Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology最新文献

Heat shock protein 70 (HSP70) and HSP70-based vaccine constructs are perspective tools for anticancer therapy. One of the established anticancer effects of HSP70 is activating natural killer (NK) cells. Moreover, HSP70-activated NK cells demonstrate effectiveness in preclinical glioblastoma and lung cancer models. It is thought that the expression of HSP70 on the surface of cancer cells stimulates the cytotoxic activity of NK cells. Regarding this, the spatial aspects of NK cells with cancer cells in the presence of HSP70 are essential but not properly investigated. Using confocal laser scanning microscopy, we have recently shown that exogenously added HSP70 is absorbed on the surface of dying NK cells, while live NK cells internalize the protein. In the present study, we visualized the spatial aspects of NK cell interactions with the target breast cancer cell line BT-20 in the presence of exogenous HSP70. We traced the uptake of HSP70 by BT-20 cells and investigated NK cell behavior and contacts with BT-20 cells in the presence of HSP70. The data obtained in the present study on the interaction of BT-20 cells, NK cells, and HSP70 revealed novel aspects of HSP70-mediated anticancer efficacy of NK cells.

Surface plasmon resonance (SPR) is a relevant method for studying interactions involving membrane proteins, mitochondrial proteins in particular. Currently, there is only a little information on the interaction of mitochondrial proteins with their partners, such as electron carriers, in lipid microenvironment. In this work, using cytochrome P450 11β-hydroxylase (CYP11B1) and adrenodoxin (Adx) as an example, we showed that SPR analysis can be used for studying protein–protein interactions in lipid microenvironment. Kinetic and equilibrium parameters of the CYP11B1-Adx complexes in lipid bilayers of different compositions were determined. Our results reveal that the CYP11B1-Adx complex in cardiolipin-containing bilayer demonstrates increased stability: nearly tenfold reduction in the equilibrium dissociation constant (Kd) compared to the bilayer without cardiolipin. The interaction between redox proteins is highly sensitive to the lipid structure. The immobilized protein and the lipid composition should be selected in each case, but overall, the obtained data are highly complementary for studying membrane proteins.

Dendritic cells belong to a heterogenic population of innate immune cells distributed across the tissues. These cells are considered to be potential therapeutic targets due to their ability to sense exogenous antigens and self-tissue damage. In the lungs, two subpopulations of conventional dendritic cells, cDC1 and cDC2, were identified. Distinguishing the populations is commonly performed by flow cytometry based on the different expressions of transcription factors and surface markers. Thus, after the exclusion of precursors and the cells rather than dendritic cells, G protein-coupled receptor XCR1 and membrane glycoprotein SIRPA (CD172a) are used to characterize cDC1 as XCR1^hiCD172a^lo and cDC2 as XCR1^loCD172a^hi. Analysis by flow cytometry permits the accurate identification of the subpopulations; however, to define the precise location of cDC1 and cDC2 in relation to the structural tissue cells, confocal laser scanning microscopy (CLSM) is required. Together with the advantage of spatial cell distribution identification, CLSM has two limitations compared to flow cytometry. The former is the limited number of antibodies that can be simultaneously applied. Besides, not all the antibodies suitable for cell staining and flow cytometry show stable antigen recognition during the tissue staining and CLSM. In this study, we applied anti-XCR1 and anti-CD172a to visualize cDC1 and cDC2 in conducting airway mucosa of mice. We detect the distribution of these cells in a steady state and upon the airway inflammation.

LOV (Light Oxygen Voltage) domains are ubiquitous blue light sensors widespread in all three domains of life. They mediate signal transduction in diverse biological processes and can be useful for practical applications in optogenetics and fluorescence microscopy. The canonical mechanism of LOV domains signal transduction is based on formation of the covalent adduct between flavin chromophore and the highly conserved cysteine in the active site of the protein leading to downstream signaling cascade. However, previous studies showed that there are natural cysteine-less LOV domains enabling signal transduction via flavin photoreduction. In the absence of the conserved cysteine, photoexcitation causes flavin one electron reduction to the neutral semiquinone radical state and subsequent structural reorganizations as in the canonical photocycle. In this work, previously uncharacterized natural cysteine-less LOV domain from thermophilic haloarchaeon Halanaeroarchaeum sulfurireducens, named HsuLOV, was successfully expressed in Escherichia coli and purified in soluble form. Although HsuLOV bound a small amount of flavin when overexpressed in E. coli, the chromophore load was increased by protein incubation with flavin mononucleotide in vitro. It was demonstrated that fluorescence spectra of purified and chromophore-loaded HsuLOV are typical for LOV domains. Finally, the comparison of absorption spectra of dark- and light-adapted states of HsuLOV revealed flavin photoreduction to the neutral semiquinone radical state, thus confirming photoactivity of HsuLOV.

IRR is a member of the insulin receptor (IR) family that has no known endogenous peptide agonists but can be activated by weak alkaline environments and has thus been proposed to function as an extracellular pH sensor. IRR activation by alkali is determined by its N-terminal extracellular region. Using recently published Cryo-EM structure of IRR ectodomain we identify three amino acids (R87, K143 and S516) that may be involved in the IRR activation by alkali. In this report we show that the triple substitution of these amino acids with alanine leads to complete disappearance of the IRR pH sensitivity. Thus, these amino acids can form one of the possible pH sensor sites in the IRR molecule.

Receptor for advanced glycation endproducts (RAGE) plays an important role in the development of inflammation and neurodegenerative diseases. There is no expression of RAGE in healthy cells, but it increases during inflammation processes that leads to tissue damage. RAGE has many ligands of different classes but with similar properties, so RAGE acts as a pattern-recognition receptor. The structure of RAGE lacks information about the transmembrane domain which is necessary for signal transduction by the receptor. In this work, for the first time, using the cell-free expression method, we obtained and purified isotope-labeled fragment of RAGE (residues 335–368, corresponding to the RAGE transmembrane domain flanked by short juxtamembrane regions). For investigation of oligomerization processes we introduced a point oncogenic mutation G349R that is located in the conservative oligomerization motif GxxxG. Nuclear magnetic resonance (NMR) studies of both peptides incorporated into dodecylphosphocholine (DPC) micelles indirectly reveal changes in protein structure and oligomerization properties following the introduction of the oncogenic G349R mutation, which is thought to affect RAGE signaling.

Unnatural amino acids (UAAs) are powerful tools for investigating protein structure and dynamics. Their inclusion in a protein can help reveal important details about its functions and structural features, especially with respect to membrane proteins. However, efficient incorporation of UAAs into membrane proteins remains a challenge. In this review, we explore using Leishmania tarentolae (LEXSY) as a host system to express membrane proteins with incorporated UAAs, aiming to develop a versatile platform for future research and drug discovery.

Rhodopsins are photosensitive transmembrane proteins found in bacteria, archaea, and eukaryotes. Upon photon absorption, they undergo conformational changes critical for their physiological functions, such as ion transport and signal transduction. Microbial rhodopsins are key tools in optogenetics, with applications in biomedical research and clinical treatments. To advance optogenetic techniques, it is crucial to understand the molecular mechanisms of the rhodopsin photocycle. The key approach for studying these mechanisms, cryotrapping, has a setback associated with possible deterioration in resolution due to the unexpectedly large light-induced structural changes. To estimate the scale of these changes, small-angle X-ray scattering (SAXS) can be used. In this study, we applied SAXS to the investigation of light-induced structural changes in purple membranes (PMs) as their two-dimensional crystal organization makes them a suitable model object for studying light-induced changes in 3D rhodopsin crystals. SAXS data from illuminated and non-illuminated PM samples revealed detectable changes, including variations in membrane thickness and planar unit cell dimensions. Particularly, we observed a statistically significant increase in the radius of gyration for flat particles (Rₜ) of ~0.2 Å for the PMs with mutant HsBR_E204Q, while no significant change was detected for wild-type HsBR. However, wild-type HsBR data showed a statistically significant shift in the (1, 1) Bragg peak position (Δ q ~ –10^–4 Å^–1; ~3% of the peak width) upon illumination. For HsBR_E204Q data, the shift was two times greater; however, in this case due to the lower sample concentration ε_Δq was ~50%, making it difficult to compare the wild-type and mutant cases. Future experiments should aim for better signal-to-noise ratios using synchrotron radiation, higher sample concentrations, and longer exposure times.

Nowadays, only a few structures of rhodopsins bound with carotenoid were obtained and functionally characterized. Nevertheless, rhodopsins-carotenoids complexes are promising molecular systems for understanding the light-harvesting process in bacteria since such complexes show ability to absorb a significant part of solar energy coming to the Earth surface. The understanding of energy absorption and transfer mechanisms in the carotenoid-retinal complex of rhodopsin would provide significant progress in different areas of modern bioscience such as ecology, protein engineering, optogenetics, etc. Here we present a long (1 µs) molecular dynamical investigation of the structure of microbial rhodopsin from an Antarctic bacterium Hymenobacter psoromatis (strain PAMC26554) (HbR1) and zeaxanthin. The structure of rhodopsin was predicted with a new multi-modal foundation for molecular structure prediction Chai-1 and used for classical MD validation in a 10 × 10 × 12 nm box in a hydrated explicit lipid bilayer. Our simulations show tight binding of the carotenoid and rhodopsin with the average distance equal to 0.25 nm between the closest atoms of carotenoid and retinal.

Here we explore capabilities of size exclusion chromatography small-angle X-ray scattering method in case of horse spleen apoferritin solution as an example of a system with high oligomerization heterogeneity. For interpretation of the experimental data, we considered models of polydisperse solution containing monomers, dimers, and trimers of apoferritin globules. Dimers were defined only by intercenter distance L, while trimers were characterized by both L and apical corner value φ. We showed that a reasonable description of the major part of the chromatogram could be reached using the model containing monomers, dimers and one type of trimers with φ about 110°–120°. In the case of two types of trimers in the model, optimal φ values were 110°–140° and 90°–110°; however, the approximation results were considered as ambiguous. Validation of the preferred model (L = 12.5 nm, φ = 120°) showed insufficient description of the experimental data for the left part of the chromatogram (elution volume <12.2 mL) indicating the presence of aggregates in these fractions. Application of the method provided the opportunity to calculate concentrations for oligomers consisting of less than four apoferritin globules, and select averaging range for different fractions properly.