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BBA - Biomembranes (v.1714, #1)
A spectroscopic study of the membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39) by A. James Mason; Jakob J. Lopez; Michael Beyermann; Clemens Glaubitz (pp. 1-10).
The membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39), which selectively activates the parathyroid hormone 2 (PTH2) receptor (PTH2-R), has been studied by fluorescence and NMR spectroscopic techniques. Membrane binding would be the first step of a potential membrane-bound activation pathway which has been discussed for a number of neuropeptides and G-protein coupled receptors (GPCRs). Here, the orientation of TIP39 on the surface of membrane mimicking dodecyl-phosphocholine (DPC) micelles was monitored by Photo-CIDNP (chemically-induced dynamic nuclear polarization) NMR which indicates that both Trp25 and Tyr29 face the membrane surface. However, the PTH2 receptor is located in the hypothalamus membrane, for which a more realistic model is required. Therefore, liposomes containing different mixtures of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphatidylserine (POPS) and cholesterol were used for fluorescence and solid-state NMR spectroscopy. Fluorescence spectroscopy showed that a large proportion of TIP39 added to these liposomes binds to the membrane surface. Proton-decoupled31P-MAS NMR is used to investigate the potential role of individual lipid headgroups in peptide binding. Significant line-broadening in POPC/cholesterol and POPC/POPS liposomes upon TIP39 association supports a surface binding model and indicates an interaction which is slightly mediated by the presence of POPS and cholesterol. Furthermore, smoothed order parameter profiles obtained from2H powder spectra of liposomes containing POPC-d31 as bulk lipid in addition to POPS and cholesterol show that TIP39 does not penetrate beyond the headgroup region. Spectra of similar bilayers with POPS-d31 show a small increase in segmental chain order parameters which is interpreted as a small but specific interaction between the peptide and POPS. Our data demonstrate that TIP39 belongs to a class of signaling peptides that associate weakly with the membrane surface but do not proceed to insert into the membrane hydrophobic compartment.
Keywords: GPCR; PTH-2 receptor; TIP39; Solid-State NMR; Peptide–lipid interaction; Photo-CIDNP
Charge patch attraction and reentrant condensation in DNA–liposome complexes by S. Sennato; F. Bordi; C. Cametti; M. Diociaiuti; P. Malaspina (pp. 11-24).
We investigated the formation of complexes between cationic liposomes built up by DOTAP and three linear anionic polyions, with different charge density and flexibility, such as a single-stranded ssDNA, a double-stranded dsDNA and the polyacrylate sodium salt [NaPAA] of three different molecular weights. Our aim is to gain further insight into the formation mechanism of polyion–liposome aggregates of different sizes (lipoplexes), by comparing the behavior of DNA with a model polyelectrolyte, such as NaPAA, with approximately the same charge density but with a higher flexibility. We employed dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements, in order to explore both the hydrodynamic and structural properties of the aggregates resulting from polyion–liposome interaction and to present a comprehensive picture of the complexation process. The phenomenology can be summarized in a charge ratio-dependent scenario, where the main feature is the formation of large equilibrium clusters due to the aggregation of intact polyion-coated vesicles. At increasing polyion–liposome ratio, the size of the clusters continuously increases, reaching a maximum at a well-defined value of this ratio, and then decreases (“reentrant� condensation). The aggregation mechanism and the role of the polyion charge density in the complex formation are discussed in the light of the recent theories on the correlated adsorption of polyelectrolytes at charged interfaces. Within this framework, the phenomena of charge inversion and the reentrant condensation, peaked at the isoelectric point, finds a simple explanation.
Keywords: DNA; Lipoplexes; Charge inversion; Reentrant condensation
Regulation of erythrocyte Na–K–2Cl cotransport by threonine phosphorylation by Ioulia Matskevich; Karen L. Hegney; Peter W. Flatman (pp. 25-34).
A method is described to measure threonine phosphorylation of the Na–K–2Cl cotransporter in ferret erythrocytes using readily available antibodies. We show that most, if not all, cotransporter in these cells is NKCC1, and this was immunoprecipitated with T4. Cotransport rate, measured as86Rb influx, correlates well with threonine phosphorylation of T4-immunoprecipitated protein. The cotransporter effects large fluxes and is significantly phosphorylated in cells under control conditions. Transport and phosphorylation increase 2.5- to 3-fold when cells are treated with calyculin A or Na+ arsenite. Both fall to 60% control when cell [Mg2+] is reduced below micromolar or when cells are treated with the kinase inhibitors, 4-amino-5-(4-methylphenyl)-7-( t-butyl)pyrazolo[3,4-d]pyrimidine or staurosporine. Importantly, these latter interventions do not abolish either phosphorylation or transport suggesting that a phosphorylated form of the cotransporter is responsible for residual fluxes. Our experiments suggest protein phosphatase 1 (PrP-1) is extremely active in these cells and dephosphorylates key regulatory threonine residues on the cotransporter. Examination of the effects of kinase inhibition after cells have been treated with high concentrations of calyculin indicates that residual PrP-1 activity is capable of rapidly dephosphorylating the cotransporter. Experiments on cotransporter precipitation with microcystin sepharose suggest that PrP-1 binds to a phosphorylated form of the cotransporter.
Keywords: Sodium-potassium-chloride symporters; Erythrocytes; Threonine phosphorylation; Protein phosphatase 1; Calyculin A; Arsenite; PP1
Cholesterol modulates the antagonist-binding function of hippocampal serotonin1A receptors by Thomas J. Pucadyil; Amitabha Chattopadhyay (pp. 35-42).
The serotonin1A receptor is the most extensively studied member of the family of seven transmembrane domain G-protein coupled serotonin receptors. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions such as sleep, mood, pain, addiction, locomotion, sexual activity, depression, anxiety, alcohol abuse, aggression and learning. Since a significant portion of the protein lies embedded in the membrane and the ligand-binding pocket is defined by the transmembrane stretches in such receptors, membrane composition and organization represent a crucial parameter in the structure–function analysis of G-protein coupled receptors. In this paper, we have monitored the role of membrane cholesterol in the ligand-binding function of the hippocampal serotonin1A receptor. Our results demonstrate that the reduction of membrane cholesterol significantly attenuates the antagonist-binding function of the serotonin1A receptor. Based on prior pharmacological knowledge regarding the requirements for the antagonist to bind the receptor, our results indicate that membrane cholesterol modulates receptor function independently of its ability to interact with G-proteins. These effects on ligand-binding function of the receptor are predominantly reversed upon cholesterol-replenishment of cholesterol-depleted membranes. When viewed in the light of our earlier results on the effect of cholesterol depletion on the serotonin1A receptor/G-protein interaction, these results comprehensively demonstrate the importance of cholesterol in the serotonin1A receptor function and form the basis for understanding lipid–protein interactions involving this important neuronal receptor.
Keywords: Abbreviations; BCA; bicinchoninic acid; DMPC; dimyristoyl-; sn; -glycero-3-phosphocholine; 5-HT; 5-hydroxytryptamine; MβCD; methyl-β-cyclodextrin; p; -MPPF; 4-(2′-methoxy)-phenyl-1-[2′-(; N; -2ʺ-pyridinyl)-; p; -fluorobenzamido]ethyl-piperazine; p; -MPPI; 4-(2′-methoxy)-phenyl-1-[2′-(; N; -2ʺ-pyridinyl)-; p; -iodobenzamido]ethyl-piperazine; PMSF; phenylmethylsulfonyl fluoride5-HT; 1A; receptor; Hippocampus; 5-HT; 1A; receptor antagonist; Cholesterol; Methyl-β-cyclodextrin
Monitoring the organization and dynamics of bovine hippocampal membranes utilizing Laurdan generalized polarization by Soumi Mukherjee; Amitabha Chattopadhyay (pp. 43-55).
Organization and dynamics of cellular membranes in the nervous system are crucial for the function of neuronal membrane receptors. The lipid composition of neuronal cells is unique and has been correlated with the increased complexity in the organization of the nervous system during evolution. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors such as the G-protein coupled serotonin1A receptor. In this paper, we have explored the organization and dynamics of bovine hippocampal membranes using the amphiphilic environment-sensitive fluorescent probe Laurdan. Our results show that the emission spectra of Laurdan display an additional red shifted peak as a function of increasing temperature in native as well as cholesterol-depleted membranes and liposomes made from lipid extracts of the native membrane. Interestingly, wavelength dependence of Laurdan generalized polarization (GP) in native membranes indicates the presence of an ordered gel-like phase at low temperatures, whereas characteristics of the liquid-ordered phase are observed at high temperatures. Similar experiments performed using cholesterol-depleted membranes show fluidization of the membrane with increasing cholesterol depletion. In addition, results from fluorescence polarization of DPH indicate that the hippocampal membrane is fairly ordered even at physiological temperature. The temperature dependence of Laurdan excitation GP provides a measure of the apparent thermal transition temperature and extent of cooperativity in these membranes. Analysis of time-resolved fluorescence measurements of Laurdan shows reduction in mean fluorescence lifetime with increasing temperature due to change in environmental polarity. These results constitute novel information on the dynamics of hippocampal membranes and its modulation by cholesterol depletion monitored using Laurdan fluorescence.
Keywords: Abbreviations; Laurdan; 6-dodecanoyl-2-(; N,N; -dimethylamino)naphthalene; BCA; bicinchoninic acid; DPH; 1,6-diphenyl-1,3,5-hexatriene; MβCD; methyl-β-cyclodextrin; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; PMSF; phenylmethylsulfonyl fluorideBovine hippocampal membranes; Cholesterol; Laurdan generalized polarization; Temperature
Calmodulin-dependent protein kinases phosphorylate gp130 at the serine-based dileucine internalization motif by Robin M. Gibson; George S. Laszlo; Neil M. Nathanson (pp. 56-62).
The receptor for leukemia inhibitory factor (LIF) consists of two polypeptides, the low affinity LIF receptor (LIFR) and gp130. We previously demonstrated that LIF stimulation caused phosphorylation of gp130 at Ser782, adjacent to a dileucine internalization motif, and that transient expression of a mutant receptor lacking Ser782 resulted in increased cell surface expression and increased LIF-stimulated gene expression compared to wild-type receptor. Phosphorylation of Ser782 on gp130 fusion protein by LIF-stimulated 3T3-L1 cell extracts was inhibited 61% by autocamtide-2-related inhibitory peptide (AIP), a highly specific and highly effective inhibitor of calmodulin-dependent protein kinase type II (CaMKII). Purified rat forebrain CaMKII was also able to phosphorylate gp130 fusion protein at Ser782 in vitro. Furthermore, antibodies targeting CaMKII and CaMKIV were able to immunoprecipitate gp130 phosphorylating activity from LIF-stimulated 3T3-L1 lysates. While pretreatment of cells with the MAPKK inhibitors PD98059 and U0126 blocked phosphorylation of Ser782 prior to LIF stimulation, these inhibitors did not block Ser782 phosphorylation by LIF-stimulated 3T3-L1 cell extracts in vitro. These results show that CaMKII and possibly CaMKIV phosphorylate Ser782 in the serine-based dileucine internalization motif of gp130 via a MAPK-dependent pathway.
Keywords: gp130; LIF; CaMKII; Serine-based internalization motif; Granulocyte colony stimulating factor; Phosphorylation
Lipid transfer between cationic vesicles and lipid–DNA lipoplexes: Effect of serum by Rumiana Koynova; Robert C. MacDonald (pp. 63-70).
Differential scanning calorimetry was used to examine the lipid exchange between model lipid systems, including vesicles of the cationic lipoids ethyldimyristoylphosphatidylcholine (EDMPC), ethyldipalmitoylphosphatidylcholine (EDPPC) or their complexes with DNA (lipoplexes), and the zwitterionic lipids (DMPC, DPPC). The changes of the lipid phase transition parameters (temperature, enthalpy, and cooperativity) upon consecutive temperature scans was used as an indication of lipid mixing between aggregates. A selective lipid transfer of the shorter-chain cationic lipoid EDMPC into the longer-chain aggregates was inferred. In contrast, transfer was hindered when EDMPC (but not EDPPC) was bound to DNA in the lipoplexes. These data support a simple molecular lipid exchange mechanism, but not lipid bilayer fusion. Exchange via lipid monomers is considerably more facile for the cationic ethylphosphatidylcholines than for zwitterionic phosphatidylcholines, presumably due to the higher monomer solubility of the charged lipids. With the cationic liposomes, lipid transfer was strongly promoted by the presence of serum in the dispersing medium. Serum proteins are presumed to be responsible for the accelerated transfer, since the effect was strongly reduced upon heating the serum to 80 °C. The effect of serum indicates that even though much lipoplex lipid is inaccessible due to the multilayered structure, the barrier due to buried lipid can be easily overcome. Serum did not noticeably promote the lipid exchange of zwitterionic liposomes. The phenomenon is of potential importance for the application of cationic liposomes to nonviral gene delivery, which often involves the presence of serum in vitro, and necessarily involves serum contact in vivo.
Keywords: Liposome; Lipid exchange; cmc; Monomer; Cationic lipid; Serum