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BBA - Biomembranes (v.1713, #2)

Editorial Board (pp. ii).

Interaction of a pseudosubstrate peptide of protein kinase C and its myristoylated form with lipid vesicles: Only the myristoylated form translocates into the lipid bilayer by Avaronnan Harishchandran; Ramakrishnan Nagaraj (pp. 73-82).

Lipopeptides derived from protein kinase C (PKC) pseudosubstrates have the ability to cross the plasma membrane in cells and modulate the activity of PKC in the cytoplasm. Myristoylation or palmitoylation appears to promote translocation across membranes, as the non-acylated peptides are membrane impermeant. We have investigated, by fluorescence spectroscopy, how myristoylation modulates the interaction of the PKC pseudosubstrate peptide KSIYRRGARRWRKL with lipid vesicles and translocation across the lipid bilayer. Our results indicate that myristoylated peptides are intimately associated with lipid vesicles and are not peripherally bound. When visualized under a microscope, myristoylation does appear to facilitate translocation across the lipid bilayer in multilamellar lipid vesicles. Translocation does not involve large-scale destabilization of the bilayer structure. Myristoylation promotes translocation into the hydrophobic interior of the lipid bilayer even when the non-acylated peptide has only weak affinity for membranes and is also only peripherally associated with lipid vesicles.

Keywords: Abbreviations; CD; circular dichroism; CF; 5-(and6-)-carboxyfluorescein; DNS; 5-(dimethylamino) naphthalene-1-sulfonyl); DNS PE; (5-(dimethylamino) naphthalene-1-sulfonyl)-; l; -α-phosphatidylethanolamine (Egg); GUV; giant unilamellar vesicles; FPLC; fast performance liquid chromatography; FRET; fluorescence resonance energy transfer; HEPES; N; -(2-hydroxyethyl) piperazine-; N′; -(2-ethanesulfonic acid); LUV; large unilamellar vesicles; MLV; multilamellar vesicles; PC; 1-palmitoyl-2-oleoyl-phosphatidylcholine; PG; 1-palmitoyl-2-oleoyl-phosphatidylglycerol; PKC; protein kinase C; SUV; small unilamellar vescles; TFA; trifluoroacetic acidFatty acylated peptide; Lipid vesicle; Fluorescence spectroscopy; Translocation across membrane; Membrane binding; Membrane perturbation

Observation of the main phase transition of dinervonoylphosphocholine giant liposomes by fluorescence microscopy by Antti J. Metso; Hongxia Zhao; Ilkka Tuunainen; Paavo K.J. Kinnunen (pp. 83-91).

The phase heterogeneity of giant unilamellar dinervonoylphosphocholine (DNPC) vesicles in the course of the main phase transition was investigated by confocal fluorescence microscopy observing the fluorescence from the membrane incorporated lipid analog, 1-palmitoyl-2-( N-4-nitrobenz-2-oxa-1,3-diazol)aminocaproyl- sn-glycero-3-phosphocholine (NBDPC). These data were supplemented by differential scanning calorimetry (DSC) of DNPC large unilamellar vesicles (LUV, diameter ∼0.1 and 0.2 μm) and multilamellar vesicles (MLV). The present data collected upon cooling reveal a lack of micron-scale gel and fluid phase coexistence in DNPC GUVs above the temperature of 20.5 °C, this temperature corresponding closely to the heat capacity maxima ( T_em) of DNPC MLVs and LUVs ( T_em ≈21 °C), measured upon DSC cooling scans. This is in keeping with the model for phospholipid main transition inferred from our previous fluorescence spectroscopy data for DMPC, DPPC, and DNPC LUVs. More specifically, the current experiments provide further support for the phospholipid main transition involving a first-order process, with the characteristic two-phase coexistence converting into an intermediate phase in the proximity of T_em. This at least macroscopically homogenous intermediate phase would then transform into the liquid crystalline state by a second-order process, with further increase in acyl chain trans→ gauche isomerization.

Keywords: Abbreviations; C; _p; excess heat capacity; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DNPC; 1,2-dinervonoyl-; sn; -glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; GUV; giant unilamellar vesicle; Δ; H; enthalpy change; LUV; large unilamellar vesicle; MLV; multilamellar vesicle; NBDPC; 1-palmitoyl-2-(; N; -4-nitrobenz-2-oxa-1,3-diazol)aminocaproyl-; sn; -glycero-3-phosphocholine; PE; phosphatidylethanolamine; PC; phosphatidylcholine; PPDPC; 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-; sn; -glycero-3-phosphocholine; T; temperature; T; _em; heat capacity maximum; T; _m; main phase transition temperature (corresponding to 50% of the transition enthalpy); X; _lipid; mole fraction of the indicated lipidGiant liposome; Phase transition

Protein secretion systems in Fusobacterium nucleatum: Genomic identification of Type 4 piliation and complete Type V pathways brings new insight into mechanisms of pathogenesis by Micka�l Desvaux; Arshad Khan; Scott A. Beatson; Anthony Scott-Tucker; Ian R. Henderson (pp. 92-112).

Recent genomic analyses of the two sequenced strains F. nucleatum subsp. nucleatum ATCC 25586 and F. nucleatum subsp. vincentii ATCC 49256 suggested that the major protein secretion systems were absent. However, such a paucity of protein secretion systems is incongruous with F. nucleatum pathogenesis. Moreover, the presence of one or more such systems has been described for every other Gram-negative organism sequenced to date. In this investigation, the question of protein secretion in F. nucleatum was revisited. In the current study, the absence in F. nucleatum of a twin-arginine translocation system (TC #2.A.64.), a Type III secretion system (TC #3.A.6.), a Type IV secretion system (TC #3.A.7.) and a chaperone/usher pathway (TC #1.B.11.) was confirmed. However, contrary to previous findings, our investigations indicated that a Type I protein secretion system was also absent from F. nucleatum. In contrast, members of the holin family (TC #1.E) and the machinery required for a Type 4 piliation/fimbriation system (TC #3.A.15.2.) were identified using a variety of bioinformatic tools. Furthermore, a complete range of proteins resembling members of the Type V secretion pathway, i.e., the Type Va (autotransporter; TC #1.B.12.), Type Vb (two-partner secretion system; TC #1.B.20.) and Type Vc (YadA-like trimeric autotransporter; TC #1.B.42.), was found. This work provides new insight into the protein secretion and virulence mechanisms of F. nucleatum.

Keywords: Protein secretion; Type V secretion pathway; Autotransporter; Two-partner secretion system; YadA-like autotransporter; Fusobacterium; Type 4 pili

Structure-dependent and receptor-independent increase in osmotic fragility of rat erythrocytes by short-chain fatty acids by Hitoshi Mineo; Hiroshi Hara (pp. 113-117).

We examined short-chain fatty acids (SCFAs) with 1 (C1) to 5 (C5) carbon atoms for osmotic fragility (OF) in isolated red blood cells (RBCs) in rats. The RBCs were used as prototypical plasma membrane model. The dense packed RBC was incubated in a phosphate–NaCl buffer solution containing each SCFA at 0 to 100 mM. The RBC suspensions were transferred into the OF test tubes containing NaCl from 0.2 to 0.9%. The hemoglobin concentration was determined and the EC50 in hemolysis was calculated. The OF in RBCs was dose-dependently increased by exposure to SCFAs, except for C1, with an increasing number of carbon atoms. Branched-chain fatty acids (isomers of C4 and C5) have a smaller effect on OF than straight-chain fatty acids (C4 and C5). The SCFA-induced increases in OF were not affected by pretreatment of RBCs with trypsin. The response of the RBC membrane to SCFAs depends on their concentration, carbon chain length and chain structure (straight or branched). The SCFAs probably disturb the lipid bilayer of the RBC membrane and result in a decrease in osmotic resistance. The plasma membrane in rat RBCs could respond to the structure of the SCFAs in detail by using the OF as an indicator.

Keywords: Short-chain fatty acids; Erythrocyte; Membrane; Lipid bilayer; Osmotic fragility; Rat

Interaction of the N-terminal segment of pulmonary surfactant protein SP-C with interfacial phospholipid films by Ines Plasencia; Kevin M.W. Keough; Jesus Perez-Gil (pp. 118-128).

Pulmonary surfactant protein SP-C is a 35-residue polypeptide composed of a hydrophobic transmembrane alpha-helix and a polycationic, palmitoylated-cysteine containing N-terminal segment. This segment is likely the only structural motif the protein projects out of the bilayer in which SP-C is inserted and is therefore a candidate motif to participate in interactions with other bilayers or monolayers. In the present work, we have detected intrinsic ability of a peptide based on the sequence of the N-terminal segment of SP-C to interact and insert spontaneously into preformed zwitterionic or anionic phospholipid monolayers. The peptide expands the π–A compression isotherms of interfacial phospholipid/peptide films, and perturbs the lipid packing of phospholipid films during compression-driven liquid-expanded to liquid-condensed lateral transitions, as observed by epifluorescence microscopy. These results demonstrate that the sequence of the SP-C N-terminal region has intrinsic ability to interact with, insert into, and perturb the structure of zwitterionic and anionic phospholipid films, even in the absence of the palmitic chains attached to this segment in the native protein. This effect has been related with the ability of SP-C to facilitate reinsertion of surface active lipid molecules into the lung interface during respiratory compression–expansion cycling.

Keywords: Pulmonary surfactant; SP-C; Monolayer; Air–liquid interface; Lipid–protein interaction

Coenzyme Q distribution in HL-60 human cells depends on the endomembrane system by Daniel J.M. Fern�ndez-Ayala; Gloria Brea-Calvo; Guillermo L�pez-Lluch; Pl�cido Navas (pp. 129-137).

Coenzyme Q (Q) is an essential factor in the mitochondrial electron chain but also exerts important antioxidant functions in the rest of cell membranes of aerobic organisms. However, the mechanisms of distribution of Q among cell membranes are largely unclear. The aim of the present work is to study the mechanisms of distribution of endogenous Q₁₀ and exogenous Q₉ among cell membranes in human HL-60 cells. Endogenous Q₁₀ synthesized using the radiolabelled precursor [¹⁴C]-pHB was first detected in mitochondria, and it was later incorporated into mitochondria-associated membranes and endoplasmic reticulum (ER). Plasma membrane was the last location to incorporate [¹⁴C]-Q₁₀. Brefeldin A prevented Q₁₀ incorporation in plasma membrane. Exogenous Q₉ was preferably accumulated into the endo-lysosomal fraction but a significant amount was distributed among other cell membranes also depending on the brefeldin-A-sensitive endomembrane system. Our results indicate that mitochondria are the first location for new synthesized Q. Exogenous Q is mainly incorporated into an endo-lysosomal fraction, which is then rapidly incorporated to cell membranes mainly to MAM and mitochondria. We also demonstrate that both endogenous and dietary Q is distributed among endomembranes and plasma membrane by the brefeldin A-sensitive endo-exocytic pathway.

Keywords: Abbreviations; BFA; brefeldin A; Q; coenzyme Q; ubiquinone; COX; cytochrome; c; oxidase; ECD; electrochemical detector; ER; endoplasmic reticulum; ETC; electron transport chain; MAM; mitochondria-associated membrane; pHB; para; -hidroxibenzoic acid; PM; plasma membrane; TPA; 12-; O; -tetradecanoyl phorbol-13-acetateCoenzyme Q; CoQ uptake; Endomembrane; CoQ distribution

Transepithelial transport of artepillin C in intestinal Caco-2 cell monolayers by Yutaka Konishi (pp. 138-144).

The absorption characteristics of artepillin C (AC), an active ingredient of Brazilian propolis, were examined by measuring permeation across Caco-2 cell monolayers. The permeation rate in the basolateral-to-apical direction, Jbl→_ap, in the presence of proton gradient was 0.14 nmol/min/mg protein, whereas Jbl→_ap in the absence of proton gradient was 1.14 nmol/min/mg protein. The latter value is nearly the same as the permeation rate in the apical-to-basolateral direction, Jap→_bl, both in the presence and absence of proton gradient. In the presence of proton gradient, Jap→_bl was almost constant, irrespective of NaN₃ or benzoic acid. However, Jbl→_ap dramatically increased upon the addition of NaN₃ or benzoic acid specifically to the apical side. In both the presence and absence of proton gradient, Jap→_bl also appeared to be constant irrespective of the paracellular permeability of Caco-2 cells. After AC was loaded apically in the presence of proton gradient, the intracellular AC increased with time. This accumulation was inhibited by apically loaded NaN₃. These indicate that AC transport occurs mainly via transcellular passive diffusion, although a considerable amount of AC was taken up intracellularly by monocarboxylic acid transporter (MCT) on the apical side and not transported out across the basolateral membrane, suggesting that different subtypes of MCT are involved.

Keywords: Abbreviations; AC; artepillin C; HBSS; Hanks' balanced salt solution; TER; transepithelial electrical resistance; MCT; monocarboxylic acid transporter; ECD; electrochemical detectorArtepillin C; Monocarboxylic acid transporter; Transcellular passive diffusion; Caco-2

Biomembranes Cumulative Contents (pp. 145-145).

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BBA - Biomembranes (v.1713, #2)