Biology subjects

Zabrodskaya, Y.

Publications and source records attributed to Zabrodskaya, Y..

10 recordsLinked to original sources

bioRxiv2024

Stick of Sticks: Structural Features of the Amyloidogenic Peptide-DNA Complex

The search for peptides that can specifically bind to regulatory regions in DNA is a necessary step for creating drugs that can regulate gene expression. The study is dedicated to the peculiarities of binding of a model peptide, which carries an ionic self-complementary motif and can form amyloid-like fibrils [1], with model double-stranded DNAs. The stoichiometric ratios of the components of the complex were found using the retardation method in agarose gel. Using microscale thermophoresis, it was shown that the peptide in the amyloid-like state is capable of binding to model 45-bp double-stranded DNA, with a micromolar equilibrium dissociation constant. Using cryo-electron, transmission electron, and atomic force microscopy, the morphology of peptide-DNA complexes was studied. Using dynamic light scattering and nanoparticle tracking analysis, as well as small-angle neutron scattering, the spatial parameters of the resulting DNA-peptide complexes were characterized. Molecular dynamics simulations showed that the arginine side chains of the peptide are prone to interact with guanine nitrogenous bases. It was shown that the formation of peptide-dsDNA complexes interferes with the operation of restriction endonucleases that have guanine-cytosine pairs in the recognition center, which is consistent with the results of prediction of interaction sites obtained using computer modeling. The results of the work can be used in the development of peptides capable of interacting with functional regions of DNA, as well as in the development of new carriers for transfection of DNA constructs.

biophysics↗

bioRxiv2023

Peptide fibrils as vaccine: a proof of concept

The potential of amyloid-like fibrils formed by peptides as vaccine candidates was investigated using a fragment of the Ebola virus glycoprotein. Peptide in fibrillar form were found to induce an immune response to the full-length protein without causing cellular toxicity or significant changes in hematological studies. The ability of the studied peptide fragment to oligomerize and form amyloid-like fibrils and intermediates suggests potential implications for the viruss mechanisms of action on cells, particularly those of the immune system. Additionally, if native GP2 epitopes are retained in the peptide fibrils, they may serve as effective immunization agents due to their autoadjuvant properties; however, it is important to consider the possibility of cross-reactivity with human proteins. These findings provide valuable insights into the potential use of amyloid-forming peptide as vaccine candidates and highlight the need for further research into their immunogenic and adjuvant properties.

biophysics↗

bioRxiv2023

Cell-penetrating peptide and cationic liposomes mediated siRNA delivery to arrest growth of chronic myeloid leukemia cells in vitro

Gene silencing through RNA interference (RNAi) is a promising therapeutic approach for a wide range of disorders, including cancer. Non-viral gene therapy, using specific siRNAs against BCR-ABL, can be a supportive or alternative measure to traditional chronic myeloid leukemia (CML) tyrosine kinase inhibitor (TKIs) therapies, given the prevalence of clinical TKI resistance. The main challenge for such approaches remains the development of the effective delivery system for siRNA tailored to the specific disease model. The purpose of this study was to examine and compare the efficiency of endosomolytic cell penetrating peptide (CPP) EB1 and PEG2000-decorated cationic liposomes composed of polycationic lipid 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2X3) and helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) for anti-bcr-abl siRNA delivery into the K562 human CML cell line. We show that both EB1 and 2X3-DOPE-DSPE-PEG2000 (0.62% mol.) liposomes effectively deliver siRNA into K562 cells by endocytic mechanisms, and the use of liposomes leads to more effective inhibition of expression of the targeted gene (BCR-ABL) and cancer cell proliferation. Taken together, these findings suggest that PEG-decorated cationic liposomes mediated siRNA delivery allows an effective antisense suppression of certain oncogenes, and represents a promising new class of therapies for CML.

molecular biology↗

bioRxiv2023

How the immune mousetrap works: structural evidence for the immunomodulatory action of a peptide from influenza NS1 protein

One of the critical stages of the T-cell immune response is the dimerization of the intramembrane domains of T-cell receptors (TCR). Structural similarities between the immunosuppressive domains of viral proteins and the transmembrane domains of TCR have led several authors to hypothesize the mechanism of immune response suppression by highly pathogenic viruses: viral proteins embed themselves in the membrane and act on the intramembrane domain of the TCRalpha subunit, hindering its functional oligomerization. It has also been suggested that this mechanism is used by influenza A virus in NS1-mediated immunosuppression. We have shown that the peptide corresponding to the primary structure of the potential immunosuppressive domain of NS1 protein (G51) can reduce concanavalin A-induced proliferation of PBMC cells, as well as in vitro, G51 can affect the oligomerization of the core peptide corresponding to the intramembrane domain of TCR, using AFM and small-angle neutron scattering. The results obtained using in cellulo and in vitro model systems suggest the presence of functional interaction between the NS1 fragment and the intramembrane domain of the TCR alpha subunit. We have proposed a possible scheme for such interaction obtained by computer modeling. This suggests the existence of another NS1-mediated mechanism of immunosuppression in influenza. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/573289v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@12b4ce4org.highwire.dtl.DTLVardef@1c53557org.highwire.dtl.DTLVardef@30f519org.highwire.dtl.DTLVardef@2b71bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Clean and folded: optimized production of high quality recombinant human interferon-{lambda}1

Type III interferons exhibit antiviral activity against influenza viruses, coronaviruses, rotaviruses, and others. In addition, this type of interferon theoretically has therapeutic advantages, in comparison with type I interferons, due to its ability to activate a narrower group of genes in a relatively small group of target cells. Hence, it can elicit more targeted antiviral or immunomodulatory responses. Obtaining biologically-active interferon lambda (hIFN-{lambda}1) is fraught with difficulties at the stage of expression in soluble form or, in the case of expression in the form of inclusion bodies, at the stage of refolding. In this work, hIFN-{lambda}1 was expressed in the form of inclusion bodies, and a simple, effective refolding method was developed. Efficient and scalable methods for chromatographic purification of recombinant hIFN-{lambda}1 were also developed. High-yield, high-purity product was obtained through optimization of several processes including: recombinant protein expression; metal affinity chromatography; cation exchange chromatography; and an intermediate protein refolding stage. The obtained protein was shown to have expected, specific biological activity in line with published effects: induction of MxA gene expression in A549 cells.

bioengineering↗

bioRxiv2020

Quench me if you can: Alpha-2-macroglobulin trypsin complexes enable serum biomarker analysis by MALDI mass spectrometry

One of the main functions of alpha-2-macroglobulin (A2M) in human blood serum is the binding of all classes of protease. It is known that trypsin, after such interaction, possesses modified proteolytic activity. Trypsin first hydrolyzes two bonds in A2Ms bait region, and the peptide 705VGFYESDVMGR715 is released from A2M. In this work, specifics of the A2M-trypsin interaction were used to determine A2M concentration directly in human blood serum using MALDI mass-spectrometry. Following exogenous addition of trypsin to human blood serum in vitro, the concentration of the VGFYESDVMGR peptide was measured, using its isotopically-labeled analogue (18O), and A2M concentration was calculated. The optimized mass spectrometric approach was verified using a standard method for A2M concentration determination (ELISA) and the relevant statistical analysis methods. It was also shown that trypsins modified proteolytic activity in the presence of serum A2M can be used to analyze other serum proteins, including potential biomarkers of pathological processes. Thus, this work describes a promising approach to serum biomarker analysis that can be technically extended in several useful directions.

molecular biology↗

bioRxiv2020

Old Dog, New Tricks: Influenza A Virus NS1 and In Vitro Fibrillogenesis

The influenza NS1 protein is involved in suppression of the host immune response. Recently, there is growing evidence that prion-like protein aggregation plays an important role in cellular signaling and immune responses. In this work, we obtained a recombinant, influenza A NS1 protein and showed that it is able to form amyloid-like fibrils in vitro. Using proteolysis and subsequent mass spectrometry, we showed that regions resistant to protease hydrolysis highly differ between the native NS1 form (NS1-N) and fibrillar form (NS1-F); this indicates that significant structural changes occur during fibril formation. The discovery of the ability of NS1 to form amyloid-like fibrils may be relevant to uncovering relationships between influenza A infection and modulation of the immune response.

molecular biology↗

bioRxiv2019

Cold and distant: structural features of the nucleoprotein complex of a cold-adapted influenza A virus strain

Two influenza A nucleoprotein variants (wt: G102R; and mutant: G102R and E292G) were studied with regard to macro-molecular interactions in oligomeric form (24-mers). The E292G mutation has been previously shown to provide cold adaptation. Molecular dynamics simulations of these complexes and trajectory analysis showed that the most significant difference between the obtained models was distance differences between nucleoprotein complex strands. Influenza virus nucleoprotein complexes were isolated from strains bearing the corresponding NP amino acid substitutions. The isolated complexes were characterized by transmission electron microscopy and differential scanning fluorimetry (DSF). Presence of the E292G substitution was shown by DSF to affect nucleoprotein complex melting temperature. In the filament interface peptide model, it was shown that the peptide corresponding in primary structure to the wild-type NP (SGYDFEREGYS, wild type peptide) is prone to temperature-dependent self-association, unlike the peptide carrying the substitution corresponding to E292G (SGYDFGREGYS, mutant peptide). It was also shown that the SGYDFEREGYS peptide (wt) is capable of interacting with a recombinant full-size monomeric nucleoprotein (with primary structure corresponding to wild type); this interactions equilibrium dissociation constant is five orders of magnitude lower than for the SGYDFGREGYS peptide. Using small-angle neutron scattering (SANS), the supramolecular structures of isolated complexes of these proteins was studied at temperatures of 15, 32, and 37{degrees}C. SANS data show that the structures of the studied complexes (mutant or normal proteins with RNA) at elevated temperature differ from the rod-like particle model and react differently to temperature changes. The data suggest that the mechanism behind cold adaptation with E292G is associated with a weakening of the interaction between strands of the ribonucleoprotein complex and, as a result, the appearance of inter-chain interface flexibility necessary for complex function at low temperature.

biophysics↗

bioRxiv2019

Effect of Alpha-Lactalbumin and Lactoferrin Oleic Acid Complexes on Chromatin Structural Organization

This work focuses on the study of multimeric alpha-lactalbumin oleic acid and lactoferrin oleic acid complexes. The purpose of the research is to study possible mechanisms involved in their pro-apoptotic activities, as seen in some tumor cell cultures. Complexes featuring oleic acid (OA) with human alpha-lactalbumin (hAl) or with bovine alpha-lactalbumin (bAl), and human lactoferrin (hLf) were investigated using small-angle neutron scattering (SANS). It was shown that while alpha-lactalbumin protein complexes were formed on the surface of polydisperse OA micelles, the lactoferrin complexes comprised a monodisperse system of nanoscale particles. Both hAl and hLf complexes appeared to interact with the chromatin of isolated nuclei affecting chromatin structural organization. The possible roles of these processes in the specific anti-tumor activity of these complexes are discussed.

biophysics↗

bioRxiv2017

Triazavirine supramolecular complexes as modifiers of the peptide oligomeric structure

In this study we present molecular dynamics simulations of the antiviral drug triazavirine, that affects formation of amyloid-like fibrils of the model peptide (SI). According to our simulations, triazavirine is able to form linear supramolecular structures which can act as shields and prevent interactions between SI monomers. This model, as validated by simulations, provides an adequate explanation of triazavirines mechanism of action as it pertains to SI peptide fibril formation.

biophysics↗