Andrey V. Vasin

Molecular mechanisms enhancing the proteome of influenza A viruses: an overview of recently discovered proteins.

Influenza A virus is one of the major human pathogens. Despite numerous efforts to produce absolutely effective anti-influenza drugs or vaccines, no such agent has been developed yet. One of the main reasons for this complication is the high mutation rate and the specific structure of influenza A viruses genome. For more than 25 years since the first mapping of the viral genome, it was believed that its 8 genome segments encode 10 proteins. However, the proteome of influenza A viruses has turned out to be much more complex than previously thought. In 2001, the first accessory protein, PB1-F2, translated from the alternative open reading frame, was discovered. Subsequently, six more proteins, PB1-N40, PA-X, PA-N155, PA-N182, M42, and NS3, have been found. It is important to pay close attention to these novel proteins in order to evaluate their role in the pathogenesis of influenza, especially in the case of outbreaks of human infections with new avian viruses, such as H5N1 or H7N9. In this review we summarize the data on the molecular mechanisms used by influenza A viruses to expand their proteome and on the possible functions of the recently discovered viral proteins.

Porous silicon and its applications in biology and medicine

Development of safe container materials for targeted and controlled drug delivery to the right site in the body is one of the most important aspects of modern biotechnologies. In the last decade, a significant progress has been achieved in the study of nanostructured drug carriers, but the use of many nanomaterials is fraught with the enormous risk because of their high toxicity. The real breakthrough became the use of porous silicon, which has such important properties as biocompatibility, bioavailability, and biodegradability, which makes it possible to use it for solving a wide range of biological and medical problems in the field of diagnosis and treatment of diseases, implantology, and biomolecular screening.

[Age-related features of ceruloplasmin biosynthesis and distribution in rats].

In vivo biosynthesis of ceruloplasmin (Cp), a copper-containing glycoprotein, which plays an important role in copper transfer between organs and bidirectional iron transport in vertebrates, was studied in 7-day old rats, which are characterized by the embryonic type of copper metabolism. In addition to the liver, Cp is synthesized in the lungs, brain, and kidneys. In “pulse-chase” experiments it was demonstrated that [14C]-Cp appearance in the blood coincides with the secretion ofde novo synthesized Cp from the liver. [14C]-Cp is taken up from the blood stream by cells of the heart, lung, and kidneys and binds to red blood cells, while Cp polypeptide chain is not taken up by the brain cells. Immunoreactive polypeptides of the Cp receptor were found using immunoblotting in plasma membranes of the heart, liver, kidneys, and red blood cells, rather than in the brain. Using the RT-PCR method with selective primers, it was shown that these cells contain molecular forms of Cp-mRNAs programming the synthesis of both secretory Cp and Cp bound to the plasma membrane via a glycosyl phosphatidylinositol anchor. After switching to the adult type of copper metabolism, the blood serum contents of copper and Cp sharply increase, while the Cp content in the cerebrospinal fluid, as measured according to the oxidase and antigen activities, and copper concentration, as determined by atomic absorption spectroscopy, remain low. Ontogenetic features of the system ensuring the copper homeostasis in mammals are discussed.

Adenosine A2A receptor as a drug target for treatment of sepsis

Sepsis is a generalized infection accompanied by response of the body that manifests in a clinical and laboratory syndrome, namely, in the systemic inflammatory response syndrome (SIRS) from the organism to the infection. Although sepsis is a widespread and life-threatening disease, the assortment of drugs for its treatment is mostly limited by antibiotics. Therefore, the search for new cellular targets for drug therapy of sepsis is an urgent task of modern medicine and pharmacology. One of the most promising targets is the adenosine A2A receptor (A2AAR). The activation of this receptor, which is mediated by extracellular adenosine, manifests in almost all types of immune cells (lymphocytes, monocytes, macrophages, and dendritic cells) and results in reducing the severity of inflammation and reperfusion injury in various tissues. The activation of adenosine A2A receptor inhibits the proliferation of T cells and production of proinflammatory cytokines, which contributes to the activation of the synthesis of anti-inflammatory cytokines, thereby suppressing the systemic response. For this reason, various selective A2AAR agonists and antagonists may be considered to be drug candidates for sepsis pharmacotherapy. Nevertheless, they remain only efficient ligands and objects of pre-clinical and clinical trials. This review examines the molecular mechanisms of inflammatory response in sepsis and the structure and functions of A2AAR and its role in the pathogenesis of sepsis, as well as examples of using agonists and antagonists of this receptor for the treatment of SIRS and sepsis.

Hybrid inorganic-organic capsules for efficient intracellular delivery of novel siRNAs against influenza A (H1N1) virus infection

The implementation of RNAi technology into the clinical practice has been significantly postponing due to the issues regarding to the delivery of naked siRNA predominantly to target cells. Here we report the approach to enhance the efficiency of siRNA delivery by encapsulating the siRNA into new carrier systems which are obtained via the combination of widely used layer-by-layer technique and in situ modification by sol-gel chemistry. We used three types of siRNAs (NP-717, NP-1155 and NP-1496) in encapsulated form as new therapeutic agents against H1N1 influenza virus infection. By employing the hybrid microcontainers for the siRNA encapsulation we demonstrate the reduction of viral nucleoprotein (NP) level and inhibition of influenza virus production in infected cell lines (MDCK and A549). The obtained hybrid carriers based on assembled biodegradable polyelectrolytes and sol-gel coating possess several advantages such as a high cell uptake efficiency, low toxicity, efficient intracellular delivery of siRNAs and the protection of siRNAs from premature degradation before reaching the target cells. These findings underpin a great potential of versatile microencapsulation technology for the development of anti-viral RNAi delivery systems against influenza virus infection.

Synthesis and antiviral activity of PB1 component of the influenza A RNA polymerase peptide fragments.

This study is devoted to the antiviral activity of peptide fragments from the PB1 protein - a component of the influenza A RNA polymerase. The antiviral activity of the peptides synthesized was studied in MDCK cell cultures against the pandemic influenza strain A/California/07/2009 (H1N1) pdm09. We found that peptide fragments 6-13, 6-14, 26-30, 395-400, and 531-540 of the PB1 protein were capable of suppressing viral replication in cell culture. Terminal modifications i.e. N-acetylation and C-amidation increased the antiviral properties of the peptides significantly. Peptide PB1 (6-14) with both termini modified showed maximum antiviral activity, its inhibitory activity manifesting itself during the early stages of viral replication. It was also shown that the fluorescent-labeled analog of this peptide was able to penetrate into the cell. The broad range of virus-inhibiting activity of PB1 (6-14) peptide was confirmed using a panel of influenza A viruses of H1, H3 and H5 subtypes including those resistant to oseltamivir, the leading drug in anti-influenza therapy. Thus, short peptide fragments of the PB1 protein could serve as leads for future development of influenza prevention and/or treatment agents.

Phylogenetic analysis of six-domain multi-copper blue proteins.

Multicopper blue proteins, composed of several repetitive copper-binding domains similar to one-domain cupredoxin-like proteins, were found in almost all organisms. They are classified into the three different groups, based on their two-, three- or six-domain organization. We found orthologs of chordate six-domain copper-binding proteins in animals, plants, bacteria and archea. The phylogenetic analysis of 183 multicopper blue proteins and their copper-binding sites comparison make us think that all the modern six-domain blue proteins have originated from the common ancestral six-domain protein in the process of gene duplication and copper-binding sites loss as a result of amino acid substitutions.

Relationships between CTR1 Activity and Copper Status in Different Rat Organs

The putative product of CTR1 (SLC31A1 according to the Entrez data base) is regarded as the main candidate for an eukaryotic transmembrane copper importer. The tissue-specific function of mammalian CTR1 is still unknown. A quasi-steady-state level of the CTR1 mRNA was assayed by semiquantitative RT-PCR and compared with the copper status in rat organs (liver, cerebellum, choroid plexus, and mammary gland), which differed in copper metabolism during development and differentiation. The CTR1 activity correlated with production of intracellular and extracellular cuproenzymes and deceased in nuclei of mesenchymal cells at high copper concentrations when copper metabolism followed the embryonic pattern. According to phylogenetic analysis, the most conserved regions of CTR1 are transmembrane domains (TMD) 2 and 3, which together contain seven amino acid residues capable of coordinating a copper atom. A model of the cuprophylic channel formed by TMD2 and TMD3 of the CTR1 homotrimer was proposed. In this model, copper is transported through the channel to the cytosolic C-terminal motif His-Cys-His. The ability of His-Cys-His to coordinate Cu(I) was evaluated by molecular modeling (MM+, ZINDO/1). Potential copper transfer from His-Cys-His to the Cys-X-X-Cys Cu (I) motif, present in all cytosol Cu-chaperones, was shown. The role of CTR1 as a donor and an acceptor of copper in higher eukaryotes is discussed.

Mitochondrial ceruloplasmin of mammals

Immunoblotting has been used to detect ceruloplasmin (Cp) polypeptides in rat mitochondria isolated from the brain, liver, testes, and mammary glands. It has been found that Cp is contained in the matrix and inner membranes of mitochondria, and that its molecular weight corresponds to the nonglycosylated form of the protein. Computer analysis has not identified sequences encoding the signal peptide for mitochondria protein import (SPMPI) in the chromosomal Cp gene of rats. However, the analysis of Cp-encoding sequences from databases has shown the presence of SPMPI nucleotide sequences in the human processed Cp pseudogene. A 984-nt region of the Cp pseudogene is translated in the only reading frame to yield a polypeptide consisting of 328 amino acid residues (aa) that contains a 66-aa SPMPI at its N-terminus. The predicted protein (without SPMPI) is 92% identical to the corresponding Cp fragment and contains 20 amino acid substitutions, including eight significant substitutions. The His—X—His motif characteristic of copper-containing ferroxidases is in the middle of the molecule. The substitution of proline by histidine at position 923 of Cp results in a potential copper-binding site. The transcription product of the Cp pseudogene has been found in cultured HepG2 and HuTu 80 human cells with the use of RT—PCR. A 30-kDa polypeptide interacting with antibodies to human Cp has been found in the mitochondria of HuTu 80 cells. The possible biological role of mitochondrial Cp is discussed.

Structural Features of the Peptide Homologous to 6-25 Fragment of Influenza A PB1 Protein.

A mirror-symmetry motif was discovered in the N-terminus of the influenza virus PB1 protein. Structure of peptide comprised of the corresponding part of PB1 (amino acid residues 6-25) was investigated by circular dichroism and in silico modeling. We found that peptide PB1 (6-25) in solution assumes beta-hairpin conformation. A truncated peptide PB1 (6-13), containing only half of the mirror-symmetry motif, appeared to stabilize the beta-structure of the original peptide and, at high concentrations, was capable of reacting with peptide to form insoluble aggregates in vitro. Ability of PB1 (6-13) peptide to interact with the N-terminal domain of PB1 protein makes it a potential antiviral agent that inhibits PA-PB1 complex formation by affecting PB1 N-terminus structure.