Vladislav Victorovich Khrustalev

Initial contact with Influenza virus happens at temperatures lower than body temperature for both human and non-human hosts. Temperature induced structural changes in these epitopes may contribute to immune escape by altering antibody recognition. To monitor temperature-dependent changes in secondary structures, we used synthetic peptides conjugated with Bovine serum albumin (BSA) in order to avoid interpeptide contacts. Circular dichroism (CD) spectra of synthetic peptides and their conjugates with BSA in 0.01M phosphate buffer pH=7.4 were recorded during the heating; observed temperature-dependent structural shifts have been illustrated with the help of molecular modeling. The CC27 peptide, derived from Influenza A virus neuraminidase N6 conjugated to BSA, underwent a transition from lower to a higher beta-sheet content upon heating from 5°C to 30°C. This structural shift is associated with repositioning of a catalytic tyrosine residue within its active site, accompanied by conformational changes in the loop located above it. The NY25 peptide conjugate, derived from Influenza A virus hemagglutinin H1, underwent a transition from lower to higher beta-sheet content upon heating from 20°C to 25°C. This conformational change at the base of the hemagglutinin stalk may alter the local environment of the disulfide bond connecting the HA1 and HA2 subunits, potentially affecting its stability and function. Structural changes in fragments of viral antigens can happen at room temperature. These findings highlight the importance of considering temperature-dependent conformational changes when interpreting crystallographic structures of viral proteins. Furthermore, antibody binding affinities for these epitopes may vary between room temperature and physiological temperature. CD spectroscopy demonstrated beta-strand elongation in peptides derived from neuraminidase N6 and hemagglutinin H1 epitopes upon heating from room temperature to physiological temperature.

Spectroscopic studies on domains and peptides of large proteins are complicated because of the tendency of short peptides to form oligomers in aquatic buffers, but conjugation of a peptide with a carrier protein may be helpful. In this study we approved that a fragment of SK30 peptide from phospholipase A2 domain of VP1 Parvovirus B19 capsid protein (residues: 144–159; 164; 171–183; sequence: SAVDSAARIHDFRYSQLAKLGINPYTHWTVADEELLKNIK) turns from random coil to alpha helix in the acidic medium only in case if it had been conjugated with BSA (through additional N-terminal Cys residue, turning it into CSK31 peptide, and SMCC linker) according to CD-spectroscopy results. In contrast, unconjugated SK30 peptide does not undergo such shift because it forms stable oligomers connected by intermolecular antiparallel beta sheet, according to IR-spectroscopy, CD-spectroscopy, blue native gel electrophoresis and centrifugal ultrafiltration, as, probably, the whole isolated phospholipase domain of VP1 protein does. However, being a part of the long VP1 capsid protein, phospholipase domain may change its fold during the acidification of the medium in the endolysosome by the way of the formation of contacts between protonated His153 and Asp175, promoting the shift from random coil to alpha helix in its N-terminal part. This study opens up a perspective of vaccine development, since rabbit polyclonal antibodies against the conjugate of CSK31 peptide with BSA, in which the structure of the second alpha helix from the phospholipase A2 domain should be reproduced, can bind epitopes of the complete recombinant unique part of VP1 Parvovirus B19 capsid (residues: 1-227).

Background: Resistance to anti-tumor agents targeting the epidermal growth factor receptor (EGFR) reduces treatment response and requires the development of novel EGFR antagonists. Mutant epidermal growth factor (EGF) forms with reduced agonistic activity could be promising agents in cancer treatment. Methods: EGF D46G affinity to EGFR domain III was assessed with affinity chromatography. EGF D46G acute toxicity in Af albino mice at 320 and 3200 μg/kg subcutaneous doses was evaluated. EGF D46G activity in human epidermoid carcinoma cells at 10 ng/mL concentration in serum-free medium and in subcutaneous Ehrlich ascites carcinoma mice model at 320 μg/kg dose was studied. Results: The D46G substitution decreases the thermal stability of EGF complexes with EGFR domain III by decreasing the ability of the C-terminus to be released from the intermolecular β- sheet. However, with remaining binding sites for EGFR domain I, EGF D46G effectively competes with other EGF-like growth factors for binding to EGFR and does not demonstrate toxic effects in mice. EGF D46G inhibits the proliferation of human epidermoid carcinoma cells compared to native EGF. A single subcutaneous administration of EGF D46G along with Ehrlich carcinoma cells injection inhibits the proliferation of these cells and delays tumor formation for up to seven days. Conclusion: EGF D46G can be defined as a partial EGFR agonist as this mutant form demonstrates reduced agonistic activity compared to native EGF. The study emphasizes the role of the EGF C-terminus in establishing interactions with EGFR domain III, which are necessary for EGFR activation and subsequent proliferation of cells.

Background:: Binding appropriate cellular receptors is a crucial step of a lifecycle for any virus. Structure of receptor-binding domain for a viral surface protein has to be determined before the start of future drug design projects. Objective:: Investigation of pH-induced changes in the secondary structure for a capsid peptide with loss of function mutation can shed some light on the mechanism of entrance. Methods:: Spectroscopic methods were accompanied by electrophoresis, ultrafiltration, and computational biochemistry. Results:: In this study, we showed that a peptide from the receptor-binding domain of Parvovirus B19 VP1 capsid (residues 13-31) is beta-structural at pH=7.4 in 0.01 M phosphate buffer, but alpha- helical at pH=5.0, according to the circular dichroism (CD) spectroscopy results. Results of infra- red (IR) spectroscopy showed that the same peptide exists in both alpha-helical and beta-structural conformations in partial dehydration conditions both at pH=7.4 and pH=5.0. In contrast, the peptide with Y20W mutation, which is known to block the internalization of the virus, forms mostly alpha-helical conformation in partial dehydration conditions at pH=7.4. According to our hypothesis, an intermolecular antiparallel beta structure formed by the wild-type peptide in its tetramers at pH=7.4 is the prototype of the similar intermolecular antiparallel beta structure formed by the corresponding part of Parvovirus B19 receptor-binding domain with its cellular receptor (AXL). Conclusion:: Loss of function Y20W substitution in VP1 capsid protein prevents the shift into the beta-structural state by way of alpha helix stabilization and the decrease of its ability to turn into the disordered state.

Эпидермальный фактор роста (EGF) – пептид семейства EGF-подобных факторов роста с общим консервативным EGF-подобным доменом, формируемым тремя внутримолекулярными дисульфидными связями. В данной работе описаны изменения в пространственной структуре EGF и его мутантной форме с заменой D46G в C-концевом фрагменте, наблюдаемые при восстановлении дисульфидных связей соответствующих синтетических пептидов в 0,01 М фосфатном буфере (рН = 7,4). Для анализа структуры использовались спектроскопия кругового дихроизма, спектрофлуориметрия, нативный электрофорез в полиакриламидном геле, центрифужная ультрафильтрация. Показано, что восстановление дисульфидных связей изменяет геометрию EGF-подобного домена в сторону увеличения содержания бета-структуры при сохранении димерной формы пептидов. Согласно результатам молекулярного моделирования, при этом может произойти удлинение главной бета-шпильки EGF-подобного домена, межмолекулярной бета-структуры или образование новой бета-структуры между N- и C-концевым фрагментами молекулы, что изменит поверхность межмолекулярного контакта в составе димера. Доказано, что восстановление дисульфидных связей препятствует диссоциации димеров EGF до мономеров. В физиологических условиях это может привести к неспособности EGF формировать сайты связывания с рецептором EGFR и вызывать его активацию.

Influenza A virus hemagglutinin (HA) is a major virus antigen. No cryo-electron microscopy or X-ray data can be obtained for the HA intraviral (cytoplasmic) domain (CT) post-translationally modified with long fatty acid residues bound to three highly conserved cysteines. We recently proposed a model of HA CT of Influenza A/H1N1 virus possessing an antiparallel beta structure based on the experimental secondary structure analysis of four 14–15 amino acid long synthetic peptides, corresponding to the HA CT sequence, with free or acetaminomethylated cysteines. To dispel doubts about possible non-specific “amyloid-like” aggregation of those synthetic peptides in phosphate buffer solution, we have determined the order of oligomers based on blue native gel electrophoresis, membrane filtration, fluorescence spectroscopy and molecular modeling approaches. We have found that unmodified peptides form only low molecular weight oligomers, while modified peptides form both oligomers of low order similar to those found for unmodified peptides and high order conglomerates, which however are not of beta-amyloid-like fold. This study confirms that the beta structure previously detected by circular dichroism spectroscopy analysis is more likely the result of intrinsic propensity of the HA CT amino acid sequence than the consequence of aggregation. The structures of low order oligomers of the synthetic peptides were used for in silico experiments on modeling of HA CT interactions with matrix protein M1 at physiological and acidic pH levels and revealed two different areas of binding. Finally, tripeptides capable of blocking interactions between HA CT and M1 were proposed.

All available full-length nucleotide sequences of avian Influenza A virus neuraminidase coding region were analyzed and compared with those of N1 and N2 isolated from viruses infecting human and swine. Nucleotide usage bias analysis showed that in most of the coding regions of N3, N4, N5, N6, N7, N8, and N9 isolated from viruses infecting birds, as well as in those of N1 isolated from viruses infecting human and swine, asymmetric A-bias in fourfold degenerated sites is stronger than in coding regions of N1 isolated from birds and in those of N2 isolated from birds, human, and swine, where the level of U is close to the level of A. In twofold degenerated sites from third codon positions, there is a strong asymmetric U-bias in coding regions of N4, N5, and N8 isolated from viruses infecting birds, as well as in those of N1 and N2 isolated from viruses infecting human, swine, and birds, while in coding regions of N9 isolated from birds, there is strong C-bias, and in sequences of N3, N6, and N7 the usage of C is quite close to the level of U. Asymmetric mutational pressure was one of the sources of amino acid replacements that resulted in an equal percentage of sites with appeared and disappeared linear B-cell epitopes in N1, N2, N4, and N5 (33.62–35.33% vs. 32.41–36.45%), but in a stronger tendency to disappear for B-cell epitopes of N3, N6, N7, N8, and N9 of avian viruses (8.74–28.77% vs. 28.96–38.89%).

Editorial: Antiviral drugs and vaccines against important human respiratory viruses

Amyloid-beta precursor protein (APP) is highly conserved in mammals. This feature allowed us to compare nucleotide usage biases in fourfold degenerated sites along the length of its coding region for 146 species of mammals and birds in search of fragments with significant deviations. Even though cytosine usage has the highest value in fourfold degenerated sites in APP coding region from all tested placental mammals, in contrast to marsupial mammals with the bias toward thymine usage, the most frequent germline and somatic mutations in human APP coding region are C to T and G to A transitions. The same mutational AT-pressure is characteristic for germline mutations in introns of human APP gene. However, surprisingly, there are several exceptional introns with deviations in germline mutations rates. The most of those introns surround exons with exceptional biases in nucleotide usage in fourfold degenerated sites. Existence of such fragments in exons 4 and 5, as well as in exon 14, can be connected with the presence of lncRNA genes in complementary strand of DNA. Exceptional nucleotide usage bias in exons 16 and 17 that contain a sequence encoding amyloid-beta peptides can be explained either by the presence of yet unmapped lncRNA(s), or by the autonomous expression of a short mRNA that encodes just C-terminal part of the APP providing an alternative source of amyloid-beta peptides. This hypothesis is supported by the increased rate of T to C transitions in introns 16–17 and 17–18 of Human APP gene relatively to other introns.