Transmembrane Polypeptide Research Articles

Overview of organic anion transporters and organic anion transporter polypeptides and their roles in the liver.

Organic anion transporters (OATs) and organic anion transporter polypeptides (OATPs) are classified within two SLC superfamilies, namely, the SLC22A superfamily and the SLCO superfamily (formerly the SLC21A family), respectively. They are expressed in many tissues, such as the liver and kidney, and mediate the absorption and excretion of many endogenous and exogenous substances, including various drugs. Most are composed of 12 transmembrane polypeptide chains with the C-terminus and the N-terminus located in the cell cytoplasm. OATs and OATPs are abundantly expressed in the liver, where they mainly promote the uptake of various endogenous substrates such as bile acids and various exogenous drugs such as antifibrotic and anticancer drugs. However, differences in the locations of glycosylation sites, phosphorylation sites, and amino acids in the OAT and OATP structures lead to different substrates being transported to the liver, which ultimately results in their different roles in the liver. To date, few articles have addressed these aspects of OAT and OATP structures, and we study further the similarities and differences in their structures, tissue distribution, substrates, and roles in liver diseases.

Tuning the Photophysical Features of Self-Assembling Photoactive Polypeptides for Light-Harvesting

The LH1 complex is the major light-harvesting antenna of purple photosynthetic bacteria. Its role is to capture photons, and then store them and transfer the excitation energy to the photosynthetic reaction center. The structure of LH1 is modular and it cooperatively self-assembles from the subunits composed of short transmembrane polypeptides that reversibly bind the photoactive cofactors: bacteriochlorophyll and carotenoid. LH1 assembly, the intra-complex interactions and the light-harvesting features of LH1 can be controlled in micellar media by varying the surfactant concentration and by adding carotenoid and/or a co-solvent. By exploiting this approach, we can manipulate the size of the assembly, the intensity of light absorption, and the energy and lifetime of its first excited singlet state. For instance, via the introduction of Ni-substituted bacteriochlorophyll into LH1, the lifetime of this electronic state of the antenna can be shortened by almost three orders of magnitude. On the other hand, via the exchange of carotenoid, light absorption in the visible range can be tuned. These results show how in a relatively simple self-assembling pigment-polypeptide system a sophisticated functional tuning can be achieved and thus they provide guidelines for the construction of bio-inspired photoactive nanodevices.

Solid-State NMR Investigations of the MHC II Transmembrane Domains: Topological Equilibria and Lipid Interactions

The major histocompatibility complex class II (MHC II) membrane proteins are key players in the adaptive immune response. An aberrant function of these molecules is associated with a large number of autoimmune diseases such as diabetes type I and chronic inflammatory diseases. The MHC class II is assembled from DQ alpha 1 and DQ beta 1 which come together as a heterodimer through GXXXG-mediated protein-protein interactions and a highly specific protein-sphingomyelin-C18 interaction motif located on DQA1. This association can have important consequences in regulating the function of these membrane proteins. Here, we investigated the structure and topology of the DQA1 and DQB1 transmembrane helical domains by CD-, oriented 2H and 15N solid-state NMR spectroscopies. The spectra at peptide-to-lipid ratios of 0.5 to 2mol% are indicative of a topological equilibrium involving a helix crossing the membrane with a tilt angle of about 20° and another transmembrane topology with around 30° tilt. The latter is probably representing a dimer. Furthermore, at the lowest peptide-to-lipid ratio, a third polypeptide population becomes obvious. Interestingly, the DQB1 and to a lesser extent the DQA1 transmembrane helical domains exhibit a strong fatty acyl chain disordering effect on the inner segments of the 2H-labelled palmitoyl chain of POPC bilayers. This phosphatidylcholine disordering requires the presence of sphingomyelin-C18 suggesting that the ensemble of transmembrane polypeptide and sphingolipid exerts positive curvature strain.

Bacteria-Based Production of Thiol-Clickable, Genetically Encoded Lipid Nanovesicles.

Despite growing research efforts on the preparation of (bio)functional liposomes, synthetic capsules cannot reach the densities of protein loading and the control over peptide display that is achieved by natural vesicles. Herein, a microbial platform for high-yield production of lipidic nanovesicles with clickable thiol moieties in their outer corona is reported. These nanovesicles show low size dispersity, are decorated with a dense, perfectly oriented, and customizable corona of transmembrane polypeptides. Furthermore, this approach enables encapsulation of soluble proteins into the nanovesicles. Due to the mild preparation and loading conditions (absence of organic solvents, pH gradients, or detergents) and their straightforward surface functionalization, which takes advantage of the diversity of commercially available maleimide derivatives, bacteria-based proteoliposomes are an attractive eco-friendly alternative that can outperform currently used liposomes.

Bacteria‐Based Production of Thiol‐Clickable, Genetically Encoded Lipid Nanovesicles

AbstractDespite growing research efforts on the preparation of (bio)functional liposomes, synthetic capsules cannot reach the densities of protein loading and the control over peptide display that is achieved by natural vesicles. Herein, a microbial platform for high‐yield production of lipidic nanovesicles with clickable thiol moieties in their outer corona is reported. These nanovesicles show low size dispersity, are decorated with a dense, perfectly oriented, and customizable corona of transmembrane polypeptides. Furthermore, this approach enables encapsulation of soluble proteins into the nanovesicles. Due to the mild preparation and loading conditions (absence of organic solvents, pH gradients, or detergents) and their straightforward surface functionalization, which takes advantage of the diversity of commercially available maleimide derivatives, bacteria‐based proteoliposomes are an attractive eco‐friendly alternative that can outperform currently used liposomes.

37P - Pooling signaling and costimulatory domains in a flexible CARpool design

The present application relates to the field of immunotherapy, more particularly to the field of chimeric antigen receptors (CARs). Currently, second and third generation CAR designs are quite rigid in that they combine fixed costimulatory domains in cis on the same intracellular protein domain. Trans signaling is not equivalent as costimulatory receptors have different expression levels or stoichiometry. Here, a ‘mix and match’ approach is proposed where different signaling and costimulatory domains are present on separate chains within the same CAR complex, allowing increased flexibility and control of the nature and strength of the CAR-generated signal. Also proposed are polynucleotides, vectors encoding the transmembrane polypeptide chains and cells expressing such CARs. These cells are particularly suitable for use in immunotherapy, and strategies to treat diseases such as cancer using these cells are also provided.

Overexpression of PMA1 enhances tolerance to various types of stress and constitutively activates the SAPK pathways in Saccharomyces cerevisiae.

PMA1 encodes a transmembrane polypeptide that functions to pump protons out of the cell. Ectopic PMA1 overexpression in Saccharomyces cerevisiae enhances tolerance to weak acids, reactive oxygen species (ROS) and ethanol, and changes the following physiological properties: better proton efflux, lower membrane permeability, and lessened internal hydrogen peroxide production. The enhanced stress tolerance was dependent on the mitogen-activated protein kinase (MAPK) Hog1 of the high osmolarity glycerol (HOG) pathway, but not the MAPK Slt2 of the cell wall integrity (CWI) pathway; however, a PMA1 overexpression constitutively activated both Hog1 and Slt2. The constitutive Hog1 activation required the MAPK kinase kinase (MAP3K) Ssk2 of the HOG pathway, but not Ste11 and Ssk22, two other MAP3Ks of the same pathway. The constitutive Slt2 activation did not require Rom2 and the membrane sensors of the CWI pathway, whereas Bck1 was indispensable. The PMA1 overexpression activated the stress response element but not the cyclic AMP response element and the Rlm1 transcription factor. PMA1 overexpression may facilitate the construction of industrial strains with simultaneous tolerance to weak acids, ROS, and ethanol.

The evolution of the AMOA, PMOA and BMOA genes measured by ka/ks ratios.

The copper-containing membrane-bound monooxygenase enzyme family consists of enzymes facilitating the oxidation of methane (catalyzed by particulate methane monooxygenase, pMMO) and ammonia (catalyzed by ammonia monooxygenase, AMO), in both aerobic and anaerobic methanotrophs and nitrifiers, respectively, and the oxidation of C2-10 alkanes (catalyzed by particulate butane monooxygenase, pBMO). Despite catalyzing different substrates, pMMO, AMO and pBMO form catabolic enzymes related structurally and evolutionarily to each other, and are encoded by the genes pmoA, amoA and bmoA, respectively. It has been suggested that oxygen-dependent methane and ammonia monooxygenases evolved from a substrate-promiscuous ancestor after horizontal transfer(s) into new hosts, which eventually became methanotrophs and nitrifiers. However, no extensive studies have been conducted on molecular adaptation of the beta-peptide structure of pmoA, amoA and bmoA gene products, which form transmembrane polypeptides, and on selective pressures acting on them. Over 80 near-complete pmoA, amoA and bmoA gene from database were downloaded and analyzed to assess whether the combined amoA, pmoA and bmoA gene tree is congruent to the 16S rRNA gene tree for these microbes. The gene was separated into 15 segments and pairwise Ka/Ks values were calculated to assess the evolutionary and selection pressure exerted on these taxa, based on the hypothesis that they evolved from a methanotrophic ancestor, as well as calculate Ka/Ks values between members of each family.Our analyses indicated a high level of negative (or purifying) selection between and amongst all taxa. Ka/Ks values were highest between methanotroph pmoA and the anammox, and that the pmoA and amoA genes are under purifying selection their clusters, and supports the hypothesis that one of these genes likely arose from the other via horizontal gene transfer.

African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes.

African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs.

Division of labor among oxidoreductases: TMX1 preferentially acts on transmembrane polypeptides.

The endoplasmic reticulum (ER) is the site of maturation for secretory and membrane proteins in eukaryotic cells. The lumen of the mammalian ER contains >20 members of the protein disulfide isomerase (PDI) superfamily, which ensure formation of the correct set of intramolecular and intermolecular disulfide bonds as crucial, rate-limiting reactions of the protein folding process. Components of the PDI superfamily may also facilitate dislocation of misfolded polypeptides across the ER membrane for ER-associated degradation (ERAD). The reasons for the high redundancy of PDI family members and the substrate features required for preferential engagement of one or the other are poorly understood. Here we show that TMX1, one of the few transmembrane members of the family, forms functional complexes with the ER lectin calnexin and preferentially intervenes during maturation of cysteine-containing, membrane-associated proteins while ignoring the same cysteine-containing ectodomains if not anchored at the ER membrane. As such, TMX1 is the first example of a topology-specific client protein redox catalyst in living cells.

Rhodopsin gene expression regulated by the light dark cycle, light spectrum and light intensity in the dinoflagellate Prorocentrum.

The proton pump rhodopsin is widely found in marine bacteria and archaea, where it functions to capture light energy and convert it to ATP. While found in several lineages of dinoflagellates, this gene has not been studied in Prorocentrales species and whether it functionally tunes to light spectra and intensities as in bacteria remains unclear. Here we identified and characterized this gene in the bloom-forming Prorocentrum donghaiense. It is a 7-helix transmembrane polypeptide containing conserved domains and critical amino acid residues of PPR. This gene is phylogenetically affiliated to the xanthorhodopsin clade, but seems to have a distinct evolutionary origin. Quantitative reverse transcription PCR showed that in regular cultures, the transcript abundance of the gene exhibited a clear diel pattern, high abundance in the light period and low in the dark. The same diel pattern was observed for protein abundance with a Western blot using specific antiserum. The rhythm was dampened when the cultures were shifted to continuous dark or light condition, suggesting that this gene is not under circadian clock control. Rhodopsin transcript and protein abundances varied with light intensity, both being highest at a moderate illumination level. Furthermore, the expression of this gene responded to different light spectra, with slightly higher transcript abundance under green than blue light, and lowest abundance under red light. Transformed Escherichia coli over-expressing this rhodopsin gene also exhibited an absorption maximum in the blue–green region with slightly higher absorption in the green. These rhodopsin-promoting light conditions are similar to the relatively turbid marine habitat where the species forms blooms, suggesting that this gene may function to compensate for the light-limited photosynthesis in the dim environment.

Lipid Composition Influences the Insertion and Folding of pHLIP Peptides

The study of polypeptide insertion into biological membranes can inform our understanding of membrane protein stability and folding, and has potential practical applications. This has been hard to investigate, since peptides hydrophobic enough to form transmembrane helices are likely to be insoluble in aqueous buffers, and soluble peptides are unlikely to insert. pH-Low Insertion Peptides (pHLIPs) provide an opportunity to study insertion since they are soluble monomers that bind to bilayer surfaces at neutral pH, and can be triggered to form monomeric transmembrane helices in acidic conditions.This set of unusual properties allows the study of spontaneous insertion and exit of a transmembrane polypeptide by changing the pH of its microenvironment. Previous studies showed that pHLIP inserts into a POPC liposomes through rapid formation of an interfacial helix (∼ 0.1s), followed by a slow insertion pathway. The time-course of pHLIP insertion can be changed by varying the number of protonatable groups at the inserting end of the peptide, which need to be moved across the bilayer.We have employed various biophysical methods: fluorescence spectroscopy, anisotropy, CD, OCD, and stopped-flow fluorescence, to study the pHLIP insertion into bilayers composed of different monounsaturated lipids (diC(14:1)PC, diC(16:1)PC, diC(18:1)PC, diC(20:1)PC, and diC(22:1)PC). We found that pHLIP can form a TM helix in bilayers of different thickness. The kinetics of pHLIP insertion vary, and correlate with bilayer thickness and fluidity. Further, pHLIP association with bilayer surfaces also depends on the lipid composition. The activation energy of pHLIP insertion increases with the membrane thickness but the process of inserted helix formation does not significantly depend the membrane type. A model for membrane-associated insertion/folding is discussed. This work was supported by grants from National Institute of Health, GM073857, and CA133890.

Dynamics in the Transmembrane Segment of the Influenza a M2 Proton Channel

Influenza A virus, responsible for the flu, infects respiratory tract epithelial cells. M2 is a tetrameric proton channel essential for the virus life cycle. The antiviral drug amantadine (Amt) used to block M2 channel prior to the recent S31N mutation. With the known backbone structure of the M2 transmembrane (TM) domain, polypeptide backbone and side chains dynamics can be characterized.Peptides corresponding to M2TM wild-type (M2TM) and M2TM S31N (S31N_TM) domains were chemically synthesized. M2TM peptides were labeled at: d4-Ala29/15N-Ile42, or d8-Val27/15N-Ala29, or d2-Gly34/15N2-Trp41, or d5-Trp41/15N-Ile39. An S31N_TM peptide was labeled at d8-Val27/15N-Ala29. 2H and 15N static NMR spectra of the peptides reconstituted in DOPC:DOPE bilayer at pH 7.5 were acquired.Analysis of the spectra of M2TM(d4-Ala29) at various temperature and echo times indicates that above the phase transition temperature of the lipids M2TM channel is undergoing rotation motion about the bilayer normal and channel axis on the ms time scale. The spectra of M2TM(d4-Ala29 and d8-Val27) in the absence and presence of Amt indicate that drug binding doesn't affect spectral line shape and is proposed to have no significant influence on backbone and side chain dynamics of these sites. The S31N mutation lead to narrowing of the spectrum of S31N_TM(d8-Val27) in comparison to M2TM(d8-Val27). This mutation was reported to increase the splay between helices which could lead to a less tight packing of Val27 side chains and an increase of side chain dynamics. The collapsed 2H powder patterns of M2TM(d2-Gly34 and d5-Trp41) at 288 K indicates that both sites are undergoing motions on µs/ms time scale. Moreover, at 297 K the collapsed powder pattern of 15Ne-Trp41 indicates that the indole ring is undergoing large amplitude motions, possibly about χ1 and χ2.

Transmembrane protein PERP is a component of tessellate junctions and of other junctional and non-junctional plasma membrane regions in diverse epithelial and epithelium-derived cells

Protein PERP (p53 apoptosis effector related to PMP-22) is a small (21.4 kDa) transmembrane polypeptide with an amino acid sequence indicative of a tetraspanin character. It is enriched in the plasma membrane and apparently contributes to cell-cell contacts. Hitherto, it has been reported to be exclusively a component of desmosomes of some stratified epithelia. However, by using a series of newly generated mono- and polyclonal antibodies, we show that protein PERP is not only present in all kinds of stratified epithelia but also occurs in simple, columnar, complex and transitional epithelia, in various types of squamous metaplasia and epithelium-derived tumors, in diverse epithelium-derived cell cultures and in myocardial tissue. Immunofluorescence and immunoelectron microscopy allow us to localize PERP predominantly in small intradesmosomal locations and in variously sized, junction-like peri- and interdesmosomal regions (“tessellate junctions”), mostly in mosaic or amalgamated combinations with other molecules believed, to date, to be exclusive components of tight and adherens junctions. In the heart, PERP is a major component of the composite junctions of the intercalated disks connecting cardiomyocytes. Finally, protein PERP is a cobblestone-like general component of special plasma membrane regions such as the bile canaliculi of liver and subapical-to-lateral zones of diverse columnar epithelia and upper urothelial cell layers. We discuss possible organizational and architectonic functions of protein PERP and its potential value as an immunohistochemical diagnostic marker.

Emission spectra of LH2 complex: full Hamiltonian model

In the present contribution we study the absorption and steady-state fluorescence spectra for ring molecular system, which can model B850 ring of peripheral light-harvesting complex LH2 from purple bacterium Rhodopseudomonas acidophila (Rhodoblastus acidophilus). LH2 is a highly symmetric ring of nine pigment-protein subunits, each containing two transmembrane polypeptide helixes and three bacteriochlorophylls (BChl). The uncorrelated diagonal static disorder with Gaussian distribution (fluctuations of local excitation energies) simultaneously with the diagonal dynamic disorder (interaction with a bath) in Markovian approximation is used in our simulations. We compare calculated absorption and steady state fluorescence spectra obtained within the full Hamiltonian model of the B850 ring with our previous results calculated within the nearest neighbour approximation model and also with experimental data.

In silico partitioning and transmembrane insertion of hydrophobic peptides under equilibrium conditions.

Nascent transmembrane (TM) polypeptide segments are recognized and inserted into the lipid bilayer by the cellular translocon machinery. The recognition rules, described by a biological hydrophobicity scale, correlate strongly with physical hydrophobicity scales that describe the free energy of insertion of TM helices from water. However, the exact relationship between the physical and biological scales is unknown, because solubility problems limit our ability to measure experimentally the direct partitioning of hydrophobic peptides across lipid membranes. Here we use microsecond molecular dynamics (MD) simulations in which monomeric polyleucine segments of different lengths are allowed to partition spontaneously into and out of lipid bilayers. This approach directly reveals all states populated at equilibrium. For the hydrophobic peptides studied here, only surface-bound and transmembrane-inserted helices are found. The free energy of insertion is directly obtained from the relative occupancy of these states. A water-soluble state was not observed, consistent with the general insolubility of hydrophobic peptides. The approach further allows determination of the partitioning pathways and kinetics. Surprisingly, the transfer free energy appears to be independent of temperature, which implies that surface-to-bilayer peptide insertion is a zero-entropy process. We find that the partitioning free energy of the polyleucine segments correlates strongly with values from translocon experiments but reveals a systematic shift favoring shorter peptides, suggesting that translocon-to-bilayer partitioning is not equivalent but related to spontaneous surface-to-bilayer partitioning.

The inhibitory effect of polypeptide cSN50 on alcoholic hepatic injuries through blocking the binding of NF-κB to importin α

Objective. To study whether alcohol and endotoxin induce inflammation, apoptosis, and expression of downstream genes TNF-α and caspase-3 through the nuclear transcription factor NF-κB, i.e., the IκB–NF-κB–importin α pathway. Methods. Flow cytometry was used to observe the apoptosis rate in human hepatoma cells (HepG2) and murine macrophages (RAW264.7) after being stimulated by alcohol and endotoxin at different concentrations. The caspase-3 activity was determined by spectrophotometry and TNF-α level in cell culture supernatant by ELISA. Western blot was used to examine the expression level of P50, importin α3 and IκBα, and indirect immunofluorescence to determine the activation level of P50, importin α3 and IκBα. Results. In human hepatoma cells, a transmembrane polypeptide, cSN50, inhibited the elevation in the level of TNF-α and caspase-3 and the expression of nuclear transport receptor importin α3 stimulated by alcohol and endotoxin. Immunofluorescence showed the nuclear translocation of NF-κB and importin α3 and cytosolic degradation of IκBα. In murine mononuclear macrophages, addition of alcohol or endotoxin or both resulted in a significant elevation of NF-κB and its downstream factors TNF-α and caspase-3 and apoptosis of RAW264.7 cells. These effects were remarkably suppressed by cSN50. However, the expression of importin α3 was not detected by Western blotting or immunofluorescence in this experiment, indicating the existence of other pathways. Conclusion. The nuclear translocation of NF-κB plays an important role in acute alcoholic hepatic injuries and the induced nuclear NF-κB activity, and its downstream gene expression can be partially suppressed by cSN50 in HepG2 and RAW264.7 cells.

Elevated Cytosolic Cyclic AMP Inhibits Platelet Adhesion to Von Willebrand Factor by Disengaging the Glycoprotein Ib-IX-V Complex From Lipid Rafts

AbstractAbstract 3204A high pressure circulatory system has two diametrically opposed requirements for its function: it must be able to rapidly gel to prevent blood loss when the integrity of the vasculature is compromised while simultaneously maintaining fluidity when the vasculature is intact. The endothelium is primarily responsible for maintaining blood fluidity, producing rapidly acting labile substances that inhibit both the clotting of blood and the adhesion and aggregation of platelets. Among these substances are the prostaglandins (PGE1, PGI2, PGD2), which bind platelet membrane receptors, raise concentrations of intracellular cyclic adenosine monophosphate (cAMP), and inhibit platelet functions. The major effector of increased cAMP is the serine/threonine kinase protein kinase A (PKA). Of the numerous targets for PKA, one of the most highly phosphorylated upon cAMP increase is glycoprotein (GP) Ibβ, a component of the GPIb-IX-V complex, the platelet receptor for VWF that mediates the initial adhesion of platelet to the vessel wall at sites of injury. The GPIb-IX-V complex consists of 4 type I transmembrane polypeptides, GPIbα, GPIbβ, GPV and GPIX. GPIbα and GPIbβ are disulfide linked in a 1:2 ratio, and the resulting GPIb is non-covalently associated with GPIX and GPV in a 2:2:1 ratio. The VWF-binding site resides within the N-terminal 300 amino acids of GPIbα 500 Å above the platelet surface. Although current data indicate that PKA phosphorylation of the GPIbβ cytoplasmic domain (at Ser166) inhibits the ability of GPIbα to bind VWF, the molecular mechanism(s) have yet to be elucidated. The cytoplasmic domain of GPIbβ associates with calmodulin (in the juxtamembrane 20 amino acids) in resting platelets; calmodulin dissociates upon platelet activation. With elevated cytosolic cAMP, GPIbβ Ser166 becomes phosphorylated and associates with 14-3-3ζ. An interesting feature of the cytoplasmic sequence N-terminal to Ser166 is its extreme cationic nature, containing 8 Arg residues in a stretch of 17 amino acids. Other cytosolic proteins with similar polybasic sequence (MARCKS, GAP43) function as organizers of the signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), and promote the formation of lipid rafts; we reasoned that the polybasic region of GPIbβ might function similarly, organizing rafts when unbound by protein, but not when occupied by calmodulin or 14-3-3ζ. Platelet activation increases raft-associated GPIb-IX-V two fold, with concomitant dissociation of calmodulin from GPIbβ. Here we present evidence that the cytoplasmic domain of GPIbβ plays a role in the localization of the GPIb-IX-V complex to lipid rafts. Treatment of platelets with agents that increase cAMP (PGI2 or forskolin) inhibited GPIb-IX-V-dependent platelet functions, including ristocetin-induced aggregation, shear-induced aggregation and adhesion to VWF under flow. This effect was prevented by the cell-permeable PKA-specific inhibitor H-89. Consistent with the functional importance of GPIb-IX-V localization to lipid rafts, PGI2 and forskolin reduced the raft content of GPIb-IX-V by 35%, and this effect was reversed by H-89. We have thus uncovered a mechanism for long-observed inhibition of platelet adhesion by agents that elevate cytosolic cAMP concentrations, which depends on modulating the quantity of GPIb-IX-V complexes associated with lipid rafts. “Resting” platelets ex vivo are relatively quiescent because calmodulin occupies the GPIbβ polybasic region. The situation changes rapidly when platelets are activated, with more of the complex assuming a ligand-competent state as calmodulin dissociates and the complex organizes rafts. Elevations of cAMP promote phosphorylation of GPIbβ, enabling 14-3-3ζ association, which also displaces the GPIbβ tail from the membrane, disrupting raft association and adhesive function. Disclosures:No relevant conflicts of interest to declare.

Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing

Laurdan and di-4-ANEPPDHQ are used as probes for membrane order, with a blue shift in emission for membranes in liquid-ordered (lo) phase relative to membranes in liquid-disordered (ld) phase. Their use as membrane order probes requires that their spectral shifts are unaffected by membrane proteins, which we have examined by using membrane inserting peptides and large unilamellar vesicles (LUVs). The transmembrane polypeptides, mastoparan and bovine prion protein-derived peptide (bPrPp), were added to LUVs of either lo or ld phase, up to 1:10 peptide/total lipid ratio. The excitation and emission spectra of laurdan and di-4-ANEPPDHQ in both lipid phases were unaltered by peptide addition. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic light scattering. The insertion efficiency of the polypeptides into LUVs was determined by measuring their secondary structure by circular dichroism. Mastoparan had an α-helical and bPrPp a β-strand conformation compatible with insertion into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ, supporting that the dyes are appropriate probes for assessing lipid order in cells.

Differences in the Interactions between the Subunits of Photosystem II Dependent on D1 Protein Variants in the Thermophilic Cyanobacterium Thermosynechococcus elongatus

The main cofactors involved in the oxygen evolution activity of Photosystem II (PSII) are located in two proteins, D1 (PsbA) and D2 (PsbD). In Thermosynechococcus elongatus, a thermophilic cyanobacterium, the D1 protein is encoded by either the psbA(1) or the psbA(3) gene, the expression of which is dependent on environmental conditions. It has been shown that the energetic properties of the PsbA1-PSII and those of the PsbA3-PSII differ significantly (Sugiura, M., Kato, Y., Takahashi, R., Suzuki, H., Watanabe, T., Noguchi, T., Rappaport, F., and Boussac, A. (2010) Biochim. Biophys. Acta 1797, 1491-1499). In this work the structural stability of PSII upon a PsbA1/PsbA3 exchange was investigated. Two deletion mutants lacking another PSII subunit, PsbJ, were constructed in strains expressing either PsbA1 or PsbA3. The PsbJ subunit is a 4-kDa transmembrane polypeptide that is surrounded by D1 (i.e. PsbA1), PsbK, and cytochrome b(559) (Cyt b(559)) in existing three-dimensional models. It is shown that the structural properties of the PsbA3/ΔPsbJ-PSII are not significantly affected. The polypeptide contents, the Cyt b(559) properties, and the proportion of PSII dimer were similar to those found for PsbA3-PSII. In contrast, in PsbA1/ΔPsbJ-PSII the stability of the dimer is greatly diminished, the EPR properties of the Cyt b(559) likely indicates a decrease in its redox potential, and many other PSII subunits are lacking. These results shows that the 21-amino acid substitutions between PsbA1 and PsbA3, which appear to be mainly conservative, must include side chains that are involved in a network of interactions between PsbA and the other PSII subunits.