Membrane-interacting Molecules Research Articles

Perspective on the role of the physical properties of membranes in neurodegenerative and infectious diseases

Cell membranes are dynamic and complex structures, and their composition and structure are major determinants of pathology. It is now commonly accepted that the membranes' physical properties, such as fluidity and thickness, are determining factors for permeability, partitioning of drug molecules, and protein aggregation. Membrane-interacting molecules can in some instances be expected to have a greater therapeutic potential than traditional therapies targeting receptors or enzymes. Alzheimer's disease is an example where traditional approaches thus far have been proven unsuccessful. With bacteria becoming resistant to more and more antibiotics, potential membrane based antibiotics provide an alternative route with great potential. Here, we provide a perspective on the basic mechanisms how physical membrane properties can affect diseases and the therapeutic potential of changing membrane lipid composition and properties to target those diseases. Neurodegenerative diseases, such as Alzheimer's disease, and infectious diseases, are prime examples among many others where the so-called Membrane-Lipid Therapy shows great potential for the development of new drugs and new therapies.

Contributions and Limitations of Biophysical Approaches to Study of the Interactions between Amphiphilic Molecules and the Plant Plasma Membrane.

Some amphiphilic molecules are able to interact with the lipid matrix of plant plasma membranes and trigger the immune response in plants. This original mode of perception is not yet fully understood and biophysical approaches could help to obtain molecular insights. In this review, we focus on such membrane-interacting molecules, and present biophysically grounded methods that are used and are particularly interesting in the investigation of this mode of perception. Rather than going into overly technical details, the aim of this review was to provide to readers with a plant biochemistry background a good overview of how biophysics can help to study molecular interactions between bioactive amphiphilic molecules and plant lipid membranes. In particular, we present the biomimetic membrane models typically used, solid-state nuclear magnetic resonance, molecular modeling, and fluorescence approaches, because they are especially suitable for this field of research. For each technique, we provide a brief description, a few case studies, and the inherent limitations, so non-specialists can gain a good grasp on how they could extend their toolbox and/or could apply new techniques to study amphiphilic bioactive compound and lipid interactions.

Detecting Single‐Molecule Dynamics on Lipid Membranes with Quenchers‐in‐a‐Liposome FRET

Tracking membrane-interacting molecules and visualizing their conformational dynamics are key to understanding their functions. It is, however, challenging to accurately probe the positions of a molecule relative to a membrane. Herein, a single-molecule method, termed LipoFRET, is reported to assess interplay between molecules and liposomes. It takes advantage of FRET between a single fluorophore attached to a biomolecule and many quenchers in a liposome. This method was used to characterize interactions between α-synuclein (α-syn) and membranes. These results revealed that the N-terminus of α-syn inserts into the membrane and spontaneously transitions between different depths. In contrast, the C-terminal tail of α-syn is regulated by calcium ions and floats in solution in two conformations. LipoFRET is a powerful tool to investigate membrane-interacting biomolecules with sub-nanometer precision at the single-molecule level.

Gene Expression Signatures of Lymph Node Metastasis in Oral Cancer: Molecular Characteristics and Clinical Significances.

Even though lymph node metastasis accounts for the vast majority of cancer death in patients with oral cancer (OC), the molecular mechanisms of lymph node metastasis remain elusive. Genome-wide microarray analyses and functional studies in vitro and in vivo, along with detailed clinical observations, have identified a number of molecules that may contribute to lymph node metastasis. These include lymphangionenic cytokines, cell adhesion molecules, basement membrane-interacting molecules, matrix enzymes and relevant downstream signaling pathways. However, defined gene signatures from different studies are highly variable, which hinders their translation to clinically relevant applications. To date, none of the identified signatures or molecular biomarkers has been successfully implemented as a diagnostic or prognostic tool applicable to routine clinical practice. In this review, we will first introduce the significance of lymph node metastasis in OC, and clinical/experimental evidences that support the underlying molecular mechanisms. We will then provide a comprehensive review and integrative analysis of the existing gene expression studies that aim to identify the metastasis-related signatures in OC. Finally, the remaining challenges will be discussed and our insights on future directions will be provided.

Inhibitory activity of synthetic peptide antibiotics on feline immunodeficiency virus infectivity in vitro.

Natural peptide antibiotics are part of host innate immunity against a wide range of microbes, including some viruses. Synthetic peptides modeled after natural peptide antibiotics interfere with microbial membranes and are termed peptidyl membrane-interactive molecules (peptidyl-MIM [Demegen Inc, Pittsburgh, Pa.]). Sixteen peptidyl-MIM candidates were tested for activity against feline immunodeficiency virus (FIV) on infected CrFK cells. Three of them (D4E1, DC1, and D1D6) showed potent anti-FIV activity in chronically infected CrFK cells as measured by decreased reverse transcriptase (RT) activity, having 50% inhibitory concentrations of 0.46, 0.75, and 0.94 micro M, respectively, which were approximately 10 times lower than their direct cytotoxic concentrations. Treatment of chronically infected CrFK cells with 2 micro M D4E1 for 3 days completely reversed virus-induced cytopathic effect. Immunofluorescence revealed reduced p26 staining in these cells. Treatment of chronically infected CrFK cells with 2 micro M D4E1 suppressed virus production ( approximately 50%) for up to 7 days, The virions from the D4E1-treated culture had impaired infectivity, as measured by the 50% tissue culture infectious dose and nested PCR analysis of proviral DNA. However, these noninfectious virions were able to bind and internalize, suggesting a defect at some postentry step. After chronically infected CrFK cells were treated with D4E1 for 24 h, increased cell-associated mature p26 Gag and decreased extracellular virus-associated p26 Gag were observed by Western blot analysis, suggesting that virus assembly and/or release may be blocked by D4E1 treatment, whereas virus binding, penetration, RNA synthesis, and protein synthesis appear to be unaffected. Synthetic peptide antibiotics may be useful tools in the search for antiviral drugs having a wide therapeutic window for host cells.

Prediction of membrane protein orientation in lipid bilayers: a theoretical approach

Over the past few years, several three-dimensional (3-D) structures of membrane proteins have been described with increasing accuracy, but their relationship with membranes are still not well understood. Recently, we have developed an empirical method, Integral Membrane Protein and Lipid Association (IMPALA), to predict the insertion of molecules (lipids, drugs) into lipid bilayers (Proteins 30 (1998) 357). The IMPALA uses a Monte Carlo minimisation procedure to calculate the depth and the angle of insertion of membrane-interacting molecules taking into account the restraints dictated by a lipid bilayer. In this paper, we use IMPALA to test the insertion of 23 integral membranous proteins (IMPs) and 2 soluble proteins into membranes. Four IMP are studied in detail: OmpA, maltoporin, MsCl channel and bacteriorhodopsin. The 3-D structures of the proteins are kept constant and the insertion into membrane is monitored by minimising the value of the restraint representing the sum of two terms, one for lipid perturbation and the other for hydrophobicity. The two soluble proteins are rejected from the membrane whereas, under the same conditions, all the membrane proteins remain inside, if the solvent accessible surface of the amino acids located inside the pore of porins is ignored. The results give the tilt angle of the IMP helices or strands with respect to the membrane surface and the depth of the protein mass centre insertion. We conclude that the restraint terms of IMPALA could be used to study the insertion of model structures or complexes of proteins within membranes.

In vitro toxicity of natural and designed peptides to tree pathogens and pollen

The toxicities of four candidate peptides, which have potential for engineering disease resistance into poplars and conifers, were tested and compared in vitro. Cecropin B, (Ala8,13,18)-magainin II amide, and the two synthetic membrane interactive molecules (Peptidyl MIMs(tm)) D2A21 and D4E1 inhibited germination of spores of the fungal pathogens Cronartium ribicola J.C. Fisch., Gremmeniella abietina (Lagerberg) Morelet, Melampsora medusae Thuem., Nectria galligena Bres. in Strass., Ophiostoma ulmi (Buisman) Nannf., andSeptoria musiva Peck. Minimal inhibitory concentrations (MICs) of peptides required to achieve >95% inhibition of germination of conidia and urediniospores ranged from 0.3 to >5 µM. Permutation analyses on MICs confirmed that peptides significantly reduced germination of fungus spores (P = 0.0038) and that MICs of the two synthetic peptides were, on average, lower than those of the two natural peptides (P = 0.0012). In contrast, peptides had much less of an effect on the pollen of Picea, Pinus, and Populus species and seeds of white pine; MICs from 10 µM to >50 µM of peptide were needed to arrest germination. Peptidyl MIM(tm) D2A21 combined high antifungal activity with low pollen toxicity. Similar to some antimicrobial peptides from plants, Peptidyl MIM(tm) D2A21 could not prevent germination of fungal spores in the presence of potato dextrose broth or chloride salts with divalent cations. This may explain why the peptide had only a limited effect on fungal mycelium grown in potato dextrose broth medium.Key words: antimicrobial peptides (AMP), white pine blister rust, scleroderris canker, poplar leaf rust, Dutch elm disease, septoria canker.

In vitro toxicity of natural and designed peptides to tree pathogens and pollen

The toxicities of four candidate peptides, which have potential for engineering disease resistance into poplars and conifers, were tested and compared in vitro. Cecropin B, (Ala 8,13,18 )-magainin II amide, and the two synthetic membrane interactive molecules (Peptidyl MIMs™) D2A21 and D4E1 inhibited germination of spores of the fungal pathogens Cronartium ribicola J.C. Fisch., Gremmeniella abietina (Lagerberg) Morelet, Melampsora medusaeThuem., Nectria galligena Bres. in Strass., Ophiostoma ulmi (Buisman) Nannf., and Septoria musiva Peck. Minimal inhibitory concentrations (MICs) of peptides required to achieve >95% inhibition of germination of conidia and urediniospores ranged from 0.3 to >5 μM. Permutation analyses on MICs confirmed that peptides significantly reduced germination of fungus spores (P = 0.0038) and that MICs of the two synthetic peptides were, on average, lower than those of the two natural peptides (P = 0.0012). In contrast, peptides had much less of an effect on the pollen of Picea, Pinus, and Populus species and seeds of white pine; MICs from 10 μ Mt o >50μM of peptide were needed to arrest germination. Peptidyl MIM™ D2A21 combined high antifungal activity with low pollen toxicity. Similar to some antimicrobial pep- tides from plants, Peptidyl MIM™ D2A21 could not prevent germination of fungal spores in the presence of potato dextrose broth or chloride salts with divalent cations. This may explain why the peptide had only a limited effect on fungal mycelium grown in potato dextrose broth medium.

Orientation of the Pore-Forming Peptide GALA in POPC Vesicles Determined by a BODIPY-Avidin/Biotin Binding Assay

We determined the orientation of a biotinylated version of the pore-forming peptide GALA (WEAALAEALAEALAEHLAEALAEALEALAA) at pH 5.0 in large unilamellar phosphatidylcholine vesicles, using the enhancement of BODIPY-avidin fluorescence subsequent to its irreversible binding to a biotin moiety. GALA and its variants were biotinylated at the N- or C-terminus. BODIPY-avidin was either added externally or was pre-encapsulated in vesicles to assess the fraction of liposome-bound biotinylated GALA that exposed its labeled terminus to the external or internal side of the bilayer, respectively. Under conditions where most of the membrane-bound peptides were involved in transmembrane aggregates and formed aqueous pores (at a lipid/bound peptide molar ratio of 2500/1), the head-to-tail (N- to C-terminus) orientation of the membrane-inserted peptides was such that 3 4 of the peptides exposed their N-terminus on the inside of the vesicle and their C-terminus on the outside. Under conditions resulting in reduced pore formation (at higher lipid/peptide molar ratios), we observed an increase in the fraction of GALA termini exposed to the outside of the vesicle. These results are consistent with a model (Parente et al., Biochemistry, 29:8720, 1990) that requires a critical number of peptides ( M) in an aggregate to form a transbilayer structure. When the peptides form an aggregate of size i, with i < M = 4 to 6, the orientation of the peptides is mostly parallel to the membrane surface, such that both termini of the biotinylated peptide are exposed to external BODIPY-avidin. This BODIPY-avidin/biotin binding assay should be useful to determine the orientation of other membrane-interacting molecules.

Improvement in survival with peptidyl membrane interactive molecule D4B treatment after burn wound infection.

To examine the effects of peptidyl membrane interactive molecule D4B in a murine model of lethal burn wound infection. Four experiments were performed: (1) growth inhibition assays of Pseudomonas aeruginosa treated with D4B, 0 to 100 micromol/L; (2) in vitro coculture of bone marrow cells with D4B, 0 to 100 micromol/L; (3) D4B treatment survival studies after burn injury only or burn wound infection in mice; and (4) peripheral white blood cell count, burn wound tissue bacterial culture, and burn wound morphological analysis at days 1, 2, and 3 after injury. University medical center laboratory. Groups of B6D2F1 male mice (20 each) were studied. Full-thickness scald burn, 15% of total body surface area, with P aeruginosa topical infection, and subeschar injections of D4B at 200 microg or 0.25 mL of placebo per mouse at 2 and 24 hours after injury. Animal survival after thermal burn wound bacterial infection, circulating leukocyte numbers, in vitro clonal cell culture of granulocyte-macrophage progenitor cells, and wound histopathological analysis. The survival rate in the D4B-treated group was nearly 2-fold greater than that in controls (P<.01) during 14 days of study. Bacterial quantitative wound cultures disclosed significant reductions in bacterial numbers at days 1, 2, and 3 in D4B-treated animals as compared with controls (P<.05 to <.01). D4B induced a dose-dependent inhibition of bacterial cell growth when added to in vitro P aeruginosa cultures (P<.01). Granulocyte-macrophage progenitor cell growth in culture was not altered by D4B treatment. D4B-treated animals displayed no signs of toxic effects or impairment in wound healing. The peptidyl membrane interactive molecule D4B had the ability to improve survival after gram-negative burn wound sepsis via direct antimicrobial effects. Peptidyl membrane interactive molecules may offer the potential of alternative treatments to standard topical agents or in patients with drug-resistant microbes.

Transcellular permeability of chlorpromazine demonstrating the roles of protein binding and membrane partitioning.

Transcellular permeability of the neuroleptic-anesthetic chlorpromazine (CPZ) was examined using a cell type (MDCK) that forms a confluent monolayer of polarized cells resulting in distinct apical (AP) and basolateral (BL) membrane domains separated by tight junctions. Because CPZ is membrane interactive, transmonolayer flux was analyzed as two kinetic events: cell uptake from the AP donor solution and efflux into the BL side receiver. Using the rate of cell uptake in the presence of different concentrations of BSA, an intrinsic cell partition coefficient of 3700 +/- 130 and an operational dissociation binding constant of 0.4 +/- 0.05 mM were calculated. In contrast to uptake, efflux of CPZ from either the AP or the BL side of the cell monolayer was approximately 10(4)-fold slower and was dependent upon the avidity of CPZ for the protein acceptor in the receiver solution. These results emphasized the importance of simultaneously measuring disappearance of a lipophilic molecule from the donor solution and its appearance in the receiver and demonstrated how interactions with proteins on either side of the cellular barrier influence permeability. Appearance kinetics showed that the composition of the receiving environment is critical to model a particular in vivo situation and implied that the intrinsic permeability of membrane-interactive molecules in vitro does not necessarily predict penetration beyond the initial cellular barrier in vivo.

Slow motions in oriented phospholipid bilayers and effects of cholesterol or gramicidin. A 19F-NMR T1 rho study

In an extension of our earlier work (Peng, Z.-y., V. Simplaceanu, I. J. Lowe, and C. Ho. 1988. Biophys. J. 54:81-95), the rotating-frame nuclear spin-lattice relaxation (T1 rho) technique has been used to investigate the slow molecular motions (10(-4) - 10(-6) s) in lipid bilayers prepared from protonated or perdeuterated 19F-labeled phospholipids in the absence and presence of cholesterol or gramicidin as membrane-interacting molecules. Complications caused by the 19F-1H cross-polarization observed previously can be removed by the substitution of 2H for 1H in the acyl chains. Only a weak dependence of the T-1(1 rho) on the locking field strength is found for a phospholipid molecule with perdeuterated acyl chains, indicating that there are no slow motions with a single, well-defined correlation time between 5 x 10(-6) and 4 x 10(-5) s. However, the orientation dependences of the T-1(1 rho) can be well fitted by motional models with either one slow motion having an unspecified geometry or with a superposition of two specific types of slow motions. Cholesterol and gramicidin show distinct effects in altering either the geometry or the weighting of slow motions in phospholipid bilayers, as reflected by changes in the orientation dependence. These two additives also exhibit quite different label-position specificities. A qualitative understanding of the induced effects of cholesterol and gramicidin on the dynamics of phospholipid bilayers will be discussed.