Surface Of Cellular Membranes Research Articles

PHLIP-fused PD-L1 engages avelumab to elicit NK cytotoxicity under acidic conditions

Natural killer (NK) cells represent key player in immune surveillance to eliminate transformed or malignant cells. One of mechanisms of action of NK cells is antibody-dependent cell-mediated cytotoxicity (ADCC) by recognizing tumor antigens on the surface of cancer cells. However, the heterogeneity of tumor antigens and the scarcity of membrane surface targets significantly restrict this strategy. Recently, we constructed a new cargo by tethering a low pH insertion peptide (pHLIP) to the C terminus of the ectodomain of programed death ligand-1 (PD-L1) and demonstrated its ability to modulate immune responses. Herein, the potential application of PD-L1-pHLIP in cancer therapy was determined. pHLIP tethering had no effect on the binding capacity of PD-L1 protein to an anti-PD-L1 antibody (i.e. avelumab). Association of pHLIP rendered PD-L1 segment display on the surface of cellular membrane in the acidic buffer instead of the neutral solution. Importantly, plate-coated or beads-coupled PD-L1-pHLIP enable robust activation and expression of cytotoxic mediators of NK cells via engaging avelumab. Overall, this work provides proof of concept that recombinant PD-L1 protein decorated on the cellular membrane driven by pHLIP in combination with appropriate monoclonal antibody has potentials to elicit NK cytotoxicity, which may represent a novel and promising therapeutic avenue in cancer.

Phosphatidylserine externalization as immune checkpoint in cancer.

Cancer is the second leading cause of mortality worldwide. Despite recent advances in cancer treatment including immunotherapy with immune checkpoint inhibitors, new unconventional biomarkers and targets for the detection, prognosis, and treatment of cancer are still in high demand. Tumor cells are characterized by mutations that allow their unlimited growth, program their local microenvironment to support tumor growth, and spread towards distant sites. While a major focus has been on altered tumor genomes and proteomes, crucial signaling molecules such as lipids have been underappreciated. One of these molecules is the membrane phospholipid phosphatidylserine (PS) that is usually found at cytosolic surfaces of cellular membranes but can be rapidly and massively shuttled to the extracellular leaflet of the plasma membrane during apoptosis to serve as a limiting factor for immune responses. These immunosuppressive interactions are exploited by tumor cells to evade the immune system. In this review, we describe mechanisms of immune regulation in tumors, discuss if PS may constitute an inhibitory immune checkpoint, and describe current and future strategies for targeting PS to reactivate the tumor-associated immune system.

Transmembrane coupling of liquid-like protein condensates

Liquid-liquid phase separation of proteins occurs on both surfaces of cellular membranes during diverse physiological processes. In vitro reconstitution could provide insight into the mechanisms underlying these events. However, most existing reconstitution techniques provide access to only one membrane surface, making it difficult to probe transmembrane phenomena. To study protein phase separation simultaneously on both membrane surfaces, we developed an array of freestanding planar lipid membranes. Interestingly, we observed that liquid-like protein condensates on one side of the membrane colocalized with those on the other side, resulting in transmembrane coupling. Our results, based on lipid probe partitioning and mobility of lipids, suggest that protein condensates locally reorganize membrane lipids, a process which could be explained by multiple effects. These findings suggest a mechanism by which signals originating on one side of a biological membrane, triggered by protein phase separation, can be transferred to the opposite side.

Preliminary assessment of toxicity of aerosol samples from central-west Brazil using Artemia spp. bioassays

The present study reports the development of a bioassay using Artemia spp. to analyse the preliminary ecotoxicity of atmospheric aerosols (PM), which can affect the environment and human health. Herein, PM samples were collected in the city of Goiânia (Brazil) in 2016, extracted with ultrapure water and subsequently filtered through membranes with different pore sizes (100, 0.8, and 0.22 μm), and the extracts employed in the bioassays. The mortality rates (endpoint analysed) declined to membranes with smaller pore sizes (15 ± 4%, 47 ± 10% and 43 ± 9% for pore sizes of 100 μm, 0.8 μm and 0.22 μm, respectively). In general, the toxicity of the extract depended on its concentration, except for the sample with a higher negative particle surface charge, which presents a lower affinity for the negatively charged surfaces of cellular membranes. Moreover, although the PM concentration was higher for the sample collected during the dry season (September), the mortality rate was not significantly different to that determined for a sample with similar physical and chemical characteristics collected in the rainy season (December). This result demonstrates the importance of monitoring PM toxicities and their chemical and physical characteristics, in addition to their concentrations. Therefore, the new protocol to provide a preliminary analysis of the toxicity of the extracts of aerosol emerges as a useful, accessible, and fast tool for monitoring possible environmental hazards, and can simplify fieldwork.

Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine Sulfonamides as Novel Potential Anticancer Agents: Apoptosis, Oxidative Stress, and Cell Cycle Analysis.

The current study continues the evaluation of the anticancer potential of three de novo synthesized pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides-MM129, MM130, and MM131-against human cancer cells of HeLa, HCT 116, PC-3, and BxPC-3 lines. The pro-apoptotic activity of the investigated sulfonamides was shown by observations of changes in the mitochondrial transmembrane potential of the tested cells, externalization of phosphatidylserine on the cellular membrane surface, and cell morphology in microscopic imaging. The computational studies have shown that MM129 exhibited the lowest binding energy values when docked against CDK enzymes. In addition, the highest stability was shown for complexes formed between MM129 and CDK5/8 enzymes. All examined compounds induced cell cycle arrest in the G0/G1 phase in the BxPC-3 and PC-3 cells and simultaneously caused the accumulation of cells in the S phase in the HCT 116 cells. In addition, the increase in the subG1 fraction was observed in PC-3 and HeLa cells. The application of a fluorescent H2DCFDA probe revealed the high pro-oxidative properties of the tested triazine derivatives, especially MM131. In conclusion, the obtained results suggest that MM129, MM130, and MM131 exhibited strong pro-apoptotic properties towards investigated cells, mainly against the HeLa and HCT 116 cell lines, and high pro-oxidative potential as well. Moreover, it is suggested that the anticancer activity of the tested compounds may be associated with their ability to inhibit CDK enzymes activities.

Epitope Imprinting of Phospholipids by Oriented Assembly at the Oil/Water Interface for the Selective Recognition of Plasma Membranes

AbstractPhospholipids, as fundamental building blocks of the cell membrane, play important roles for molecule transportation, cell recognition, etc. However, due to the structural diversity and amphipathic nature, there are few methods for the specific recognition of lipids as compared to other biomolecules such as proteins and glycans. Herein, we developed a molecular imprinting strategy for controllable imprinting toward the polar head of phospholipid exposed on the surface of cellular membranes for recognition. Phosphatidylserine, as unique lipid on the outer membrane leaflet of exosome and also hallmark for cell apoptosis, was imprinted with the developed method. The phosphatidylserine imprinted materials showed high efficiency and specific targeting capability not only for apoptotic cell imaging but also for the isolation of exosomes. Collectively, the synthesized molecularly imprinted materials have great potential for selective plasma membrane recognition for targeted drug delivery and biomarker discovery.

Epitope Imprinting of Phospholipids by Oriented Assembly at the Oil/Water Interface for the Selective Recognition of Plasma Membranes.

Phospholipids, as fundamental building blocks of the cell membrane, play important roles for molecule transportation, cell recognition, etc. However, due to the structural diversity and amphipathic nature, there are few methods for the specific recognition of lipids as compared to other biomolecules such as proteins and glycans. Herein, we developed a molecular imprinting strategy for controllable imprinting toward the polar head of phospholipid exposed on the surface of cellular membranes for recognition. Phosphatidylserine, as unique lipid on the outer membrane leaflet of exosome and also hallmark for cell apoptosis, was imprinted with the developed method. The phosphatidylserine imprinted materials showed high efficiency and specific targeting capability not only for apoptotic cell imaging but also for the isolation of exosomes. Collectively, the synthesized molecularly imprinted materials have great potential for selective plasma membrane recognition for targeted drug delivery and biomarker discovery.

Biofabrication of nanovesicles for brain diseases.

Biofabrication of nanovesicles for brain diseases.

Geometric–Statistical Model for Middle-Ear Anatomy and Ventilation

The ventilation of the middle-ear (ME) is achieved by the mucosa covering the bony cavities of this segment, which we have previously defined as consisting of two distinct epithelial areas, each representing an independent organ with characteristic function: the D-Organ and the F-Organ. The D-Organ corresponds to the epithelium covering the Antrum walls (belonging to the central cavities of the middle-ear) and the walls of mastoid and petrous cavities (peripheral cavities of the ME); it ensures the D-Function, the biophysical process comparable to that of energy-consuming ionic membrane pumps, works against electrical trans-membrane gradients to transfer gas molecules against trans-membrane and trans-cellular pressure gradients. The F-Organ corresponds to the epithelium covering the Protympanum, Tympanic Cavity and Aditus ad Antrum (central cavities of ME). The F-Function is represented by the permeability of cell membranes for respiratory gases. This is a general function of all cells and the size of the cellular membrane surface (luminal and basal) and the height of the cell (distance between the two membranes) determines the diffusion flow for each molecular type of gas. The present work aims to give an original point of view on middle-ear geometry and precedence over ME mucosa affliction or structural-anatomic type of the mastoid (pneumatic, pneumato-diploic, diploic, sclerotic). This type of approach to the problem has never been attempted since the two organs have never been previously defined. We aim to establish a clear topographic structure for these two organs within the reference system represented by the anatomy of ME cavities and to establish the reasons why the mastoid and petrous cavitary system grow or stop growing at a certain point in the life of an individual.

Dual film-like organelles enable spatial separation of orthogonal eukaryotic translation

SummaryEngineering new functionality into living eukaryotic systems by enzyme evolution or de novo protein design is a formidable challenge. Cells do not rely exclusively on DNA-based evolution to generate new functionality but often utilize membrane encapsulation or formation of membraneless organelles to separate distinct molecular processes that execute complex operations. Applying this principle and the concept of two-dimensional phase separation, we develop film-like synthetic organelles that support protein translation on the surfaces of various cellular membranes. These sub-resolution synthetic films provide a path to make functionally distinct enzymes within the same cell. We use these film-like organelles to equip eukaryotic cells with dual orthogonal expanded genetic codes that enable the specific reprogramming of distinct translational machineries with single-residue precision. The ability to spatially tune the output of translation within tens of nanometers is not only important for synthetic biology but has implications for understanding the function of membrane-associated protein condensation in cells.

Deciphering the Molecular Terms of Arp2/3 Allosteric Regulation from All-Atom Simulations and Dynamical Network Theory.

The Arp2/3 molecular machine stimulates the generation of branched actin networks at the cytosolic surface of cellular membranes. Arp2/3 is thus pivotal for cell motility and migration, and its aberrant function is implicated in cancer invasion and metastasis. Here, all-atom multi μs-long molecular dynamics simulations and dynamical NetWork Analysis (NWA) unprecedentedly disclose the molecular terms of Arp2/3 regulation (activation/inhibition) by positive/negative allosteric modulators. After identifying the crucial structural elements underlying Arp2/3's conformational transition toward its active actin-polymerization-competent state, we decrypt the activating signaling paths heading from the allosteric effector (ATP) binding sites to these pivotal regions, also elucidating how small-molecule inhibitors scramble this signal-exchange. As a result, while ATP-induced signaling triggers a harmonious conformational transition toward active Arp2/3, the inhibitors disturb these information channels, desynchronizing Arp2/3 functional movements, thus hindering its activation. Our outcomes supply a conceptual basis for devising small-molecule inhibitors to block infiltrative cancer migration.

Diabetes Insipidus: Pathogenesis, Diagnosis, and Clinical Management.

Diabetes insipidus (DI) is an endocrine condition involving the posterior pituitary peptide hormone, antidiuretic hormone (ADH). ADH exerts its effects on the distal convoluted tubule and collecting duct of the nephron by upregulating aquaporin-2 channels (AQP2) on the cellular apical membrane surface. DI is marked by expelling excessive quantities of highly dilute urine, extreme thirst, and craving for cold water. The two main classifications of DI are central diabetes insipidus (CDI), characterized by a deficiency of the posterior pituitary gland to release ADH, and nephrogenic diabetes insipidus (NDI), characterized by the terminal distal convoluted tubule and collecting duct resistance to ADH. The two less common classifications include dipsogenic DI, characterized by excessive thirst due to a low osmotic threshold, and gestational DI, characterized by increased concentration of placental vasopressinase during pregnancy. Treatment of DI is dependent on the disease classification, but severe complications may arise if not tended to appropriately. The most important step in symptom management is maintaining fluid intake ahead of fluid loss with emphasis placed on preserving the quality of life. The most common treatment of CDI and gestational DI is the administration of synthetic ADH, desmopressin (DDAVP). Nephrogenic treatment, although more challenging, requires discontinuation of medications as well as maintaining a renal-friendly diet to prevent hypernatremia. Treatment of dipsogenic DI is mainly focused on behavioral therapy aimed at regulating water intake and/or administration of antipsychotic pharmaceutical therapy. Central and nephrogenic subtypes of DI share a paradoxical treatment in thiazide diuretics.

Creating Contacts Between Replication and Movement at Plasmodesmata - A Role for Membrane Contact Sites in Plant Virus Infections?

To infect their hosts and cause disease, plant viruses must replicate within cells and move throughout the plant both locally and systemically. RNA virus replication occurs on the surface of various cellular membranes, whose shape and composition become extensively modified in the process. Membrane contact sites (MCS) can mediate non-vesicular lipid-shuttling between different membranes and viruses co-opt components of these structures to make their membrane environment suitable for replication. Whereas animal viruses exit and enter cells when moving throughout their host, the rigid wall of plant cells obstructs this pathway and plant viruses therefore move between cells symplastically through plasmodesmata (PD). PD are membranous channels connecting nearly all plant cells and are now viewed to constitute a specialized type of endoplasmic reticulum (ER)-plasma membrane (PM) MCS themselves. Thus, both replication and movement of plant viruses rely on MCS. However, recent work also suggests that for some viruses, replication and movement are closely coupled at ER-PM MCS at the entrances of PD. Movement-coupled replication at PD may be distinct from the main bulk of replication and virus accumulation, which produces progeny virions for plant-to-plant transmission. Thus, MCS play a central role in plant virus infections, and may provide a link between two essential steps in the viral life cycle, replication and movement. Here, we provide an overview of plant virus-MCS interactions identified to date, and place these in the context of the connection between viral replication and cell-to-cell movement.

Sulfur-containing histidine compounds inhibit γ-glutamyl transpeptidase activity in human cancer cells

γ-Glutamyl transpeptidase (GGT) is an enzyme located on the surface of cellular membranes and involved in GSH metabolism and maintenance of redox homeostasis. High GGT expression on tumor cells is associated with increased cell proliferation and resistance against chemotherapy. GGT inhibitors evaluated so far in clinical trials are too toxic for human use. In this study, using enzyme kinetics analyses, we demonstrate that ovothiols, 5(Nπ)-methyl thiohistidines of marine origin, act as noncompetitive inhibitors of GGT, with an apparent Ki of 21 μm, when we fixed the concentrations of the donor substrate. We found that these compounds are more potent than the known GGT inhibitor 6-diazo-5-oxo-l-norleucine and are not toxic toward human embryonic cells. In particular, cellular process-specific fluorescence-based assays revealed that ovothiols induce a mixed cell-death phenotype of apoptosis and autophagy in GGT-overexpressing cell lines, including human liver cancer and chronic B leukemic cells. The findings of our study provide the basis for further development of 5-thiohistidines as therapeutics for GGT-positive tumors and highlight that GGT inhibition is involved in autophagy.

Dynamic Imaging of Transferrin Receptor Molecules on Single Live Cell with Bridge Gaps-Enhanced Raman Tags.

A metal nanoparticles-based surface-enhanced Raman scattering (SERS) technique has been developed for biosensing and bioimaging due to its advantages in ultra-narrow line width for multiplexing, ultra-high sensitivity and excellent photostability. However, the “hotspots” effect between nanoparticles usually leads to unstable and nonuniform Raman enhancement, and this will greatly reduce the quality of SERS imaging. In this study, we employ the bridge gaps-enhanced Raman tags (BGERTs) to perform SERS imaging, in which BGERTs can not only reduce the influence of the “hotspots” effect between nanoparticles on Raman signal intensity but provide a great Raman enhancement when the Gold (Au) shell is thick enough. Based on BGERTs and its conjugation with the thiol-terminated polyethylene glycol (PEG) and transferrin, we construct a targeted Transferrin (TF)-PEG-BGERTs SERS nanoprobe and achieve the dynamic imaging of transferrin receptor (TfR) molecules on a single live cell, in which the role of transferrin-conjugated PEG-BGERT is for targeting TfR molecules located in cellular membrane surface. Importantly, this BGERTs-based SERS imaging could potentially provide a useful tool for studying the precise mechanism during the receptor-mediated nanoparticles endocytosis or cell proliferation, apoptosis, and other complicated molecular events.

Lipid Bilayer-like Mixed Self-Assembled Monolayers with Strong Mobility and Clustering-Dependent Lectin Affinity.

Glycans at the surface of cellular membranes modulate biological activity via multivalent association with extracellular messengers. The lack of tuneable simplified models mimicking this dynamic environment complicates basic studies of these phenomena. We here present a series of mixed reversible self-assembled monolayers (rSAMs) that addresses this deficiency. Mixed rSAMs were prepared in water by simple immersion of a negatively charged surface in a mixture of sialic acid- and hydroxy-terminated benzamidine amphiphiles. Surface compositions derived from infrared reflection-absorption spectroscopy (IRAS) and film thickness information (atomic force microscopy, ellipsometry) suggest the latter to be statistically incorporated in the monolayer. These surfaces' affinity for the lectin hemagglutinin revealed a strong dependence of the affinity on the presentation, density, and mobility of the sialic acid ligands. Hence, a spacer length of 4 ethylene glycol and a surface density of 15% resulted in a dissociation constant Kd,multi of 1.3 × 10-13 M, on par with the best di- or tri-saccharide-based binders reported to date, whereas a density of 20% demonstrated complete resistance to hemagglutinin binding. These results correlated with ligand mobility measured by fluorescence recovery after photobleaching which showed a dramatic drop in the same interval. The results have a direct bearing on biological cell surface multivalent recognition involving lipid bilayers and may guide the design of model surfaces and sensors for both fundamental and applied studies.

Targeting the Tetraspanins with Monoclonal Antibodies in Oncology: Focus on Tspan8/Co-029.

Tetraspanins are exposed at the surface of cellular membranes, which allows for the fixation of cognate antibodies. Developing specific antibodies in conjunction with genetic data would largely contribute to deciphering their biological behavior. In this short review, we summarize the main functions of Tspan8/Co-029 and its role in the biology of tumor cells. Based on data collected from recently reported studies, the possibilities of using antibodies to target Tspan8 in immunotherapy or radioimmunotherapy approaches are also discussed.

IL-2 induces apoptosis, increases the production and expression of cytochrome-C and inhibits COX IV in cervical cancer cells

It is known that cervical cancer cells express IL-2 receptor (IL-2R) and those high doses induce cell death. To identify the type of cell death two cervical cancer cell lines, CALO and INBL, were cultured with 100 IU/ml of IL-2. Our results showed the presence of apoptotic cell death by the significant expression of phosphatidylserine on the external surface of cellular membranes, the presence of a typical DNA fragmentation and the activation of caspase 3. We also observed that the expression of COX I, COX II and COX III was not significantly altered while that of COX IV was completely inhibited. An increased expression of cytochrome-C by confocal microscopy was observed. Finally, we speculate that the clinical effect and toxicities of IL-2 used in cancer therapies is mostly due to its apoptotic effect on the cancer cells themselves rather than, as thought, the cytotoxic contribution of leukocytes.

Molecular snapshots of dynamic membrane-bound metabolons.

Numerous biosynthetic pathways have been shown to assemble at the surface of cellular membranes into efficient dynamic supramolecular assemblies termed metabolons. In response to environmental stimuli, metabolons assemble on-demand making them highly dynamic and fragile. This transient nature has previously hampered isolation and molecular characterization of dynamic metabolons. In contrast to conventional detergents, which tend to disrupt weak protein-protein interactions and remove lipids, the competence of a styrene maleic acid copolymer to carve out discrete lipid nanodisc from membranes offers immense potential for isolation of intact protein assemblies. Here, we present a method to extract the entire membrane-bound dhurrin pathway directly from microsomal fractions of the cereal Sorghum bicolor. This detergent-free nanodisc approach may be generally transposed for isolation of entire plant biosynthetic metabolons. This method provides a simple practical toolkit for the study of membrane protein complexes.

The Effect of Dextran Sulfate\u2014as Model Glycosaminoglycan Analogue\u2014on Membrane Lipids: DPPC, Cholesterol, and DPPC\u2013Cholesterol Mixture. The Monolayer Study

Glycosaminoglycans (GAGs) are essential components of the extracellular matrices (ECMs) located on the outer surface of cellular membranes. They belong to the group of polysaccharides involved in diverse biological processes acting on the surface and across natural lipid membranes. Recently, particular attention has been focused on possible role of GAGs in the amyloid deposits. The amyloid formation is related to a disorder in protein folding, causing that soluble—in normal conditions—peptides become deposited extracellularly as insoluble fibrils, impairing tissue structure and its function. One of the hypothesis holds that GAGs may inhibit amyloid formation by interacting with the lipid membrane by blocking the accumulation of protein aggregates on the membrane surface. Although the biophysical properties of GAGs are described rather well, little is known about the nature of association between these polysaccharides and components of natural cell membranes. Therefore, a study of GAGs influence on membrane lipids is of particular importance. The aim of the present work is to get insight into the effect of hydrophilic dextran sulfate (DS)—that can be considered as GAG analogue—on membrane lipids organization. This study was based on examining interactions between DS sodium salt of molecular weight equal to about 40 kDa (DS40), dissolved in water subphase, and a model membrane, mimicked as Langmuir monolayer, formed by representative natural membrane lipids: cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as well as their mixtures. Due to the fact that calcium ions in excess may accumulate in the lipid membrane, attracting high molecular weight molecules to their surface, the influence of calcium ions present in the subphase on the DS40 activity has also been examined. It has been found that negatively charged DS, forming a sublayer underneath the monolayer, barely interacts with membrane lipids; however, in the presence of calcium ions the electrostatic interactions between DS40 and lipid membrane are significantly enhanced, leading to the formation of network-like crystalline structures at the surface of model membrane, which can prevent incorporation and interaction with other extracellular molecules, e.g., proteins.