TY - JOUR AB - Antimicrobial peptides are a promising class of potential antibiotics that interact selectively with negatively charged lipid bilayers. This paper presents the structural characterization of the antimicrobial peptides myxinidin and WMR associated with bacterial membrane mimetic micelles and bicelles by NMR, CD spectroscopy, and molecular dynamics simulations. Both peptides adopt a different conformation in the lipidic environment than in aqueous solution. The location of the peptides in micelles and bicelles has been studied by paramagnetic relaxation enhancement experiments with paramagnetic tagged 5- and 16-doxyl stearic acid (5-/16-SASL). Molecular dynamics simulations of multiple copies of the peptides were used to obtain an atomic level of detail on membrane-peptide and peptide-peptide interactions. Our results highlight an essential role of the negatively charged membrane mimetic in the structural stability of both myxinidin and WMR. The peptides localize predominantly in the membrane's headgroup region and have a noticeable membrane thinning effect on the overall bilayer structure. Myxinidin and WMR show a different tendency to self-aggregate, which is also influenced by the membrane composition (DOPE/DOPG versus DOPE/DOPG/CL) and can be related to the previously observed difference in the ability of the peptides to disrupt different types of model membranes. AU - Cherniavskyi, Y.K.* AU - Oliva, R.* AU - Stellato, M.* AU - Del Vecchio, P.* AU - Galdiero, S.* AU - Falanga, A.* AU - Dames, S.A. AU - Tieleman, D.P.* C1 - 69761 C2 - 55259 CY - Radarweg 29, 1043 Nx Amsterdam, Netherlands TI - Structural characterization of the antimicrobial peptides myxinidin and WMR in bacterial membrane mimetic micelles and bicelles. JO - Biochim. Biophys. Acta-Biomembr. VL - 1866 IS - 3 PB - Elsevier PY - 2024 SN - 0005-2736 ER - TY - JOUR AB - A membrane protein's oligomeric state modulates its functionality in various cellular processes. Since membrane proteins have to be solubilized in an appropriate membrane mimetic, the use of classical biophysical methods to analyze protein oligomers is challenging. We here present a method to determine the number of membrane proteins inserted into lipid nanodiscs. It is based on the ability to selectively quantify the amount of a small and robust fusion protein that can be proteolytically cleaved off from a membrane protein after incorporation into lipid nanodiscs. A detailed knowledge of the number of membrane proteins per nanodisc at defined assembly conditions is essential to estimate the tendency for oligomerization, but also for guiding sample optimization for structural investigations that require the presence of a homogenous oligomeric state. We show that this method can efficiently be used to determine the number of VDAC1 channels in nanodiscs at various assembly conditions, as confirmed by negative stain EM. The presented method is suitable in particular for membrane proteins that cannot be probed easily by other methods such as single span transmembrane helices. This assay can be applied to any membrane protein that can be incorporated into a nanodisc without the requirement for special instrumentation and will thus be widely applicable and complementary to other methods that quantify membrane protein insertion in lipid nanodiscs. AU - Häusler, E. AU - Fredriksson, K. AU - Goba, I. AU - Peters, C.* AU - Raltchev, K. AU - Sperl, L.E. AU - Steiner, A. AU - Weinkauf, S.* AU - Hagn, F. C1 - 57893 C2 - 48141 CY - Radarweg 29, 1043 Nx Amsterdam, Netherlands TI - Quantifying the insertion of membrane proteins into lipid bilayer nanodiscs using a fusion protein strategy. JO - Biochim. Biophys. Acta-Biomembr. VL - 1862 IS - 4 PB - Elsevier PY - 2020 SN - 0005-2736 ER - TY - JOUR AB - The cell-toxic bile salts glycochenodeoxycholic acid (GCDCA) and taurochenodeoxycholic acid (TCDCA) are responsible for hepatocyte demise in cholestatic liver diseases, while tauroursodeoxycholic acid (TUDCA) is regarded hepatoprotective. We demonstrate the direct mitochondrio-toxicity of bile salts which deplete the mitochondrial membrane potential and induce the mitochondrial permeability transition (MPT). The bile salt mediated mechanistic mode of destruction significantly differs from that of calcium, the prototype MPT inducer. Cell-toxic bile salts initially bind to the mitochondrial outer membrane. Subsequently, the structure of the inner boundary membrane disintegrates. And it is only thereafter that the MPT is induced. This progressive destruction occurs in a dose- and time-dependent way. We demonstrate that GCDCA and TCDCA, but not TUDCA, preferentially permeabilize liposomes containing the mitochondrial membrane protein ANT, a process resembling the MPT induction in whole mitochondria. This suggests that ANT is one decisive target for toxic bile salts. To our knowledge this is the first report unraveling the consecutive steps leading to mitochondrial destruction by cell-toxic bile salts. AU - Schulz, S. AU - Schmitt, S. AU - Wimmer, R.* AU - Aichler, M. AU - Eisenhofer, S. AU - Lichtmannegger, J. AU - Eberhagen, C. AU - Artmann, R.* AU - Toókos, F. AU - Walch, A.K. AU - Krappmann, D. AU - Brenner-Jan, C.* AU - Rust, C.* AU - Zischka, H. C1 - 24525 C2 - 31568 SP - 2121-2133 TI - Progressive stages of mitochondrial destruction caused by cell toxic bile salts. JO - Biochim. Biophys. Acta-Biomembr. VL - 1828 IS - 9 PB - Elsevier Science PY - 2013 SN - 0005-2736 ER - TY - JOUR AB - The protein- and/or lipid-mediated association of chaperone proteins to membranes is a widespread phenomenon and implicated in a number of physiological and pathological events that were earlier partially or completely overlooked. A temporary association of certain HSPs with membranes can re-establish the fluidity and bilayer stability and thereby restore the membrane functionality during stress conditions. The fluidity and microdomain organization of membranes are decisive factors in the perception and transduction of stresses into signals that trigger the activation of specific HS genes. Conversely, the membrane association of HSPs may result in the inactivation of membrane-perturbing signals, thereby switch off the heat shock response. Interactions between certain HSPs and specific lipid microdomains ("rafts") might be a previously unrecognized means for the compartmentalization of HSPs to specific signaling platforms, where key signaling proteins are known to be concentrated. Any modulations of the membranes, especially the raft-lipid composition of the cells can alter the extracellular release and thus the immuno-stimulatory activity of certain HSPs. Reliable techniques, allowing mapping of the composition and dynamics of lipid microdomains and simultaneously the spatio-temporal localization of HSPs in and near the plasma membrane can provide suitable means with which to address fundamental questions, such as how HSPs are transported to and translocated through the plasma membrane. The possession of such information is critical if we are to target the membrane association principles of HSPs for successful drug development in most various diseases. AU - Horváth, I.* AU - Multhoff, G. AU - Sonnleitner, A.* AU - Vigh, L.* C1 - 721 C2 - 25347 SP - 1653-1664 TI - Membrane-associated stress proteins: More than simply chaperones. JO - Biochim. Biophys. Acta-Biomembr. VL - 1778 IS - 7-8 PB - Elsevier PY - 2008 SN - 0005-2736 ER - TY - JOUR AB - Recently, we reported that 1,2-dipalmitoyl-sn-glycero-3-phosphoglyceroglycerol (DPPGOG) prolongs the circulation time of thermosensitive liposomes (TSL). Since the only TSL formulation in clinical trials applies DSPE-PEG2000 and lysophosphatidylcholine (P-lyso-PC), the objective of this study was to compare the influence of these lipids with DPPGOG on in vitro stability and heat-induced drug release properties of TSL. The content release rate was significantly increased by incorporating DPPGOG or P-lyso-PC in TSL formulations. DPPC/DSPC/DPPGOG 50:20:30 (m/m) and DPPC/P-lyso-PC/DSPE-PEG2000 90:10:4 (m/m) did not differ significantly in their release rate of carboxyfluorescein with >70% being released within the first 10s at their phase transition temperature. Furthermore, DPPC/DSPC/DPPGOG showed an improved stability at 37 degrees C in serum compared to the PEGylated TSL. The in vitro properties of DPPGOG-containing TSL remained unchanged when encapsulating doxorubicin instead of carboxyfluorescein. The TSL retained 89.1+/-4.0% of doxorubicin over 3 h at 37 degrees C in the presence of serum. The drug was almost completely released within 120s at 42 degrees C. In conclusion, DPPGOG improves the in vitro properties in TSL formulations compared to DSPE-PEG2000, since it not only increases the in vivo half-life, it even increases the content release rate without negative effect on TSL stability at 37 degrees C which has been seen for DSPE-PEG2000/P-lyso-PC containing TSL. AU - Hossann, M.* AU - Wiggenhorn, M.* AU - Schwerdt, A.* AU - Wachholz, K.* AU - Teichert, N.* AU - Eibl, H.* AU - Issels, R.D. AU - Lindner, L.H. C1 - 2690 C2 - 24974 SP - 2491-2499 TI - In vitro stability and content release properties of phosphatidylglyceroglycerol containing thermosensitive liposomes. JO - Biochim. Biophys. Acta-Biomembr. VL - 1768 IS - 10 PB - Elsevier PY - 2007 SN - 0005-2736 ER - TY - JOUR AB - The nicotinic acetylcholine receptor (nAcChoR) has an absolute requirement for cholesterol if agonist-stimulated channel opening is to occur [Biochemistry 25 (1986) 830]. Certain non-polar analogs could replace cholesterol in vectorial vesicle permeability assays. Using a stopped-flow fluorescence assay to avoid the limitations of permeability assays imposed by vesicle morphology, it was shown that polar conjugates of cholesterol could also satisfy the sterol requirement [Biochim. Biophys. Acta 1370 (1998) 299]. Here this assay is used to explore the chemical specificity of sterols. Affinity-purified nAcChoRs from Torpedo were reconstituted into bilayers at mole ratios of 58:12:30 [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phosphate (DOPA)/steroid]. When the enantiomer of cholesterol was used, or when the stereochemistry at the 3-hydroxy group was changed from β to α by substituting epicholesterol for cholesterol, activation was still supported. The importance of cholesterol's planar ring structure was tested by comparing planar cholestanol (5α-cholestan-3β-ol) with nonplanar coprostanol (5β-cholestan-3β-ol). Both supported activation. Thus, these steroids support activation independent of structural features known to be important for modulation of lipid bilayer properties. This provides indirect support for a steroid binding site possessing very lax structural requirements. AU - Addona, G.H.* AU - Sandermann, H. AU - Kloczewiak, M.A.* AU - Miller, K.W.* C1 - 22252 C2 - 21014 SP - 177-182 TI - Low chemical specificity of the nicotinic acetylcholine receptor sterol activation site. JO - Biochim. Biophys. Acta-Biomembr. VL - 1609 IS - 2 PY - 2003 SN - 0005-2736 ER - TY - JOUR AB - We have isolated and functionally characterized an additional murine member of the organic-anion-transporting polypeptide (Oatp) family of membrane transport proteins from mouse liver. The 3.6 kb cDNA insert contains an open reading frame of 2010 bp coding for a 670 amino acid protein. Based on its amino acid identity of 88% to the rat Oatp2, it is considered the mouse Oatp2 orthologue. Functional expression in Xenopus laevis oocytes demonstrated that mouse Oatp2 transports several general Oatp substrates such as estrone-3-sulfate, dehydroepiandrosterone sulfate (DHEAS), ouabain and BQ-123 but hardly any taurocholate nor rocuronium or deltorphin II. The high-affinity rat Oatp2 substrate digoxin is transported with a rather low affinity with an apparent Km value of 5.7 μM. Bromosulfophthalein (BSP), a substrate not transported by the rat Oatp2, is transported very well by mouse Oatp2. Northern blot analysis demonstrated a predominant expression in the liver with additional signals in kidney and brain. Using fluorescence in situ hybridization, the Oatp2 gene (gene symbol Slc21a5) was mapped to chromosome 6G1–G3. AU - van Montfoort, J.E.* AU - Schmid, T.E. AU - Adler, I.-D. AU - Meier, P.J.* AU - Hagenbuch, B.* C1 - 21957 C2 - 20476 SP - 183-188 TI - Functional characterization of the mouse organic-anion-transporting polypeptide 2. JO - Biochim. Biophys. Acta-Biomembr. VL - 1564 IS - 1 PY - 2002 SN - 0005-2736 ER - TY - JOUR AB - Many membrane-active xenobiotics, such as organic solvents or general anesthetics, are without specific target sites. A common, but as yet ill-defined physical mechanism of action is usually assumed. Displacement of 'boundary' lipid activators from functional membrane proteins by the induction of lipid/protein mismatch is now established as a candidate common physical mechanism. Multiple binding site kinetics demonstrate that xenobiotics can be strongly inhibitory at realistic membrane concentrations of below 4 mol%. A general equation for inhibition is derived. AU - Sandermann, H.J. C1 - 40403 C2 - 40033 SP - 130-133 TI - Induction of lipid-protein mismatch by xenobiotics with general membrane targets. JO - Biochim. Biophys. Acta-Biomembr. VL - 1150 IS - 2 PY - 1993 SN - 0005-2736 ER - TY - JOUR AB - A previously developed kinetic theory for lipid-dependent membrane enzymes (Sandermann, H. (1982) Eur. J. Biochem. 127, 123-138) is used to examine the activation of protein kinase C by phosphatidylserine. Hill-coefficients ranging up to 11 have been reported for activation in mixed micelles with Triton X-100. On the basis of this uniquely high degree of cooperativity, protein kinase C has been postulated to represent a new class of lipid-dependent membrane enzymes (Newton, A. and Koshland, D.E., Jr. (1989) J. Biol. Chem. 264, 14909-14915). In contrast, activation in the absence of Triton X-100 has led to Hill-coefficients of only ≤ 2.6. In order to resolve the apparent discrepancy, activation is now considered to involve binding of PS monomers to interacting sites on the enzyme, a non-activating PS trapping process also occurring in the presence of Triton X-100. Estimates for trapping are made for several sets of published data for micellar activation. The kinetic model developed here successfully fits each data set using a Hill-coefficient of only 3.0. An influence of Ca2+/ions or of a two-step mechanism of lipid-protein interaction are considered as possible molecular explanations. It is concluded (i) that lipid activation of protein kinase C may proceed without unique cooperativity and (ii) that ligand trapping could provide another means for 'threshold-type' kinetic regulation of membrane enzyme and receptor systems. AU - Sandermann, H.J. AU - Duncan, T.M. C1 - 34136 C2 - 40164 SP - 235-240 TI - Lipid-dependent membrane enzymes. Kinetic modelling of the activation of protein kinase C by phosphatidylserine. JO - Biochim. Biophys. Acta-Biomembr. VL - 1069 IS - 2 PY - 1991 SN - 0005-2736 ER - TY - JOUR AB - Fusion of nuclei was studied in electrofused cells using staining procedures and DNA flow cytometry. Homogeneous and heterogeneous electrofusion of Ehrlich ascites tumor cells. Muntjac cells and V79-S181 cells were performed in balanced-salt solutions at low temperature. Incubation of the cells subjected to electrofusion in fusion media for about 2 h was required to complete cell fusion and, in particular, nuclear membrane fusion. Under optimum electrofusion conditions it was found that fusion of nuclei is a very frequent event. Half of the fused cells (about 30 to 50% of the field-exposed cells) underwent nuclear membrane fusion. It is shown that the high frequency of nuclear membrane fusion in electrofused, unsynchronised cells resulted from intracellular dielectrophoresis occurring during cell alignment. In accordance with theory, maximum nuclear membrane fusion was observed using alignment fields of between 1 and 4 MHz (depending on the cell species), that is above the frequencies at which the plasmalemma capacity no longer shielded the cell interior from participation in the conduction process. In this frequency range a potential difference can be built up across the nuclear membrane leading to repositioning of the nuclei into the contact zone of the plasmalemmas of two attached cells. This intracellular dielectrophoresis apparently facilitated fusion of nuclei once intermingling of the plasma membranes had occurred. It was further demonstrated that exponentially growing cells showed higher cell fusion rates than cells taken from the unfed plateau phase. One, but not the only reason, might be the higher ATP content of exponentially growing cells compared to cells of the plateau phase. Addition of external ATP to plateau phase cells during electrofusion resulted, in accordance with this assumption, in an increase of fusion frequency, whereas ATP had apparently no effect on the fusion yield of exponentially growing cells. G1 cells obtained by mitotic selection after nocodazole-induced blockage in metaphase also showed higher cellular and nuclear membrane fusion yields than exponentially growing cells. Most importantly, it could be demonstrated both experimentally and theoretically that electrofusion of cells in a dielectrophoretically aligned chain is controlled by a simple law of probability resulting predominantly in fusion of two cells independent of the number of cells in the chain. The likelihood of fusion of various numbers of cells in a chain is given by the appropriate power of the probability of two-cell fusion. These experimental and theoretical findings could explain why electrofusion leads to a high number of hybrids despite the fact that cell chains normally consist of more than two cells. AU - Bertsche, U. AU - Mader, A.C.L.* AU - Zimmermann, U.Z.* C1 - 33864 C2 - 36193 SP - 509-522 TI - Nuclear membrane fusion in electrofused mammalian cells. JO - Biochim. Biophys. Acta-Biomembr. VL - 939 IS - 3 PY - 1988 SN - 0005-2736 ER - TY - JOUR AB - The resting membrane potential, E(m), and the cell input resistance R(i)n(p), of cultured human Chang liver cells were measured using the single electrode 'double-pulse' current clamp technique, following exposure of the cells to the insecticide DDT (20 μM). In control (unexposed) cells, the mean E(m) was -24 mV, and the mean R(inp) was 30 MΩ. Neither parameter was significantly impaired after 1 h of cell exposure to DDT. But after 7 and 48 h, the E(m) was depolarized by 15 and 25 mV, respectively, in parallel with a decrease of the cell input resistance. The strongly time-delayed effect of DDT on Chang liver cell membranes may indicate a mode of interaction different from excitable membranes. AU - Schefczik, K. AU - Buff, K. C1 - 41610 C2 - 38494 SP - 337-339 TI - The insecticide DDT decreases membrane potential and cell input resistance of cultured human liver cells. JO - Biochim. Biophys. Acta-Biomembr. VL - 776 IS - 2 PY - 1984 SN - 0005-2736 ER -