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Article ; Online: Interferon-

Krachmarova, Elena / Petkov, Peicho / Lilkova, Elena / Stoynova, Dayana / Malinova, Kristina / Hristova, Rossitsa / Gospodinov, Anastas / Ilieva, Nevena / Nacheva, Genoveva / Litov, Leandar

International journal of molecular sciences

2024 Volume 25, Issue 4

Abstract: 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 ...

Abstract	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.
MeSH term(s)	Humans ; COVID-19 ; Interferon-gamma/pharmacology ; Interferons/metabolism ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; SARS-CoV-2/metabolism ; Viral Proteins/drug effects ; Viral Proteins/metabolism
Chemical Substances	Interferon-gamma (82115-62-6) ; Interferons (9008-11-1) ; RNA, Messenger ; ORF6 protein, SARS-CoV-2 ; Viral Proteins
Language	English
Publishing date	2024-02-10
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms25042155
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Interferon-γ as a Potential Inhibitor of SARS-CoV-2 ORF6 Accessory Protein

Krachmarova, Elena / Petkov, Peicho / Lilkova, Elena / Stoynova, Dayana / Malinova, Kristina / Hristova, Rossitsa / Gospodinov, Anastas / Ilieva, Nevena / Nacheva, Genoveva / Litov, Leandar

bioRxiv

Abstract: 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 ... ...

Abstract	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 hIFNγ 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 hIFNγ 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 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.
Keywords	covid19
Language	English
Publishing date	2024-01-25
Publisher	Cold Spring Harbor Laboratory
Document type	Article ; Online
DOI	10.1101/2024.01.24.577015
Database	COVID19

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Article ; Online: Self-Association of Antimicrobial Peptides

Peicho Petkov / Elena Lilkova / Nevena Ilieva / Leandar Litov

International Journal of Molecular Sciences, Vol 20, Iss 21, p

A Molecular Dynamics Simulation Study on Bombinin

2019 Volume 5450

Abstract: 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 ... ...

Abstract	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.
Keywords	antimicrobial peptides ; self-association ; aggregation ; promotion of folding ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	612
Language	English
Publishing date	2019-11-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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

Petkov, Peicho / Lilkova, Elena / Ilieva, Nevena / Litov, Leandar

International journal of molecular sciences

2019 Volume 20, Issue 21

Abstract	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.
MeSH term(s)	Antimicrobial Cationic Peptides/chemistry ; Antimicrobial Cationic Peptides/metabolism ; Membrane Lipids/chemistry ; Membrane Lipids/metabolism ; Molecular Dynamics Simulation ; Protein Binding ; Protein Structure, Secondary ; Water/chemistry
Chemical Substances	Antimicrobial Cationic Peptides ; Membrane Lipids ; bombinin H2 ; Water (059QF0KO0R)
Language	English
Publishing date	2019-11-01
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms20215450
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Heparan Sulfate Facilitates Binding of hIFN

Miladinova, Elisaveta / Lilkova, Elena / Krachmarova, Elena / Malinova, Kristina / Petkov, Peicho / Ilieva, Nevena / Nacheva, Genoveva / Litov, Leandar

International journal of molecular sciences

2022 Volume 23, Issue 16

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Cell Membrane/metabolism ; Heparitin Sulfate/metabolism ; Humans ; Oligosaccharides ; Proteoglycans/metabolism ; Receptors, Cell Surface
Chemical Substances	Oligosaccharides ; Proteoglycans ; Receptors, Cell Surface ; Heparitin Sulfate (9050-30-0)
Language	English
Publishing date	2022-08-20
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms23169415
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Article ; Online: Insights into the SARS-CoV-2 ORF6 Mechanism of Action.

Krachmarova, Elena / Petkov, Peicho / Lilkova, Elena / Ilieva, Nevena / Rangelov, Miroslav / Todorova, Nadezhda / Malinova, Kristina / Hristova, Rossitsa / Nacheva, Genoveva / Gospodinov, Anastas / Litov, Leandar

International journal of molecular sciences

2023 Volume 24, Issue 14

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Humans ; Active Transport, Cell Nucleus ; COVID-19/genetics ; COVID-19/metabolism ; Cytoplasm ; SARS-CoV-2/genetics ; SARS-CoV-2/metabolism ; SARS-CoV-2/pathogenicity
Language	English
Publishing date	2023-07-18
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms241411589
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Article ; Online: Molecular modeling of the effects of glycosylation on the structure and dynamics of human interferon-gamma.

Lilkova, Elena / Petkov, Peicho / Ilieva, Nevena / Krachmarova, Elena / Nacheva, Genoveva / Litov, Leandar

Journal of molecular modeling

2019 Volume 25, Issue 5, Page(s) 127

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Glycoproteins/chemistry ; Glycosylation ; Humans ; Interferon-gamma/chemistry ; Interferon-gamma/genetics ; Models, Molecular ; Molecular Dynamics Simulation ; Polysaccharides/chemistry ; Protein Conformation, alpha-Helical ; Proteolysis
Chemical Substances	Glycoproteins ; IFNG protein, human ; Polysaccharides ; Interferon-gamma (82115-62-6)
Language	English
Publishing date	2019-04-25
Publishing country	Germany
Document type	Journal Article
ZDB-ID	1284729-X
ISSN	0948-5023 ; 1610-2940
ISSN (online)	0948-5023
ISSN	1610-2940
DOI	10.1007/s00894-019-4013-8
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Molecular Mechanism of the Anti-Inflammatory Action of Heparin.

Litov, Leandar / Petkov, Peicho / Rangelov, Miroslav / Ilieva, Nevena / Lilkova, Elena / Todorova, Nadezhda / Krachmarova, Elena / Malinova, Kristina / Gospodinov, Anastas / Hristova, Rossitsa / Ivanov, Ivan / Nacheva, Genoveva

International journal of molecular sciences

2021 Volume 22, Issue 19

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Anti-Inflammatory Agents/pharmacology ; Anticoagulants/pharmacology ; COVID-19/immunology ; Cell Line ; Heparin, Low-Molecular-Weight/pharmacology ; Humans ; Interferon-gamma/immunology ; Interleukin-6/immunology ; Models, Molecular ; Receptors, Interleukin-6/immunology ; SARS-CoV-2/drug effects ; SARS-CoV-2/immunology ; COVID-19 Drug Treatment
Chemical Substances	Anti-Inflammatory Agents ; Anticoagulants ; Heparin, Low-Molecular-Weight ; Interleukin-6 ; Receptors, Interleukin-6 ; Interferon-gamma (82115-62-6)
Language	English
Publishing date	2021-10-03
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms221910730
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Article ; Online: Molecular Mechanism of the Anti-Inflammatory Action of Heparin

Leandar Litov / Peicho Petkov / Miroslav Rangelov / Nevena Ilieva / Elena Lilkova / Nadezhda Todorova / Elena Krachmarova / Kristina Malinova / Anastas Gospodinov / Rossitsa Hristova / Ivan Ivanov / Genoveva Nacheva

International Journal of Molecular Sciences, Vol 22, Iss 10730, p

2021 Volume 10730

Abstract	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.
Keywords	molecular dynamics ; molecular modelling ; cytokine storm ; inflammation ; low-molecular-weight heparin (LMWH) ; IFNγ ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	540
Language	English
Publishing date	2021-10-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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

Krachmarova, Elena / Tileva, Milena / Lilkova, Elena / Petkov, Peicho / Maskos, Klaus / Ilieva, Nevena / Ivanov, Ivan / Litov, Leandar / Nacheva, Genoveva

BioMed research international

2017 Volume 2017, Page(s) 3018608

Abstract: In order to obtain glycosylated human interferon-gamma ( ... ...

Abstract	In order to obtain glycosylated human interferon-gamma (hIFN
Language	English
Publishing date	2017
Publishing country	United States
Document type	Journal Article
ZDB-ID	2698540-8
ISSN	2314-6141 ; 2314-6133
ISSN (online)	2314-6141
ISSN	2314-6133
DOI	10.1155/2017/3018608
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