suggest the administration of LMWH to COVID-19 patients in the initial stages of the acute phase. The beginning of the treatment and the dosage should be based on a careful follow-up of the platelet count and the D-dimer, IL-6, IFN, T-cells, and B-cells levels.

molecular biology↗

bioRxiv2019

Why Do Long Zinc Finger Proteins have Short Motifs? A case study of ZFY and CTCF reveals non-independent recognition of tandem zinc finger proteins.

The human genome contains around 800 C2H2 Zinc Finger Proteins (ZFPs), and many of them are composed of long tandem arrays of zinc fingers. Current motif prediction models assume longer finger arrays correspond to longer DNA-binding motifs and higher specificity. However, recent experimental efforts to identify ZFP binding sites in vivo contradict this assumption, with many having short motifs. Here, we systematically test how multiple zinc fingers contribute to binding for three model ZFPs: Zinc Finger Y (ZFY), CTCF, and ZNF343. Using ZFY, which contains 13 fingers, we quantitatively characterize its binding specificity with several methods, including Affinity-seq, HT-SELEX, Spec-seq and fluorescence anisotropy, and find evidence for dependent recognition where downstream fingers can recognize some extended motifs only in the presence of an intact core site. For the genomic insulator CTCF, additional high-throughput affinity measurements reveal that its upstream specificity profile depends on the strength of the core, violating presumed additivity and positionindependence. Moreover, the effect of different epigenetic modifications within the core site depends on the strength of flanking upstream site, providing new insight into how the previously identified intellectual disability-causing and cancer-related mutant R567W disrupts upstream recognition and deregulates CTCFs methylation sensitivity. Lastly, we used ZNF343 as example to show that a simple iterative motif analysis strategy based on a small set of prefixed cores can reveal the dependent relationship between cores and upstream motifs. These results establish that the current underestimation of ZFPs motif lengths is due to our lack of understanding of intrinsic properties of tandem zinc finger recognition, including irregular motif structure, variable spacing, and dependent recognition between sub-motifs. These results also motivate a need for better recognition models beyond additive, position-weight matrix to predict ZFP specificities, occupancies, and the molecular mechanisms of disease mutations.

genetics↗