Search

Melanin binding of drugs is known to increase drug concentrations and retention in pigmented eye tissues. Even though the correlation between melanin binding in vitro and exposure to pigmented eye in vivo has been shown, there is a discrepancy between rapid drug release from melanin particles in vitro and the long in vivo retention in the pigmented tissues. We investigated mechanisms and kinetics of pigment-related drug retention experimentally using isolated melanin particles from porcine retinal pigment epithelium and choroid, isolated porcine eye melanosomes, and re-pigmented ARPE-19 cells in a dynamic flow system. The experimental studies were supplemented with kinetic simulations. Affinity and capacity of levofloxacin, terazosin, papaverine, and timolol binding to melanin revealed Kd values of asymptotic to 50-150 mu M and B-max asymptotic to 40-112 nmol.mg(-1). The drugs were released from melanin in < 1 h (timolol) or in 6-12 h (other drugs). The drugs were released slower from the melanosomes than from melanin; the experimental differences ranged from 1.2-fold (papaverine) to 7.4-fold (timolol). Kinetic simulations supported the role of the melanosomal membrane in slowing down the release of melanin binders. In release studies from the pigmented ARPE-19 cells, drugs were released from the cellular melanin to the extra -cellular space in asymptotic to 1 day (timolol) and asymptotic to 11 days (levofloxacin), i.e., much slower than the release from melanin or melanosomes. Simulations of drug release from pigmented cells in the flow system matched the experimental data and enabled further sensitivity analyses. The simulations demonstrated a significant prolongation of drug retention in the cells as a function of decreasing drug permeability in the melanosomal membranes and increasing melanin content in the cells. Overall, we report the impact of cellular factors in prolonging drug retention and release from melanin-containing cells. These data and simulations will facilitate the design of melanin binding drugs with prolonged ocular actions.

The retinal pigment epithelial (RPE) cell monolayer forms the outer blood−retinal barrier and has a crucial role in ocular pharmacokinetics. Although several RPE cell models are available, there have been no systematic comparisons of their barrier properties with respect to drug permeability. We compared the barrier properties of several RPE secondary cell lines (ARPE19, ARPE19mel, and LEPI) and both primary (hfRPE) and stem-cell derived RPE (hESC-RPE) cells by investigating the permeability of nine drugs (aztreonam, ciprofloxacin, dexamethasone, fluconazole, ganciclovir, ketorolac, methotrexate, voriconazole, and quinidine) across cell monolayers. ARPE19, ARPE19mel, and hfRPE cells displayed a narrow Papp value range, with relatively high permeation rates (5.2−26 × 10−6 cm/s. In contrast, hESC-RPE and LEPI cells efficiently restricted the drug flux, and displayed even lower Papp values than those reported for bovine RPE-choroid, with the range of 0.4−32 cm−6/s (hESC-RPE cells) and 0.4−29 × 10−6 cm/s, (LEPI cells). Therefore, ARPE19, ARPE19mel, and hfRPE cells failed to form a tight barrier, whereas hESC-RPE and LEPI cells restricted the drug flux to a similar extent as bovine RPE-choroid. Therefore, LEPI and hESC-RPE cells are valuable tools in ocular drug discovery.

The mechanisms of Pb(II) toxicity have been studied in human red blood cells using confocal microscopy, immunolabeling, fluorescence-activated cell sorting and atomic force microscopy. The process follows a sequence of events, starting with calcium entry, followed by potassium release, morphological change, generation of ceramide, lipid flip-flop and finally cell lysis. Clotrimazole blocks potassium channels and the whole process is inhibited. Immunolabeling reveals the generation of ceramide-enriched domains linked to a cell morphological change, while the use of a neutral sphingomyelinase inhibitor greatly delays the process after the morphological change, and lipid flip-flop is significantly reduced. These facts point to three major checkpoints in the process: first the upstream exchange of calcium and potassium, then ceramide domain formation, and finally the downstream scramblase activation necessary for cell lysis. In addition, partial non-cytotoxic cholesterol depletion of red blood cells accelerates the process as the morphological change occurs faster. Cholesterol could have a role in modulating the properties of the ceramide-enriched domains. This work is relevant in the context of cell death, heavy metal toxicity and sphingolipid signaling. AGA was a predoctoral student supported by the Basque Government and later by the University of the Basque Country (UPV/EHU). This work was also supported in part by grants from the Spanish Government (FEDER/MINECO BFU 2015-66306-P to F.M.G. and A.A.) and the Basque Government (IT849-13 to F.M.G. and IT838-13 to A.A.), and by the Swiss National Science Foundation.

International audience; Sphingomyelin (SM) is a major sphingolipid in mammalian cells whereas its analog, ceramide phosphoethanolamine (CPE) is found in trace amounts in mammalian cells and in larger amounts in invertebrates such as insect cells like Drosophila melanogaster. To visualize endogenous SM or CPE, we need specific probes able to recognize the chemical structure of the lipid, rather than its physical property. A limited number of proteins is known to specifically and strongly bind SM or CPE. These proteins are either toxins produced by non-mammalian organisms, subunits or fragments of toxins or a protein that has similar structure to a toxin. These proteins labeled with small fluorophore (e.g. Alexa Fluor) or conjugated to fluorescent proteins (e.g. mCherry) or other types of markers (e.g. I-125, maltose-binding protein) are used to detect SM or CPE. Here we summarize the characteristics of specific SM-binding proteins, lysenin and equinatoxin II; CPE- and SM/cholesterol (Chol) binding aegerolysin proteins, pleurotolysin A(2), ostreolysin and erylysin A and SM/Chol-binding protein, nakanori. Then we give examples of their applications including their limitations related not only to their lipid specificity and binding constants, but also to the lipid organization in the membrane.

The microscopic viscosity plays an essential role in cellular biophysics by controlling the rates of diffusion and bimolecular reactions within the cell interior. While several approaches have emerged that have allowed the measurement of viscosity and diffusion on a single cell level in vitro, the in vivo viscosity monitoring has not yet been realized. Here we report the use of fluorescent molecular rotors in combination with Fluorescence Lifetime Imaging Microscopy (FLIM) to image microscopic viscosity in vivo, both on a single cell level and in connecting tissues of subcutaneous tumors in mice. We find that viscosities recorded from single tumor cells in vivo correlate well with the in vitro values from the same cancer cell line. Importantly, our new method allows both imaging and dynamic monitoring of viscosity changes in real time in live animals and thus it is particularly suitable for diagnostics and monitoring of the progress of treatments that might be accompanied by changes in microscopic viscosity.

Objective. Wegener's granulomatosis (WG) is strongly associated with antineutrophil cytoplasmic autoantibodies (ANCAs) directed against proteinase 3 (PR3). Recent studies have shown that membrane-bound PR3 (mPR3) is differentially expressed and colocalizes with CD177/NB1 on circulating neutrophils. We undertook this study to assess the differential expression of CD177 on neutrophils from patients with ANCA-associated systemic vasculitis (ASV) in comparison with patients with systemic lupus erythematosus (SLE), patients with rheumatoid arthritis (RA), and healthy individuals, and to investigate whether colocalization of mPR3 and CD177 affects anti-PR3-mediated neutrophil activation.Methods. Expression of CD177 and mPR3 was analyzed by flow cytometry on isolated neutrophils from patients with ASV (n = 53), those with SLE (n = 30), those with RA (n = 26), and healthy controls (n = 31). Neutrophil activation mediated by anti-PR3 antibodies was assessed by measuring the oxidative burst with a dihydrorbodamine assay.Results. Percentages of CD177-expressing neutrophils were significantly higher in patients with ASV and those with SLE than in healthy controls. In 3 healthy donors, CD177 expression was not detected. After priming with tumor necrosis factor a, neutrophils remained negative for CD177 while mPR3 expression was induced. Neutrophils from CD177-negative donors or CD177-neutrophils sorted from donors with bimodal expression were susceptible to anti-PR3-mediated oxidative burst. Variation in the extent of anti-PR3-mediated neutrophil activation among different donors occurred independent of the percentage of CD177-expressing neutrophils.Conclusion. Membrane expression of CD177 on circulating neutrophils is increased in patients with ASV and in those with SLE, but not in RA patients. However, primed neutrophils from CD177-negative individuals also express mPR3 and are susceptible to anti-PR3-mediated oxidative burst, suggesting that recruitment of CD177-independent mPR3 is involved in anti-PR3-induced neutrophil activation.

Atomistic molecular dynamics simulations were performed with 13 non-peptidic neurotransmitters (NTs) in three different membrane environments. The results provide compelling evidence that NTs are divided into membrane-binding and membrane-nonbinding molecules. NTs adhere to the postsynaptic membrane surface whenever the ligand-binding sites of their synaptic receptors are buried in the lipid bilayer. In contrast, NTs that have extracellular ligand-binding sites do not have a similar tendency to adhere to the membrane surface. This finding is a seemingly simple yet important addition to the paradigm of neurotransmission, essentially dividing it into membrane-independent and membrane-dependent mechanisms. Moreover, the simulations also indicate that the lipid composition especially in terms of charged lipids can affect the membrane partitioning of NTs. The revised paradigm, highlighting the importance of cell membrane and specific lipids for neurotransmission, should to be of interest to neuroscientists, drug industry and the general public alike.

Sphingolipids are a diverse group of lipids found in all eukaryotes and some bacteria, consisting of a hydrophobic ceramide and a hydrophilic head group. We have summarised the contemporary understanding of the structure of plant sphingolipids with an emphasis on glucosylceramides and inositolphosphorylceramides. Plant glucosylceramides are important structural components of plasma and vacuole membranes. inositolphosphorylceramides have been identified as moieties of the glycosylphosphorylinositol (GPI) anchors of plant proteins targeted to the plasma membrane. in the last few years, progress has been made in the cloning of plant genes coding for enzymes involved in sphingolipid metabolism. As found in yeast and mammals, the plant sphingolipid pathway is a potential generator of powerful cell signals. The role of plant sphingolipid metabolites in programmed cell death and calcium influx is discussed.

International audience; Although dietary lipid quality markedly affects fatty acid (FA) composition of mitochondrial membranes from rainbow trout red muscle (Oncorhynchus mykiss), mitochondrial processes are relatively unchanged. As certain classes of phospholipids interact more intimately with membrane proteins than others, we examined whether specific phospholipid classes from these muscle mitochondria were more affected by dietary FA composition than others. To test this hypothesis, we fed trout with two diets differing only in their FA composition: Diet 1 had higher levels of 18:1n-9 and 18:2n-6 than Diet 2, while 22:6n-3 and 22:5n-6 were virtually absent from Diet 1 and high in Diet 2. After 5 months, trout fed Diet 2 had higher proportions of phosphatidylcholine (PC) and less phosphatidylethanolamine (PE) in mitochondrial membranes than those fed Diet 1. The FA composition of PC, PE and cardiolipin (CL) showed clear evidence of regulated incorporation of dietary FA. For trout fed Diet 2, 22:6n-3 was the most abundant FA in PC, PE and CL. The n-6 FA were consistently higher in all phospholipid classes of trout fed Diet 1, with shorter n-6 FA being favoured in CL than in PC and PE. Despite these marked changes in individual FA levels with diet, general characteristics such as total polyunsaturated FA, total monounsaturated FA and total saturated FA were conserved in PE and CL, confirming differential regulation of the FA composition of PC, PE and CL. The regulated changes of phospholipid classes presumably maintain critical membrane characteristics despite varying nutritional quality. We postulate that these changes aim to protect mitochondrial function. J. Exp. Zool. 323A: 60-71, 2015. © 2014 Wiley Periodicals, Inc.

Line-active molecules (“linactants”) that bind to the boundary interface between different fluid lipid domains in membranes have a strong potential as regulators of the lateral heterogeneity that is important for many biological processes. Here, we use molecular dynamics simulations in combination with a coarse-grain model that retains near-atomic resolution to identify lipid species that can act as linactants in a model membrane that is segregated into two lipid domains of different fluidity. Our simulations predict that certain hybrid saturated/unsaturated chain lipids can bind to the interface and lower the line tension, whereas cone-shaped lysolipids have a less pronounced effect.

Background & Aims: Wilson's disease is an autosomal-recessive disorder of copper metabolism that results from the absence or dysfunction of a copper-transporting P-type adenosine triphosphatase that leads to impaired biliary copper excretion and disturbed holoceruloplasmin synthesis, To gain further insight into the role of the Wilson's disease protein in hepatic copper handling, its localization in human liver was investigated, Methods: By use of a specific antibody, localization of the Wilson's disease protein was studied in liver membrane fractions and liver sections by immunoblotting, immunohistochemistry, and double-label confocal scanning laser microscopy, Results: The 165-kilodalton protein, found by immunoblotting, was most abundant mainly in isolated plasma membrane fractions enriched in canalicular domains, Immunohistochemistry revealed intracellular punctuate staining of hepatocytes in certain regions of the liver, whereas a canalicular membrane staining pattern was observed in other regions. Double-labeling studies showed that in the latter regions the transporter is present mainly in vesicular structures just underneath the canalicular membrane that are positive for markers of the trans-Golgi network. A weak staining of the canalicular membrane, identified by staining for P-glycoprotein, was observed, Conclusions: These results show that in human liver the Wilson's disease protein is predominantly present in trans-Golgi vesicles in the pericanalicular area, whereas relatively small amounts of the protein appear to localize to the canalicular membrane, consistent with a dual function of the protein in holoceruloplasmin synthesis and biliary copper excretion.

In industrial fermentations, Penicillium chrysogenum uses sulfate as the source of sulfur for the biosynthesis of penicillin. By a PCR-based approach, two genes, sutA and sutB, whose encoded products belong to the SulP superfamily of sulfate permeases were isolated. Transformation of a sulfate uptake-negative sB3 mutant of Aspergillus nidulans with the sutB gene completely restored sulfate uptake activity. The sutA gene did not complement the A. nidulans sB3 mutation, even when expressed under control of the sutB promoter. Expression of both sutA and sutB in P. chrysogenum is induced by growth under sulfur starvation conditions. However, sutA is expressed to a much lower level than is sutB. Disruption of sutB resulted in a loss of sulfate uptake ability. Overall, the results show that SutB is the major sulfate permease involved in sulfate uptake by P. chrysogenum.

Sperm undergo maturation acquiring progressive motility and the ability to fertilize oocytes through exposure to the components of the epididymal fluid (EF). Although the establishment of a calcium (Ca-2) gradient along the epididymis has been described, its direct effects on epididymal function remain poorly explored. Regucalcin (RGN) is a Ca-2-binding protein, regulating the activity of Ca-2-channels and Ca-2-ATPase, for which a role in male reproductive function has been suggested. This study aimed at comparing the morphology, assessed by histological analysis, and function of epididymis, by analysis of sperm parameters, antioxidant potential and Ca-2 fluxes, between transgenic rats overexpressing RGN (Tg-RGN) and their wild-type littermates. Tg-RGN animals displayed an altered morphology of epididymis and lower sperm counts and motility. Tissue incubation with Ca-45(2) showed also that epididymis of Tg-RGN displayed a diminished rate of Ca-2-influx, indicating unbalanced Ca-2 concentrations in the epididymal lumen. Sperm viability and the frequency of normal sperm, determined by the one-step eosin-nigrosin staining technique and the Diff-Quik staining method, respectively, were higher in Tg-RGN. Moreover, sperm of Tg-RGN rats showed a diminished incidence of tail defects. Western blot analysis demonstrated the presence of RGN in EF as well as its higher expression in the corpus region. The results presented herein demonstrated the importance of maintaining Ca-2-levels in the epididymal lumen and suggest a role for RGN in sperm maturation. Overall, a new insight into the molecular mechanisms driving epididymal sperm maturation was obtained, which could be relevant to development of better approaches in male infertility treatment and contraception. info:eu-repo/semantics/publishedVersion

In the present study we compared, in vivo in rats, the hepatobiliary transport of monovalent (silver:Ag) and divalent metals (zinc:Zn; cadmium:Cd) with that of copper (Cu). Cu can have two oxidation states in vivo, i.e. Cu(I) and Cu(II). Studies were performed in normal Wistar (NW) rats and mutant GY Wistar rats. The latter express defective canalicular ATP-dependent glutathione-conjugate transport (cMOAT); reduced glutathione (GSH) is virtually absent in bile of these mutants. Cd (400 nmol/100g body wt, i.v.) was rapidly secreted into bile in NW rats concommitant with a 4-fold increase in biliary GSH secretion. In contrast, biliary Cd concentrations remained below detection limits in GY rats. Injection of Zn (1500 nmol/100g body wt) did not affect Zn secretion in GY rats and resulted only in a very small increase in NW rats (recovery

We studied compositionally heterogeneous multi-component model membranes comprised of saturated lipids, unsaturated lipids, cholesterol, and a-helical TM protein models using coarse-grained molecular dynamics simulations. Reducing the mismatch between the length of the saturated and unsaturated lipid tails reduced the driving force for segregation into liquid-ordered (l(o)) and liquid-disordered (l(d)) lipid domains. Cholesterol depletion had a similar effect, and binary lipid mixtures without cholesterol did not undergo large-scale phase separation under the simulation conditions. The phase-separating ternary dipalmitoyl-phosphatidylcholine (DPPC)/dilinoleoyl-PC (DLiPC)/cholesterol bilayer was found to segregate into l(o) and l(d) domains also in the presence of a high concentration of TM helices. The l(d) domain was highly crowded with TM helices (protein-to-lipid ratio -1:5), slowing down lateral diffusion by a factor of 5-10 as compared to the dilute case, with anomalous (sub)-diffusion on the mu s time scale. The membrane with the less strongly unsaturated palmitoyl-linoleoyl-PC instead of DLiPC, which in the absence of TM alpha-helices less strongly deviated from ideal mixing, could be brought closer to a miscibility critical point by introducing a high concentration of TM helices. Finally, the 7-TM protein bacteriorhodopsin was found to partition into the l(d) domains irrespective of hydrophobic matching. These results show that it is possible to directly study the lateral reorganization of lipids and proteins in compositionally heterogeneous and crowded model biomembranes with coarse-grained molecular dynamics simulations, a step toward simulations of realistic, compositionally complex cellular membranes. This article is part of a Special Issue entitled: Protein Folding in Membranes. (C) 2011 Elsevier B.V. All rights reserved.

Enveloped animal viruses, such as Semliki Forest virus (SFV), utilize a membrane fusion strategy to deposit their genome into the cytosol of the host cell. SFV enters cells through receptor-mediated endocytosis, fusion of the viral envelope occurring subsequently from within acidic endosomes. Fusion of SFV has been demonstrated before to be strictly dependent on the presence of cholesterol in the target membrane. Here, utilizing a variety of membrane fusion assays, including an on-line fluorescence assay involving pyrene-labeled virus, we demonstrate that low-pa-induced fusion of SFV with cholesterol-containing liposomal model membranes requires the presence of sphingomyelin or other sphingolipids in the target membrane. The minimal molecular characteristics essential for supporting SFV fusion are encompassed by a ceramide. The action of the sphingolipids is confined to the actual fusion event, cholesterol being necessary and sufficient for low-pH-dependent binding of the virus to target membranes. Complex formation of the sphingolipids with cholesterol is unlikely to be important for the induction of SFV-liposome fusion, as sphingolipids that do not interact appreciably with cholesterol, such as galactosylceramide, effectively support the process. The remarkably low levels of sphingomyelin required for half-maximal fusion (1-2 mole%) suggest that sphingolipids do not play a structural role in the SFV fusion process, but rather act as a cofactor, possibly activating the viral fusion protein in a specific manner.

We have studied the effects of dietary corn and olive oil versus coconut fat on bile formation and fluidity of hepatic plasma membranes in rats. After 4 weeks of feeding the purified diets containing 9% (w/w) of the test fats, there was no difference in plasma cholesterol concentration between the dietary groups. The amount of free and esterified cholesterol in the liver was significantly higher in rats fed either corn oil or olive oil as compared with coconut fat. In the rats fed olive oil, but not in those fed corn oil this was associated with lower rates of biliary phospholipid excretion. Bile flow was not differently influenced by the three dietary fats. Hepatic plasma membranes of the rats fed corn or olive oil contained more cholesterol and less phospholipids than those on coconut fat, which was, however, not accompanied by changes in fluidity of the membranes. These results indicate that in rats the type of dietary fat can induce considerable changes in hepatic cholesterol metabolism without affecting plasma cholesterol concentrations, and without consistent effects on biliary cholesterol secretion.

Rapid conduction of nerve impulses requires coating of axons by myelin sheaths, which are multilamellar, lipid-rich membranes produced by oligodendrocytes in the central nervous system. To act as an insulator, myelin has to form a stable and firm membrane structure. In this study, we have analyzed the biophysical properties of myelin membranes prepared from wild-type mice and from mouse mutants that are unable to form stable myelin. Using C-Laurdan and fluorescence correlation spectroscopy, we find that lipids are tightly organized and highly ordered in myelin isolated from wild-type mice, but not from shiverer and ceramide synthase 2 null mice. Furthermore, only myelin lipids from wild-type mice laterally segregate into physically distinct lipid phases in giant unilamellar vesicles in a process that requires very long chain glycosphingolipids. Taken together, our findings suggest that oligodendrocytes exploit the potential of lipids to self-segregate to generate a highly ordered membrane for electrical insulation of axons.

It has often been suggested that the high curvature of transport intermediates in cells may be a sufficient means to segregate different lipid populations based on the relative energy costs of forming bent membranes. In this review, we present in vitro experiments that highlight the essential physics of lipid sorting at thermal equilibrium: It is driven by a trade-off between bending energy, mixing entropy, and interactions between species. We collect evidence that lipid sorting depends strongly on lipid-lipid and protein-lipid interactions, and hence on the underlying composition of the membrane and on the presence of bound proteins.

Azido procainamide methoiodide (APM), a photolabile derivative of the transport model compound procainamide ethobromide (PAEB), shows a close resemblance to PAEB from a physicochemical point of view. Like PAEB it is effectively taken up by the liver and excreted into bile. Kinetics of the uptake of APM in isolated hepatocytes revealed that in addition to a non-saturable process, two saturable uptake systems are involved (K(m1) = 3-mu-M, V(max1)) = 80 pmol/min/10(6) cells, K(m2) = 100-mu-M, V(max2) = 130 pmol/min x 10(6) cells). The uptake rate of APM was inhibited markedly in the presence of other organic cations. Organic anions and uncharged compounds generally had no inhibitory effect on the APM uptake. These results support the theory that there is a separate hepatic uptake system for organic cations like APM. Photoaffinity labeling of intact hepatocytes as well as plasma membrane sub-fractions enriched with sinusoidal domains disclosed two major binding polypeptides with apparent M(r) of 48,000 and 72,000. Such labeling patterns were not observed in membranes from hepatoma cells that are deficient in organic solute uptake. Differential photoaffinity labeling with other cationic compounds such as tributylmethyl ammonium and d-tubocurarine reduced the incorporation of AMP in these polypeptides. The 48- and 72-kDa proteins might be involved in carrier-mediated transport of type I organic cations at the hepatic uptake level.

Objective. Wegener's granulomatosis (WG) is strongly associated with antineutrophil cytoplasmic autoantibodies (ANCAs) directed against proteinase 3 (PR3). Recent studies have shown that membrane-bound PR3 (mPR3) is differentially expressed and colocalizes with CD177/NB1 on circulating neutrophils. We undertook this study to assess the differential expression of CD177 on neutrophils from patients with ANCA-associated systemic vasculitis (ASV) in comparison with patients with systemic lupus erythematosus (SLE), patients with rheumatoid arthritis (RA), and healthy individuals, and to investigate whether colocalization of mPR3 and CD177 affects anti-PR3-mediated neutrophil activation. Methods. Expression of CD177 and mPR3 was analyzed by flow cytometry on isolated neutrophils from patients with ASV (n = 53), those with SLE (n = 30), those with RA (n = 26), and healthy controls (n = 31). Neutrophil activation mediated by anti-PR3 antibodies was assessed by measuring the oxidative burst with a dihydrorbodamine assay. Results. Percentages of CD177-expressing neutrophils were significantly higher in patients with ASV and those with SLE than in healthy controls. In 3 healthy donors, CD177 expression was not detected. After priming with tumor necrosis factor a, neutrophils remained negative for CD177 while mPR3 expression was induced. Neutrophils from CD177-negative donors or CD177-neutrophils sorted from donors with bimodal expression were susceptible to anti-PR3-mediated oxidative burst. Variation in the extent of anti-PR3-mediated neutrophil activation among different donors occurred independent of the percentage of CD177-expressing neutrophils. Conclusion. Membrane expression of CD177 on circulating neutrophils is increased in patients with ASV and in those with SLE, but not in RA patients. However, primed neutrophils from CD177-negative individuals also express mPR3 and are susceptible to anti-PR3-mediated oxidative burst, suggesting that recruitment of CD177-independent mPR3 is involved in anti-PR3-induced neutrophil activation.

International audience; Sorting of lipids and proteins is a key process allowing eukaryotic cells to execute efficient and accurate intracellular transport and to maintain membrane homeostasis. It occurs during the formation of highly curved transport intermediates that shuttle between cell compartments. Protein sorting is reasonably well described, but lipid sorting is much less understood. Lipid sorting has been proposed to be mediated by a physical mechanism based on the coupling between membrane composition and high curvature of the transport intermediates. To test this hypothesis, we have performed a combination of fluorescence and force measurements on membrane tubes of controlled diameters pulled from giant unilamellar vesicles. A model based on membrane elasticity and nonideal solution theory has also been developed to explain our results. We quantitatively show, using 2 independent approaches, that a difference in lipid composition can build up between a curved and a noncurved membrane. Importantly, and consistent with our theory, lipid sorting occurs only if the system is close to a demixing point. Remarkably, this process is amplified when even a low fraction of lipids is clustered upon cholera toxin binding. This can be explained by the reduction of the entropic penalty of lipid sorting when some lipids are bound together by the toxin. Our results show that curvature-induced lipid sorting results from the collective behavior of lipids and is even amplified in the presence of lipid-clustering proteins. In addition, they suggest a generic mechanism by which proteins can facilitate lipid segregation in vivo.

Deckblatt-Impressum persönlicher Dank Inhaltsverzeichnis Verzeichnis der Abbildungen Abkürzungsverzeichnis Einleitung und Fragestellung Schrifttum Material und Methoden Ergebnisse Diskussion Zusammenfassung Summary Literaturverzeichnis Veröffentlichungen Danksagung Selbständigkeitserklärung, Unter dem Begriff der Palmitoylierung wird eine posttranslationale Modifikation von Proteinen verstanden, wobei in den meisten Fällen ein Palmitinsäurerest über eine Thioesterbindung an einen Cysteinrest des Proteins gebunden wird. Diese Modifikation ist in nahezu allen eukaryotischen Lebensformen weit verbreitet. Während laufend weitere Proteine identifiziert werden können, die in dieser Weise modifiziert sind, bleiben noch immer viele Fragen hinsichtlich der zugrunde liegenden molekularen Mechanismen offen. In den letzten fünf Jahren konnten große Fortschritte bei der Entschlüsselung der zugrunde liegenden Enzyme und dynamischen Regelmechanismen erzielt werden. Spezifische Sequenzmuster für eine Palmitoylierung konnten bislang allerdings nur für cytoplasmatisch vorliegende Proteine entdeckt werden. In integralen Membranproteinen liegt die Palmitoylierungsstelle im Allgemeinen nahe der Grenze zwischen cytoplasmatischer Domäne und Transmembranregion. Ein Vergleich der Aminosäuresequenzen palmitoylierter Proteine zeigt aber keine deutliche Konsensus-Sequenz, was vermuten lässt, dass acylierte Proteine in der cytoplasmatischen Domäne und der Transmembranregion komplexere Informationen für die Palmitoylierung beinhalten. Im Rahmen dieser Arbeit sollte der Einfluss der Transmembranregion und speziell dort vorliegender Aminosäuren auf die Palmitoylierung untersucht werden. Hierzu wurden Chimären aus palmitoylierten und nicht palmitoylierbaren Proteinen hergestellt. Diese Versuche zeigten, dass sowohl der Ersatz von Transmembranregion und cytoplasmatischer Domäne zusammen als auch nur der Transmembranregion des nicht palmitoylierbaren Fusionsproteins des Sendaivirus allein gegen die entsprechenden Domänen der palmitoylierten Proteine CD4, CD8 oder Influenza A-Virus Hämagglutinin eine befriedigende Palmitoylierung der Chimären ermöglichte. Die Transmembranregion muss demnach molekulare Informationen für die Palmitoylierung enthalten. Auch nicht-hydrophobe Aminosäure-Muster scheinen eine Palmitoylierung zu unterstützen. Ein Sequenzvergleich bekannter palmitoylierter Transmembranproteine zeigt ein häufiges Vorliegen der Aminosäure Glycin im cytoplasma-nahen Bereich der Transmembranregion. Austausch der membrannahen Glycin- und Phenylalaninreste in der Transmembranregion führte zu gravierendem Abfall des Palmitoylierungsniveaus, was sich durch die veränderte Ausrichtung des Proteins in der Membran und so gestörte Interaktion mit der membranständigen Palmitoyltransferase erklären lässt. Weiter entfernt liegende Glycinreste hatten keinen Einfluss auf die Acylierung. Nicht-hydrophobe Cytoplasma-nahe Motive in der ansonsten strikt hydrophoben Transmembrandomäne stellen demnach molekulare Signale für eine Palmitoylierung dar., The covalent attachment of fatty acids - commonly palmitic acid - in a thioester-type linkage is a widespread modification of viral and cellular polypeptides. While constantly further palmitoylated proteins are being identified, many questions remain open regarding the molecular mechanisms for palmitoylation. So far specific sequence motives for palmitoylation could be discovered only for cytoplasmic proteins. With integral membrane proteins the hydrocarbon chain is usually bound to cysteine residues located close to the boundary between the transmembrane region and the cytoplasmic tail. Inspection of the amino acids in the vicinity of the acylated cysteine residues reveals no obvious consensus-signal for palmitoylation. This implies that acylated membrane proteins contain complex conformational signals for palmitoylation that are mainly located within the cytoplasmic half of the transmembrane domain, but also involve the cytoplasmic region. Therefore the aim of this study was to resolve the influence of the transmembrane region for palmitoylation as well as the particular contribution of amino acid residues within the transmembrane domain using a series of new chimeric and mutant proteins derived from the acylated proteins Influenza virus haemagglutinin, CD4 and CD8 receptor protein as well as from the non-acylated Sendai virus fusion protein. While Sendai virus fusion protein is not palmitoylated even after introducing of a cysteine residue into the potential acylation region, substitution of the transmembrane domain or both transmembrane and cytoplasmic region for the corresponding regions of Influenza virus haemagglutinin or CD4- or CD8 receptor protein is sufficient for palmitoylation. Non-hydrophobic amino acid residues on the hydrophilic face of the TMD-helix may support palmitoylation of membrane proteins. Sequence alignment of some palmitoylated proteins shows frequent occurrence of glycine in the transmembrane region close to the cytoplasmic tail. Replacement of cytoplasma-near glycine and phenylalanine residues in the transmembrane region leads to a lower acylation rate of the chimerae, while distant glycine residues have no significant influence on palmitoylation rate. This may be due to changes in the protein adjustment in the membrane and therefore disturbed interaction with the membrane-based enzyme palmitoyltransferase. Non-hydrophobic motives in the otherwise strictly hydrophobic transmembrane helix therefore represent molecular signals for palmitoylation.

The N-terminal portion of the large envelope protein of the human hepatitis B virus (HBV), the preS1 domain, plays a fundamental role in cell attachment and infectivity. Recent investigations have suggested that myristylation of preS1 Gly(2) residue is essential for viral infectivity, but the importance of this post-translational modification on HBV-receptor interaction has not been elucidated completely. In this study we produced, using stepwise solid-phase chemical synthesis, the entire preS1[1-119] domain (adw2 subtype), and compared its receptor binding activity with the myristylated form, myristyl-preS1[2-119] in order to define the importance of fatty acid modification. Both synthetic proteins were fully characterized in terms of structural identity using TOF-MALDI mass spectrometry and analysis of tryptic fragments. Circular dichroism measurements indicated a low content of ordered structure in the preS1 protein, while the propensity of the myristylated derivative to assume a conformationally defined structure was more evident. HBV-receptor binding assays performed with plasma membranes preparations from the hepatocyte carcinoma cell line HepG2 clearly showed that the preS1[1-119] domain recognizes the HBV receptor, and confirmed that binding is occurring through the 21-47 region. The myristylated derivative recognized HBV receptor preparations with higher affinity than the preS1 domain, suggesting that the conformational transitions induced in the preS1 moiety by fatty acid post-translational modification are important for efficient attachment of viral particles to HBV receptors.

In the present study we compared, in vivo in rats, the hepatobiliary transport of monovalent (silver:Ag) and divalent metals (zinc:Zn; cadmium:Cd) with that of copper (Cu). Cu can have two oxidation states in vivo, i.e. Cu(I) and Cu(II). Studies were performed in normal Wistar (NW) rats and mutant GY Wistar rats. The latter express defective canalicular ATP-dependent glutathione-conjugate transport (cMOAT); reduced glutathione (GSH) is virtually absent in bile of these mutants. Cd (400 nmol/100g body wt, i.v.) was rapidly secreted into bile in NW rats concommitant with a 4-fold increase in biliary GSH secretion. In contrast, biliary Cd concentrations remained below detection limits in GY rats. Injection of Zn (1500 nmol/100g body wt) did not affect Zn secretion in GY rats and resulted only in a very small increase in NW rats (recovery

Enveloped animal viruses, such as Semliki Forest virus (SFV), utilize a membrane fusion strategy to deposit their genome into the cytosol of the host cell. SFV enters cells through receptor-mediated endocytosis, fusion of the viral envelope occurring subsequently from within acidic endosomes. Fusion of SFV has been demonstrated before to be strictly dependent on the presence of cholesterol in the target membrane. Here, utilizing a variety of membrane fusion assays, including an on-line fluorescence assay involving pyrene-labeled virus, we demonstrate that low-pa-induced fusion of SFV with cholesterol-containing liposomal model membranes requires the presence of sphingomyelin or other sphingolipids in the target membrane. The minimal molecular characteristics essential for supporting SFV fusion are encompassed by a ceramide. The action of the sphingolipids is confined to the actual fusion event, cholesterol being necessary and sufficient for low-pH-dependent binding of the virus to target membranes. Complex formation of the sphingolipids with cholesterol is unlikely to be important for the induction of SFV-liposome fusion, as sphingolipids that do not interact appreciably with cholesterol, such as galactosylceramide, effectively support the process. The remarkably low levels of sphingomyelin required for half-maximal fusion (1-2 mole%) suggest that sphingolipids do not play a structural role in the SFV fusion process, but rather act as a cofactor, possibly activating the viral fusion protein in a specific manner.

Azido procainamide methoiodide (APM), a photolabile derivative of the transport model compound procainamide ethobromide (PAEB), shows a close resemblance to PAEB from a physicochemical point of view. Like PAEB it is effectively taken up by the liver and excreted into bile. Kinetics of the uptake of APM in isolated hepatocytes revealed that in addition to a non-saturable process, two saturable uptake systems are involved (K(m1) = 3-mu-M, V(max1)) = 80 pmol/min/10(6) cells, K(m2) = 100-mu-M, V(max2) = 130 pmol/min x 10(6) cells). The uptake rate of APM was inhibited markedly in the presence of other organic cations. Organic anions and uncharged compounds generally had no inhibitory effect on the APM uptake. These results support the theory that there is a separate hepatic uptake system for organic cations like APM. Photoaffinity labeling of intact hepatocytes as well as plasma membrane sub-fractions enriched with sinusoidal domains disclosed two major binding polypeptides with apparent M(r) of 48,000 and 72,000. Such labeling patterns were not observed in membranes from hepatoma cells that are deficient in organic solute uptake. Differential photoaffinity labeling with other cationic compounds such as tributylmethyl ammonium and d-tubocurarine reduced the incorporation of AMP in these polypeptides. The 48- and 72-kDa proteins might be involved in carrier-mediated transport of type I organic cations at the hepatic uptake level.

In industrial fermentations, Penicillium chrysogenum uses sulfate as the source of sulfur for the biosynthesis of penicillin. By a PCR-based approach, two genes, sutA and sutB , whose encoded products belong to the SulP superfamily of sulfate permeases were isolated. Transformation of a sulfate uptake-negative sB3 mutant of Aspergillus nidulans with the sutB gene completely restored sulfate uptake activity. The sutA gene did not complement the A. nidulans sB3 mutation, even when expressed under control of the sutB promoter. Expression of both sutA and sutB in P. chrysogenum is induced by growth under sulfur starvation conditions. However, sutA is expressed to a much lower level than is sutB . Disruption of sutB resulted in a loss of sulfate uptake ability. Overall, the results show that SutB is the major sulfate permease involved in sulfate uptake by P. chrysogenum .

We have studied the effects of dietary corn and olive oil versus coconut fat on bile formation and fluidity of hepatic plasma membranes in rats. After 4 weeks of feeding the purified diets containing 9% (w/w) of the test fats, there was no difference in plasma cholesterol concentration between the dietary groups. The amount of free and esterified cholesterol in the liver was significantly higher in rats fed either corn oil or olive oil as compared with coconut fat. In the rats fed olive oil, but not in those fed corn oil this was associated with lower rates of biliary phospholipid excretion. Bile flow was not differently influenced by the three dietary fats. Hepatic plasma membranes of the rats fed corn or olive oil contained more cholesterol and less phospholipids than those on coconut fat, which was, however, not accompanied by changes in fluidity of the membranes. These results indicate that in rats the type of dietary fat can induce considerable changes in hepatic cholesterol metabolism without affecting plasma cholesterol concentrations, and without consistent effects on biliary cholesterol secretion.