High-affinity Membrane Research Articles

Melatonin as an inducer of Th17 cell differentiation: new mechanisms

Cells of the immune system are sensitive to the action of melatonin, and the most obvious target of the hormone is the Th17 T helper subset: in addition to two high-affinity membrane receptors for melatonin, MT1 and MT2, these cells express a nuclear receptor, RORα. Among the secondary messengers involved in the transmission of signals from melatonin receptors, proteins of the sirtuin family are currently of particular interest. It is known that in non-tumor cells, sirtuin 1 (SIRT1) not only increases its expression in response to melatonin, but is also involved in the implementation of melatonin effects, as indicated by inhibitory assays using a specific SIRT1 blocker or corresponding siRNA/shRNA. This relates to the regulation of circadian oscillators, as well as the anti-inflammatory, antioxidant and anti-apoptotic effects of melatonin. Further mechanisms of SIRT1 activity in the cell include transcriptional and posttranscriptional regulation of gene expression through deacetylation of histones and non-histone proteins, and the apparent target of SIRT1 is the transcription factor RORα. It is this factor that mediates classical melatonin/SIRT1-dependent transcriptional control of key circadian regulators, the genes of which have ROR-binding sequences in their promoters. These data raise questions about the functions of SIRT1 in other cells expressing RORα, in particular in Th17 T helper cells, for which it is one of two key differentiation factors, along with RORγt. And if for RORα such a connection still remains hypothetical, for RORγt it has been convincingly demonstrated in studies both in vivo and in vitro: it has been shown that SIRT1 directly binds to RORγt and stimulates the development of Th17 cells, and blockade of sirtuin suppresses the differentiation of these cells in normal and prevents the development of Th17-associated pathology in mice. Summarizing these data, we can confidently predict the existence of a new mechanism for the regulation of the T helper Th17 population by melatonin, through the activation of sirtuin SIRT1, and this mechanism must be taken into account when interpreting data on the immunoregulatory activity of melatonin.

Thiamine status and genes encoding intestinal thiamine transporters and transcription factors in obese subjects

Background and aimsThe inconsistent data on thiamine status in obese subjects necessitates an examination of genes associated with intestinal absorption of thiamine. We aimed to reveal thiamine status in obese subjects and examine the expression of SLC19A2/3 genes encoding thiamine transporters and Sp1 transcription factor. Methods and resultsThirty-five adult obese subjects and 11 healthy controls were included in this cross-sectional study. Small intestine epithelial cells were used for quantitative RT-PCR analysis of the gene expression. The daily thiamine and energy intake were assessed with a food frequency questionnaire. Thiamine phosphate esters were hydrolyzed to free thiamine, and liquid chromatography with a tandem mass spectrometry-based method was used to measure total thiamine in whole blood. Daily energy intake according to body weight and daily carbohydrate intake were not significantly different between groups after adjustment for sex. Although daily thiamine intake was significantly lower in the obesity group (p = 0.015), obese subjects had significantly higher whole blood thiamine levels than controls (44.96 ± 14.6 ng/mL and 33.05 ± 8.6 ng/mL, p = 0.002). There was a significant positive correlation between whole blood thiamine and BMI (r = 0.342, p = 0.020). SLC19A2 gene expression was lower in those with BMI ≥35 kg/m2 (p = 0.036). A significant positive correlation was found between SLC19A2 expression and whole blood thiamine level (r = 0.310, p = 0.038). ConclusionA possible association between intestinal thiamine intake and total thiamine in whole blood was determined. The transcriptional changes of genes encoding the high-affinity membrane thiamine transporters, especially SLC19A2, probably play a role in this relationship.

Effect of Conformational Variability on the Drug Resistance of Candida auris ERG11p and FKS1.

Candida auris infection has been recognized as an urgent threat to antifungal drug resistance, and the Eagle effect of C. auris FKS1 (1,3-β-d-glucan synthase) wild-type isolates has also been noted. The Eagle effect, namely, where higher concentrations of antifungals reduce fungicidal activity relative to lower concentrations, is a confounding factor of apparent antifungal resistance, but the detailed mechanism remains unclear. Here, we present the conformational variability of mutation sites for ERG11p (lanosterol 14α-demethylase) and FKS1 from deep neural network-based prediction along with the reported X-ray crystallographic and cryo-electron microscopy (cryo-EM) structures of antifungals. The sequence variability maps provide valuable insights into the inconsistent correlation between azole resistance and the mysterious Eagle effect with the dispersion of minimal inhibitory concentration (MIC) for echinocandin resistance. The conformational variability prediction supports the hypothesis that mutations K143R of clade I, VF125AL of clade III, and Y132F of clade IV for C. auris ERG11p make the corresponding site variable and that an increased population of invisible variable conformations potentially contributes to triazole resistance. In contrast, the predicted rigid conformation by the S639F mutation of hot spot region 1 (HS1) for FKS1 suggests that caspofungin (CAS) is involved in an uncompetitive inhibition, and a decreased population of the CAS-bound state of FKS1 with Rho1 leads to drug resistance. The predicted variable HS1 region for FKS1 WT isolates and the rigid one for FKS1 S639F mutants support the in vivo drug response and the in vitro MIC dispersion. A plausible mechanism of the Eagle effect is hereby proposed, namely, that a high concentration of CAS with a high membrane affinity reduces the population of the CAS-bound state of FKS1 with Rho1, as well as accompanying events such as aggregation or association depending on the conformational variability of HS1.

Fluorescence lifetime imaging and phasor analysis of intracellular porphyrinic photosensitizers applied with different polymeric formulations

The fluorescence lifetime of a porphyrinic photosensitizer (PS) is an important parameter to assess the aggregation state of the PS even in complex biological environments. Aggregation-induced quenching of the PS can significantly reduce the yield of singlet oxygen generation and thus its efficiency as a medical drug in photodynamic therapy (PDT) of diseased tissues. Hydrophobicity and the tendency to form aggregates pose challenges on the development of efficient PSs and often require carrier systems. A systematic study was performed to probe the impact of PS structure and encapsulation into polymeric carriers on the fluorescence lifetime in solution and in the intracellular environment. Five different porphyrinic PSs including chlorin e6 (Ce6) derivatives and tetrakis(m-hydroxyphenyl)-porphyrin and -chlorin were studied in free form and combined with polyvinylpyrrolidone (PVP) or micelles composed of triblock-copolymers or Cremophor. Following incubation of HeLa cells with these systems, fluorescence lifetime imaging combined with phasor analysis and image segmentation was applied to study the lifetime distribution in the intracellular surrounding. The data suggest that for free PSs, the structure-dependent cell uptake pathways determine their state and emission lifetimes. PS localization in the plasma membrane yielded mostly monomers with long fluorescence lifetimes whereas the endocytic pathway with subsequent lysosomal deposition adds a short-lived component for hydrophilic anionic PSs. Prolonged incubation times led to increasing contributions from short-lived components that derive from aggregates mainly localized in the cytoplasm. Encapsulation of PSs into polymeric carriers led to monomerization and mostly fluorescence emission decays with long fluorescence lifetimes in solution. However, the efficiency depended on the binding strength that was most pronounced for PVP. In the cellular environment, PVP was able to maintain monomeric long-lived species over prolonged incubation times. This was most pronounced for Ce6 derivatives with a logP value around 4.5. Micellar encapsulation led to faster release of the PSs resulting in multiple components with long and short fluorescence lifetimes. The hydrophilic hardly aggregating PS exhibited a mostly stable invariant lifetime distribution over time with both carriers. The presented data are expected to contribute to optimized PDT treatment protocols and improved PS-carrier design for preventing intracellular fluorescence quenching. In conclusion, amphiphilic and concurrent hydrophobic PSs with high membrane affinity as well as strong binding to the carrier have best prospects to maintain their photophysical properties in vivo and serve thus as efficient photodynamic diagnosis and PDT drugs.

Advancing Photodynamic Antimicrobial Strategy: Sustainable Fabrication of Novel Lauryl Gallate-Chitosan Hydrophobic Films with Rapid Bacterial Capture and Biofilms Elimination Capabilities for Promoting Wound Healing.

Bioinspired photoactive composites, in terms of photodynamic inactivation, cost-effectiveness, and biosafety, are promising alternatives to antibiotics for combating bacterial infections while avoiding antibacterial resistance. However, the weak bacterial membrane affinity of the photoactive substrate and the lack of synergistic antibacterial effect remain crucial shortcomings for their antibacterial applications. Herein, we developed a hydrophobic film from food antioxidant lauryl gallate covalently functionalized chitosan (LG-g-CS conjugates) through a green radical-induced grafting reaction that utilizes synergistic bacteria capture, contact-killing, and photodynamic inactivation activities to achieve enhanced bactericidal and biofilm elimination capabilities. Besides, the grafting reaction mechanism between LG and CS in the ascorbic acid (AA)/H2O2 redox system was further proposed. The LG-g-CS films feature hydrophobic side chains and photoactive phenolic hydroxyl groups, facilitating dual bactericidal activities through bacteria capture and contact-killing via strong hydrophobic and electrostatic interactions with bacterial membranes as well as blue light (BL)-driven photodynamic bacterial eradication through the enhanced generation of reactive oxygen species. As a result, the LG-g-CS films efficiently capture and immobilize bacteria and exhibit excellent photodynamic antibacterial activity against model bacteria (Escherichia coli and Staphylococcus aureus) and their biofilms under BL irradiation. Moreover, LG-g-CS films could significantly promote the healing process of S. aureus-infected wounds. This research demonstrates a new strategy for designing and fabricating sustainable bactericidal and biofilm-removing materials with a high bacterial membrane affinity and photodynamic activity.

A high‐performance cell‐labeling NIR‐II dye for in vivo cell tracking

AbstractFluorescent dyes that emit in the second near‐infrared (NIR‐II, 1000–3000 nm) region have provided significant advances toward real‐time and high‐resolution imaging of vessel and lymphatic system. However, in vivo NIR‐II tracking of the fate of labeled cells still remains challenging. Here, we develop a shielding unit–donor–acceptor–donor–shielding unit (S‐D‐A‐D‐S) NIR‐II fluorophore (FE‐4ZW) with zwitterionic terminal groups for high‐efficiency cell labeling without using cell‐penetrating peptides, which provides for enhanced non‐invasive in vivo determination of the location of cell migration. The tethering terminal sulfoammonium inner salts are featured with its high affinity for cell membranes, thereby enabling the stable labeling even for fixed cells. The fate of transplanted stem cell and the tumor cell migration along lymphatic system in brain or periphery tissues are clearly monitored by the cell‐internalized FE‐4ZW. We also confirmed that a clinically used surfactant, D‐α‐tocopheryl polyethylene glycol‐1000 succinate, can reduce the liver and spleen uptake of FE‐4ZW. The fluorophore design strategy and cell‐labeling technology reported here open a new realm in the visualization of cell migration and insight into the relocation process, thereby ultimately providing an opportunity to investigate in greater detail of the underlying mechanisms of stem cell therapy and tumor metastasis.

A versatile food packaging film fabricated by gallic acid/octanoic acid grafted chitosan: Preparation, characterization, high-efficient cells capture and photodynamic eradication of bacteria and biofilms

This study aims to construct a versatile film fabricated by gallic acid/octanoic acid-grafted-chitosan (GA/OA-g-CS) for high photodynamic antibacterial and antibiofilm efficacy with blue light (BL, 420 nm) irradiation. First, the GA/OA-g-CS conjugates were synthesized by a green radical-induced grafting reaction in the redox system composed of ascorbic acid/hydroxyl peroxide (AA/H2O2). Then, the molecular structural and physicochemical characteristics of the conjugates, as well as the mechanisms of the free radical grafting reaction were further studied. Ascorbate radical (Asc•−) and hydroxyl radical (•OH) were produced in the AA/H2O2 system, and •OH reacted with CS to produce CS macro-radicals (•CS). UV–vis, Fourier transform infrared, nuclear magnetic resonance spectroscopy, X-ray diffraction (XRD), and thermogravimetric analysis confirmed that GA/OA was attached onto CS through •OH. Notably, the films constructed by GA/OA-g-CS exhibited robust multi-modal functionalities, including intriguing bacteria-capturing and contact-killing, as well as BL-driven photodynamic sterilization, ascribed to the inherent merits based on the massive photoactive GA, hydrophobic and high membrane affinity of OA within the CS chains. As a result, the GA/OA-g-CS (2:1) film plus BL irradiation can efficiently eradicate Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and their biofilms. After BL irradiation for 30 min, the GA/OA-g-CS (2:1) film showed a reduction of 5.2 Log CFU/mL against E. coli. More importantly, the film with BL irradiation can efficiently not only inhibit the formation of biofilms but also jeopardize the matured biofilms. This research provides a potential platform and promises for applications in active and intelligent food packaging.

Abstract 16947: Sotagliflozin, a Dual SGLT 1 and 2 Inhibitor, Has High Membrane Affinity and Hydrocarbon Core Location Compared With Empagliflozin

Background: The dual SGLT-1 and 2 inhibitor sotagliflozin (sota) reduced atherothrombotic events and hospitalization for worsening heart failure in patients with diabetes in clinical trials (SCORED, SOLOIST). Unlike selective SGLT2 inhibitors such as empagliflozin (empa), sota binds with higher affinity to SGLT1, which is located in the intestine, kidney, heart, brain, and vascular endothelial cells. To elucidate the physico-chemical basis for differences in tissue distribution and receptor affinity, we compared the effects of these drugs on membrane binding and molecular location. Methods: Membranes reconstituted from cardiac phospholipid and cholesterol were used to measure drug partitioning across a range of cholesterol concentrations. Multilamellar vesicles were prepared as mixtures of cardiac phospholipid (CPL), cholesterol, and each agent at 30 μM or vehicle. Drug-membrane partitioning was determined by rapid centrifugation and UV/Vis spectrophotometry. In parallel experiments, we used small angle x-ray scattering (SAXS) to identify drug location in model membranes containing phosphatidylcholine and cholesterol. Results: Sota had a membrane partition coefficient (8590 ± 2078) that was 16-fold higher than empa (519 ± 195, p<0.001) in CPL membranes. This high membrane affinity was preserved over a range of cholesterol levels compared to empa (p<0.001). Consistent with high membrane affinity, SAXS analysis showed that sota had a location evidenced by a broad increase in electron density in the hydrocarbon core extending to approximately 20 Å from the membrane center. By contrast, empa had a location limited to the polar headgroup region, consistent with its lower lipophilicity and tissue penetration. Conclusions: Sota had high membrane affinity and distinct location in the hydrocarbon core compared to empa. This may contribute to the effect of its dual SGLT-1 and 2 inhibition in vivo across various tissues including the heart. These distinct membrane interactions may also partially explain the reduced atherothrombotic risk as demonstrated in outcome trials with sota but not selective SGLT-2 inhibitors.

Conformational ensemble-dependent lipid recognition and segregation by prenylated intrinsically disordered regions in small GTPases

We studied diverse prenylated intrinsically disordered regions (PIDRs) of Ras and Rho family small GTPases using long timescale atomistic molecular dynamics simulations in an asymmetric model membrane of phosphatidylcholine (PC) and phosphatidylserine (PS) lipids. Here we show that conformational plasticity is a key determinant of lipid sorting by polybasic PIDRs and provide evidence for lipid sorting based on both headgroup and acyl chain structures. We further show that conformational ensemble-based lipid recognition is generalizable to all polybasic PIDRs, and that the sequence outside the polybasic domain (PBD) modulates the conformational plasticity, bilayer adsorption, and interactions of PIDRs with membrane lipids. Specifically, we find that palmitoylation, the ratio of basic to acidic residues, and the hydrophobic content of the sequence outside the PBD significantly impact the diversity of conformational substates and hence the extent of conformation-dependent lipid interactions. We thus propose that the PBD is required but not sufficient for the full realization of lipid sorting by prenylated PBD-containing membrane anchors, and that the membrane anchor is not only responsible for high affinity membrane binding but also directs the protein to the right target membrane where it participates in lipid sorting.

Antileishmanial activities of three chalcone derivatives and their association with plasma membrane rigidity as assessed by EPR spectroscopy

Novel chalcone-like compounds, LQFM332 (4), LQFM333 (5), and LQFM341 (6), were synthesized and their activity against Leishmania (L.) amazonensis was evaluated and compared. Compound 4 was synthesized from the molecular hybridization of vanillin (9) and butylated hydroxytoluene (BHT), forming a chalcone scaffold. BHT was replaced by another apocynin to give compound 5, while for compound 6 the Michael acceptor double bond from compound 4 was reduced. The IC50 values for compounds 4, 5, and 6 against the Leishmania parasite were 7, 20, and 16 µM for the promastigote form, and 19, 11, and 1 µM for the amastigote form inside infected macrophages, respectively. Electron paramagnetic resonance (EPR) spectroscopy of a lipid spin label incorporated into the parasite plasma membrane demonstrated that treatment with these compounds in culture medium causes membrane rigidity in a concentration-dependent manner, owing to oxidative processes. From the IC50 values with different cell concentrations, the membrane-water partition coefficient (KM/W) of the compounds could be estimated, as well as their concentrations in the membrane (cm50) and in the aqueous culture medium (cw50) that reduce antiproliferative activity by 50%. Values for these biophysical parameters were similar to those reported for the antileishmanial drug, miltefosine. KM/W values indicated that the compounds have high affinity for the parasite membrane, with the compound containing the BHT group having the highest affinity. Compound 5, which contained a two 4-OH-3-methoxybenzene scaffold, showed the lowest values of cw50 and cm50. Compound 6 showed that the reduction of the double bond decreased the membrane affinity and the cw50 value, but maintained the cm50, demonstrating the same activity as compound 4 for assays with higher concentration of cells. As cytotoxic activities were modulated by effects on the cell membrane, our results suggest that the primary action of the compounds is on the parasite membrane.

Antiviral activity of singlet oxygen-photogenerating perylene compounds against SARS-CoV-2: Interaction with the viral envelope and photodynamic virion inactivation

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has prompted great interest in novel broad-spectrum antivirals, including perylene-related compounds. In the present study, we performed a structure–activity relationship analysis of a series of perylene derivatives, which comprised a large planar perylene residue, and structurally divergent polar groups connected to the perylene core by a rigid ethynyl or thiophene linker. Most of the tested compounds did not exhibit significant cytotoxicity towards multiple cell types susceptible to SARS-CoV-2 infection, and did not change the expressions of cellular stress-related genes under normal light conditions. These compounds showed nanomolar or sub-micromolar dose-dependent anti-SARS-CoV-2 activity, and also suppressed the in vitro replication of feline coronavirus (FCoV), also termed feline infectious peritonitis virus (FIPV). Perylene compounds exhibited high affinity for liposomal and cellular membranes, and efficiently intercalated into the envelopes of SARS-CoV-2 virions, thereby blocking the viral–cell fusion machinery. Furthermore, the studied compounds were demonstrated to be potent photosensitizers, generating reactive oxygen species (ROS), and their anti-SARS-CoV-2 activities were considerably enhanced after irradiation with blue light. Our results indicated that photosensitization is the major mechanism underlying the anti-SARS-CoV-2 activity of perylene derivatives, with these compounds completely losing their antiviral potency under red light. Overall, perylene-based compounds are broad-spectrum antivirals against multiple enveloped viruses, with antiviral action based on light-induced photochemical damage (ROS-mediated, likely singlet oxygen-mediated), causing impairment of viral membrane rheology.

Lipophilic Anchors that Embed Bioconjugates in Bilayer Membranes: A Review.

A wide range of biomaterials and engineered cell surfaces are composed of bioconjugates embedded in liposome membranes, surface-immobilized bilayers, or the plasma membranes of living cells. This review article summarizes the various ways that Nature anchors integral and peripheral proteins in a cell membrane and describes the strategies devised by chemical biologists to label a membrane protein in living cells. Also discussed are modern synthetic and semisynthetic methods to produce lipidated proteins. Subsequent sections describe methods to anchor a three-component synthetic construct that is composed of a lipophilic membrane anchor, hydrophilic linker, and exposed functional component. The surface exposed payload can be a fluorophore, aptamer, oligonucleotide, polypeptide, peptide nucleic acid, polysaccharide, branched dendrimer, or linear polymer. Hydrocarbon chains are commonly used as the membrane anchor, and a general experimental trend is that a two chain lipid anchor has higher membrane affinity than a cholesteryl or single chain lipid anchor. Amphiphilic fluorescent dyes are effective molecular probes for cell membrane imaging and a zwitterionic linker between the fluorophore and the lipid anchor promotes high persistence in the plasma membrane of living cells. A relatively new advance is the development of switchable membrane anchors as molecular tools for fundamental studies or as technology platforms for applied biomaterials.

Identification of a CyaA region exhibiting high affinity for membranes mimicking the lipid composition of the inner membrane of Bordetella pertussis.

The CyaA toxin, a 1706-residue long protein, is one of the major virulence factors produced by Bordetella pertussis. The secretion of CyaA is initiated by its C-terminal extremity through a dedicated Type 1 secretion system (T1SS). We are interested to decipher how CyaA is recognized, uptake and secreted by the T1SS. We have identified a C-terminal membrane-interacting region (MIR), which exhibits high affinity for membranes composed of lipids mimicking the inner leaflet of Bordetella pertussis (ilbp)inner membrane. We show by a combination of approaches that MIR undergoes conformational changes upon interactions with ilbp membranes. We further characterized the parameters tuning MIR/membrane interactions such as pH, ionic strength, and lipid composition. Moreover, key residues of MIR involved in membrane partitioning have been identified. These results allow us to propose a model of MIR/membrane interactions, which may help to recruit CyaA at the surface of the inner membrane, at proximity of the entry gate of the T1SS. These data pave the way to characterize the interaction of the full length CyaA toxin with the inner membrane of Bordetella pertussis prior its secretion from the bacterium.

How 1,n-Bis(3-alkylimidazolium-1-yl) Alkane Interacts with the Phospholipid Membrane and Impacts the Toxicity of Dicationic Ionic Liquids.

Ionic liquids based on doubly charged cations, often termed dicationic ionic liquids (DILs), offer robust physicochemical properties and low toxicity than conventional monocationic ionic liquids. In this design-based study, we used solid-state NMR spectroscopy to provide the interaction mechanism of two DILs, 1,n-bis(3-alkylimidazolium-1-yl) alkane dibromide ([C2n(C7-nIM)2]2+·2Br-, n = 1, 6), with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) phospholipid membranes, to explain the low toxicity of DILs toward HeLa, Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae cell lines. Dications with a short linker and long terminal chains cause substantial perturbation to the bilayer structure, making them more membrane permeabilizing, as shown by fluorescence-based dye leakage assays. The structural perturbation is even higher than [C12(MIM)]+ monocations, which carry a single 12-carbon long chain and exhibit a much higher membrane affinity, permeability, and cytotoxicity. These structural details are a crucial contribution to the design strategies aimed at harnessing the biological activity of ionic liquids.

Efficient production of the β-ionone aroma compound from organic waste hydrolysates using an engineered Yarrowia lipolytica strain.

This study demonstrates the feasibility of establishing a natural compound supply chain in a biorefinery. The process starts with the biological or chemical hydrolysis of food and agricultural waste into simple and fermentative sugars, followed by their fermentation into more complex molecules. The yeast strain, Yarrowia lipolytica, was modified by introducing high membrane affinity variants of the carotenoid cleavage dioxygenase enzyme, PhCCD1, to increase the production of the aroma compound, β-ionone. The initial hydrolysis process converted food waste or sugarcane bagasse into nutrient-rich hydrolysates containing 78.4 g/L glucose and 8.3 g/L fructose, or 34.7 g/L glucose and 20.1 g/L xylose, respectively. During the next step, engineered Y. lipolytica strains were used to produce β-ionone from these feedstocks. The yeast strain YLBI3120, carrying a modified PhCCD1 gene was able to produce 4 g/L of β-ionone with a productivity of 13.9 mg/L/h from food waste hydrolysate. This is the highest yield reported for the fermentation of this compound to date. The integrated process described in this study could be scaled up to achieve economical large-scale conversion of inedible food and agricultural waste into valuable aroma compounds for a wide range of potential applications.

Anionic membrane phospholipids: A New Class of Chemokine‐Binding Site Important for both Apoptotic Cell Clearance and Antibiotic Activity by Chemokines

Chemokines are small cytokines that orchestrate immune cell migration by binding to cell surface glycosaminoglycans (GAGs) and activating G protein‐couple receptors (GPCRs) expressed by leukocytes. We have discovered that chemokines selectively bind with high affinity to anionic membrane phospholipids, including phosphatidylserine (PS) and cardiolipin (CL). This adds to GPCRs and GAGs a third class of high affinity membrane binding site for chemokines and expands the scope of chemokine action to new areas of biology dictated by the specific biological niches occupied by their phospholipid binding partners. PS, for example, is usually restricted to the cytosolic face of the phospholipid bilayer of the plasma membrane of healthy eukaryotic cells, but it is flipped to the outer leaflet of the membrane of apoptotic cells and apoptotic extracellular vesicles (ApoEVs), where it acts as an ‘eat‐me’ signal recognized by phagocytes for apoptotic cell clearance. In this regard, we have recently demonstrated that ApoEVs generated in vivo from mouse thymus induce phagocyte migration using endogenous chemokines bound to the vesicle surface via PS. Our results indicate that PS‐bound chemokines on ApoEVs may serve as ‘find‐me’ signals that recruit phagocytes toward apoptotic cells for the removal of dying cells. This is a new PS‐dependent mechanism for chemokine delivery by extracellular vesicles. On the other hand, CL is absent in the plasma membrane of eukaryotic cells but it is a major and naturally exposed anionic phospholipid in the bacterial plasma membrane. As such, we have found that chemokines act as potent direct bacteria‐killing agents by binding to bacterial CL. This finding identifies the molecular mechanism for antimicrobial action by chemokines and launch new avenues of research in antimicrobial therapies and bacteria‐host interactions. In short, we have identified chemokines as the first example of a family of soluble cytokines whose functions are regulated by direct interaction with membrane phospholipids.

Recent Advances in IL-13Rα2-Directed Cancer Immunotherapy.

Interleukin-13 receptor subunit alpha-2 (IL-13Rα2, CD213A), a high-affinity membrane receptor of the anti-inflammatory Th2 cytokine IL-13, is overexpressed in a variety of solid tumors and is correlated with poor prognosis in glioblastoma, colorectal cancer, adrenocortical carcinoma, pancreatic cancer, and breast cancer. While initially hypothesized as a decoy receptor for IL-13-mediated signaling, recent evidence demonstrates IL-13 can signal through IL-13Rα2 in human cells. In addition, expression of IL-13Rα2 and IL-13Rα2-mediated signaling has been shown to promote tumor proliferation, cell survival, tumor progression, invasion, and metastasis. Given its differential expression in tumor versus normal tissue, IL-13Rα2 is an attractive immunotherapy target, as both a targetable receptor and an immunogenic antigen. Multiple promising strategies, including immunotoxins, cancer vaccines, and chimeric antigen receptor (CAR) T cells, have been developed to target IL-13Rα2. In this mini-review, we discuss recent developments surrounding IL-13Rα2-targeted therapies in pre-clinical and clinical study, including potential strategies to improve IL-13Rα2-directed cancer treatment efficacy.

Alkyl esters of umbelliferone-4-acetic acid as protonophores in bilayer lipid membranes and ALDH2-dependent soft uncouplers in rat liver mitochondria

A great variety of coumarin-related compounds, both natural and synthetic, being often brightly fluorescent, have shown themselves beneficial in medicine for both therapeutic and imaging purposes. Here, in search for effective uncouplers of oxidative phosphorylation, we synthesized a series of 7-hydroxycoumarin (umbelliferone, UB) derivatives combining rather high membrane affinity with the presence of a hydroxyl group deprotonable at physiological pH - alkyl esters of umbelliferone-4-acetic acid (UB-4 esters) differing in alkyl chain length. Addition of UB-4 esters to isolated rat liver mitochondria (RLM) resulted in their rapid depolarization, unexpectedly followed by membrane potential recovery on a minute time scale. According to TLC and HPLC data, incubation of RLM with UB-4 esters caused their hydrolysis, which led to disappearance of the uncoupling activity (recoupling). Both mitochondrial recoupling and hydrolysis of UB-4 esters were suppressed by inhibitors of mitochondrial aldehyde dehydrogenase (ALDH2), disulfiram and daidzin, thus pointing to the involvement of this enzyme in the recoupling of RLM incubated with UB-4 esters. The protonophoric mechanism of mitochondrial uncoupling by UB-4 esters was proved in experiments with artificial bilayer lipid membranes (BLM): these compounds induced proton-selective electrical current across planar BLM and caused dissipation of pH gradient on liposomes. UB-4 esters showed antibacterial activity against Bacillus subtilis, Staphylococcus aureus and Mycobacterium smegmatis.

Supramolecular Amphiphiles Based on Pillar[5]arene and Meroterpenoids: Synthesis, Self-Association and Interaction with Floxuridine.

In recent years, meroterpenoids have found wide biomedical application due to their synthetic availability, low toxicity, and biocompatibility. However, these compounds are not used in targeted drug delivery systems due to their high affinity for cell membranes, both healthy and in cancer cells. Using the approach of creating supramolecular amphiphiles, we have developed self-assembling systems based on water-soluble pillar[5]arene and synthetic meroterpenoids containing geraniol, myrtenol, farnesol, and phytol fragments. The resulting systems can be used as universal drug delivery systems. It was shown by turbidimetry that the obtained pillar[5]arene/synthetic meroterpenoid systems do not interact with the model cell membrane at pH = 7.4, but the associates are destroyed at pH = 4.1. In this case, the synthetic meroterpenoid is incorporated into the lipid bilayer of the model membrane. The characteristics of supramolecular self-assembly, association constants and stoichiometry of the most stable pillar[5]arene/synthetic meroterpenoid complexes were established by UV-vis spectroscopy and dynamic light scattering (DLS). It was shown that supramolecular amphiphiles based on pillar[5]arene/synthetic meroterpenoid systems form monodisperse associates in a wide range of concentrations. The inclusion of the antitumor drug 5-fluoro-2′-deoxyuridine (floxuridine) into the structure of the supramolecular associate was demonstrated by DLS, 19F, 2D DOSY NMR spectroscopy.

Altered Membrane Mechanics Provides a Receptor-Independent Pathway for Serotonin Action.

Serotonin, an important signaling molecule in humans, has an unexpectedly high lipid membrane affinity. The significance of this finding has evoked considerable speculation. Here we show that membrane binding by serotonin can directly modulate membrane properties and cellular function, providing an activity pathway completely independent of serotonin receptors. Atomic force microscopy shows that serotonin makes artificial lipid bilayers softer, and induces nucleation of liquid disordered domains inside the raft‐like liquid‐ordered domains. Solid‐state NMR spectroscopy corroborates this data at the atomic level, revealing a homogeneous decrease in the order parameter of the lipid chains in the presence of serotonin. In the RN46A immortalized serotonergic neuronal cell line, extracellular serotonin enhances transferrin receptor endocytosis, even in the presence of broad‐spectrum serotonin receptor and transporter inhibitors. Similarly, it increases the membrane binding and internalization of oligomeric peptides. Our results uncover a mode of serotonin–membrane interaction that can potentiate key cellular processes in a receptor‐independent fashion.