A novel requirement for C. elegans Alix/ALX-1 in RME-1-mediated membrane transport

BACKGROUND: Alix/Bro1p family proteins have recently been identified as important components of multivesicular endosomes (MVEs) and are involved in the sorting of endocytosed integral membrane proteins, interacting with components of the ESCRT complex, the unconventional phospholipid LBPA, and other known endocytosis regulators. During infection, Alix can be co-opted by enveloped retroviruses, including HIV, providing an important function during virus budding from the plasma membrane. In addition, Alix is associated with the actin cytoskeleton and might regulate cytoskeletal dynamics. RESULTS: Here we demonstrate a novel physical interaction between the only apparent Alix/Bro1p family protein in C. elegans, ALX-1, and a key regulator of receptor recycling from endosomes to the plasma membrane, called RME-1. The analysis of alx-1 mutants indicates that ALX-1 is required for the endocytic recycling of specific basolateral cargo in the C. elegans intestine, a pathway previously defined by the analysis of rme-1 mutants. The expression of truncated human Alix in HeLa cells disrupts the recycling of major histocompatibility complex class I, a known Ehd1/RME-1-dependent transport step, suggesting the phylogenetic conservation of this function. We show that the interaction of ALX-1 with RME-1 in C. elegans, mediated by RME-1/YPSL and ALX-1/NPF motifs, is required for this recycling process. In the C. elegans intestine, ALX-1 localizes to both recycling endosomes and MVEs, but the ALX-1/RME-1 interaction appears to be dispensable for ALX-1 function in MVEs and/or late endosomes. CONCLUSIONS: This work provides the first demonstration of a requirement for an Alix/Bro1p family member in the endocytic recycling pathway in association with the recycling regulator RME-1.

Modulation of adipocyte G-protein expression in cancer cachexia by a lipid-mobilizing factor (LMF)

Adipocytes isolated from cachectic mice bearing the MAC 16 tumour showed over a 3-fold increase in lipolytic response to both low concentrations of isoprenaline and a tumour-derived lipid mobilizing factor (LMF). This was reflected by an enhanced stimulation of adenylate cyclase in plasma membrane fractions of adipocytes in the presence of both factors. There was no up-regulation of adenylate cyclase in response to forskolin, suggesting that the effect arose from a change in receptor number or G-protein expression. Immunoblotting of adipocyte membranes from mice bearing the MAC16 tumour showed an increased expression of Gαs up to 10% weight loss and a reciprocal decrease in Gα. There was also an increased expression of Gαs and a decrease in Gα in adipose tissue from a patient with cancer-associated weight loss compared with a non-cachectic cancer patient. The changes in G-protein expression were also seen in adipose tissue of normal mice administered pure LMF as well as in 3T3L1 adipocytes in vitro. The changes in G-protein expression induced by LMF were attenuated by the polyunsaturated fatty acid, eicosapentaenoic acid (EPA). This suggests that this tumour-derived lipolytic factor acts to sensitize adipose tissue to lipolytic stimuli, and that this effect is attenuated by EPA, which is known to preserve adipose tissue in cancer cachexia. © 2001 Cancer Research Campaign.

The activity of pH membrane disruptive pseudopeptides and their subcellular fate in mammalian cells cultured in-vitro

This thesis describes investigations upon pseudopeptides which were conducted to improve our understanding of the fate of synthetic macromolecules in cells and to develop approaches to influence that fate. The low uptake of molecules across the external cellular membrane is the principal barrier against effective delivery of therapeutic products to within the cell structure. In nature, disruption of this membrane by amphiphilic peptides plays a central role in the pathogenesis by bacterial and toxin infections. These amphiphilic peptides contain both hydrophobic and weakly charged hydrophilic amino acid residues and upon activation they become integrated into the lipid bilayers of the extracellular or endosomal membranes. The architectures of the pseudopeptides described here were designed to display similar pH dependent membrane rupturing activity to that of peptides derived from the influenza virus hemagglutinin HA-2. This HA protein promotes fusion of the influenza virus envelope with the cell endosome membrane due to a change in conformation in response to the acidic pH of the endosome lumen (pH 5.0-6.0). The pseudopeptides were obtained by the copolymerisation of L-lysine and L-lysine ethyl-ester with various dicarboxylic acid moieties. In this way a linear polyamide comprising of alternating pendant carboxylic acids and pendant hydrophobic moieties was made. At physiological pH (pH 7.4), electrostatic repulsion of pendant anionic carboxyl groups along the polymer backbone is sufficient to overcome the intramolecular association of the hydrophobic groups resulting in an extended conformation. At low pH (typically pH 4.8) loss of charge results in increased intramolecular hydrophobic association and the polymer chain collapses to a compact conformation, leading to precipitation of the polymer. Consequently, a conformation dependent functional property could be made to respond to small changes in the environmental pH. Pseudopepides were investigated for their cytoxicity towards a well known cell line, namely C26 (colorectal adenocarcinoma) and were shown through the use of a cell viability assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) to be well tolerated by C26 cells over a range of concentrations (2-500,μg/ml) at physiological pH (pH 7.4). A modified version of a shorter 30-minute coupled enzymatic assay, the LDH (lactate dehydrogenase) assay was used to evaluate the ability of the pseudopeptides to disrupt the membrane of two different cell lines (COS-1; African green monkey, kidney and A2780; human ovarian carcinoma) at low pH (pH 5.5). The cell membrane disruption property of the pseudopeptides was successfully demonstrated for COS-I and A2780 cell lines at this pH (pH 5.5). A variety of cell lines were chosen owing to limited availability and to compare the cytotoxic action of these pH responsive psudopeptides towards normal and tumorogenic cell lines. To investigate the intracellular delivery of one of the pseudopeptides, poly (L-lysine iso-phthalamide) and its subcellular location, a Cy3 bisamine fluorophore was conjugated into its backbone, at ratios of dye:lysine of 1:20, 1:30, 1:40, 1:60 and 1:80. Native polyacrylacrylamide gel electrophoresis (PAGE) and high voltage paper electrophoresis (HVPE) studies of the polydyes were conducted and provided evidence that that the Cy3 bisamine fluorophore was conjugated into the backbone of the polymer, poly (L-lysine iso-phthalamide). The subcellular fate of the fluorescentlylabelled "polydye" (hereafter PD20) was monitored by laser scanning confocal microscopy (LSCM) in CHO (Chinese hamster ovary) cells cultured in-vitro at various pH values (pH 7.4 and 5.0). LSCM images depicting time-dependent internalisation of PD20 indicated that PD20 traversed the extracellular membrane of CHO cells cultured in-vitro within ten minutes and migrated towards the endosomal regions where the pH is in the region of 5.0 to 6.0. Nuclear localisation of PD20 was demonstrated in a subpopulation of CHO cells. A further study was completed in CHO and HepG2 (hepatocellular carcinoma) cells cultured in-vitro using a lower molecular weight polymer to demonstrate that the molecular weight of "polydye" could be tailored to attain nuclear trafficking in cells. Prospective use of this technology encompasses a method of delivering a payload into a living cell based upon the hypercoiling nature of the pseudopeptides studied in this thesis and has led to a patent application (GB0228525.2; 20(2).

A novel silicon membrane-based biosensing platform using distributive sensing strategy and artificial neural networks for feature analysis

A novel biosensing system based on a micromachined rectangular silicon membrane is proposed and investigated in this paper. A distributive sensing scheme is designed to monitor the dynamics of the sensing structure. An artificial neural network is used to process the measured data and to identify cell presence and density. Without specifying any particular bio-application, the investigation is mainly concentrated on the performance testing of this kind of biosensor as a general biosensing platform. The biosensing experiments on the microfabricated membranes involve seeding different cell densities onto the sensing surface of membrane, and measuring the corresponding dynamics information of each tested silicon membrane in the form of a series of frequency response functions (FRFs). All of those experiments are carried out in cell culture medium to simulate a practical working environment. The EA.hy 926 endothelial cell lines are chosen in this paper for the bio-experiments. The EA.hy 926 endothelial cell lines represent a particular class of biological particles that have irregular shapes, non-uniform density and uncertain growth behaviour, which are difficult to monitor using the traditional biosensors. The final predicted results reveal that the methodology of a neural-network based algorithm to perform the feature identification of cells from distributive sensory measurement has great potential in biosensing applications.

Molecular dynamics study of solvent transport in nanoscale osmosis

An ideal of osmotic equilibrium between an ideal solution and pure solvent separated by a semi-permeable membrane is studied numerically using the method of molecular dynamics. The osmotic flow is observed as the inflow of the solvent across the membrane from the dilute to the concentrated side. The validity of van't Hoff's law for osmotic pressure is confirmed over a wide range of concentrations. It is found that the law is established by a balance between non-uniform partial pressures of solute and solvent. Furthermore, the present model permits an understanding of the mechanism of the osmotic flow in the relaxation process as the liquids evolve from the initial state to the equilibrium state. We focus in particular on the interaction between solute and solvent. ©2008 The Physical Society of Japan.

Role of fluctuations in membrane models:thermal versus nonthermal

We study the comparative importance of thermal to nonthermal fluctuations for membrane-based models in the linear regime. Our results, both in 1+1 and 2+1 dimensions, suggest that nonthermal fluctuations dominate thermal ones only when the relaxation time τ is large. For moderate to small values of τ, the dynamics is defined by a competition between these two forces. The results are expected to act as a quantitative benchmark for biological modeling in systems involving cytoskeletal and other nonthermal fluctuations. © 2011 American Physical Society.

Oxidised LDL-lipids increase beta amyloid production by SH-SY5Y cells through glutathione depletion and lipid raft formation

Elevated total cholesterol in midlife has been associated with increased risk of dementia in later life. We have previously shown that low-density lipoprotein (LDL) is more oxidized in the plasma of dementia patients, although total cholesterol levels are not different from those of age-matched controls. β-Amyloid (Aβ) peptide, which accumulates in Alzheimer disease (AD), arises from the initial cleavage of amyloid precursor protein by β-secretase-1 (BACE1). BACE1 activity is regulated by membrane lipids and raft formation. Given the evidence for altered lipid metabolism in AD, we have investigated a mechanism for enhanced Aβ production by SH-SY5Y neuronal-like cells exposed to oxidized LDL (oxLDL). The viability of SH-SY5Y cells exposed to 4 μg oxLDL and 25 μM 27-hydroxycholesterol (27OH-C) was decreased significantly. Lipids, but not proteins, extracted from oxLDL were more cytotoxic than oxLDL. In parallel, the ratio of reduced glutathione (GSH) to oxidized glutathione was decreased at sublethal concentrations of lipids extracted from native and oxLDL. GSH loss was associated with an increase in acid sphingomyelinase (ASMase) activity and lipid raft formation, which could be inhibited by the ASMase inhibitor desipramine. 27OH-C and total lipids from LDL and oxLDL independently increased Aβ production by SH-SY5Y cells, and Aβ accumulation could be inhibited by desipramine and by N-acetylcysteine. These data suggest a mechanism whereby oxLDL lipids and 27OH-C can drive Aβ production by GSH depletion, ASMase-driven membrane remodeling, and BACE1 activation in neuronal cells. © 2014 The Authors.

Overcoming bottlenecks in the membrane protein structural biology pipeline

Membrane proteins account for a third of the eukaryotic proteome, but are greatly under-represented in the Protein Data Bank. Unfortunately, recent technological advances in X-ray crystallography and EM cannot account for the poor solubility and stability of membrane protein samples. A limitation of conventional detergent-based methods is that detergent molecules destabilize membrane proteins, leading to their aggregation. The use of orthologues, mutants and fusion tags has helped improve protein stability, but at the expense of not working with the sequence of interest. Novel detergents such as glucose neopentyl glycol (GNG), maltose neopentyl glycol (MNG) and calixarene-based detergents can improve protein stability without compromising their solubilizing properties. Styrene maleic acid lipid particles (SMALPs) focus on retaining the native lipid bilayer of a membrane protein during purification and biophysical analysis. Overcoming bottlenecks in the membrane protein structural biology pipeline, primarily by maintaining protein stability, will facilitate the elucidation of many more membrane protein structures in the near future.

GPCR-styrene maleic acid lipid particles (GPCR-SMALPs):their nature and potential

G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a 'molecular pastry cutter' to 'solubilize' GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR-SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR.

Detergent-free membrane protein purification

Membrane proteins are localised within a lipid bilayer; in order to purify them for functional and structural studies the first step must involve solubilising or extracting the protein from these lipids. To date this has been achieved using detergents which disrupt the bilayer and bind to the protein in the transmembrane region. However finding conditions for optimal extraction, without destabilising protein structure is time consuming and expensive. Here we present a recently-developed method using a styrene maleic acid (SMA) co-polymer instead of detergents. The SMA co-polymer extracts membrane proteins in a small disc of lipid bilayer which can be used for affinity chromatography purification, thus enabling the purification of membrane proteins while maintaining their native lipid bilayer environment.

Organic matter dynamics in the Florida coastal Everglades: A molecular marker and isotopic approach

Everglades National Park (ENP) is about to undergo the world's largest wetland restoration with the aim of improving the quality, timing and distribution of water flow. The changes in water flow are hypothesized to alter the nutrient fluxes and organic matter (OM) dynamics within ENP, especially in the estuarine areas. This study used a multi-proxy approach of molecular markers and stable δ 13C isotope measurements, to determine the present day OM dynamics in ENP. ^ OM dynamics in wetland soils/sediments have proved to be difficult to understand using traditional geochemical approaches. These are often inadequate to describe the multitude of OM sources (e.g. higher land plant, emergent vegetation, submerged vegetation) to the soils/sediments and the complex diagenetic processes that can alter the OM characteristics. A multi-proxy approach, however, that incorporates both molecular level and bulk parameter information is ideal to comprehend complex OM dynamics in aquatic environments. Therefore, biomass-specific molecular markers or proxies can be useful in tracing the sources and processing of OM. This approach was used to examine the OM dynamics in the two major drainage basins, Shark River Slough and Taylor River Slough, of ENP. Freshwater to marine transects were sampled in both systems for soils/sediments and suspended particulate organic matter (SPOM) to be characterized through bulk OM analyses, lipid biomarker determinations (e.g. sterols, fatty acids, hydrocarbons and triterpenoids) and compound-specific stable carbon isotope (δ 13C) determinations. ^ One key accomplishment of the research was the assessment of a molecular marker proxy (Paq) to distinguish between emergent/higher plant vegetation from submerged vegetation within ENP. This proxy proved to be quite useful at tracing OM inputs to the soils/sediments of ENP. A second key accomplishment was the development of a 3-way model using vegetation specific molecular markers. This novel, descriptive model was successfully applied to the estuarine areas of Taylor and Shark River sloughs, providing clear evidence of mixing of freshwater, estuarine and marine derived OM in these areas. In addition, diagenetic transformations of OM in these estuaries were found to be quite different between Taylor and Shark Rivers, and are likely a result of OM quality and hydrological differences. ^

Membrane protein production in the Yeast, S. cerevisiae

The first crystal structures of recombinant mammalian membrane proteins were solved in 2005 using protein that had been produced in yeast cells. One of these, the rabbit Ca2+-ATPase SERCA1a, was synthesized in Saccharomyces cerevisiae. All host systems have their specific advantages and disadvantages, but yeast has remained a consistently popular choice in the eukaryotic membrane protein field because it is quick, easy and cheap to culture, whilst being able to post-translationally process eukaryotic membrane proteins. Very recent structures of recombinant membrane proteins produced in S. cerevisiae include those of the Arabidopsis thaliana NRT1.1 nitrate transporter and the fungal plant pathogen lipid scramblase, TMEM16. This chapter provides an overview of the methodological approaches underpinning these successes.

Lipid analytic of environmental and cultural samples

Membrane lipids of marine planktonic archaea have provided unique insights into archaeal ecology and paleoceanography. However, past studies of archaeal lipids in suspended particulate matter (SPM) and sediments mainly focused on a small class of fully saturated glycerol dibiphytanyl glycerol tetraether (GDGT) homologues identified decades ago. The apparent low structural diversity of GDGTs is in strong contrast to the high diversity of metabolism and taxonomy among planktonic archaea. Furthermore, adaptation of archaeal lipids in the deep ocean remains poorly constrained. We report the archaeal lipidome in SPM from diverse oceanic regimes. We extend the known inventory of planktonic archaeal lipids to include numerous unsaturated archaeal ether lipids (uns-AELs). We further reveal i) different thermal regulations and polar headgroup compositions of membrane lipids between the epipelagic (<= 100 m) and deep (> 100 m) populations of archaea; ii) stratification of unsaturated GDGTs with varying redox conditions; and iii) enrichment of tetra-unsaturated archaeol and fully saturated GDGTs in epipelagic and deep oxygenated waters, respectively. Such stratified lipid patterns are consistent with the typical distribution of archaeal phylotypes in marine environments. We thus provide an ecological context for GDGT-based paleoclimatology and bring about the potential use of uns-AELs as biomarkers for planktonic Euryarchaeota. This article is protected by copyright. All rights reserved.

Développement d'une méthode de prédiction de l'orientation et de l'insertion des protéines dans une membrane modèle

Les protéines membranaires intégrales jouent un rôle indispensable dans la survie des cellules et 20 à 30% des cadres de lectures ouverts codent pour cette classe de protéines. La majorité des protéines membranaires se trouvant sur la Protein Data Bank n’ont pas une orientation et une insertion connue. L’orientation, l’insertion et la conformation que les protéines membranaires ont lorsqu’elles interagissent avec une bicouche lipidique sont importantes pour la compréhension de leur fonction, mais ce sont des caractéristiques difficiles à obtenir par des méthodes expérimentales. Des méthodes computationnelles peuvent réduire le temps et le coût de l’identification des caractéristiques des protéines membranaires. Dans le cadre de ce projet de maîtrise, nous proposons une nouvelle méthode computationnelle qui prédit l’orientation et l’insertion d’une protéine dans une membrane. La méthode est basée sur les potentiels de force moyenne de l’insertion membranaire des chaînes latérales des acides aminés dans une membrane modèle composèe de dioléoylphosphatidylcholine.

Interactions of Cytochrome P450 3A4 with Phospholipid Bilayer Nanodiscs

Thesis (Ph.D.)--University of Washington, 2016-08