Showing papers by "Andreas Herrmann published in 2009"

The Lipid Modifications of Ras that Sense Membrane Environments and Induce Local Enrichment

Abstract: The transduction of an external stimulus from the outside of a cell into its nucleus is one of the most important mechanisms for the regulation of numerous biological processes. External signals activate receptors that transmit the information across the membrane, where it is transducted by a set of proteins that activate ion channels, phosphokinases, or other downstream effectors. GTP binding proteins that pick up the signal at the receptor, such as heterotrimeric G proteins or Ras, are membrane-associated by post-translationally acquired lipid modifications. These lipid chains provide the hydrophobic free energy for membrane association and their lack releases the proteins to the cytosol, rendering them inactive. Thus, through membrane binding, Ras increases its effective concentration to optimize the interaction both with the receptor and downstream effectors. Ras is an important molecular switch that regulates cell proliferation, differentiation, and growth. The highly specific membrane binding of Ras can be appreciated by comparing the members of the Ras family: Two lipid modifications are required for N-Ras and K-Ras4A, whereas H-Ras carries three lipid chains. In contrast, K-Ras4B requires the concerted action of one lipid chain and favorable electrostatics for membrane binding. Although inserted into the membrane, the lipid modifications experience a high degree of motional freedom that is also transmitted to the adjacent polypeptide chain. Although the highly homologous Ras proteins interact with the same effectors in vitro, they produce distinctly different output signals in vivo, which suggests that these differences are imparted by the lipid-modified C termini of the proteins, where the homology is very low. Moreover, depending on the nucleotide binding state, the localization of Ras in liquid-crystalline or raft domains of the membrane appears to be regulated. Only active H-Ras*GTP interacts with the respective set of effectors; the non-activated form, H-Ras*GDP, is constrained to rafts, where the signal is not further transmitted. An alternative model suggests that the difference in signaling of the Ras isoforms is imparted from the altered access and residence time in a specific compartment. 10] This model suggests that interactions of Ras and its lipid modifications with rafts or fluid membrane domains determines the membrane localization and the biological function of the molecule, which is investigated herein. H NMR is a useful tool for the investigation of lipid rafts. It is applicable to each component of a lipid mixture, and only requires the synthesis of the relevant molecule with a deuterated chain. First, we investigated the adaptation of the lipid modifications of a N-Ras heptapeptide, which was hexadecylated at Cys181 and Cys186, to the membrane thickness. Four different membranes composed of lipids with varying hydrocarbon chains were chosen to constitute the host membrane. Membrane thicknesses studied by H NMR varied from 21.0 (DLPC) to 38.8 (DPPC/cholesterol 10:6, Table 1). The high cholesterol content leads to condensation of the lipids, which increases their length and abolishes the phase transitions of DPPC such that all lipid mixtures could be studied at 30 8C.

Lipid membranes carrying lipophilic cholesterol-based oligonucleotides--characterization and application on layer-by-layer coated particles.

Abstract: Cholesterol-based lipophilic oligonucleotides incorporated into lipid membranes were studied using solid-state NMR, differential scanning calorimetry, and fluorescence methods. Lipophilic oligonucleotides can be used to build nanotechnological structures on membrane surfaces, taking advantage of the specific Watson−Crick base pairing. We used a cholesteryl-TEG anchor first described by Pfeiffer and Hook (J. Am. Chem. Soc. 2004, 126, 10224−10225). The cholesterol-based anchor molecules were found to incorporate well into lipid membranes without disturbing the bilayer structure and dynamics. In contrast to cholesterol, which is known to induce significant condensation of the membrane lipids, the cholesteryl-TEG anchor does not display this property. When the cholesteryl-TEG moiety was covalently bound to an oligonucleotide, the resulting lipophilic DNA molecules inserted spontaneously into lipid membranes without altering their structure. The duplex formed by two complementary cholesteryl-TEG oligonucleotid...

Functional implications of the influence of ABCA1 on lipid microenvironment at the plasma membrane: a biophysical study

Abstract: The ABCA1 transporter orchestrates cellular lipid homeostasis by promoting the release of cholesterol to plasmatic acceptors. The molecular mechanism is, however, unknown. We report here on the biophysical analysis in living HeLa cells of the ABCA1 lipid microenvironment at the plasma membrane. The modifications of membrane attributes induced by ABCA1 were assessed at both the outer and inner leaflet by monitoring either the lifetime of membrane inserted fluorescent lipid analogues by fluorescence lifetime imaging microscopy (FLIM) or, respectively, the membrane translocation of cationic sensors. Analysis of the partitioning of dedicated probes in plasma membrane blebs vesiculated from these cells allowed visualization of ABCA1 partitioning into the liquid disordered-like phase and corroborated the idea that ABCA1 destabilizes the lipid arrangement at the membrane. Specificity was demonstrated by comparison with cells expressing an inactive transporter. The physiological relevance of these modifications was finally demonstrated by the reduced membrane mobility and function of transferrin receptors under the influence of an active ABCA1. Collectively, these data assess that the control of both transversal and lateral lipid distribution at the membrane is the primary function of ABCA1 and positions the effluxes of cholesterol from cell membranes downstream to the redistribution of the sterol into readily extractable membrane pools.

Controlled Assembly of Vesicle‐Based Nanocontainers on Layer‐by‐Layer Particles via DNA Hybridization

Abstract: Layer-by-layer (LbL) particles represent versatile microstructures for numerous applications such as encapsulation and controlled release of proteins, sensors for biomolecules, and chemical reactors on a micrometer scale. Advantages of these structures are the simple functionalization of the capsule wall, high stability, large size range (from 100 nm to 15mm), monodispersity, tunable permeability, and biodegradability. Successful incorporation of various polymers, and even intact lipid vesicles, into LbL assemblies have been demonstrated. LbL particles carrying nanocontainers with reactants to be released on demand would offer novel attractive applications. LbL particles coated with different types of vesicles containing specific reactants could be used to generate microenvironments, in which distinct reactions at a specific time and place could be triggered.

Myristoylation of the arterivirus E protein: the fatty acid modification is not essential for membrane association but contributes significantly to virus infectivity

Abstract: The envelope of equine arteritis virus (EAV) contains two glycoprotein complexes (GP2b/GP3/GP4 and GP5/M) and the small, non-glycosylated E protein As E is essential for the production of infectious progeny but dispensable for assembly and release of virus-like particles, it probably mediates virus entry into cells, putatively in concert with the GP2b/GP3/GP4 complex The E protein contains a central hydrophobic domain and a conserved potential site for N-terminal myristoylation, a hydrophobic modification usually pivotal for membrane targeting of the modified protein Here, it was shown by radiolabelling that E is myristoylated at glycine-2, both in transfected cells as a fusion protein with yellow fluorescent protein (YFP) and in virus particles Biochemical fractionation revealed that E–YFP with an inactivated acylation site was still completely membrane-bound, indicating that the putative transmembrane domain of E mediates membrane targeting Confocal microscopy showed that both myristoylated and non-myristoylated E–YFP were localized to the endoplasmic reticulum and Golgi complex, the membranes from which EAV buds The presence of a myristoylation inhibitor during replication of EAV, whilst completely blocking E acylation, reduced virus titres by 15 log10 Similarly, a mutant EAV with non-myristoylatable E grew to a titre five- to sevenfold lower than that of the wild-type virus and exhibited a reduced plaque size Western blotting of cell-culture supernatants showed that N and M, the major structural proteins of EAV, are released in similar amounts by cells transfected with wild-type and mutant genomes Thus, E myristoylation is not required for budding of particles and probably has a function during virus entry

Parameters Influencing the Release of Tertiary Alcohols from the Surface of Spherical Dendrimers and Linear Stylomers by Neighbouring-Group-Assisted Hydrolysis of 2-Carbamoylbenzoates

Abstract: The influence of structural and physico-chemical parameters on the release of a volatile tertiary alcohol (2-methyl-1-phenyl-2-propanol) by neighbouring-group-assisted cyclisation of 2-carbamoylbenzoates at neutral pH was investigated by comparing the covalent-bond cleavage from the surface of linear, comblike poly(propylene imine) "stylomers" and their corresponding spherical, globular dendrimers. Determination of the kinetic rate constants for the stepwise intramolecular cyclisation of the 2-carbamoylbenzoate moiety by using HPLC showed that the polarity of the conjugates, and thus their solubility in the aqueous reaction medium, has a stronger influence on the rates of hydrolysis than the size (generation) or shape (linear or spherical) of the macromolecules. Furthermore, structural modifications in close proximity to the release unit, such as the presence of functionalities with catalytic activity, have a strong impact on the release efficiency of the active molecules. An understanding of the physico-chemical parameters determining the local environment of the covalent-bond cleavage site is therefore an important prerequisite to transfer the characteristics of small molecules to larger structures such as oligomers and polymers and thus to design efficient macromolecular conjugates for the controlled delivery of bioactive compounds.

Molecular rods with oligospiroketal backbones as anchors in biological membranes.

Abstract: Getting stuck in: A hydrophobic molecular rod with terminal fluorescent moieties has been synthesized. The insertion of the rod into membranes was investigated and shown to incorporate efficiently into model and biological membranes (see picture; gray C, blue N, red O). Those rods can be used as stable membrane-associated anchors for functionalization of membrane surfaces.

Energetics of the loop-to-helix transition leading to the coiled-coil structure of influenza virus hemagglutinin HA2 subunits.

Abstract: Fusion of influenza virus with the endosomal membrane of the host cell is mediated by the homotrimer-organized glycoprotein hemagglutinin (HA). Its fusion activity is triggered by a low pH-mediated conformational change affecting the structure of the HA1 and HA2 subunits. The HA2 subunits undergo a loop-to-helix transition leading to a coiled-coil structure, a highly conserved motif for many fusion mediating viral proteins. However, experimental studies showed that the HA2 coiled-coil structure is stable at neutral and low pH, implying that there is no direct relationship between low pH and the HA2 loop-to-helix transition. To interpret this observation, we used a computational approach based on the dielectric continuum solvent model to explore the influence of water and pH on the free energy change of the transition. The computations showed that the electrostatic interaction between HA2 fragments and water is the major driving force of the HA2 loop-to-helix transition leading to the coiled-coil structure, as long as the HA1 globular domain covering the HA2 subunits in the nonfusion competent conformation is reorganized and thereby allows water molecules to interact with the whole loop segments of the HA2 subunits. Moreover, we show that the energy released by the loop-to-helix transition may account for those energies required for driving the subsequent steps of membrane fusion. Such a water-driven process may resemble a general mechanism for the formation of the highly conserved coiled-coil motif of enveloped viruses.

Characterization of lipid bilayers adsorbed on spherical LbL-support

Abstract: Structural and dynamic properties of membranes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) on layer-by-layer (LbL) polyelectrolyte coated particles were investigated using solid-state nuclear magnetic resonance (NMR) and fluorescence methods. These spherically supported membranes showed structural, dynamic, and elastic properties similar to free-standing membranes as proved by 31P and 2H NMR. Small differences between behaviour of PC and PS on LbL support due to interaction with the polyelectrolyte were observed. Fluorescence lifetime imaging microscopy (FLIM) using 7-nitro-2-1,3-benzoxadiazol (NBD) labeled PC and PS showed a stronger impact of the outermost polyelectrolyte (PAH) on the fluorescence lifetimes of NBD-PS compared to NBD-PC. Although small defects in nm range allowing passage of Mn2+ to both layers of the membrane coat were present, a rather homogeneous coating observed by fluorescence microscopy, complete fluorescence recovery after photobleaching, and NMR results reveal that somewhat continuous lipid bilayers were formed around the LbL particles.

Molekulare Stäbe mit Oligospiroketal‐Rückgrat als Anker in Biomembranen

Abstract: Anker im Nanomas-Stab: Ein hydrophober molekularer Stab mit terminalen fluoreszierenden Gruppen wurde synthetisiert und lies sich effizient in Modellmembranen und biologische Membranen einbauen (siehe Bild; grau C, blau N, rot O). Derartige Stabe konnen als stabile membranassoziierte Anker fur die Funktionalisierung von Membranoberflachen eingesetzt werden.

Methods and materials for the reproducible generation of high producer cell lines for recombinant proteins

Abstract: The invention lies in the field of production of recombinant gene products in eukaryotic cells. The invention refers to methods and materials for the fast and reproducible generation of production cells lines suitable for large scale production of recombinant gene products. The invention encompasses specific vector systems, genetic engineered host-cells and methods of use.