Tumor angiogenesis promotes tumor growth, metastasis and disease progression. Different cancer types vary in their angiogenic potential, which may influence prognosis and response to therapy. In the present work, we established xenograft models of three of the most aggressive types of human cancers: glioblastoma U87MG, gastric cancer MKN-45, and pancreatic cancer MIA PaCa-2, in immunodeficient mice. The study of vascular network by optoacoustic microangiography revealed the highest degree of vascularization in U87MG xenografts, and the lowest in MIA PaCa-2 xenografts. As shown by PAS-CD31 dual staining, U87MG-derived tumors also showed the highest expression of the endothelial marker CD31 as well as the highest vasculogenic mimicry capacity. In line with this, metabolic imaging by fluorescence Lifetime Imaging Microscopy (FLIM) of nicotinamide adenine dinucleotide (NADH) revealed that MIA PaCa-2 xenografts were the most glycolytic, whereas U87MG had higher levels of oxidative phosphorylation, and MKN-45 showed intermediate values. Therefore, when creating animal models with xenografted tumors, it is important to understand the angiogenic potential of cancer cells, especially for studying drug candidates with an antiangiogenic effect. Also, the combination of optoacoustics and immunohistochemical analysis with FLIM imaging allows for a comprehensive assessment of both vascularization and the metabolic state of the tumor, which can help predict the therapeutic response.

Anti-angiogenic therapy is a clinically validated method for cancer treatment. It was previously revealed that concurrent targeting of angiogenic and death receptor signaling pathways by a multivalent DR5-specific cytokine TRAIL variant DR5-B genetically fused with the effector peptides, SRH-DR5-B-iRGD, enhances solid tumor suppression and prolongs survival. The SRH peptide is aimed at blocking the tumor neoangiogenesis by preventing activation of the VEGFR2 receptor, while the iRGD peptide interferes with the activation of integrin αvβ3, and enhances the tumor penetration. Here, we investigated how the antiangiogenic activity of the SRH-DR5-B-iRGD fusion protein contributes to its antitumor effects. An integrated approach has been applied involving molecular modeling of SRH-DR5-B-iRGD binding to DR5 receptor, optoacoustic (OA) and optical coherence tomography-based microangiography (OCT-MA) imaging of the vessel networks in xenografts of human glioblastoma and pancreatic adenocarcinoma in nude mice, supported by immunohistochemical (IHC) staining for vascularization marker CD31, and in vitro and in vivo bioactivity studies. Molecular modeling has demonstrated that genetic fusion of DR5-B with the SRH and iRGD peptides not only enables the engagement of additional tumor targets VEGFR2 and integrin αvβ3/NRP-1, but also improves the interaction with DR5 receptor. OA imaging of the vessel network in xenograft tumor nodes of human glioblastoma and pancreatic adenocarcinoma displayed a decrease in the vessel fraction in DR5-B-treated xenograft tumors, with the effect being even more pronounced in SRH-DR5-B-iRGD-treated tumor nodes. This data was consistent with the reduction in the number of perfused vessels in DR5-B and SRH-DR5-B-iRGD-treated tumors as quantified by OCT-MA, and also correlate well with the data obtained by IHC staining and tumor growth inhibition. Ameliorated interaction with the DR5 receptor and imparting antiangiogenic properties to the multivalent fusion protein SRH-DR5-B-iRGD resulted in improved antitumor activity compared to DR5-B. Thereby, SRH-DR5-B-iRGD can be considered as a promising candidate for the treatment of vascularized solid tumors.

Solubilization in detergents is a widely used technique for the isolation of membrane proteins and the study of their properties. Unfortunately, protein stability in detergent micelles can sometimes be compromised. We encountered this issue with xanthorhodopsin (XR) from Salinibacter ruber, which had been previously engineered for expression in Escherichia coli cells. To explore the factors affecting stability and to enhance thermal stability of recombinant XR preparations following solubilization of membranes using n-dodecyl-β-D-maltopyranoside and nickel-affinity chromatography, we developed a series of hybrid proteins based on the homology between XR and a stable rhodopsin from Gloeobacter violaceus (GR). Functional studies of these hybrids and measurements of their melting temperatures revealed the structural elements of XR that account for its notable difference in stability compared to GR, despite their high overall homology of approximately 50 % identical residues. In particular, XR variants with an engineered loop between transmembrane helices D and E, similar to that in GR, demonstrated enhanced stability. However, we found that replacing the DE loop affects carotenoid binding. Additionally, two hybrid proteins containing the C and D helices from GR exhibited increased stability as well as improved photocycle and proton transport rates. In conclusion, we have demonstrated that optimizing the amino acid sequence of xanthorhodopsin from S. ruber based on its homology with Gloeobacter rhodopsin is an effective approach to enhance its thermal stability in vitro and improve its potential for optogenetic applications.

The cytokine TRAIL is distinguished by its remarkable ability to preferentially induce apoptosis in transformed, but not in normal, cells. The recombinant TRAIL extracellular domain and other first-generation agonists of DR4 and DR5 death receptors (DRs) have shown very limited antitumor activity in clinical trials. To enhance the antitumor effect, we developed the multitarget recombinant fusion protein SRH-DR5-B-p48 based on the DR5-selective TRAIL variant DR5-B to simultaneously affect tumor cells (DR5-B-mediated apoptosis) and tumor microenvironment, in particular, to suppress angiogenesis. For this purpose, we modeled and produced the recombinant SRH-DR5-B-p48 fusion protein containing antagonistic synthetic peptides (SRH and p48) to VEGFR2 and FGFR1 receptors, respectively. Analysis of molecular trajectories using molecular dynamics methods showed that the SRH and p48 peptides form non-specific temporary contacts with the DR5-B domain. Using enzyme-linked immunosorbent assay, we showed that SRH-DR5-B-p48 was similar to DR5-B in its affinity for the death receptor DR5 and demonstrated a high affinity for VEGFR2 and FGFR1 with nanomolar dissociation constants. SRH-DR5-B-p48 killed tumor cells of various origin more efficiently than DR5-B and destroyed tumor-like structures in 3D cell models, as well as inhibited FGF2-mediated stimulation of fibroblast proliferation. Therefore, the SRH-DR5-B-p48 fusion protein can be considered as a promising agent for the therapy of solid tumors of various origin.

Redox interaction between heme proteins - neuroglobin (Ngb) and cytochrome c (Cyt c) is considered to contribute to the Ngb-mediated neuroprotection which mechanism is still unclear. We developed a new methodological approach based on resonance Raman spectroscopy to study redox interaction between Ngb and Cyt c and their mutants with amino acid substitutions in their putative interaction surface or Ω-loop. Spectral features of heme c conformational changes during electron transfer (ET) between wild types (WT) of Ngb and Cyt c were established, such as increase of CS bond vibration mode and altering of Cβ-CH3 groups' vibrations indicating displacement of Cyt c heme in its crevice. Furthermore, the probability of the planar conformation of Cyt c heme increases under ET between Ngb and Cyt c. ET was disrupted in following redox pairs: Ngb with substitutions E60K/E87K, E60K and WT Cyt c; WT Ngb and Cyt c with substitutions K25E, T78S/K79P and K8E/K27E/K72E/K86E/K87E/E62K/E69K/E90K. Our results suggest that disruption of ET occurs due to both deterioration of proteins' electrostatic interactions and heme conformational changes. We propose the mechanism of Ngb-Cyt c redox interaction: 1) transient complex formation mainly due to electrostatic interactions; 2) heme conformational tuning; 3) electron transfer; 4) complex dissociation.

TRAIL (Tumor Necrosis Factor-Related Apoptosis Inducing Ligand) receptor pathway is an important component of the immune system participating in surveillance and selective elimination of transformed cells. Therefore, TRAIL-based proteins and other agonists of TRAIL death receptors are promising drug candidates for the treatment of malignant tumors and autoimmune diseases. Initially, in the first series of clinical trials, drugs targeting the death receptors DR4 or DR5, did not reveal significant anticancer activity. The reasons for this were multiple TRAIL resistance mechanisms that tumors evolve to evade the efficient induction of apoptotic signaling. However, a wide range of novel TRAIL death receptor-targeted formulations are currently being developed, mainly aimed at improving stability, enhancing death receptor clustering and engagement of additional tumor targets. Over the past two decades, several dozens of multitargeted fusion proteins with either TRAIL protein or DR5-specific agonistic monoclonal antibodies have been created to improve therapeutic efficacy. These include fusions with either short peptide tags or large functional proteins, as well as antibody fragments targeting molecular pathways involved in angiogenesis or proliferative signaling such as EGFR, VEGFR, PD-L1, etc. Collectively, these multimodal proteins deal with enhancing the activation of extrinsic and intrinsic apoptotic pathways in transformed cells, as well as affecting the tumor microenvironment. This comprehensive review aims to systematize the bispecific and multivalent fusion proteins and conjugates targeting TRAIL death receptors, analyze the molecular mechanisms by which they overcome tumor resistance to TRAIL, and assess clinical prospects. Bibliography — 242 references.

Destroying tumor vasculature is a relevant therapeutic strategy due to its involvement in tumor progression. However, adaptive resistance to approved antiangiogenic drugs targeting VEGF/VEGFR pathway requires the recruitment of additional targets. In this aspect, targeting TRAIL pathway is promising as it is an important component of the immune system involved in tumor immunosurveillance. For dual targeting of malignant cells and tumor vascular microenvironment, we designed a multivalent fusion protein SRH-DR5-B-iRGD with antiangiogenic VEGFR2-specific peptide SRH at the N-terminus and a tumor-targeting and -penetrating peptide iRGD at the C-terminus of receptor-selective TRAIL variant DR5-B. SRH-DR5-B-iRGD obtained high affinity for DR5, VEGFR2 and αvβ3 integrin in nanomolar range. Fusion of DR5-B with effector peptides accelerated DR5 receptor internalization rate upon ligand binding. Antitumor efficacy was evaluated in vitro in human tumor cell lines and primary patient-derived glioblastoma neurospheres, and in vivo in xenograft mouse model of human glioblastoma. Multivalent binding of SRH-DR5-B-iRGD fusion efficiently stimulated DR5-mediated tumor cell death via caspase-dependent mechanism, suppressed xenograft tumor growth by >80 %, doubled the lifespan of xenograft animals, and inhibited tumor vascularization. Therefore, targeting DR5 and VEGFR2 molecular pathways with SRH-DR5-B-iRGD protein may provide a novel therapeutic approach for treatment of solid tumors.

Objective: To study the transport activity of bacterial rhodopsins, proteoliposomes with the same orientation of the protein molecules should be obtained. Methods: The genes of fusion proteins containing Exiguobacterium sibiricum proteorhodopsin (ESR) and various N-terminal soluble domains have been constructed. Results and Discussion: Effective synthesis in Escherichia coli cells was observed only in the case of fusion with chaperone Caf1M and maltose-binding protein MBP expressed as precursors with their own signal sequences. The study of the isolated MBP-ESR protein in micelles and proteoliposomes demonstrated formation and decay of the main photocycle intermediates at pH > 8. The photoelectric response of the fusion proteins Caf-ESR and MBP-ESR is comparable in amplitude to the wild-type ESR response, indicating their homogeneous orientation in the proteoliposome membrane. Conclusions: The obtained constructs can be used to create bacterial expression systems for various retinal proteins, ensuring their uniform incorporation into proteoliposomes.

In the original publication [...].

Objective: Rhodopsin from the eubacterium Exiguobacterium sibiricum (ESR) performs the lightdependent proton pumping function. The operation of ESR is based on the ultrafast photochemical reaction of isomerization of the retinal chromophore, which triggers dark processes closed in a photocycle. Many parameters of the photocycle are determined by the presence of a hydrogen bond between the primary counterion Asp85 and the chromophore. ESR in detergent micelles pumps protons most efficiently at pH > 9, when such a bond is most probable. Methods: In the present study, the photochemical reaction of ESR at pH 9.5 was investigated by femtosecond laser absorption spectroscopy. Results and Discussion: It was shown that photoisomerization of the chromophore group occurs in 0.51 ps, with the contribution from the reactive excited state being ca. 80

This article is devoted to the construction and study of a model cell line expressing neuroglobin. The obtained data show the manifestation of anti-apoptotic properties by neuroglobin under condi-tions of apoptosis via the cytochrome C-dependent pathway

Cytochrome c (CytC), a one-electron carrier, transfers electrons from complex bc1 to cytochrome c oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex bc1 and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at the periphery of the UBS for charge compensation. All mutants showed a 4–6 times increased peroxidase activity and accelerated binding of cyanide to the ferric heme of CytC. In contrast, decomposition of the cyanide complex with ferrous CytC, as monitored by magnetic circular dichroism spectroscopy, was slower in mutants compared to WT. Molecular dynamic simulations revealed the increase in the fluctuations of Cα atoms of individual residues of mutant CytC compared to WT, especially in the Ω-loop (70–85), which can cause destabilization of the Fe…S(Met80) coordination link, facilitation of the binding of exogenous ligands cyanide and peroxide, and an increase in peroxidase activity. It was found that only one substitution K72E is enough to induce all these changes, indicating the significance of K72 and the Ω-loop (70–85) for the structure and physiology of mitochondrial CytC. In this work, we also propose using a ferro-ferricyanide buffer as a substrate to monitor the peroxidase activity of CytC. This new approach allows us to determine the rate of peroxidase activity at moderate (200 µM) concentrations of H2O2 and avoid complications of radical formation during the reaction.

This paper is dedicated to the memory of Oleg B. Ptitsyn (1929-1999) and presents an answer to his question: “What is the role of conserved non-functional residues in protein folding?”. This answer follows from the experimental works of three labs. The role of non-functional but conserved residues of apomyoglobin (apoMb) in the formation of the native protein fold in the molten globule state has been experimentally revealed. This research proves that the non-functional but conserved residues of apoMb are necessary for the formation and maintenance of the correct topological arrangement of the main elements in the apoMb secondary structure already in the early folding intermediate.

Neuroglobin (Ngb) is a cytosolic heme protein that plays an important role in protecting cells from apoptosis through interaction with oxidized cytochrome c (Cyt c) released from mitochondria. The interaction of reduced Ngb and oxidized Cyt c is accompanied by electron transfer between them and the reduction in Cyt c. Despite the growing number of studies on Ngb, the mechanism of interaction between Ngb and Cyt c is still unclear. Using Raman spectroscopy, we studied the effect of charged amino acid substitutions in Ngb and Cyt c on the conformation of their hemes. It has been shown that Ngb mutants E60K, K67E, K95E and E60K/E87K demonstrate changed heme conformations with the lower probability of the heme planar conformation compared to wild-type Ngb. Moreover, oxidized Cyt c mutants K25E, K72E and K25E/K72E demonstrate the decrease in the probability of methyl-radicals vibrations, indicating the higher rigidity of the protein microenvironment. It is possible that these changes can affect electron transfer between Ngb and Cyt c.

Cell-surface display using anchor motifs of outer membrane proteins allows exposure of target peptides and proteins on the surface of microbial cells. Previously, we obtained and characterized highly catalytically active recombinant oligo-α-1,6-glycosidase from the psychrotrophic bacterium Exiguobacterium sibiricum (EsOgl). It was also shown that the autotransporter AT877 from Psychrobacter cryohalolentis and its deletion variants efficiently displayed type III fibronectin ( 10 Fn3) domain 10 on the surface of Escherichia coli cells. The aim of the work was to obtain an AT877-based system for displaying EsOgl on the surface of bacterial cells. The genes for the hybrid autotransporter EsOgl877 and its deletion mutants EsOgl877Δ239 and EsOgl877Δ310 were constructed, and the enzymatic activity of EsOgl877 was investigated. Cells expressing this protein retained ~90% of the enzyme maximum activity within a temperature range of 15-35°C. The activity of cells expressing EsOgl877Δ239 and EsOgl877Δ310 was 2.7 and 2.4 times higher, respectively, than of the cells expressing the full-size AT. Treatment of cells expressing EsOgl877 deletion variants with proteinase K showed that the passenger domain localized to the cell surface. These results can be used for further optimization of display systems expressing oligo-α-1,6-glycosidase and other heterologous proteins on the surface of E. coli cells.

Retinal-containing light-sensitive proteins - rhodopsins - are found in many microorganisms. Interest in them is largely explained by their role in light energy storage and photoregulation in microorganisms, as well as the prospects for their use in optogenetics to control neuronal activity, including treatment of various diseases. One of the representatives of microbial rhodopsins is ESR, the retinal protein of Exiguobacterium sibiricum. What distinguishes ESR from homologous proteins is the presence of a lysine residue (Lys96) as a proton donor for the Schiff base. This feature, along with the hydrogen bond of the proton acceptor Asp85 with the His57 residue, determines functional characteristics of ESR as a proton pump. This review examines the results of ESR studies conducted using various methods, including direct electrometry. Comparison of the obtained data with the results of structural studies and with other retinal proteins allows us to draw conclusions about the mechanisms of transport of hydrogen ions in ESR and similar retinal proteins.

BACKGROUND:The biopharmaceutical industry is significantly growing worldwide, and the Chinese hamster ovary (CHO) cells are used as a main expression host for the production of recombinant monoclonal antibodies. Various metabolic engineering approaches have been investigated to generate cell lines with improved metabolic characteristics for increasing longevity and mAb production. A novel cell culture method based on the 2-stage selection makes it possible to develop a stable cell line with high-quality mAb production.RESULTS:We have constructed several design options of mammalian expression vectors for the high production of recombinant human IgG antibodies. Versions for bipromoter and bicistronic expression plasmids different in promoter orientation and cistron arrangements were generated. The aim of the work presented here was to assess a high-throughput mAb production system that integrates the advantages of high-efficiency cloning and stable cell clones to stage strategy selection reducing the time and effort required to express therapeutic monoclonal mAbs. Development of a stable cell line using bicistronic construct with EMCV IRES-long link gave an advantage in high mAb expression and long-term stability. Two-stage selection strategies allowed the elimination of low-producer clones by using metabolic level intensity to estimate the IgG production in the early steps of selection. The practical application of the new method allows to reduce time and costs during stable cell line development.

Curcumin attracts huge attention because of its biological properties: it is antiproliferative, antioxidant, anti-inflammatory, immunomodulatory and so on. However, its usage has been limited by poor water solubility and low bioavailability. Herein, to solve these problems, we developed curcumin-loaded nanoparticles based on end-capped amphiphilic poly(N-vinylpyrrolidone). Nanoparticles were obtained using the solvent evaporation method and were characterized by dynamic and electrophoretic light scattering, transmission electron (TEM) and atomic force (AFM) microscopy. The average particle size was 200 nm, and the ζ-potential was −4 mV. Curcumin-release studies showed that nanoparticles are stable in aqueous solutions. An in vitro release study showed prolonged action in gastric, intestinal and colonic fluids, consistently, and in PBS. In vitro studies on epidermoid carcinoma and human embryonic kidney cells showed that the cells absorbed more curcumin in nanoparticles compared to free curcumin. Nanoparticles are safe for healthy cells and show high cytotoxicity for glioblastoma cells in cytotoxicity studies in vitro. The median lethal dose was determined in an acute toxicity assay on zebrafish and was 23 μM. Overall, the curcumin-loaded nanoparticles seem promising for cancer treatment.

— An efficient system for the biosynthesis, isolation and purification of recombinant human neuroglobin has been developed and optimized, which makes it possible to produce protein in quantities sufficient to study its properties. According to UV-visible, IR-, CD-, and NMR spectroscopy data, recombinant neuroglobin is a structured protein in the holoform state. The data of chromato-mass-spectrometric analysis made it possible to conclude that there is a correctly formed disulfide bond in the structure of the oxidized form of the protein. Using Raman and surface-enhanced Raman spectroscopy with laser excitation at 532 nm, it was shown that heme in the reduced and oxidized forms of neuroglobin has vibrational degrees of freedom typical of b -type hemes, and the iron atom is hexacoordinated. Using Raman spectroscopy with laser excitation at 633 nm, it was found that reduced –SH-groups were present in reduced neuroglobin, while in oxidized neuroglobin a disulfide bridge was formed. The results obtained serve as the basis for detailed studies of the functioning of neuroglobin as a neuroprotector, in particular, during its interaction with oxidized cytochrome c , which is released from mitochondria in violation of their functioning and/or morphology.

Cytochrome c (CytC) is a single-electron carrier between complex bc1 and cytochrome c-oxidase (CcO) in the electron transport chain (ETC). It is also known as a good radical scavenger but its participation in electron flow through the ETC makes it impossible to use CytC as a radical sensor. To solve this problem, a series of mutants were constructed with substitutions of Lys residues in the universal binding site (UBS) which interact electrostatically with negatively charged Asp and Glu residues at the binding sites of CytC partners, bc1 complex and CcO. The aim of this study was to select a mutant that had lost its function as an electron carrier in the ETC, retaining the structure and ability to quench radicals. It was shown that a mutant CytC with substitutions of five (8Mut) and four (5Mut) Lys residues in the UBS was almost inactive toward CcO. However, all mutant proteins kept their antioxidant activity sufficiently with respect to the superoxide radical. Mutations shifted the dipole moment of the CytC molecule due to seriously changed electrostatics on the surface of the protein. In addition, a decrease in the redox potential of the protein as revealed by the redox titrations of 8Mut was detected. Nevertheless, the CD spectrum and dynamic light scattering suggested no significant changes in the secondary structure or aggregation of the molecules of CytC 8Mut. Thus, a variant 8Mut with multiple mutations in the UBS which lost its ability to electron transfer and saved most of its physico-chemical properties can be effectively used as a detector of superoxide generation both in mitochondria and in other systems.