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Membranes May 2023In this work, we have developed a method for the preparation of pH-responsive track-etched membranes (TeMs) based on poly(ethylene terephthalate) (PET) with pore...
In this work, we have developed a method for the preparation of pH-responsive track-etched membranes (TeMs) based on poly(ethylene terephthalate) (PET) with pore diameters of 2.0 ± 0.1 μm of cylindrical shape by RAFT block copolymerization of styrene (ST) and 4-vinylpyridine (4-VP) to be used in the separation of water-oil emulsions. The influence of the monomer concentration (1-4 vol%), the molar ratio of RAFT agent: initiator (1:2-1:100) and the grafting time (30-120 min) on the contact angle (CA) was studied. The optimal conditions for ST and 4-VP grafting were found. The obtained membranes showed pH-responsive properties: at pH 7-9, the membrane was hydrophobic with a CA of 95°; at pH 2, the CA decreased to 52°, which was due to the protonated grafted layer of poly-4-vinylpyridine (P4VP), which had an isoelectric point of pI = 3.2. The obtained membranes with controlled hydrophobic-hydrophilic properties were tested by separating the direct and reverse "oil-water" emulsions. The stability of the hydrophobic membrane was studied for 8 cycles. The degree of purification was in the range of 95-100%.
PubMed: 37233585
DOI: 10.3390/membranes13050523 -
Theranostics 2023Cancer is generally considered a result of genetic mutations that cause epigenetic changes, leading to anomalous cellular behavior. Since 1970s, an increasing... (Review)
Review
Cancer is generally considered a result of genetic mutations that cause epigenetic changes, leading to anomalous cellular behavior. Since 1970s, an increasing understanding of the plasma membrane and specifically the lipid alterations in tumor cells have provided novel insights for cancer therapy. Moreover, the advances in nanotechnology offer a potential opportunity to target the tumor plasma membrane while minimizing side effects on normal cells. To further develop membrane lipid perturbing tumor therapy, the first section of this review demonstrates the association between plasma membrane physicochemical properties and tumor signaling, metastasis, and drug resistance. The second section highlights existing nanotherapeutic strategies for membrane disruption, including lipid peroxide accumulation, cholesterol regulation, membrane structure disruption, lipid raft immobilization, and energy-mediated plasma membrane perturbation. Finally, the third section evaluates the prospects and challenges of plasma membrane lipid perturbing therapy as a therapeutic strategy for cancers. The reviewed membrane lipid perturbing tumor therapy strategies are expected to bring about necessary changes in tumor therapy in the coming decades.
Topics: Humans; Neoplasms; Membrane Lipids; Cell Membrane; Membrane Microdomains; Nanotechnology
PubMed: 37215569
DOI: 10.7150/thno.82189 -
BioRxiv : the Preprint Server For... Nov 2023CD4 T cell activation is driven by 5-module receptor complexes. The T cell receptor (TCR) is the receptor module that binds composite surfaces of peptide antigens...
CD4 T cell activation is driven by 5-module receptor complexes. The T cell receptor (TCR) is the receptor module that binds composite surfaces of peptide antigens embedded within MHCII molecules (pMHCII). It associates with three signaling modules (CD3γε, CD3δε, and CD3ζζ) to form TCR-CD3 complexes. CD4 is the coreceptor module. It reciprocally associates with TCR-CD3-pMHCII assemblies on the outside of a CD4 T cells and with the Src kinase, LCK, on the inside. Previously, we reported that the CD4 transmembrane GGXXG and cytoplasmic juxtamembrane (C/F)CV+C motifs found in eutherian (placental mammal) CD4 have constituent residues that evolved under purifying selection (Lee, et al., 2022). Expressing mutants of these motifs together in T cell hybridomas increased CD4-LCK association but reduced CD3ζ, ZAP70, and PLCγ1 phosphorylation levels, as well as IL-2 production, in response to agonist pMHCII. Because these mutants preferentially localized CD4-LCK pairs to non-raft membrane fractions, one explanation for our results was that they impaired proximal signaling by sequestering LCK away from TCR-CD3. An alternative hypothesis is that the mutations directly impacted signaling because the motifs normally play an LCK-independent role in signaling. The goal of this study was to discriminate between these possibilities. Using T cell hybridomas, our results indicate that: intracellular CD4-LCK interactions are not necessary for pMHCII-specific signal initiation; the GGXXG and (C/F)CV+C motifs are key determinants of CD4-mediated pMHCII-specific signal amplification; the GGXXG and (C/F)CV+C motifs exert their functions independently of direct CD4-LCK association. These data provide a mechanistic explanation for why residues within these motifs are under purifying selection in jawed vertebrates. The results are also important to consider for biomimetic engineering of synthetic receptors.
PubMed: 37214965
DOI: 10.1101/2023.05.05.539613 -
BioRxiv : the Preprint Server For... May 2023The inhibitory Fcγ receptor FcγRIIb is involved in immune regulation and is known to localize to specific regions of the plasma membrane called lipid rafts. Previous...
The inhibitory Fcγ receptor FcγRIIb is involved in immune regulation and is known to localize to specific regions of the plasma membrane called lipid rafts. Previous studies suggested a link between the altered lateral receptor localization within the plasma membrane and the functional impairment of the FcγRIIb-I232T variant that is associated with systemic lupus erythematosus. Here, we conducted microsecond all-atom molecular dynamics simulations and IgG binding assays to investigate the lipid nano-environment of FcγRIIb monomers and of the FcγRIIb-I232T mutant within a plasma membrane model, the orientation of the FcγRIIb ectodomain, and its accessibility to IgG ligands. In contrast to previously proposed models, our simulations indicated that FcγRIIb does not favor a cholesterol- or a sphingolipid-enriched lipid environment. Interestingly, cholesterol was depleted for all studied FcγRIIb variants within a 2-3 nm environment of the receptor, counteracting the usage of raft terminology for models on receptor functionality. Instead, the receptor interacts with lipids that have poly-unsaturated fatty acyl chains and with (poly-) anionic lipids within the cytosolic membrane leaflet. We also found that FcγRIIb monomers adopt a conformation that is not suitable for binding to its IgG ligand, consistent with a lack of detectable binding of monomeric IgG in experiments on primary immune cells. However, our results propose that multivalent IgG complexes might stabilize FcγRIIb in a binding-competent conformation. We suggest differences in receptor complex formation within the membrane as a plausible cause of the altered membrane localization or clustering and the altered suppressive function of the FcγRIIb-I232T variant.
PubMed: 37214871
DOI: 10.1101/2023.05.09.540011 -
Nature Communications May 2023The spatial organization of cell membrane glycoproteins and glycolipids is critical for mediating the binding of ligands, receptors, and macromolecules on the plasma...
The spatial organization of cell membrane glycoproteins and glycolipids is critical for mediating the binding of ligands, receptors, and macromolecules on the plasma membrane. However, we currently do not have the methods to quantify the spatial heterogeneities of macromolecular crowding on live cell surfaces. In this work, we combine experiment and simulation to report crowding heterogeneities on reconstituted membranes and live cell membranes with nanometer spatial resolution. By quantifying the effective binding affinity of IgG monoclonal antibodies to engineered antigen sensors, we discover sharp gradients in crowding within a few nanometers of the crowded membrane surface. Our measurements on human cancer cells support the hypothesis that raft-like membrane domains exclude bulky membrane proteins and glycoproteins. Our facile and high-throughput method to quantify spatial crowding heterogeneities on live cell membranes may facilitate monoclonal antibody design and provide a mechanistic understanding of plasma membrane biophysical organization.
Topics: Humans; Cell Membrane; Membrane Proteins; Phagocytosis; Antibodies, Monoclonal; Glycoproteins; Macromolecular Substances
PubMed: 37208326
DOI: 10.1038/s41467-023-38525-2 -
Cellular and Molecular Life Sciences :... May 2023Inflammation can impair intestinal barrier, while increased epithelial permeability can lead to inflammation. In this study, we found that the expression of Tspan8, a...
Inflammation can impair intestinal barrier, while increased epithelial permeability can lead to inflammation. In this study, we found that the expression of Tspan8, a tetraspanin expressed specifically in epithelial cells, is downregulated in mouse model of ulcerative disease (UC) but correlated with those of cell-cell junction components, such as claudins and E-cadherin, suggesting that Tspan8 supports intestinal epithelial barrier. Tspan8 removal increases intestinal epithelial permeability and upregulates IFN-γ-Stat1 signaling. We also demonstrated that Tspan8 coalesces with lipid rafts and facilitates IFNγ-R1 localization at or near lipid rafts. As IFN-γ induces its receptor undergoing clathrin- or lipid raft-dependent endocytosis and IFN-γR endocytosis plays an important role in Jak-Stat1 signaling, our analysis on IFN-γR endocytosis revealed that Tspan8 silencing impairs lipid raft-mediated but promotes clathrin-mediated endocytosis of IFN-γR1, leading to increased Stat1 signaling. These changes in IFN-γR1 endocytosis upon Tspan8 silencing correlates with fewer lipid raft component GM1 at the cell surface and more clathrin heavy chain in the cells. Our findings indicate that Tspan8 determines the IFN-γR1 endocytosis route, to restrain Stat1 signaling, stabilize intestine epithelium, and subsequently prevent intestine from inflammation. Our finding also implies that Tspan8 is needed for proper endocytosis through lipid rafts.
Topics: Animals; Mice; Clathrin; Endocytosis; Inflammation; Interferons; Intestinal Mucosa; Receptors, Interferon; Tetraspanins
PubMed: 37204469
DOI: 10.1007/s00018-023-04803-x -
International Journal of Molecular... May 2023Aquaporin 3 (AQP3) is a peroxiporin, a membrane protein that channels hydrogen peroxide in addition to water and glycerol. AQP3 expression also correlates with tumor...
Aquaporin 3 (AQP3) is a peroxiporin, a membrane protein that channels hydrogen peroxide in addition to water and glycerol. AQP3 expression also correlates with tumor progression and malignancy and is, therefore, a potential target in breast cancer therapy. In addition, epithelial growth factor receptor (EGFR) plays an important role in breast cancer. Therefore, we investigated whether disruption of the lipid raft harboring EGFR could affect AQP3 expression, and conversely, whether AQP3 silencing would affect the EGFR/phosphoinositide-3-kinase (PI3K)/Protein kinase B (PKB or Akt) signaling pathway in breast cancer cell lines with different malignant capacities. We evaluated HO uptake, cell migratory capacity, and expression of PI3K, pAkt/Akt in three breast cancer cell lines, MCF7, SkBr3, and SUM159PT, and in the nontumorigenic breast epithelial cell line MCF10A. Our results show different responses between the tested cell lines, especially when compared to the nontumorigenic cell line. Neither lipid raft disruption nor EGF stimuli had an effect on PI3K/Akt pathway in MCF10A cell line. AQP3-silencing in SkBr3 and SUM159PT showed that AQP3 can modulate PI3K/Akt activation in these cells. Interestingly, SUM159PT cells increase nuclear factor-E2-related factor 2 (NRF2) in response to lipid raft disruption and EGF stimuli, suggesting an oxidative-dependent response to these treatments. These results suggest that in breast cancer cell lines, AQP3 is not directly related to PI3K/Akt pathway but rather in a cell-line-dependent manner.
Topics: Female; Humans; Aquaporin 3; Breast Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Hydrogen Peroxide; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt
PubMed: 37175840
DOI: 10.3390/ijms24098133 -
International Journal of Molecular... Apr 2023The gel-phase domains in a binary supported lipid bilayer (SLB) comprising dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) were localized on...
The gel-phase domains in a binary supported lipid bilayer (SLB) comprising dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) were localized on graphene oxide (GO) deposited on a SiO/Si substrate. We investigated the distribution of the gel-phase domains and the liquid crystalline (L) phase regions in DOPC+DPPC-SLB on thermally oxidized SiO/Si substrates with GO flakes to understand the mechanism of the domain localization on GO. Fluorescence microscopy and atomic force microscopy revealed that the gel-phase domains preferably distributed on GO flakes, whereas the fraction of the L-phase increased on the bare SiO surface which was not covered with the GO flakes. The gel-phase domain was condensed on GO more effectively at the lower cooling rate. We propose that nucleation of the gel-phase domain preferentially occurred on GO, whose surface has amphiphilic property, during the gel-phase domain formation. The domains of the liquid ordered (L) phase were also condensed on GO in a ternary bilayer containing cholesterol that was phase-separated to the L phase and the liquid disordered phase. Rigid domains segregates on GO during their formation process, leaving fluid components to the surrounding region of GO.
Topics: Lipid Bilayers; Silicon Dioxide; Phosphatidylcholines; 1,2-Dipalmitoylphosphatidylcholine; Graphite; Microscopy, Atomic Force
PubMed: 37175707
DOI: 10.3390/ijms24097999 -
Biochemical and Biophysical Research... Jul 2023The interplay between membrane subregions and receptor tyrosine kinases (RTK) will influence signaling in both normal and pathological RTK conditions. In this study,...
The interplay between membrane subregions and receptor tyrosine kinases (RTK) will influence signaling in both normal and pathological RTK conditions. In this study, epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor β (PDGFR-β) internalizations were investigated by immunofluorescent microscopy following simultaneous treatment with EGF and PDGF-BB. We found that the two receptors utilize separate routes of internalization, which merges in a common perinuclear endosomal compartment after 45 min of stimulation. This is further strengthened when contrasting the recruitment of either EGFR or PDGFR-β to either clathrin or caveolin-1: PDGFR-β dissociates from caveolin-1 upon stimulation, and engages clathrin, whilst an increased recruitment of EGFR, to both clathrin and caveolin-1, was observed upon EGF stimulation. The association between EGFR and caveolin-1 is supported by the observation that EGFR was localized in lipid raft associated fractions, whereas PDGFR-β was not. We also found that disruption of lipid rafts using MβCD led to an increased EGFR dimerization and phosphorylation in response to ligand, as well as a dramatic decrease in AKT- and a smaller but robust decrease in ERK1/2 phosphorylation. This suggest that lipid rafts may be important to effectively connect the EGFR with downstream proteins to facilitate signaling. Our data implies that cholesterol depletion of the plasma membrane affect the signaling of EGFR and PDGFRβ differently.
Topics: Proto-Oncogene Proteins c-akt; Caveolin 1; Epidermal Growth Factor; MAP Kinase Signaling System; ErbB Receptors; Phosphorylation; Receptor, Platelet-Derived Growth Factor beta; Clathrin; Cholesterol
PubMed: 37163940
DOI: 10.1016/j.bbrc.2023.04.099 -
ELife May 2023Flaviviruses are enveloped viruses which include human pathogens that are predominantly transmitted by mosquitoes and ticks. Some, such as dengue virus, exhibit the...
Flaviviruses are enveloped viruses which include human pathogens that are predominantly transmitted by mosquitoes and ticks. Some, such as dengue virus, exhibit the phenomenon of antibody-dependent enhancement (ADE) of disease, making vaccine-based routes of fighting infections problematic. The pH-dependent conformational change of the envelope (E) protein required for fusion between the viral and endosomal membranes is an attractive point of inhibition by antivirals as it has the potential to diminish the effects of ADE. We examined six flaviviruses by employing large-scale molecular dynamics (MD) simulations of raft systems that represent a substantial portion of the flaviviral envelope. We utilised a benzene-mapping approach that led to a discovery of shared hotspots and conserved cryptic sites. A cryptic pocket previously shown to bind a detergent molecule exhibited strain-specific characteristics. An alternative conserved cryptic site at the E protein domain interfaces showed a consistent dynamic behaviour across flaviviruses and contained a conserved cluster of ionisable residues. Constant-pH simulations revealed cluster and domain-interface disruption under low pH conditions. Based on this, we propose a cluster-dependent mechanism that addresses inconsistencies in the histidine-switch hypothesis and highlights the role of cluster protonation in orchestrating the domain dissociation pivotal for the formation of the fusogenic trimer.
Topics: Animals; Humans; Flavivirus; Molecular Dynamics Simulation; Histidine; Hydrogen-Ion Concentration; Viral Envelope Proteins
PubMed: 37144875
DOI: 10.7554/eLife.82447