Genoveva Nacheva - Academia.edu (original) (raw)

Papers by Genoveva Nacheva

Research paper thumbnail of RESEARCH ARTICLE Open Access Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli

Background: Segregation of expression plasmids leads to loss of recombinant DNA from transformed ... more Background: Segregation of expression plasmids leads to loss of recombinant DNA from transformed bacterial cells due to the irregular distribution of plasmids between the daughter cells during cell division. Under nonselective conditions this segregational instability results in a heterogeneous population of cells, where the nonproductive plasmid-free cells overgrow the plasmid-bearing cells thus decreasing the yield of recombinant protein. Amongst the factors affecting segregational plasmid instability are: the plasmid design, plasmid copy-number, host cell genotype, fermentation conditions etc. This study aims to investigate the influence of transcription and translation on the segregation of recombinant plasmids designed for constitutive gene expression in Escherichia coli LE392 at glucose-limited continuous cultivation. To this end a series of pBR322-based plasmids carrying a synthetic human interferon-gamma (hIFNg) gene placed under the control of different regulatory elements ...

Research paper thumbnail of Segregation of bacterial plasmids expressing 3’-end truncated human interferon-gamma genes in E. coli cells

Research paper thumbnail of Effects of human interferon-gamma gene expression on ColE1-like plasmid segregation in Escherichia coli

Research paper thumbnail of Computational Modelling of the Full Length hIFN- \gamma γ Homodimer

Human interferon gamma (hIFN-\(\gamma \)) is an important signalling molecule, which plays a key ... more Human interferon gamma (hIFN-\(\gamma \)) is an important signalling molecule, which plays a key role in the formation and modulation of immune response. The controversial conclusions concerning the function of hIFN-\(\gamma \) C-termini as well as the lack of structural information about this domain motivated us to perform molecular dynamics simulations in order to model the structure of the hIFN-\(\gamma \) C-terminal part. The simulations were carried out with the CHARMM22 force field, starting from a fully extended conformation of the C-termini. They showed unambiguously that the C-termini tend to approach the globular part of the protein, so that the whole hIFN-\(\gamma \) molecule adopts a more compact conformation. The energetic favourability of the more compact conformations of the whole cytokine was also confirmed by means of free energy perturbation simulations.

Research paper thumbnail of Characterization of functional domains of human interferon gamma by specific monoclonal antibodies

Folia biologica, 2002

For the study of functional domains of hIFNgamma, two MABs were characterized. Both MABs, named 1... more For the study of functional domains of hIFNgamma, two MABs were characterized. Both MABs, named 1E11 and 5D6, were sequence-specific for hIFNgamma. According to the estimated additivity index, they recognized epitopes located at distinct non-overlapping areas of the hIFNgamma molecule. When pre-incubated with hIFNgamma, MAB 1E11 was able to neutralize the antiviral as well as the antiproliferative activity of the cytokine. This indicated that MAB 1E11 was specific either for the domain responsible for binding to the cell receptor or for a domain required for both functions. By contrast, the second MAB 5D6 did not interfere with any of the two hIFNgamma biological activities. The epitope of MAB 5D6 was located between the amino acid residues Leu 135 and Glu 143 by using different forms of C-terminally truncated hIFNgamma. These data allow the conclusion that the last nine C-terminal amino acids are not essential for the receptor binding and biological functioning of this cytokine. Th...

Research paper thumbnail of Molecular modeling of the effects of glycosylation on the structure and dynamics of human interferon-gamma

Journal of Molecular Modeling, 2019

Natural hIFNγ is a glycoprotein with two N-glycosylation sites in each monomer chain, which are i... more 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.

Research paper thumbnail of Buffer and additive thermofluor screening of wild type human interferon gamma and mutant proteins

Thermochimica Acta, 2017

Abstract Human interferon gamma (hIFNγ) plays a key role in the immune system and therefore this ... more Abstract Human interferon gamma (hIFNγ) plays a key role in the immune system and therefore this cytokine has many current and future therapeutic applications. hIFNγ is well known with its aggregation propensity and despite its clinical use, there is not much data about the stabilization of hIFNγ preparations. Nowadays, substantial evidence indicates that the pathogenesis of many autoimmune diseases is related to overproduction of hIFNγ. In this regard we have developed inactive hIFNγ analogues to act as receptor antagonists of the endogenous hIFNγ. Since they show even higher tendency for aggregation than the wild type protein, we designed two-step thermofluor screen of 61 buffer conditions to identify the best storage solution for all investigated proteins to be used in the form of research samples or biopharmaceuticals. Tris buffer pH 8.0 supplemented with NaCl and trehalose/betaine/glycerol as additives showed to be the most appropriate choice ensuring high solubility and thermal stability of both hIFNγ and its mutants.

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

BioMed research international, 2017

In order to obtain glycosylated human interferon-gamma (hIFNγ) and its highly prone to aggregatio... more 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 recepto...

Research paper thumbnail of Inhibitor of Endogenous Human Interferon-Gamma

Research paper thumbnail of 258 a New Approach to Cope with Autoimmune Diseases: Computer Simulations and Laboratory Tests

Radiotherapy and Oncology, 2012

data files for 10 incident protons with energies ranging from 100 MeV to 230 MeV, although only r... more data files for 10 incident protons with energies ranging from 100 MeV to 230 MeV, although only results for 150 MeV are reported here. Spectra of emitted prompt gamma particles as well as depth emission profiles were obtained. Those plots were filtered using different constraints to better understand the processes involved in the production of prompt gamma rays produced by proton beams. In first place, the plots were filtered by energy of the prompt gamma particles to study which energy produce better correlation with dose distributions. The energy ranges employed were Eγ 10 MeV. A second filter was the type of parent particle that led to the production of the prompt gamma, differentiating among proton, electron, positron and neutron. Finally, a filter by type of interaction that produced the prompt gamma is presented splitting profiles by inelastic, bremsstrahlung, neutron capture and annihilation. Results: Figure 1 shows the results obtained of the prompt gamma produced by a 150 MeV proton beam in the soft tissue phantom. Figure 1 shows the energy spectra (d,e) and depth profiles (a,b,c) of produced prompt gammas differentiating between range of energy (a), type of particles (b) and type of interactions (c) that produce them. Energy spectra are in very good agreement with those found in the literature. Depth profiles show the best correlation with the dose profile for higher energy photons (3 MeV < Eγ < 10 MeV). Prompt gammas produced by protons are well correlated to the dose profile while neutrons and electrons show no clear peak. Prompt gammas produced by electrons can be mostly filtered by selecting energies greater than 1 MeV while the gammas produced by neutrons have an energy similar to those produced by protons. Inelastic interactions are dominant in the production of prompt gammas and show good correlation with dose profile. Bremsstrahlung and neutron capture contributes to the background with no information of the distal falloff position.

Research paper thumbnail of Metadynamics study of mutant human interferon-gamma forms

Computers & Mathematics with Applications, 2012

Human interferon-gamma (hIFN-γ) is an important antiviral and immunomodulating signaling molecule... more Human interferon-gamma (hIFN-γ) is an important antiviral and immunomodulating signaling molecule. The upregulation of its production, however, is related to the etiology of certain autoimmune diseases. In the search for a mechanism for suppressing the hIFNγ biological activity, we investigated the possibility to obtain mutant derivatives of the protein, capable to bind to hIFN-γ cellular receptors, but lacking the ability to trigger the biological response inside the cell. In order to preserve the affinity to the receptor, the introduced mutations should not induce conformational changes in the secondary structure of the mutants. Molecular dynamics simulations were performed to study the secondary structure of 100 randomly chosen hIFN-γ derivatives with substitutions in amino acids 86-88. The stability of the local structure of all hIFN-γ forms was investigated by means of metadynamics. It was found that some of the mutated forms preserve the local secondary structure and show similar or higher stability of the mutated helix, compared to the native form. The 12 most promising mutants were suggested for experimental investigation.

Research paper thumbnail of Additional file 1 of Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma

Additional file 1: Fig. S1. UV spectra of nucleic acids isolated from purified IBs by phenol–chlo... more Additional file 1: Fig. S1. UV spectra of nucleic acids isolated from purified IBs by phenol–chloroform extraction and precipitation with ethanol. The probe was analysed by NanoDrop®. Fig. S2. UV spectra of RNA isolated from purified IBs by TRIzol® and analysed by NanoDrop®.

Research paper thumbnail of Molecular Mechanism of the Anti-Inflammatory Action of Heparin

International Journal of Molecular Sciences

Our objective is to reveal the molecular mechanism of the anti-inflammatory action of low-molecul... more 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...

Research paper thumbnail of Heparin as an Anti-Inflammatory Agent

ABSTRACTTimely control of the cytokine release syndrome (CRS) at the severe stage of COVID-19 is ... more ABSTRACTTimely 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, IFNγ 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 IFNγ 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 IFNγ 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 IFNγ and is able to inhibit fully the interaction with its cellular re...

Research paper thumbnail of Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma

Microbial Cell Factories

Background Inclusion bodies (IBs) are protein aggregates in recombinant bacterial cells containin... more Background Inclusion bodies (IBs) are protein aggregates in recombinant bacterial cells containing mainly the target recombinant protein. Although it has been shown that IBs contain functional proteins along with protein aggregates, their direct application as pharmaceuticals is hindered by their heterogeneity and hazardous contaminants with bacterial origin. Therefore, together with the production of soluble species, IBs remain the main source for manufacture of recombinant proteins with medical application. The quality and composition of the IBs affect the refolding yield and further purification of the recombinant protein. The knowledge whether nucleic acids are genuine components or concomitant impurities of the IBs is a prerequisite for the understanding of the IBs formation and for development of optimized protocols for recombinant protein refolding and purification. IBs isolated from Escherichia coli overexpressing human interferon-gamma (hIFNγ), a protein with therapeutic ap...

Research paper thumbnail of Segregational Instability in E. Coli of Expression Plasmids Carrying Human Interferon Gamma Gene and its 3’-End Truncated Variants

Biotechnology & Biotechnological Equipment, 2009

Bertani medium, where the cell cultures were maintained at an exponential growth phase. To descri... more Bertani medium, where the cell cultures were maintained at an exponential growth phase. To describe the population dynamics, a mathematical model proposed by Stewart & Levin was used and the parameters Δ (difference in specific growth rate between plasmid-free and plasmid-harbouring cells) and θ (relative plasmid loss rate) were determined for each plasmid. The obtained results showed that any variation in the 3'-end of the hIFNγ-gene strongly affected plasmid instability. We also found that the relative plasmid loss rate θ was proportional to the hIFNγ-mRNA content in the cell. Based on these results, we conclude that the structure of the constitutively expressed gene is of great importance for the segregational plasmid instability.

Research paper thumbnail of Identification of two RNA-binding proteins in Balbiani ring premessenger ribonucleoprotein granules and presence of these proteins in specific subsets of heterogeneous nuclear ribonucleoprotein particles

Molecular and Cellular Biology

Balbiani ring (BR) granules are premessenger ribonucleoprotein particles (RNPs) generated in gian... more Balbiani ring (BR) granules are premessenger ribonucleoprotein particles (RNPs) generated in giant chromosomal puffs, the BRs, in the larval salivary glands of the dipteran chironomus tentans. Monoclonal antibodies were raised against nuclear proteins collected on a single-stranded-DNA-agarose affinity column, and two of them were used to identify RNA-binding proteins in BR granules. First, in Western blots (immunoblots), one of the antibodies recognized a 36-kDa protein and the other recognized a 45-KDa protein. Second, both antibodies bound to the BRs in immunocytological experiments. It was shown in cross-linking experiments that the two proteins are associated with heterogeneous nuclear RNP (hnRNP) complexes extracted from C. tentans nuclei. By immunoelectron microscopy of isolated and partly unfolded BR RNPs, it was specifically demonstrated that the BR granules contain the two proteins and, in addition, that both proteins are distributed frequently along the RNP fiber of the p...

Research paper thumbnail of Characterization of functional domains of human interferon gamma by specific monoclonal antibodies

Folia biologica, 2002

For the study of functional domains of hIFNgamma, two MABs were characterized. Both MABs, named 1... more For the study of functional domains of hIFNgamma, two MABs were characterized. Both MABs, named 1E11 and 5D6, were sequence-specific for hIFNgamma. According to the estimated additivity index, they recognized epitopes located at distinct non-overlapping areas of the hIFNgamma molecule. When pre-incubated with hIFNgamma, MAB 1E11 was able to neutralize the antiviral as well as the antiproliferative activity of the cytokine. This indicated that MAB 1E11 was specific either for the domain responsible for binding to the cell receptor or for a domain required for both functions. By contrast, the second MAB 5D6 did not interfere with any of the two hIFNgamma biological activities. The epitope of MAB 5D6 was located between the amino acid residues Leu 135 and Glu 143 by using different forms of C-terminally truncated hIFNgamma. These data allow the conclusion that the last nine C-terminal amino acids are not essential for the receptor binding and biological functioning of this cytokine. Th...

Research paper thumbnail of Identification of two RNA-binding proteins in Balbiani ring premessenger ribonucleoprotein granules and presence of these proteins in specific subsets of heterogeneous nuclear ribonucleoprotein particles

Molecular and cellular biology, 1996

Balbiani ring (BR) granules are premessenger ribonucleoprotein particles (RNPs) generated in gian... more Balbiani ring (BR) granules are premessenger ribonucleoprotein particles (RNPs) generated in giant chromosomal puffs, the BRs, in the larval salivary glands of the dipteran chironomus tentans. Monoclonal antibodies were raised against nuclear proteins collected on a single-stranded-DNA-agarose affinity column, and two of them were used to identify RNA-binding proteins in BR granules. First, in Western blots (immunoblots), one of the antibodies recognized a 36-kDa protein and the other recognized a 45-KDa protein. Second, both antibodies bound to the BRs in immunocytological experiments. It was shown in cross-linking experiments that the two proteins are associated with heterogeneous nuclear RNP (hnRNP) complexes extracted from C. tentans nuclei. By immunoelectron microscopy of isolated and partly unfolded BR RNPs, it was specifically demonstrated that the BR granules contain the two proteins and, in addition, that both proteins are distributed frequently along the RNP fiber of the p...

Research paper thumbnail of Role of the C-terminal chain in human interferongamma stability: an electrostatic study

Proteins, 2001

Electrostatic interactions in two structures of human interferon gamma (hIFNgamma), corresponding... more Electrostatic interactions in two structures of human interferon gamma (hIFNgamma), corresponding to interferon molecule alone and bound to its receptor, were analyzed on the basis of a continuum dielectric model. It was found that a number of titratable groups, mainly basic, show large pK shifts and remain in their neutral forms at physiologically relevant pH. The fact that these groups are largely common to both structures and that most of them belong to the set of most conserved sites suggests that this is a property inherent to the hIFNgamma molecule rather than an artifact of the crystal packing. His111 was also found deprotonated at neutral pH. It was concluded that receptor recognition involving His111 is driven by aromatic coupling of His111 and Tyr52 from the receptor rather than by electrostatic interactions. The structure corresponding to hIFNgamma in complex with its receptor shows a reduction in number and in degree of desolvation of the buried titratable sites. This fi...

Research paper thumbnail of RESEARCH ARTICLE Open Access Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli

Background: Segregation of expression plasmids leads to loss of recombinant DNA from transformed ... more Background: Segregation of expression plasmids leads to loss of recombinant DNA from transformed bacterial cells due to the irregular distribution of plasmids between the daughter cells during cell division. Under nonselective conditions this segregational instability results in a heterogeneous population of cells, where the nonproductive plasmid-free cells overgrow the plasmid-bearing cells thus decreasing the yield of recombinant protein. Amongst the factors affecting segregational plasmid instability are: the plasmid design, plasmid copy-number, host cell genotype, fermentation conditions etc. This study aims to investigate the influence of transcription and translation on the segregation of recombinant plasmids designed for constitutive gene expression in Escherichia coli LE392 at glucose-limited continuous cultivation. To this end a series of pBR322-based plasmids carrying a synthetic human interferon-gamma (hIFNg) gene placed under the control of different regulatory elements ...

Research paper thumbnail of Segregation of bacterial plasmids expressing 3’-end truncated human interferon-gamma genes in E. coli cells

Research paper thumbnail of Effects of human interferon-gamma gene expression on ColE1-like plasmid segregation in Escherichia coli

Research paper thumbnail of Computational Modelling of the Full Length hIFN- \gamma γ Homodimer

Human interferon gamma (hIFN-\(\gamma \)) is an important signalling molecule, which plays a key ... more Human interferon gamma (hIFN-\(\gamma \)) is an important signalling molecule, which plays a key role in the formation and modulation of immune response. The controversial conclusions concerning the function of hIFN-\(\gamma \) C-termini as well as the lack of structural information about this domain motivated us to perform molecular dynamics simulations in order to model the structure of the hIFN-\(\gamma \) C-terminal part. The simulations were carried out with the CHARMM22 force field, starting from a fully extended conformation of the C-termini. They showed unambiguously that the C-termini tend to approach the globular part of the protein, so that the whole hIFN-\(\gamma \) molecule adopts a more compact conformation. The energetic favourability of the more compact conformations of the whole cytokine was also confirmed by means of free energy perturbation simulations.

Research paper thumbnail of Characterization of functional domains of human interferon gamma by specific monoclonal antibodies

Folia biologica, 2002

For the study of functional domains of hIFNgamma, two MABs were characterized. Both MABs, named 1... more For the study of functional domains of hIFNgamma, two MABs were characterized. Both MABs, named 1E11 and 5D6, were sequence-specific for hIFNgamma. According to the estimated additivity index, they recognized epitopes located at distinct non-overlapping areas of the hIFNgamma molecule. When pre-incubated with hIFNgamma, MAB 1E11 was able to neutralize the antiviral as well as the antiproliferative activity of the cytokine. This indicated that MAB 1E11 was specific either for the domain responsible for binding to the cell receptor or for a domain required for both functions. By contrast, the second MAB 5D6 did not interfere with any of the two hIFNgamma biological activities. The epitope of MAB 5D6 was located between the amino acid residues Leu 135 and Glu 143 by using different forms of C-terminally truncated hIFNgamma. These data allow the conclusion that the last nine C-terminal amino acids are not essential for the receptor binding and biological functioning of this cytokine. Th...

Research paper thumbnail of Molecular modeling of the effects of glycosylation on the structure and dynamics of human interferon-gamma

Journal of Molecular Modeling, 2019

Natural hIFNγ is a glycoprotein with two N-glycosylation sites in each monomer chain, which are i... more 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.

Research paper thumbnail of Buffer and additive thermofluor screening of wild type human interferon gamma and mutant proteins

Thermochimica Acta, 2017

Abstract Human interferon gamma (hIFNγ) plays a key role in the immune system and therefore this ... more Abstract Human interferon gamma (hIFNγ) plays a key role in the immune system and therefore this cytokine has many current and future therapeutic applications. hIFNγ is well known with its aggregation propensity and despite its clinical use, there is not much data about the stabilization of hIFNγ preparations. Nowadays, substantial evidence indicates that the pathogenesis of many autoimmune diseases is related to overproduction of hIFNγ. In this regard we have developed inactive hIFNγ analogues to act as receptor antagonists of the endogenous hIFNγ. Since they show even higher tendency for aggregation than the wild type protein, we designed two-step thermofluor screen of 61 buffer conditions to identify the best storage solution for all investigated proteins to be used in the form of research samples or biopharmaceuticals. Tris buffer pH 8.0 supplemented with NaCl and trehalose/betaine/glycerol as additives showed to be the most appropriate choice ensuring high solubility and thermal stability of both hIFNγ and its mutants.

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

BioMed research international, 2017

In order to obtain glycosylated human interferon-gamma (hIFNγ) and its highly prone to aggregatio... more 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 recepto...

Research paper thumbnail of Inhibitor of Endogenous Human Interferon-Gamma

Research paper thumbnail of 258 a New Approach to Cope with Autoimmune Diseases: Computer Simulations and Laboratory Tests

Radiotherapy and Oncology, 2012

data files for 10 incident protons with energies ranging from 100 MeV to 230 MeV, although only r... more data files for 10 incident protons with energies ranging from 100 MeV to 230 MeV, although only results for 150 MeV are reported here. Spectra of emitted prompt gamma particles as well as depth emission profiles were obtained. Those plots were filtered using different constraints to better understand the processes involved in the production of prompt gamma rays produced by proton beams. In first place, the plots were filtered by energy of the prompt gamma particles to study which energy produce better correlation with dose distributions. The energy ranges employed were Eγ 10 MeV. A second filter was the type of parent particle that led to the production of the prompt gamma, differentiating among proton, electron, positron and neutron. Finally, a filter by type of interaction that produced the prompt gamma is presented splitting profiles by inelastic, bremsstrahlung, neutron capture and annihilation. Results: Figure 1 shows the results obtained of the prompt gamma produced by a 150 MeV proton beam in the soft tissue phantom. Figure 1 shows the energy spectra (d,e) and depth profiles (a,b,c) of produced prompt gammas differentiating between range of energy (a), type of particles (b) and type of interactions (c) that produce them. Energy spectra are in very good agreement with those found in the literature. Depth profiles show the best correlation with the dose profile for higher energy photons (3 MeV < Eγ < 10 MeV). Prompt gammas produced by protons are well correlated to the dose profile while neutrons and electrons show no clear peak. Prompt gammas produced by electrons can be mostly filtered by selecting energies greater than 1 MeV while the gammas produced by neutrons have an energy similar to those produced by protons. Inelastic interactions are dominant in the production of prompt gammas and show good correlation with dose profile. Bremsstrahlung and neutron capture contributes to the background with no information of the distal falloff position.

Research paper thumbnail of Metadynamics study of mutant human interferon-gamma forms

Computers & Mathematics with Applications, 2012

Human interferon-gamma (hIFN-γ) is an important antiviral and immunomodulating signaling molecule... more Human interferon-gamma (hIFN-γ) is an important antiviral and immunomodulating signaling molecule. The upregulation of its production, however, is related to the etiology of certain autoimmune diseases. In the search for a mechanism for suppressing the hIFNγ biological activity, we investigated the possibility to obtain mutant derivatives of the protein, capable to bind to hIFN-γ cellular receptors, but lacking the ability to trigger the biological response inside the cell. In order to preserve the affinity to the receptor, the introduced mutations should not induce conformational changes in the secondary structure of the mutants. Molecular dynamics simulations were performed to study the secondary structure of 100 randomly chosen hIFN-γ derivatives with substitutions in amino acids 86-88. The stability of the local structure of all hIFN-γ forms was investigated by means of metadynamics. It was found that some of the mutated forms preserve the local secondary structure and show similar or higher stability of the mutated helix, compared to the native form. The 12 most promising mutants were suggested for experimental investigation.

Research paper thumbnail of Additional file 1 of Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma

Additional file 1: Fig. S1. UV spectra of nucleic acids isolated from purified IBs by phenol–chlo... more Additional file 1: Fig. S1. UV spectra of nucleic acids isolated from purified IBs by phenol–chloroform extraction and precipitation with ethanol. The probe was analysed by NanoDrop®. Fig. S2. UV spectra of RNA isolated from purified IBs by TRIzol® and analysed by NanoDrop®.

Research paper thumbnail of Molecular Mechanism of the Anti-Inflammatory Action of Heparin

International Journal of Molecular Sciences

Our objective is to reveal the molecular mechanism of the anti-inflammatory action of low-molecul... more 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...

Research paper thumbnail of Heparin as an Anti-Inflammatory Agent

ABSTRACTTimely control of the cytokine release syndrome (CRS) at the severe stage of COVID-19 is ... more ABSTRACTTimely 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, IFNγ 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 IFNγ 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 IFNγ 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 IFNγ and is able to inhibit fully the interaction with its cellular re...

Research paper thumbnail of Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma

Microbial Cell Factories

Background Inclusion bodies (IBs) are protein aggregates in recombinant bacterial cells containin... more Background Inclusion bodies (IBs) are protein aggregates in recombinant bacterial cells containing mainly the target recombinant protein. Although it has been shown that IBs contain functional proteins along with protein aggregates, their direct application as pharmaceuticals is hindered by their heterogeneity and hazardous contaminants with bacterial origin. Therefore, together with the production of soluble species, IBs remain the main source for manufacture of recombinant proteins with medical application. The quality and composition of the IBs affect the refolding yield and further purification of the recombinant protein. The knowledge whether nucleic acids are genuine components or concomitant impurities of the IBs is a prerequisite for the understanding of the IBs formation and for development of optimized protocols for recombinant protein refolding and purification. IBs isolated from Escherichia coli overexpressing human interferon-gamma (hIFNγ), a protein with therapeutic ap...

Research paper thumbnail of Segregational Instability in E. Coli of Expression Plasmids Carrying Human Interferon Gamma Gene and its 3’-End Truncated Variants

Biotechnology & Biotechnological Equipment, 2009

Bertani medium, where the cell cultures were maintained at an exponential growth phase. To descri... more Bertani medium, where the cell cultures were maintained at an exponential growth phase. To describe the population dynamics, a mathematical model proposed by Stewart & Levin was used and the parameters Δ (difference in specific growth rate between plasmid-free and plasmid-harbouring cells) and θ (relative plasmid loss rate) were determined for each plasmid. The obtained results showed that any variation in the 3'-end of the hIFNγ-gene strongly affected plasmid instability. We also found that the relative plasmid loss rate θ was proportional to the hIFNγ-mRNA content in the cell. Based on these results, we conclude that the structure of the constitutively expressed gene is of great importance for the segregational plasmid instability.

Research paper thumbnail of Identification of two RNA-binding proteins in Balbiani ring premessenger ribonucleoprotein granules and presence of these proteins in specific subsets of heterogeneous nuclear ribonucleoprotein particles

Molecular and Cellular Biology

Balbiani ring (BR) granules are premessenger ribonucleoprotein particles (RNPs) generated in gian... more Balbiani ring (BR) granules are premessenger ribonucleoprotein particles (RNPs) generated in giant chromosomal puffs, the BRs, in the larval salivary glands of the dipteran chironomus tentans. Monoclonal antibodies were raised against nuclear proteins collected on a single-stranded-DNA-agarose affinity column, and two of them were used to identify RNA-binding proteins in BR granules. First, in Western blots (immunoblots), one of the antibodies recognized a 36-kDa protein and the other recognized a 45-KDa protein. Second, both antibodies bound to the BRs in immunocytological experiments. It was shown in cross-linking experiments that the two proteins are associated with heterogeneous nuclear RNP (hnRNP) complexes extracted from C. tentans nuclei. By immunoelectron microscopy of isolated and partly unfolded BR RNPs, it was specifically demonstrated that the BR granules contain the two proteins and, in addition, that both proteins are distributed frequently along the RNP fiber of the p...

Research paper thumbnail of Characterization of functional domains of human interferon gamma by specific monoclonal antibodies

Folia biologica, 2002

For the study of functional domains of hIFNgamma, two MABs were characterized. Both MABs, named 1... more For the study of functional domains of hIFNgamma, two MABs were characterized. Both MABs, named 1E11 and 5D6, were sequence-specific for hIFNgamma. According to the estimated additivity index, they recognized epitopes located at distinct non-overlapping areas of the hIFNgamma molecule. When pre-incubated with hIFNgamma, MAB 1E11 was able to neutralize the antiviral as well as the antiproliferative activity of the cytokine. This indicated that MAB 1E11 was specific either for the domain responsible for binding to the cell receptor or for a domain required for both functions. By contrast, the second MAB 5D6 did not interfere with any of the two hIFNgamma biological activities. The epitope of MAB 5D6 was located between the amino acid residues Leu 135 and Glu 143 by using different forms of C-terminally truncated hIFNgamma. These data allow the conclusion that the last nine C-terminal amino acids are not essential for the receptor binding and biological functioning of this cytokine. Th...

Research paper thumbnail of Identification of two RNA-binding proteins in Balbiani ring premessenger ribonucleoprotein granules and presence of these proteins in specific subsets of heterogeneous nuclear ribonucleoprotein particles

Molecular and cellular biology, 1996

Balbiani ring (BR) granules are premessenger ribonucleoprotein particles (RNPs) generated in gian... more Balbiani ring (BR) granules are premessenger ribonucleoprotein particles (RNPs) generated in giant chromosomal puffs, the BRs, in the larval salivary glands of the dipteran chironomus tentans. Monoclonal antibodies were raised against nuclear proteins collected on a single-stranded-DNA-agarose affinity column, and two of them were used to identify RNA-binding proteins in BR granules. First, in Western blots (immunoblots), one of the antibodies recognized a 36-kDa protein and the other recognized a 45-KDa protein. Second, both antibodies bound to the BRs in immunocytological experiments. It was shown in cross-linking experiments that the two proteins are associated with heterogeneous nuclear RNP (hnRNP) complexes extracted from C. tentans nuclei. By immunoelectron microscopy of isolated and partly unfolded BR RNPs, it was specifically demonstrated that the BR granules contain the two proteins and, in addition, that both proteins are distributed frequently along the RNP fiber of the p...

Research paper thumbnail of Role of the C-terminal chain in human interferongamma stability: an electrostatic study

Proteins, 2001

Electrostatic interactions in two structures of human interferon gamma (hIFNgamma), corresponding... more Electrostatic interactions in two structures of human interferon gamma (hIFNgamma), corresponding to interferon molecule alone and bound to its receptor, were analyzed on the basis of a continuum dielectric model. It was found that a number of titratable groups, mainly basic, show large pK shifts and remain in their neutral forms at physiologically relevant pH. The fact that these groups are largely common to both structures and that most of them belong to the set of most conserved sites suggests that this is a property inherent to the hIFNgamma molecule rather than an artifact of the crystal packing. His111 was also found deprotonated at neutral pH. It was concluded that receptor recognition involving His111 is driven by aromatic coupling of His111 and Tyr52 from the receptor rather than by electrostatic interactions. The structure corresponding to hIFNgamma in complex with its receptor shows a reduction in number and in degree of desolvation of the buried titratable sites. This fi...