The Inseparable Relationship Between Cholesterol and Hedgehog Signaling.

Ligands of the Hedgehog (HH) pathway are paracrine signaling molecules that coordinate tissue development in metazoans. A remarkable feature of HH signaling is the repeated use of cholesterol in steps spanning ligand biogenesis, secretion, dispersal, and reception on target cells. A cholesterol molecule covalently attached to HH ligands is used as a molecular baton by transfer proteins to guide their secretion, spread, and reception. On target cells, a signaling circuit composed of a cholesterol transporter and sensor regulates transmission of HH signals across the plasma membrane to the cytoplasm. The repeated use of cholesterol in signaling supports the view that the HH pathway likely evolved by coopting ancient systems to regulate the abundance or organization of sterol-like lipids in membranes.

Atoms to Phenotypes: Molecular Design Principles of Cellular Energy Metabolism.

We report a 100-million atom-scale model of an entire cell organelle, a photosynthetic chromatophore vesicle from a purple bacterium, that reveals the cascade of energy conversion steps culminating in the generation of ATP from sunlight. Molecular dynamics simulations of this vesicle elucidate how the integral membrane complexes influence local curvature to tune photoexcitation of pigments. Brownian dynamics of small molecules within the chromatophore probe the mechanisms of directional charge transport under various pH and salinity conditions. Reproducing phenotypic properties from atomistic details, a kinetic model evinces that low-light adaptations of the bacterium emerge as a spontaneous outcome of optimizing the balance between the chromatophore's structural integrity and robust energy conversion. Parallels are drawn with the more universal mitochondrial bioenergetic machinery, from whence molecular-scale insights into the mechanism of cellular aging are inferred. Together, our integrative method and spectroscopic experiments pave the way to first-principles modeling of whole living cells.

Fluorescent Probes Based on Charge and Proton Transfer for Probing Biomolecular Environment.

Fluorescent probes for sensing fundamental properties of biomolecular environment, such as polarity and hydration, help to study assembly of lipids into biomembranes, sensing interactions of biomolecules and imaging physiological state of the cells. Here, we summarize major efforts in the development of probes based on two photophysical mechanisms: (i) an excited-state intramolecular charge transfer (ICT), which is represented by fluorescent solvatochromic dyes that shift their emission band maximum as a function of environment polarity and hydration; (ii) excited-state intramolecular proton transfer (ESIPT), with particular focus on 5-membered cyclic systems, represented by 3-hydroxyflavones, because they exhibit dual emission sensitive to the environment. For both ICT and ESIPT dyes, the design of the probes and their biological applications are summarized. Thus, dyes bearing amphiphilic anchors target lipid membranes and report their lipid organization, while targeting ligands direct them to specific organelles for sensing their local environment. The labels, amino acid and nucleic acid analogues inserted into biomolecules enable monitoring their interactions with membranes, proteins and nucleic acids. While ICT probes are relatively simple and robust environment-sensitive probes, ESIPT probes feature high information content due their dual emission. They constitute a powerful toolbox for addressing multitude of biological questions.

Cationic Proteins Rich in Lysine Residue Trigger Formation of Non-bilayer Lipid Phases in Model and Biological Membranes: Biophysical Methods of Study.

Cationic membrane-active toxins are the most abundant group of proteins in the venom of snakes and insects. Cationic proteins such as cobra venom cytotoxin and bee venom melittin are known for their pharmacological reactions including anticancer and antimicrobial effects which arise from the toxin-induced alteration in the dynamics and structure of plasma membranes and membranes of organelles. It has been established that these cationic toxins trigger the formation of non-bilayer lipid phase transitions in artificial and native mitochondrial membranes. Remarkably, the toxin-induced formation of non-bilayer lipid phase increases at certain conditions mitochondrial ATP synthase activity. This observation opens an intriguing avenue for using cationic toxins in the development of novel drugs for the treatment of cellular energy deficiency caused by aging and diseases. This observation also warrants a thorough investigation of the molecular mechanism(s) of lipid phase polymorphisms triggered by cationic proteins. This article presents a review on the application of powerful biophysical methods such as resonance spectroscopy (31P-, 1H-, 2H-nuclear magnetic resonance, and electron paramagnetic resonance), luminescence, and differential scanning microcalorimetry in studies of non-bilayer lipid phase transitions triggered by cationic proteins in artificial and biological membranes. A phenomenon of the triggered by cationic proteins the non-bilayer lipid phase transitions occurring within 10-2-10-11 s is discussed in the context of potential pharmacological applications of cationic proteins. Next to the ATP dimer is an inverted micelle made of cardiolipin that serves as a vehicle for the transport of H+ ions from the intra-crista space to the matrix. It is proposed that such inverted micelles are triggered by the high density of H+ ions and the cationic proteins rich in lysine residue which compete with the conserved lysine residues of the ATP synthase rotor for binding to cardiolipin in the inner mitochondrial membrane and perturb the bilayer lipid packing of cristae. Phospholipids with a blue polar head represent cardiolipin and those with a red polar head represent other phospholipids found in the crista membrane.

Effect of Non-Modified as Well as Surface-Modified SiO2 Nanoparticles on Red Blood Cells, Biological and Model Membranes.

Nanoparticles are extremely promising components that are used in diagnostics and medical therapies. Among them, silica nanoparticles are ultrafine materials that, due to their unique physicochemical properties, have already been used in biomedicine, for instance, in cancer therapy. The aim of this study was to investigate the cytotoxicity of three types of nanoparticles (SiO2, SiO2-SH, and SiO2-COOH) in relation to red blood cells, as well as the impact of silicon dioxide nanoparticles on biological membranes and liposome models of membranes. The results obtained prove that hemolytic toxicity depends on the concentration of nanoparticles and the incubation period. Silica nanoparticles have a marginal impact on the changes in the osmotic resistance of erythrocytes, except for SiO2-COOH, which, similarly to SiO2 and SiO2-SH, changes the shape of erythrocytes from discocytes mainly towards echinocytes. What is more, nanosilica has an impact on the change in fluidity of biological and model membranes. The research gives a new view of the practical possibilities for the use of large-grain nanoparticles in biomedicine.

The use of biological membranes for correction of congenital malformations.

Many congenital malformations often require a multidisciplinary and multistep surgical treatment, including the use of biological membranes. Aims of the study were to describe the use of these membranes for the correction of malformations, their clinical performance at follow-up, and patient's tolerance to them. The study included patients treated between 2009 and November 2020 in two referral centers. They were affected by abdominal wall defects (AWD), esophageal atresia/tracheo-esophageal fistula (EA/TEF), diaphragmatic hernia (CDH), spinal defects (SD), and anorectal malformations (ARM). The human origin membranes used during surgery were amniotic membrane, fascia lata, and pericardium provided by the local tissue bank and the porcine-derived membrane available on the market. Thirty-one patients were retrieved. The sample included 10 AWD, 7 EA/TEF, 5 CDH, 4 SD, 2 ARM, and 3 miscellaneous defects. The median age at repair was 139 days (range: 10,5-1494). The median follow-up was 1021 days (range: 485,5-1535). Two patients were lost at follow-up. The defects were successfully repaired and the membranes perfectly tolerated in 28/29 cases. In 1 case of CDH the fascia lata was replaced with a Goretex patch due to recurrence of the defect. This is the largest series on the use of biological membranes in congenital malformations. The variety of tissues allows to choose the best material for each malformation. The excellent tolerance and performance of this first series of patients encourage the use of these membranes to correct different type of malformations at any age.

Unravelling the structural complexity of protein-lipid interactions with neutron reflectometry.

Neutron reflectometry (NR) is a large-facility technique used to examine structure at interfaces. In this brief review an introduction to the utilisation of NR in the study of protein-lipid interactions is given. Cold neutron beams penetrate matter deeply, have low energies, wavelengths in the Ångstrom regime and are sensitive to light elements. High differential hydrogen sensitivity (between protium and deuterium) enables solution and sample isotopic labelling to be utilised to enhance or diminish the scattering signal of individual components within complex biological structures. The combination of these effects means NR can probe buried structures such as those at the solid-liquid interface and encode molecular level structural information on interfacial protein-lipid complexes revealing the relative distribution of components as well as the overall structure. Model biological membrane sample systems can be structurally probed to examine phenomena such as antimicrobial mode of activity, as well as structural and mechanistic properties peripheral/integral proteins within membrane complexes. Here, the example of the antimicrobial protein α1-purothionin binding to a model Gram negative bacterial outer membrane is used to highlight the utilisation of this technique, detailing how changes in the protein/lipid distributions across the membrane before and after the protein interaction can be easily encoded using hydrogen isotope labelling.

Interaction of cationic surfactants with DPPC membranes: effect of a novel Nα-benzoylated arginine-based compound.

Cationic amino acid-based surfactants are known to interact with the lipid bilayer of microorganism resulting in cell death through a disruption of the membrane topology. To elucidate the interaction of a cationic surfactant synthesized in our lab, investigations involving Nα-benzoyl-arginine decyl amide (Bz-Arg-NHC10), and model membranes composed by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were done. Bz-Arg-NHC10was able to penetrate into DPPC monolayers up to a critical pressure of 59.6 mN m-1. Differential scanning calorimetry revealed that as the concentration of Bz-Arg-NHC10 increased, the main transition temperature of DPPC slightly decreased. Atomic force microscopy (AFM) in situ experiments performed on supported DPPC bilayers on mica allowed monitoring the changes induced by Bz-Arg-NHC10. DPPC bilayer patches were partially removed, mainly in borders and bilayer defects for 50 µM Bz-Arg-NHC10 solution. Increasing the concentration to 100 µM resulted in a complete depletion of the supported bilayers. Surface plasmon resonance (SPR) experiments, carried out with fully DPPC bilayers covered chips, showed a net increase of the SPR signal, which can be explained by Bz-Arg-NHC10 adsorption. When patchy DPPC bilayers were formed on the substrate, a SPR signal net decrease was obtained, which is consistent with the phospholipids' removal observed in the AFM images. The results obtained suggest that the presence of the benzoyl group attached to the polar head of our compound would be the responsible of the increased antimicrobial activity against gram-negative bacteria when compared with other arginine-based surfactants.

Recent Experimental Developments in Studying Passive Membrane Transport of Drug Molecules.

The ability to measure the passive membrane permeation of drug-like molecules is of fundamental biological and pharmaceutical importance. Of significance, passive diffusion across the cellular membranes plays an effective role in the delivery of many pharmaceutical agents to intracellular targets. Hence, approaches for quantitative measurement of membrane permeability have been the topics of research for decades, resulting in sophisticated biomimetic systems coupled with advanced techniques. In this review, recent developments in experimental approaches along with theoretical models for quantitative and real-time analysis of membrane transport of drug-like molecules through mimetic and living cell membranes are discussed. The focus is on time-resolved fluorescence-based, surface plasmon resonance, and second-harmonic light scattering approaches. The current understanding of how properties of the membrane and permeant affect the permeation process is discussed.

The Role of P-Glycoprotein in Decreasing Cell Membranes Permeability during Oxidative Stress.

P-Glycoprotein (P-gp) is one of the most clinically significant representatives of the ABC transporter superfamily due to its participation in the transport of biotic components and xenobiotics across the plasma membrane. It is known that various chemicals, environmental factors, and pathological processes can affect P-gp activity and expression. In this study, we investigated the role of P-gp in limiting the cell membrane permeability during oxidative stress. Human adenocarcinoma colon cells (Caco-2) overexpressing P-gp were cultured for 72 h in the medium containing hydrogen peroxide (0.1-50 µM). The transport of the P-gp substrate fexofenadine was evaluated in a special Transwell system. The amounts of P-gp and Nrf2 transcription factor were analyzed by the enzyme-linked immunosorbent assay. The concentration of SH-groups in proteins and the contents of lipid peroxidation products and protein carbonyl derivatives were determined spectrophotometrically. Hydrogen peroxide at a concentration of 0.1-5 µM did not significantly affect the studied parameters, while incubation with 10 µM H2O2 decreased in the level of SH groups in cell lysates and increased in the amount of Nrf2 in the cell lysates. Nrf2, in its turn, mediated an increase in the content and activity of the P-gp transporter, thus limiting the increasing permeability of the cell membrane. Hydrogen peroxide at a concentration of 50 µM promoted oxidative stress, which was manifested as a decrease in the content of SH-groups, increase in the concentration of lipid peroxidation products and protein carbonyl derivatives, and decrease in the P-gp level, which led to a significantly increased permeability of the plasma membrane. These results show that the transport and protective roles of P-gp, in particular, reduction of the cell membrane permeability, are affected by the intensity of oxidative stress and can be manifested only if the extent of membrane damage is insignificant.

Steered Molecular Dynamics of Lipid Membrane Indentation by Carbon and Silicon-Carbide Nanotubes-The Impact of Indenting Angle Uncertainty.

Due to the semi-liquid nature and uneven morphologies of biological membranes, indentation may occur in a range of non-ideal conditions. These conditions are relatively unstudied and may alter the physical characteristics of the process. One of the basic challenges in the construction of nanoindenters is to appropriately align the nanotube tip and approach the membrane at a perpendicular angle. To investigate the impact of deviations from this ideal, we performed non-equilibrium steered molecular dynamics simulations of the indentation of phospholipid membranes by homogeneous CNT and non-homogeneous SiCNT indenters. We used various angles, rates, and modes of indentation, and the withdrawal of the relative indenter out of the membrane in corresponding conditions was simulated.

A Trifunctional Linker for Palmitoylation and Peptide and Protein Localization in Biological Membranes.

Attachment of lipophilic groups is an important post-translational modification of proteins, which involves the coupling of one or more anchors such as fatty acids, isoprenoids, phospholipids, or glycosylphosphatidyl inositols. To study its impact on the membrane partitioning of hydrophobic peptides or proteins, we designed a tyrosine-based trifunctional linker. The linker allows the facile incorporation of two different functionalities at a cysteine residue in a single step. We determined the effect of the lipid modification on the membrane partitioning of the synthetic α-helical model peptide WALP with or without here and in all cases below; palmitoyl groups in giant unilamellar vesicles that contain a liquid-ordered (Lo ) and liquid-disordered (Ld ) phase. Introduction of two palmitoyl groups did not alter the localization of the membrane peptides, nor did the membrane thickness or lipid composition. In all cases, the peptide was retained in the Ld phase. These data demonstrate that the Lo domain in model membranes is highly unfavorable for a single membrane-spanning peptide.

Phospholipase A activity of adenylate cyclase toxin mediates translocation of its adenylate cyclase domain.

Adenylate cyclase toxin (ACT or CyaA) plays a crucial role in respiratory tract colonization and virulence of the whooping cough causative bacterium Bordetella pertussis Secreted as soluble protein, it targets myeloid cells expressing the CD11b/CD18 integrin and on delivery of its N-terminal adenylate cyclase catalytic domain (AC domain) into the cytosol, generates uncontrolled toxic levels of cAMP that ablates bactericidal capacities of phagocytes. Our study deciphers the fundamentals of the heretofore poorly understood molecular mechanism by which the ACT enzyme domain directly crosses the host cell membrane. By combining molecular biology, biochemistry, and biophysics techniques, we discover that ACT has intrinsic phospholipase A (PLA) activity, and that such activity determines AC translocation. Moreover, we show that elimination of the ACT-PLA activity abrogates ACT toxicity in macrophages, particularly at toxin concentrations close to biological reality of bacterial infection. Our data support a molecular mechanism in which in situ generation of nonlamellar lysophospholipids by ACT-PLA activity into the cell membrane would form, likely in combination with membrane-interacting ACT segments, a proteolipidic toroidal pore through which AC domain transfer could directly take place. Regulation of ACT-PLA activity thus emerges as novel target for therapeutic control of the disease.

Modification of membrane lipid compositions in single-celled organisms - From basics to applications.

All intact cells, and their organelles, are surrounded by a ∼30 Šhydrophobic film that typically separates the interior from the environment. This film is composed of lipid bilayers that form from a pool of structurally highly diverse, amphipathic lipids. The specific composition and nature of these lipids strongly contributes to many different processes in the cell by influencing membrane structures, membrane protein sorting and functionalities. In this review, we discuss strategies to alter membrane lipid compositions of organelles and plasma membranes in different organisms, focusing on microbial cells. Reflecting the many essential roles of lipids in cellular regulation, we delineate diverse cellular processes affected by membrane lipid modifications and discuss possible applications in a biotechnological and biomedical context. A major motivation for membrane lipid engineering has been the improvement of expression, translocation and activity of heterologous membrane proteins, which can facilitate the biochemical and structural characterization of this challenging class of proteins. Additionally, better expression of membrane proteins or membrane lipid engineering - or a combination of both - led to improved production of high-value compounds and food additives, e.g. polyunsaturated fatty acids and glycolipids, in diverse hosts. More recently it has been shown that diverse cellular pathologies such as cancer and Alzheimer's disease are associated with lipid alterations. Hence, the progress in our understanding of membrane structure, function and protein-lipid interactions, and the resulting possibilities regarding the engineering of membrane lipid composition clearly enable novel nutraceutical and pharmaceutical interventions to be developed. Significant progress in this important area of research is highlighted in this review.

Physiological Functions of Phosphoinositide-Modifying Enzymes and Their Interacting Proteins in Arabidopsis.

The integrity of cellular membranes is maintained not only by structural phospholipids such as phosphatidylcholine and phosphatidylethanolamine, but also by regulatory phospholipids, phosphatidylinositol phosphates (phosphoinositides). Although phosphoinositides constitute minor membrane phospholipids, they exert a wide variety of regulatory functions in all eukaryotic cells. They act as key markers of membrane surfaces that determine the biological integrity of cellular compartments to recruit various phosphoinositide-binding proteins. This review focuses on recent progress on the significance of phosphoinositides, their modifying enzymes, and phosphoinositide-binding proteins in Arabidopsis.

Direct Prediction of EPR Spectra from Lipid Bilayers: Understanding Structure and Dynamics in Biological Membranes.

Of the many biophysical techniques now being brought to bear on studies of membranes, electron paramagnetic resonance (EPR) of nitroxide spin probes was the first to provide information about both mobility and ordering in lipid membranes. Here, we report the first prediction of variable temperature EPR spectra of model lipid bilayers in the presence and absence of cholesterol from the results of large scale fully atomistic molecular dynamics (MD) simulations. Three types of structurally different spin probes were employed in order to study different parts of the bilayer. Our results demonstrate very good agreement with experiment and thus confirm the accuracy of the latest lipid force fields. The atomic resolution of the simulations allows the interpretation of the molecular motions and interactions in terms of their impact on the sensitive EPR line shapes. Direct versus indirect effects of cholesterol on the dynamics of spin probes are analysed. Given the complexity of structural organisation in lipid bilayers, the advantage of using a combined MD-EPR simulation approach is two-fold. Firstly, prediction of EPR line shapes directly from MD trajectories of actual phospholipid structures allows unambiguous interpretation of EPR spectra of biological membranes in terms of complex motions. Secondly, such an approach provides an ultimate test bed for the up-to-date MD simulation models employed in the studies of biological membranes, an area that currently attracts great attention.

Imaging Proton Transport in Giant Vesicles through Cyclic Peptide-Polymer Conjugate Nanotube Transmembrane Ion Channels.

Since their discovery in 1993, interest in various aspects of cyclic peptides (CPs) has expanded rapidly. Of particular note is their potential to form artificial ion channels in lipid membranes, an attractive characteristic in supramolecular chemistry and biological research. The design and synthesis of cyclic peptide-polymer conjugates (CPPCs) that can self-assemble within lipid bilayers into nanotubes, mimicking naturally occurring membrane channels and pores, has been reported. However, methods that allow direct detection of the transport process with high levels of certainty are still lacking. This work focuses on the development of a simple but reliable approach to verify and quantify proton transport across a bilayer membrane. Giant unilamellar vesicles (GUVs) are created via the electroformation method and CPPCs are incorporated in GUV membranes at varying concentrations (0-10%). Confocal fluorescence microscopy is used to demonstrate full inclusion of fluorescein-labeled CPPCs in the GUV membranes. The pH-sensitive dye carboxyfluorescein is encapsulated within the water pool of the GUVs and used as an indicator of proton transport. This assay is versatile and can be exploited on other existing proton transporter systems, providing a consistent tool to compare their performances. It should also aid the development of novel antineoplastics and drug delivery systems.

Preparation of Enantiomeric ß-(2',5'-Dimethylphenyl)Bromolactones, Their Antiproliferative Activity and Effect on Biological Membranes.

Three novel enantiomeric pairs of bromolactones possesing a 2,5-dimethylphenyl substituent at the ß-position of the lactone ring have been synthesized from corresponding enantiomeric (E)-3-(2',5'-dimethylphenyl)hex-4-enoic acids (4) by kinetically controlled bromolactonization with N-bromosuccinimide (NBS). γ-Bromo-δ-lactones (5) were isolated as the major products. Absolute configurations of stereogenic centers of γ-bromo-δ-lactones (5) were assigned based on X-ray analysis; configurations of cis δ-bromo-γ-lactones (6) and trans δ-bromo-γ-lactones (7) were determined based on mechanism of bromolactonization. Synthesized compounds exhibited significant antiproliferative activity towards the four canine cancer cell lines (D17, CLBL-1, CLB70, and GL-1) and one human cancer line (Jurkat). Classifying the compounds in terms of activity, the most active were enantiomers of trans δ-bromo-γ-lactones (7) followed by enantiomers of cis isomer (6) and enantiomeric γ-bromo-δ-lactones (5). Higher activity was observed for all stereoisomers with S configuration at C-4 in comparison with their enantiomers with 4R configuration. Synthesized compounds did not induce hemolysis of erythrocytes. The results of the interaction of bromolactones with red blood cell membranes suggest that these compounds incorporate into biological membranes, concentrating mainly in the hydrophilic part of the bilayer but have practically no influence on fluidity in the hydrophobic region. The differences in interactions with the membrane between particular enantiomers were observed only for γ-lactones: stronger interactions were found for enantiomer 4R,5R,6S of cis γ-lactone (6) and for enantiomer 4S,5R,6S of trans γ-lactone (7).

Lipid perturbation by membrane proteins and the lipophobic effect.

Understanding how membrane proteins interact with their environment is fundamental to the understanding of their structure, function and interactions. We have performed coarse-grained molecular dynamics simulations on a series of membrane proteins in a membrane environment to examine the perturbations of the lipids by the presence of protein. We analyze these perturbations in terms of elastic membrane deformations and local lipid protein interactions. However these two factors are insufficient to describe the variety of effects that we observe and the changes caused by membranes proteins to the structure and dynamics of their lipid environment. Other factors that change the conformation available to lipid molecules are evident and are able to modify lipid structure far from the protein surface, and thus mediate long-range interactions between membrane proteins. We suggest that these multiple modifications to lipid behavior are responsible, at the molecular level, for the lipophobic effect we have proposed to account for our observations of membrane protein organization.

Towards a Unified Understanding of Lithium Action in Basic Biology and its Significance for Applied Biology.

Lithium has literally been everywhere forever, since it is one of the three elements created in the Big Bang. Lithium concentration in rocks, soil, and fresh water is highly variable from place to place, and has varied widely in specific regions over evolutionary and geologic time. The biological effects of lithium are many and varied. Based on experiments in which animals are deprived of lithium, lithium is an essential nutrient. At the other extreme, at lithium ingestion sufficient to raise blood concentration significantly over 1 mM/, lithium is acutely toxic. There is no consensus regarding optimum levels of lithium intake for populations or individuals-with the single exception that lithium is a generally accepted first-line therapy for bipolar disorder, and specific dosage guidelines for sufferers of that condition are generally agreed on. Epidemiological evidence correlating various markers of social dysfunction and disease vs. lithium level in drinking water suggest benefits of moderately elevated lithium compared to average levels of lithium intake. In contrast to other biologically significant ions, lithium is unusual in not having its concentration in fluids of multicellular animals closely regulated. For hydrogen ions, sodium ions, potassium ions, calcium ions, chloride ions, and magnesium ions, blood and extracellular fluid concentrations are closely and necessarily regulated by systems of highly selective channels, and primary and secondary active transporters. Lithium, while having strong biological activity, is tolerated over body fluid concentrations ranging over many orders of magnitude. The lack of biological regulation of lithium appears due to lack of lithium-specific binding sites and selectivity filters. Rather lithium exerts its myriad physiological and biochemical effects by competing for macromolecular sites that are relatively specific for other cations, most especially for sodium and magnesium. This review will consider what is known about the nature of this competition and suggest using and extending this knowledge towards the goal of a unified understanding of lithium in biology and the application of that understanding in medicine and nutrition.