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

The 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 to and immobilises the RAE1 protein on the cytoplasmic membranes, thereby blocking mRNA transport 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 hIFNγ binds the ORF6 protein with high affinity, thus impairing its interactions with RAE1 and, consequently, its activity in viral invasion. The in vitro studies reported here reveal a shift of the localisation of RAE1 in ORF6 overexpressing cells upon treatment with hIFNγ from predominantly cytoplasmic to mainly nuclear, resulting in the 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 hIFNγ 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 hIFNγ as a promising inhibitor of the most toxic SARS-CoV-2 protein.

Insights into the SARS-CoV-2 ORF6 Mechanism of Action.

ORF6 is responsible for suppressing the immune response of cells infected by the SARS-CoV-2 virus. It is also the most toxic protein of SARS-CoV-2, and its actions are associated with the viral pathogenicity. Here, we study in silico and in vitro the structure of the protein, its interaction with RAE1 and the mechanism of action behind its high toxicity. We show both computationally and experimentally that SARS-CoV-2 ORF6, embedded in the cytoplasmic membranes, binds to RAE1 and sequesters it in the cytoplasm, thus depleting its availability in the nucleus and impairing nucleocytoplasmic mRNA transport. This negatively affects the cellular genome stability by compromising the cell cycle progression into the S-phase and by promoting the accumulation of RNA-DNA hybrids. Understanding the multiple ways in which ORF6 affects DNA replication may also have important implications for elucidating the pathogenicity of SARS-CoV-2 and developing therapeutic strategies to mitigate its deleterious effects on host cells.

Heparan Sulfate Facilitates Binding of hIFNγ to Its Cell-Surface Receptor hIFNGR1.

Human interferon-gamma (hIFNγ) is a crucial signaling molecule with an important role in the initialization and development of the immune response of the host. However, its aberrant activity is also associated with the progression of a multitude of autoimmune and other diseases, which determines the need for effective inhibitors of its activity. The development of such treatments requires proper understanding of the interaction of hIFNγ to its cell-surface receptor hIFNGR1. Currently, there is no comprehensive model of the mechanism of this binding process. Here, we employ molecular dynamics simulations to study on a microscopic level the process of hIFNγ-hIFNGR1 complex formation in different scenarios. We find that the two molecules alone fail to form a stable complex, but the presence of heparan-sulfate-like oligosaccharides largely facilitates the process by both demobilizing the highly flexible C-termini of the cytokine and assisting in the proper positioning of its globule between the receptor subunits. An antiproliferative-activity assay on cells depleted from cell-surface heparan sulfate (HS) sulfation together with the phosphorylation levels of the signal transducer and activator of transcription STAT1 confirms qualitatively the simulation-based multistage complex-formation model. Our results reveal the key role of HS and its proteoglycans in all processes involving hIFNγ signalling.

Availability of technology for managing cancer patients in the Southeast European (SEE) region.

Background: The Southeast European (SEE) region of 10 countries and about 43 million people differs from Western Europe in that most SEE countries lack active cancer registries and have fewer diagnostic imaging devices and radiotherapy (RT) units. The main objective of this research is to initiate a common platform for gathering SEE regional cancer data from the ground up to help these countries develop common cancer management strategies. Methods: To obtain detailed on-the-ground information, we developed separate questionnaires for two SEE groups: a) ONCO - oncologists regarding cancer treatment modalities and the availability of diagnostic imaging and radiotherapy equipment; and b) REG - national radiation protection and safety regulatory bodies regarding diagnostic imaging and radiotherapy equipment in SEE facilities. Results: Based on responses from 13/17 ONCO participants (at least one from each country) and from 9/10 REG participants (all countries but Albania), cancer incidence rates are higher in those SEE countries that have greater access to diagnostic imaging equipment while cancer mortality-to-incidence (MIR) ratios are higher in countries that lack radiotherapy equipment. Conclusion: By combining unique SEE region information with data available from major global databases, we demonstrated that the availability of diagnostic imaging and radiotherapy equipment in the SEE countries is related to their economic development. While immediate diagnostic imaging and radiation therapy capacity building is necessary, it is also essential to develop both national and SEE-regional cancer registries in order to understand the heterogeneity of each country's needs and to establish regional collaborative strategies for combating cancer.

Molecular Mechanism of the Anti-Inflammatory Action of Heparin.

Our objective is to reveal the molecular mechanism of the anti-inflammatory action of low-molecular-weight heparin (LMWH) based on its influence on the activity of two key cytokines, IFNγ and IL-6. The mechanism of heparin binding to IFNγ and IL-6 and the resulting inhibition of their activity were studied by means of extensive molecular-dynamics simulations. The effect of LMWH on IFNγ signalling inside stimulated WISH cells was investigated by measuring its antiproliferative activity and the translocation of phosphorylated STAT1 in the nucleus. We found that LMWH binds with high affinity to IFNγ and is able to fully inhibit 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-6Rα, thus preventing the formation of the IL-6/IL-6Rα/gp130 signalling complex. These findings shed light on the molecular mechanism of the anti-inflammatory action of LMWH and underpin its ability to influence favourably conditions characterised by overexpression of these two cytokines. Such conditions are not only associated with autoimmune diseases, but also with inflammatory processes, in particular with COVID-19. Our results put forward heparin as a promising means for the prevention and suppression of severe CRS and encourage further investigations on its applicability as an anti-inflammatory agent.

Self-Association of Antimicrobial Peptides: A Molecular Dynamics Simulation Study on Bombinin.

Antimicrobial peptides (AMPs) are a diverse group of membrane-active peptides which play a crucial role as mediators of the primary host defense against microbial invasion. Many AMPs are found to be fully or partially disordered in solution and to acquire secondary structure upon interaction with a lipid membrane. Here, we report molecular dynamics simulations studies on the solution behaviour of a specific AMP, bombinin H2. We show that in monomeric form in water solution the peptide is somewhat disordered and preferably adopts a helix-loop-helix conformation. However, when more than a single monomer is placed in the solution, the peptides self-associate in aggregates. Within the aggregate, the peptides provide each other with an amphipathic environment that mimics the water-membrane interface, which allows them to adopt a single-helix structure. We hypothesise that this is the mechanism by which bombinin H2 and, possibly, other small linear AMPs reach the target membrane in a functional folded state and are able to effectively exert their antimicrobial action on it.

Molecular modeling of the effects of glycosylation on the structure and dynamics of human interferon-gamma.

Natural hIFNγ is a glycoprotein with two N-glycosylation sites in each monomer chain, which are independently and differentially glycosylated. Although glycosylation is not necessary for the activity of the cytokine, it was proposed that it protects the cytokine from proteolytic degradation and thus extends its circulatory half-life. Here, we report the development of model structures of glycosylated full-length native hIFNγ homodimers. Our aim is to shed light on the mechanism through which glycosylation preserves the integrity of the cytokine molecule. To this end, we employ molecular dynamics simulations to study the interaction of the carbohydrate chains with the receptor-binding sites in the cytokine and with its flexible highly positively charged C-termini. The glycans interact primarily with the globular part of the protein, but also occasionally form contacts with the solvent-exposed and sensitive to proteases C-terminal tails. We show that the glycans restrict the C-termini wagging motion into the solvent, limit their flexibility and keep them closer to the α-helical globule of hIFNγ, thus possibly protecting them from proteolytic processing.

His-FLAG Tag as a Fusion Partner of Glycosylated Human Interferon-Gamma and Its Mutant: Gain or Loss?

In order to obtain glycosylated human interferon-gamma (hIFNγ) and its highly prone to aggregation mutant K88Q, a secretory expression in insect cells was employed. To facilitate recombinant proteins purification, detection, and stability the baculovirus expression vectors were constructed to bear N-terminal His6-FLAG tag. Although the obtained proteins were glycosylated, we found that their biological activity was 100 times lower than expected. Our attempts to recover the biological properties of both proteins by tag removal failed due to enterokinase resistance of the tag. Surprisingly, the tag was easily cleaved when the proteins were expressed in E. coli cells and the tag-free proteins showed fully restored activity. To shed light on this phenomenon we performed molecular dynamics simulations. The latter showed that the tags interact with the receptor binding domains and the flexible C-termini of the fusion proteins thus suppressing their complex formation with the hIFNγ receptor. We hypothesize that in the case of glycosylated proteins the tag/C-terminal interaction positions the FLAG peptide in close proximity to the glycans thus sterically impeding the enterokinase access to its recognition site.