It is known that prolonged cell passaging may affect the outcomes of cell-based experiments. This study aimed to comprehensively investigate the consequences of long-term culture of U87MG glioblastoma cells, focusing on cell metabolism, morphology, tumorigenicity and drug response. U87MG (L) cells subjected to long-term culturing (20 passages) displayed altered morphology and significantly enhanced autofluorescence of lipofuscin, a well-established marker of oxidative stress and cellular senescence, compared with control U87MG cells maintained for only 5 passages. Fluorescence-lifetime imaging microscopy (FLIM) of NADH revealed the metabolic shift likely associated with glycolysis in long-term cultured U87MG (L) cells. Using the genetically encoded sensor HyPer7, the increase in basal intracellular levels of hydrogen peroxide in U87MG (L) cells was detected. In vivo studies employing orthotopic intracranial xenotransplantation in immunodeficient NSG mice revealed that, unlike short-term cultured U87MG cells, which formed visible tumor nodules with clearly delineated margins, long-term cultured U87MG (L) cells infiltrated diffusely into brain tissues, invaded bone tissue, and exhibited perineural growth. Such an aggressive behavior resulted in worse survival outcomes of xenograft-bearing mice. Notably, U87MG (L) cells became less susceptible to temozolomide, but acquired sensitivity to death receptor 5 (DR5)-selective variant of cytokine TRAIL in vitro and in vivo due to increased DR5 expression on the cell surface and downregulation of cFLIP expression. Our findings with the U87MG cell line indicate that culture duration can alter cellular responses, thereby impacting experimental outcomes. It should be carefully considered when establishing tumor models and evaluating efficacy of potential drug candidates.

Nicotinic acetylcholine receptor of α7 type (α7-nAChR) is a ligand-gated ion channel composed of five identical α7 subunits. Secreted lymphocyte antigen-6 urokinase-type plasminogen activator receptor (Ly6/uPAR)-related protein-1 (SLURP-1) controls carcinoma progression by negative modulation of oncogenic α7-nAChR. In this study, we observed dramatic decrease of SLURP-1 plasma level in patients with metastatic melanoma. We suggested usage of recombinant analog of human SLURP-1 (rSLURP-1) to compensate this deficiency for metastatic melanoma treatment. rSLURP-1 did not affect viability of different patient-derived metastatic melanoma cells, but reduced migration of some of them. Metastatic melanoma cells of other lines were resistant to rSLURP-1. Antimigratory rSLURP-1 effect was mediated by α7-nAChR, whereas resistance to rSLURP-1 correlated with overexpression of human-specific CHRFAM7A gene, which encodes the α7 subunit with truncated N-terminal region (dupα7) able to form hybrid α7/dupα7-nAChR channels. Electrophysiological study in Xenopus laevis oocytes showed that rSLURP-1 inhibits α7/dupα7-nAChR weaker than α7-nAChR. In contrast, "Oncotag" peptide, which mimics the loop I of SLURP-1, inhibited α7/dupα7- and α7-nAChRs with similar efficiency. Oncotag suppressed metastatic melanoma cell migration independently on dupα7 expression. Computer modeling provided rationale for altered activities of rSLURP-1 and Oncotag on α7/dupα7-nAChR. The Cancer Genome Atlas Program (TCGA) database analysis revealed correlation between CHRNΑ7 and CHRFAM7A gene expression and worse survival prognosis for patients with metastatic melanoma. Thus, 1) low plasma SLURP-1 level may be a specific marker of metastatic melanoma development, 2) metastatic melanoma progression can be controlled by α7-nAChR inhibition, and 3) dupα7 overexpression is a new molecular mechanism of melanoma resistance to internal cholinergic control and new target for melanoma treatment.NEW & NOTEWORTHY Metastatic melanoma is aggressive skin tumor often resistant to standard therapies. High α7-nAChR expression negatively correlates with survival of patients with metastatic melanoma, who are characterized by SLURP-1 drop in the plasma. Targeting α7-nAChR with recombinant SLURP-1 could significantly suppress metastatic melanoma cell migration; however, overexpression of human-specific dupα7 subunit forming hybrid α7/dupα7-nAChRs causes melanoma resistance to SLURP-1. This resistance can be overcome by the SLURP-1 mimicking peptide Oncotag, which exhibits activity against hybrid α7/dupα7-nAChRs.

To introduce noninvasive optical diagnostic methods based on detection of tissue autofluorescence signals into medical practice, it is necessary to correlate the optical response with the functional state of a particular chromophore. Diagnostics of the state of lipofuscin granule chromophores is an important task to assess pathologic changes in the visual system in various diseases, primarily age-related macular degeneration. A key feature of lipofuscin granules (LGs) is the diversity of the chromophores and their susceptibility to oxidation. Here, we used time-resolved emission spectroscopy, confocal fluorescence lifetime imaging microscopy, and high-performance liquid chromatography (HPLC) to follow changes of LG chromophore composition upon photooxidation. In both isolated LGs and LG-loaded retinal pigment epithelium cells (ARPE-19), the distributions of the short lifetime component and its amplitude of LG fluorescence shifted, and the mean lifetime increased upon photooxidation, indicating depletion of population of rapidly relaxing bisretinoids, including A2E, and accumulation of their oxidized species, as confirmed by HPLC data. We applied differential evolution algorithms to decompose LG autofluorescence parameters and identified distinct photooxidation patterns in the presence and absence of antioxidant protection. Delivery of zeaxanthin by water-soluble carotenoprotein ΔNC-AstaP attenuated photooxidation-induced changes in the decay kinetics of both isolated and intracellular LGs, suppressed the accumulation of oxidized bisretinoids, and prevented complete photooxidation. Taken together, our results establish that time-resolved lipofuscin autofluorescence provides a quantitative, label-free readout of chromophore composition and oxidative status that is compatible with functional imaging. We propose that such lifetime-based measurements can be employed for early assessment of retinal pathology and for monitoring antioxidant interventions targeting lipofuscin-induced oxidative stress in emerging clinical techniques such as fluorescence lifetime imaging ophthalmoscopy.

The full complement of chromatin-associated proteins-collectively referred to as the chromatome-enables genome functioning in eukaryotes by participating in a wide range of physico-chemical processes. These include mediating diverse specific and nonspecific intermolecular interactions, catalyzing in situ synthesis and modification of macromolecules, facilitating ATP-dependent chromatin remodeling, etc. Despite considerable progress in epigenomics and the structural characterization of many nuclear proteins and their complexes, our understanding of chromatin organization at the proteome scale remains incomplete. This gap hinders the development of a holistic view of genome regulation. In this study, we present a state-of-the-art characterization of the human chromatome based on an integrative meta-analysis of diverse data sources describing the composition, abundance, and sub-nuclear localization of chromatin proteins. This effort is complemented by original analyses of their physico-chemical properties, domain architectures, and interaction patterns. To support and streamline these analyses, we developed a reference dataset of chromatin proteins, integrated with an empirical, function-based classification ontology and an associated interactive web resource-SimChrom-accessible at https://simchrom.intbio.org/. The reference dataset was carefully curated by reconciling data among protein databases, localization, and mass spectrometry-based experimental studies. Sequence-based and AI-assisted structural analyses revealed previously unannotated domains within chromatin proteins that warrant experimental validation, as well as the widespread use of multivalent interaction strategies that underpin chromatin organization. Together, our findings establish a robust framework for future studies aimed at elucidating genome function through detailed analysis of protein-protein and protein-nucleic acid interactions within chromatin.

During various DNA-centered processes in the cell nucleus, the minimal structural units of chromatin organization, nucleosomes, are often transiently converted to hexasomes and tetrasomes missing one or both H2A/H2B histone dimers, respectively. However, the structural and functional properties of the subnucleosomes and their impact on biological processes in the nuclei are poorly understood. Here, using biochemical approaches, molecular dynamics simulations, single-particle Förster resonance energy transfer microscopy, and nuclear magnetic resonance spectroscopy, we have shown that, surprisingly, removal of both dimers from a nucleosome results in much higher mobility of both histones and DNA in the tetrasome. Accordingly, DNase I footprinting shows that DNA–histone interactions in tetrasomes are greatly compromised, resulting in formation of a much lower barrier to transcribing RNA polymerase II than nucleosomes. The data suggest that tetrasomes are remarkably dynamic structures and their formation can strongly affect various biological processes.

Heterotetramerization of Kv1.1 and Kv1.2 α-subunits expands the functional diversity of voltage-gated potassium Kv1 channels in the central nervous system (CNS), thus necessitating the study of the properties of these heterochannels, including their interactions with ligands. We report on the expression, electrophysiological, and ligand-binding properties of human heterochannels Kv(1.1-1.2)2 formed by dimeric concatemers Kv1.1-Kv1.2 fused with fluorescent protein mKate2 in Neuro-2a cells. Kv(1.1-1.2)2 is a low-voltage-activated, highly active, non-inactivating channel with a fast activation rate. Its activation rate and half-maximum activation voltage are similar to that of the Kv1.1 channel, but differ from that of Kv1.2. This suggests that the membrane expression of Kv(1.1-1.2)2 may functionally compensate for the absence of membrane presentation of homotetrameric Kv1.1 channels in CNS. Hongotoxin 1 fused with fluorescent protein GFP (HgTx-G) is shown to be a pore-blocking ligand of Kv(1.1-1.2)2 with a dissociation constant of 100 pM. Using confocal microscopy and competitive binding assay, HgTx-G and cells expressing Kv(1.1-1.2)2, the apparent dissociation constants of the complexes between Kv(1.1-1.2)2 and peptides Ce1, Ce4, hongotoxin 1, MeKTx11-1, agitoxin 2, charybdotoxin, and scyllatoxin were evaluated to be 14, 33, 40, 250, 800, and >>3300 pM, respectively. Heterotetramerization of α-subunits has a different effect on the affinity of ligands compared to those for Kv1.1 and Kv1.2 channels.

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.

Yeast +1 nucleosomes positioned at transcription start sites must be reorganized to allow transcription initiation. Nucleosome reorganization involves multiple factors including histone chaperone FACT (FAcilitates Chromatin Transcription), histone acetylation, and histone variant H2A.Z; however, the mechanism of this process is not fully understood. Here we investigated nucleosome unfolding in the presence of these factors by combining biochemical assays with single-particle Förster resonance energy transfer (spFRET) microscopy. The presence of the H3:K56Ac mimic (H3:K56Q) alone or together with H2A.Z (but not H2A.Z alone) facilitates the Nhp6-dependent unfolding of nucleosomes by FACT. In contrast to canonical nucleosomes, the unfolding of nucleosomes with the studied variant histones promotes the eviction of core histones from nucleosomal DNA. Furthermore, H2A.Z alone or in synergy with H3:K56Q facilitates transcription through a nucleosome as efficiently as FACT facilitates transcription through canonical nucleosomes. The data suggest that FACT, together with H3:K56 acetylation and H2A.Z, unfold promoter nucleosomes and participate in the eviction of histones to increase the accessibility of the transcription start site, thereby stimulating transcription initiation and possibly early elongation.

Virus-neutralizing peptides (VNPs) emerged as promising antiviral drug candidates with unprecedented specificity and cost-effectiveness during the recent COVID-19 pandemic. However, limited avidity, lack of effector functions, short circulatory half-life, and restricted administration routes make them inferior compared to neutralizing antibodies. To address these constraints, a potent VNP that targets the SARS-CoV-2 S protein is combined with Barnase, a highly active RNA-cleaving enzyme from Bacillus amyloliquefaciens. The resulting LCB1-Barnase (LCB1-Bn) chimera retains strong binding affinity for the SARS-CoV-2 S protein and demonstrates a fourfold reduction in IC50 compared to the LCB1 peptide alone in competitive ELISA and in in vitro neutralization tests. In transgenic CAG-hACE2 mice infected with wild-type SARS-CoV-2, intranasal administration of LCB1-Bn significantly improves survival and reduces viral load by 29-fold. To extend circulation life and allow systemic intravenous administration, an albumin-binding domain (ABD) from Streptococcus protein G is added to LCB1-Bn, producing LCB1-ABD-Bn fusion protein which displays a 95-fold increase in serum half-life. LCB1-ABD-Bn exhibits good tolerability at doses below 10 mg/kg and provides protection of SARS-CoV-2-infected CAG-hACE2 animals in 24-hour post-infection intraperitoneal treatment. Cryo-EM reveals the LCB1-ABD-Bn’s tight interaction with S protein RBD domains, highlighting its potential as a promising drug candidate against SARS-CoV-2.

Prostate stem cell antigen (PSCA) is a Ly6/uPAR protein that targets neuronal nicotinic acetylcholine receptors (nAChRs). It exists in membrane-tethered and soluble forms, with the latter upregulated in Alzheimer’s disease. We hypothesize that PSCA may be linked to a wider spectrum of neurological diseases and could induce neuroinflammation. Indeed, PSCA expression is significantly upregulated in the brain of patients with multiple sclerosis, Huntington’s disease, Down syndrome, bipolar disorder, and HIV-associated dementia. To investigate PSCA’s structure, pharmacology, and inflammatory function, we produced a correctly folded water-soluble recombinant analog (ws-PSCA). In primary hippocampal neurons and astrocytes, ws-PSCA differently regulates secretion of inflammatory factors and adhesion molecules and induces pro-inflammatory responses by increasing TNFβ secretion. Heteronuclear NMR and 15N relaxation measurements reveal a classical β-structural three-finger fold with conformationally disordered loops II and III. Positive charge clustering on the molecular surface suggests the functional importance of ionic interactions by these loops. Electrophysiological studies in Xenopus oocytes point on ws-PSCA inhibition of α3β2-, high-, and low-sensitive variants of α4β2- (IC50 ~50, 27, and 15 μM, respectively) but not α4β4-nAChRs, suggesting targeting of the β2 subunit. Ensemble docking and molecular dynamics simulations predict PSCA binding to high-sensitive α4β2-nAChR at α4/β2 and β2/β2 interfaces. Complexes are stabilized by ionic and hydrogen bonds between PSCA’s loops II and III and the primary and complementary receptor subunits, including glycosyl groups. This study gives new structural and functional insights into PSCA’s interaction with molecular targets and provides clues to understand its role in the brain function and mental disorders.

Recently, we found that Lystar5 protein from coelomic cells of A. rubens starfish interacts with nicotinic acetylcholine receptors (nAChRs) and integrin α8-like protein. We hypothesized that Lystar5 mediates detachment of coelomic cells from the matrix and their migration. Skin wound healing in humans is based on keratinocytes migration and is regulated by nAChRs and integrins. Here, we revealed that Lystar5 stimulates migration of human skin HaCaT keratinocytes and peripheral blood monocytes. Using ELISA, we found that Lystar5 binds to the membrane fraction of coelomic cells with its loops I and II, which form an active site of Lystar5 and resemble its pro-migratory activity. In keratinocytes and monocytes, Lystar5 and the peptides mimicking its loops I and II bound with α3, α4, and β2 nAChR and α5, αV, and β1 integrin subunits, which form molecular complexes. In keratinocytes, Lystar5 and its mimetics promoted short-term E/N cadherin switch and upregulated expression of α5 and αV integrins, EGFR, and ICAM-1. In keratinocytes and monocytes, Lystar5 and its mimetics upregulated E-selectin secretion. The ability of Lystar5 and its mimetics to stimulate skin keratinocyte migration and immune cell infiltration may be considered promising for the development of new wound-healing agents.

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.

Mutation T226R in the Kv1.1 α-subunit of voltage-gated potassium Kv1 channels is associated with episodic ataxia type 1, severe neuromyotonia, and epilepsy. In vitro, this mutation was reported to considerably distort the functioning of homotetrameric channels Kv1.1; however, in the brain, Kv1.1 α-subunits form heterochannels predominantly associating with Kv1.2 α-subunits. Using the patch-clamp technique, fluorescent and Förster resonance energy transfer confocal microscopy, we revealed that heterochannels Kv(1.1(T226R)-1.2)2 formed by concatemers Kv1.1(T226R)-Kv1.2 in Neuro-2a cells have significantly slower activation and deactivation rates, and their activation occurs at a much less negative membrane potential compared to channels Kv(1.1-1.2)2 formed by concatemers Kv1.1-Kv1.2. This mutation does not noticeably affect the formation of complexes between α-subunits Kv1.1 and Kv1.2, but it does induce a delayed and possibly decreased presentation of heterochannels Kv(1.1(T226R)-1.2)2 on the plasma membrane. At the same time, the T226R mutation has a much stronger negative effect on the membrane presentation of homotetrameric Kv1.1 channels. Since heterochannels Kv1.1-Kv1.2 but not homotetrameric channels Kv1.1 are present in the brain, the heterochannels bearing mutation T226R are most likely underlying the pathogenesis of the disease by decreasing the responsiveness of cells to mild membrane depolarization and, thus, increasing the excitability of neurons.

Fluorogen-activating proteins are powerful molecular tools for microscopy, including functional imaging. These proteins serve as an alternative to GFP-like proteins, as they do not require oxygen for chromophore maturation. However, the restricted selectivity of proteins to chromophores, combined with the limited number of spectral channels of conventional fluorescent microscopes, hinders the development of multicolor synthetic dyes. Additionally, the poor cell and tissue permeability of synthetic chromophores further limits their utility. In this work, we address these challenges by combining time-resolved methods with the rational design of the UnaG protein, which utilizes bilirubin as a natural chromophore. To turn UnaG into a palette of probes for fluorescence lifetime imaging microscopy (FLIM), we solved two practical problems: first, we determined the limits of bilirubin lifetime variations in response to changes in the protein structure and, second, we determined what minimal structural changes can be reliably distinguished by lifetime analysis in cellula. Combining classical point mutagenesis and the translational introduction of noncanonical amino acids, we generated UnaG with fluorescence lifetimes ranging from hundreds of picoseconds to nanoseconds. We explored the potential for further modification of the UnaG protein matrix to optimize spectral and temporal characteristics of bilirubin fluorescence and its quantitative detection through time-resolved approaches.

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.

One of the approaches for treatment of COVID-19 is a use of neutralizing antibodies (nAbs). The study of the mechanisms by which nAbs recognize different strains of SARS-CoV-2 may facilitate the development of new drugs and vaccines against the coronavirus infection. In this work, we present the 3.1 & Aring; resolution cryo-electron microscopy structure of a full-length trimeric spike-protein (S-protein) of the SARS-CoV-2 Alpha (B.1.1.7) variant in complex with the Fab of the REGN10987 nAb. In the complex, two receptor-binding domains (RBDs) of the Sprotein were observed in the 'up' state, whereas third RBD was in the 'down' state. This distinguishes the obtained structure from the complex of Delta (B.1.617.2) S-protein with REGN10987-Fab, where only one RBD was in the 'up' state. Probably some of the substituted residues (K478T, A570D, and S982A) located at the interprotomer interfaces are responsible for the greater Alpha S-protein opening upon the REGN10987-Fab binding. The Fab identically binds to the RBD in the both 'up' and 'down' conformations. The RBD-Fab interaction interface was refined to a resolution of 3.6 & Aring;. The antibody binds to the receptor-binding motif (RBM), which prevents the S-protein from the binding to its receptor, angiotensin-converting enzyme 2 (ACE-2). Comparison with the structures of the Wuhan (wild type) and Delta RBD variants in complex with REGN10987-Fab revealed that the N501Y and T478K/L452R mutations presented in the RBM of the Alpha and Delta variants, respectively, do not affect the mode of the RBD-Fab interaction.

Recombinant proteorhodopsin ESR of the gram-positive bacterium Exiguobacterium sibiricum isolated from permafrost deposits in northeastern Siberia binds retinal and acts as a light-dependent proton pump, but not much is known about its expression under natural conditions. In this work, expression of the esr gene in E. sibiricum cultures grown under various conditions was studied by quantitative PCR. It has been discovered that cultivation on poor media at low temperatures contributes to a significant increase in the content of the corresponding mRNA. The data obtained are confirmed by the results of the analysis of the membrane fraction of cells using label-free quantitative chromatography-mass spectrometry. Also, at 10°C, increased content of phytoene desaturases, which are involved in the biosynthesis of carotenoids, is observed. However, we were unable to detect the presence of a functional retinal-containing protein in the cells, presumably due to the lack of an enzymatic retinal synthesis system in E. sibiricum. The possible functions of ESR in E. sibiricum cells are discussed in connection with the characteristics of the extreme habitat of the bacterium. The results of this study contribute to expanding the understanding of the molecular mechanisms of microbial adaptation to environmental conditions and the potential role of microbial rhodopsins in these processes.

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.

Therapy-induced senescence plays a crucial role in cancer treatment, evolving the resistance of malignant cells to therapeutic interventions. Therefore, the discovery of safe and effective senolytics may serve to develop new promising therapeutic options. Here we have revealed the enhanced apoptotic potential of DR5 receptor agonist, receptor-selective TRAIL variant DR5-B in temozolomide-induced senescent glioblastoma cell lines U87MG and T98G and primary tumor samples from patients with diagnosed glioblastoma. Senescence features were most pronounced in p53-proficient, MGMT-deficient U87MG cells, as demonstrated by enlarged cell and nuclei size, increased β-galactosidase activity, p21 expression and lipofuscin autofluorescence. This was accompanied by a metabolic shift towards glycolysis measured by fluorescence lifetime imaging (FLIM) of NADH, and upregulation of DR5, DcR1, DcR2 and cFLIP. As a result, a strong sensitization of U87MG cells to DR5-B-mediated apoptosis was observed after TMZ pre-treatment. However, neither was observed in p53-mutated, MGMT-proficient T98G cells. Differential gene expression analysis in TMZ-treated U87MG cells showed the activation of proinflammatory and proapoptotic signaling and downregulation of genes related to DNA metabolism and cell cycle. Two of three primary patient-derived glioblastoma samples tested acquired similar senescence features and were sensitized to DR5-B-mediated apoptosis by TMZ pre-treatment. These findings suggest that TMZ-induced senescence enhances glioblastoma cell sensitivity to DR5 receptor agonists. However, when developing strategies for senolytic antitumor therapy, the heterogeneous response of tumor cells to senescence induction should be taken into account.