Lipids and membrane-associated proteins in autophagy.

Autophagy is essential for the maintenance of cellular homeostasis and its dysfunction has been linked to various diseases. Autophagy is a membrane driven process and tightly regulated by membrane-associated proteins. Here, we summarized membrane lipid composition, and membrane-associated proteins relevant to autophagy from a spatiotemporal perspective. In particular, we focused on three important membrane remodeling processes in autophagy, lipid transfer for phagophore elongation, membrane scission for phagophore closure, and autophagosome-lysosome membrane fusion. We discussed the significance of the discoveries in this field and possible avenues to follow for future studies. Finally, we summarized the membrane-associated biochemical techniques and assays used to study membrane properties, with a discussion of their applications in autophagy.

Lipid larceny: channelizing host lipids for establishing successful pathogenesis by bacteria.

Lipids are complex organic compounds made up of carbon, oxygen, and hydrogen. These play a diverse and intricate role in cellular processes like membrane trafficking, protein sorting, signal transduction, and bacterial infections. Both Gram-positive bacteria (Staphylococcus sp., Listeria monocytogenes, etc.) and Gram-negative bacteria (Chlamydia sp., Salmonella sp., E. coli, etc.) can hijack the various host-lipids and utilize them structurally as well as functionally to mount a successful infection. The pathogens can deploy with various arsenals to exploit host membrane lipids and lipid-associated receptors as an attachment for toxins' landing or facilitate their entry into the host cellular niche. Bacterial species like Mycobacterium sp. can also modulate the host lipid metabolism to fetch its carbon source from the host. The sequential conversion of host membrane lipids into arachidonic acid and prostaglandin E2 due to increased activity of cPLA-2 and COX-2 upon bacterial infection creates immunosuppressive conditions and facilitates the intracellular growth and proliferation of bacteria. However, lipids' more debatable role is that they can also be a blessing in disguise. Certain host-lipids, especially sphingolipids, have been shown to play a crucial antibacterial role and help the host in combating the infections. This review shed light on the detailed role of host lipids in bacterial infections and the current understanding of the lipid in therapeutics. We have also discussed potential prospects and the need of the hour to help us cope in this race against deadly pathogens and their rapidly evolving stealthy virulence strategies.

The importance of the membrane for biophysical measurements.

Within cell membranes numerous protein assemblies reside. Among their many functions, these assemblies regulate the movement of molecules between membranes, facilitate signaling into and out of cells, allow movement of cells by cell-matrix attachment, and regulate the electric potential of the membrane. With such critical roles, membrane protein complexes are of considerable interest for human health, yet they pose an enduring challenge for structural biologists because it is difficult to study these protein structures at atomic resolution in in situ environments. To advance structural and functional insights for these protein assemblies, membrane mimetics are typically employed to recapitulate some of the physical and chemical properties of the lipid bilayer membrane. However, extraction from native membranes can sometimes change the structure and lipid-binding properties of these complexes, leading to conflicting results and fueling a drive to study complexes directly from native membranes. Here we consider the co-development of membrane mimetics with technological breakthroughs in both cryo-electron microscopy (cryo-EM) and native mass spectrometry (nMS). Together, these developments are leading to a plethora of high-resolution protein structures, as well as new knowledge of their lipid interactions, from different membrane-like environments.

Distinct functional roles for hopanoid composition in the chemical tolerance of Zymomonas mobilis.

Hopanoids are a class of membrane lipids found in diverse bacterial lineages, but their physiological roles are not well understood. The ethanol fermenter Zymomonas mobilis features the highest measured concentration of hopanoids, leading to the hypothesis that these lipids can protect against the solvent toxicity. However, the lack of genetic tools for manipulating hopanoid composition in this bacterium has limited their further functional analysis. Due to the polyploidy (>50 genome copies per cell) of Z. mobilis, we found that disruptions of essential hopanoid biosynthesis (hpn) genes act as genetic knockdowns, reliably modulating the abundance of different hopanoid species. Using a set of hpn transposon mutants, we demonstrate that both reduced hopanoid content and modified hopanoid polar head group composition mediate growth and survival in ethanol. In contrast, the amount of hopanoids, but not their head group composition, contributes to fitness at low pH. Spectroscopic analysis of bacterial-derived liposomes showed that hopanoids protect against several ethanol-driven phase transitions in membrane structure, including lipid interdigitation and bilayer dissolution. We propose that hopanoids act through a combination of hydrophobic and inter-lipid hydrogen bonding interactions to stabilize bacterial membranes during solvent stress.

Lavage lipidomics signatures in children with cystic fibrosis and protracted bacterial bronchitis.

BACKGROUND: Balanced composition of a well-functioning pulmonary surfactant is crucial and essential for normal breathing. Here, we explored whether the composition of lipids recovered by broncho-alveolar lavage (BAL) in children with cystic fibrosis (CF) differ from children with protracted bacterial bronchitis (PBB) and controls. We wanted to differentiate, if alterations are primarily caused by the disease process or secondary due to an increased amount of cell-membrane lipids derived from inflammatory cells. METHODS: Comprehensive lipidomics profiles of BAL fluid from children diagnosed with CF, PBB and controls were generated by electrospray ionization tandem mass spectrometry analysis. BAL cell differential and numbers were examined. RESULTS: 55 children (37 patients with CF, 8 children with PBB and 10 controls) were included in this study. Results showed comparable total quantities of lipids in all groups. Phospholipids were the major lipid fraction and similar in all groups, whereas the fractions of cholesteryl esters were less and of free cholesterol were increased in children with CF. Among the phospholipids, patients with CF had higher proportion of the non-surfactant membrane-lipids in the classes phosphatidylethanolamine based plasmalogens (PE P), phosphatidylethanolmine (PE) and phosphatidylserine (PS), but a lower proportion of phosphatidylcholine (PC) compared to healthy controls. No such changes were identified in the BAL fluid of children diagnosed with PBB. No differences were observed for the surfactant lipids dipalmitoyl-phosphatidylcholin (PC 32:0) and phosphatidylglycerol (PG). CONCLUSIONS: In CF patients with neutrophilic airway inflammation the lipid composition for surfactant phospholipid components were unchanged, whereas alteration in lipid profile were characteristic for those found in membranes of inflammatory cells. We suspect that the changes in CF were caused by the prolonged inflammation in contrast to a relatively short standing process in PBB.

Butyrate and docosahexaenoic acid interact in alterations of specific lipid classes in differentiating colon cancer cells.

Docosahexaenoic acid (DHA) and sodium butyrate (NaBt) exhibit a number of interactive effects on colon cancer cell growth, differentiation, or apoptosis; however, the molecular mechanisms responsible for these interactions and their impact on cellular lipidome are still not fully clear. Here, we show that both dietary agents together induce dynamic alterations of lipid metabolism, specific cellular lipid classes, and fatty acid composition. In HT-29 cell line, a model of differentiating colon carcinoma cells, NaBt supported incorporation of free DHA into non-polar lipids and their accumulation in cytoplasmic lipid droplets. DHA itself was not incorporated into sphingolipids; however, it significantly altered representation of individual ceramide (Cer) classes, in particular in combination with NaBt (DHA/NaBt). We observed altered expression of enzymes involved in Cer metabolism in cells treated with NaBt or DHA/NaBt, and exogenous Cer 16:0 was found to promote induction of apoptosis in differentiating HT-29 cells. NaBt, together with DHA, increased n-3 fatty acid synthesis and attenuated metabolism of monounsaturated fatty acids. Finally, DHA and/or NaBt altered expression of proteins involved in synthesis of fatty acids, including elongase 5, stearoyl CoA desaturase 1, or fatty acid synthase, with NaBt increasing expression of caveolin-1 and CD36 transporter, which may further promote DHA incorporation and its impact on cellular lipidome. In conclusion, our results indicate that interactions of DHA and NaBt exert complex changes in cellular lipidome, which may contribute to the alterations of colon cancer cell differentiation/apoptotic responses. The present data extend our knowledge about the nature of interactive effects of dietary fatty acids.

Bacterial lipid diversity.

The glycerophospholipids phosphatidylethanolamine, phosphatidylglycerol (PG), and cardiolipin (CL) are major structural components of bacterial membranes. In some bacteria, phosphatidylcholine or phosphatidylinositol and its derivatives form part of the membrane. PG or CL can be modified with the amino acid residues lysine, alanine, or arginine. Diacylglycerol is the lipid anchor from which syntheses of phosphorus-free glycerolipids, such as glycolipids, sulfolipids, or homoserine-derived lipids initiate. Many membrane lipids are subject to turnover and some of them are recycled. Other lipids associated with the membrane include isoprenoids and their derivatives such as hopanoids. Ornithine-containing lipids are widespread in Bacteria but absent in Archaea and Eukarya. Some lipids are probably associated exclusively with the outer membrane of many bacteria, i.e. lipopolysaccharides, sphingolipids, or sulfonolipids. For certain specialized membrane functions, specific lipid structures might be required. Upon cyst formation in Azotobacter vinelandii, phenolic lipids are accumulated in the membrane. Anammox bacteria contain ladderane lipids in the membrane surrounding the anammoxosome organelle, presumably to impede the passage of highly toxic compounds generated during the anammox reaction. Considering that present knowledge on bacterial lipids was obtained from only a few bacterial species, we are probably only starting to unravel the full scale of lipid diversity in bacteria. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.

Roles of specific lipid species in the cell and their molecular mechanism.

Thousands of different molecular species of lipids are present within a single cell, being involved in modulating the basic processes of life. The vast number of different lipid species can be organized into a number of different lipid classes, which may be defined as a group of lipids with a common chemical structure, such as the headgroup, apart from the nature of the hydrocarbon chains. Each lipid class has unique biological roles. In some cases, a relatively small change in the headgroup chemical structure can result in a drastic change in function. Such phenomena are well documented, and largely understood in terms of specific interactions with proteins. In contrast, there are observations that the entire structural specificity of a lipid molecule, including the hydrocarbon chains, is required for biological activity through specific interactions with membrane proteins. Understanding of these phenomena represents a fundamental change in our thinking of the functions of lipids in biology. There are an increasing number of diverse examples of roles for specific lipids in cellular processes including: Signal transduction; trafficking; morphological changes; cell division. We are gaining knowledge and understanding of the underlying molecular mechanisms. They are of growing importance in both basic and applied sciences.

Fatty acids composition of inner mitochondrial membrane of rat cardiomyocytes and hepatocytes during hypoxia-hypercapnia.

We studied the influence of hypoxic-hypercapnic environment under the effect of hypothermia (artificial hibernation) on fatty acids spectrum of inner mitochondrial membrane (IMM) lipids of rat cardiomyocytes and hepatocytes. Specific for cellular organelles redistribution of IMM fatty acids was determined. It led to the reduction of total amount of saturated fatty acids (SFAs) and increase of unsaturated fatty acids (UFAs) in cardiomyocytes and to the increase of SFAs and decrease of UFAs in hepatocytes. The decrease in the content of oleic acid and increased content of arachidonic and docosahexaenoic acids in IMM were shown. This may be due to their role in the regulatory systems during hibernation, as well as following exit therefrom. It is assumed that artificial hibernation state is characterized by the stress reaction leading to optimal readjustment of fatty acids composition of membrane lipids, which supports functional activity of mitochondria in hepatocytes and cardiomyocytes.

Identification of lipids in the cuticle of the parasitic nematode Anisakis simplex and the somatic tissues of the Atlantic cod Gadus morhua.

The main aim of this work was to assign the cuticular lipids identified in a parasitic nematode and to distinguish those originating from its host. The hypothesis that long-chained fatty acids and sterols are imported by the parasite in the absence of certain enzymes was also tested. The organisms (Anisakis simplex and Gadus morhua) were extracted in petroleum ether and dichloromethane. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) was used to identify unknown components, and electrospray ionization mass spectrometry (ESI/MS) to verify recognized groups of lipids. The lipid classes identified in the surface layer were free saturated and unsaturated fatty acids, triacylglycerols, sterols and non-polar sphingolipids (ceramides, sphingoid bases). The most abundant fraction consisted of fatty acids. The predominant saturated acids were tetradecanoic acid in the petroleum ether extract of A. simplex, hexadecanoic acid in the dichloromethane extract of A. simplex, and also the polyunsaturated octadecahexaenoic and octadecatrienoic acids in both extracts of the parasitic nematode. The mass spectrum revealed the presence of fatty acids with different numbers of carbons, and with odd and even numbers of unsaturated bonds. The MALDI-TOF mass spectrum also identified triacylglycerols (TAGs). The dominant short-chain TAGs were CoCoCy:(1), CoCoPg and Bu0:0B:(6). The majority of TAGs were found in the ether and dichloromethane extracts of A. simplex. Sterols were the least common class of lipids found in the nematode extracts; most likely, this is the fraction that is entirely incorporated from the host organism because of the parasite's inability to synthesize them. MALDI-TOF also identified non-polar sphingolipids--ceramides and sphingoid bases. The signals due to N-octanoyl-D-erythro-octasphinganine (m/z 288.3) and N-tetranoyl-D-erythro-tetradecasphinganine (m/z 316.4) were dominant on the mass spectra; quite a large number of short-chain non-polar sphingolipids were also identified.

Type A and type B monogalactosyldiacylglycerol synthases are spatially and functionally separated in the plastids of higher plants.

Mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively) constitute the bulk of membrane lipids in plant chloroplasts. The final step in MGDG biosynthesis occurs in the plastid envelope and is catalyzed by MGDG synthase. In Arabidopsis, the three MGDG synthases are classified into type A (atMGD1) and type B MGD isoforms (atMGD2 and atMGD3). atMGD1 is an inner envelope membrane-associated protein of chloroplasts and is responsible for the bulk of galactolipid biosynthesis in green tissues. MGD1 function is indispensable for thylakoid membrane biogenesis and embryogenesis. By contrast, type B atMGD2 and atMGD3 are localized in the outer envelopes and have no important role in chloroplast biogenesis or plant development under nutrient-sufficient conditions. These type B MGD genes are, however, strongly induced by phosphate (Pi) starvation and are essential for alternative galactolipid biosynthesis during Pi starvation. MGD1 gene expression is up-regulated by light and cytokinins. By contrast, Pi starvation-dependent expression of atMGD2/3 is suppressed by cytokinins but induced through auxin signaling pathways. These growth factors may control the functional sharing of the inner envelope pathway by atMGD1 and the outer envelope pathway by atMGD2/3 according to the growth environment.

Membrane lipids as signaling molecules.

PURPOSE OF REVIEW: Membrane lipids play important roles in signaling reactions. They are involved in most if not all cellular signaling cascades and in a wide variety of tissue and cell types. The purpose of this review is to highlight major pathways of signaling originating in membrane lipids. Details of lipid metabolism, and its relation to protein function, will thus advance understanding of the role of lipids in health and disease. RECENT FINDINGS: Major classes of lipids including glycerophospholipids, their metabolites (eicosanoids, endocannabinoids), and sphingolipids have recently generated interest in the field of signal transduction. These lipids are tightly regulated and have an impact on various physiological functions. Importantly, aberrant lipid metabolism often leads to onset of pathology, and thus the precise balance of signaling lipids and their effectors can serve as biomarkers. SUMMARY: Membrane lipids form precursors for second messengers and functional assembly matrices on membrane domains during cellular stimulation. Many of these modifications are rapid reactions at lipid headgroups. Metabolism of the fatty acyl portion of membrane lipids leads to the generation of a bewildering complexity of lipid mediators with extended effects in space and time.

Regiospecificity profiles of storage and membrane lipids from the gill and muscle tissue of Atlantic salmon (Salmo salar L.) grown at elevated temperature.

Regiospecific and traditional analysis, of both storage and membrane lipids, was performed on gill, white muscle, and red muscle samples taken from Atlantic salmon (Salmo salar) to gauge the effect of elevated water temperature. The fish, fed a commercial diet, were held at an elevated water temperature of 19 degrees C. Total n-3 PUFA, total PUFA, and n-3/n-6 and unsaturated/saturated fatty acid (UFA/SFA) ratios in the FA profile of the total lipid extract in the white muscle were fairly low compared with fish grown at 15 degrees C. Adaptation of structural and storage lipids at elevated temperatures was shown by a significant (P < 0.01) reduction in PUFA especially in the percentage of EPA (6-8%). Further adaptation was indicated by the percentages of SFA, which were significantly (P< 0.05) higher in gill (56%) and white muscle (58%) polar lipid fractions and coincided with lower percentages of n-3, n-6, and total PUFA. The regiospecific profiles indicated a high affinity of DHA to the sn-2 position in both the TAG (61-68%) and polar lipid (35-60%) fractions. The combination of detailed regiospecific and lipid analyses demonstrated adaptation of cell membrane structure in Atlantic salmon grown at an elevated water temperature.

Strategies and data analysis techniques for lipid and phospholipid chemistry elucidation by intact cell MALDI-FTMS.

Ions attributed to lipids and phospholipids are directly observed by desorption from whole bacteria using intact cell (IC) matrix-assisted laser desorption-ionization (MALDI) Fourier transform mass spectrometry (FTMS). Saccharomyces cerevisiae are grown in rich media broth, concentrated, and applied directly to the MALDI surface without lysis or chemical treatment. FTMS of MALDI ions gives excellent signal to noise ratios with typical resolving powers of 90,000 and mass precision better than 0.002 Da. Use of accurate mass measurements and a simple set of rules allow assignment of major peaks into one of twelve expected lipid classes. Subsequently, fractional mass versus whole number mass plots are employed to enhance visual interpretation of the high-resolution data and to facilitate detection of related ions such as those representing homologous series or different degrees of unsaturation. This approach, coupled with rules based on bacterial biochemistry, is used to classify ions with m/z up to about 1000. Major spectral peaks in the range m/z 200-1000 are assigned as lipids and phospholipids. In this study, it is assumed that biologically-derived ions with m/z values lower than 1000 are lipids. This is not unreasonable in view of the facts that molecular weights of lipids are almost always less than 1000 Da, that the copy numbers for lipids in a cell are higher than those for any single protein or other component, and that lipids are generally collections of distinct homologous partners, unlike proteins or other cell components. This paper presents a new rapid lipid-profiling method based on IC MALDI-FTMS.

X-ray crystallographic analysis of lipid-protein interactions in the bacteriorhodopsin purple membrane.

The past decade has witnessed increasingly detailed insights into the structural mechanism of the bacteriorhodopsin photocycle. Concurrently, there has been much progress within our knowledge pertaining to the lipids of the purple membrane, including the discovery of new lipids and the overall effort to localize and identify each lipid within the purple membrane. Therefore, there is a need to classify this information to generalize the findings. We discuss the properties and roles of haloarchaeal lipids and present the structural data as individual case studies. Lipid-protein interactions are discussed in the context of structure-function relationships. A brief discussion of the possibility that bacteriorhodopsin functions as a light-driven inward hydroxide pump rather than an outward proton pump is also presented.

The search for Brachyspira outer membrane proteins that interact with the host.

Little is known about the outer membrane structure of Brachyspira hyodysenteriae and Brachyspira pilosicoli or the role of outer membrane proteins (OMPs) in host colonization and the development of disease. The isolation of outer membrane vesicles from B. hyodysenteriae has confirmed that cholesterol is a significant outer membrane constituent and that it may impart unique characteristics to the lipid bilayer structure, including a reduced density. Unique proteins that have been identified in the B. hyodysenteriae outer membrane include the variable surface proteins (Vsp) and lipoproteins such as SmpA and BmpB. While the function of these proteins remains to be determined, there is indirect evidence to suggest that they may be involved in immune evasion. These data may explain the ability of the organism to initiate chronic infection. OMPs may be responsible for the unique attachment of B. pilosicoli to colonic epithelial cells; however, the only B. pilosicoli OMPs that have been identified to date are involved in metabolism. In order to identify further B. pilosicoli OMPs we have isolated membrane vesicle fractions from porcine strain 95-1000 by osmotic lysis and isopycnic centrifugation. The fractions were free of contamination by cytoplasm and flagella and contained outer membrane. Inner membrane contamination was minimal but could not be completely excluded. An abundant 45-kDa, heat-modifiable protein was shown to have significant homology with B. hyodysenteriae Vsp, and monoclonal antibodies were produced that reacted with five B. pilosicoli-specific membrane protein epitopes. The first of these proteins to be characterized is a unique surface-exposed lipoprotein.

The evolution of lipids.

Living organisms on the Earth which are divided into three major domains--Archaea, Bacteria, and Eucarya, probably came from a common ancestral cell. Because there are many thermophilic microorganisms near the root of the universal phylogenetic tree, the common ancestral cell should be considered to be a thermophilic microorganism. The existence of a cell is necessary for the living organisms; the cell membrane is the essential structural component of a cell, so its amphiphilic property is vital for the molecule of lipids for cell membranes. Tetraether type glycerophospholipids with C40 isoprenoid chains are major membrane lipids widely distributed in archaeal cells. Cyclization number of C40 isoprenoid chains in thermophilic archaea influences the fluidity of lipids whereas the number of carbons and degree of unsaturation in fatty acids do so in bacteria and eucarya. In addition to the cyclization of the tetraether lipids, covalent bonding of two C40 isoprenoid chains was found in hyperthermophiles. These characteristic structures of the lipids seem to contribute to their fundamental physiological roles in hyperthermophiles. Stereochemical differences between G-1-P archaeal lipids and G-3-P bacterial and eucaryal lipids might have occurred by the function of some proteins long after the first cell was developed by the reactions of small organic molecules. We propose that the structure of lipids of the common ancestral cell may have been similar to those of hyperthermophilic archaea.

Glycerophospholipids in brain: their metabolism, incorporation into membranes, functions, and involvement in neurological disorders.

Neural membranes contain several classes of glycerophospholipids which turnover at different rates with respect to their structure and localization in different cells and membranes. The glycerophospholipid composition of neural membranes greatly alters their functional efficacy. The length of glycerophospholipid acyl chain and the degree of saturation are important determinants of many membrane characteristics including the formation of lateral domains that are rich in polyunsaturated fatty acids. Receptor-mediated degradation of glycerophospholipids by phospholipases A(l), A(2), C, and D results in generation of second messengers such as arachidonic acid, eicosanoids, platelet activating factor and diacylglycerol. Thus, neural membrane phospholipids are a reservoir for second messengers. They are also involved in apoptosis, modulation of activities of transporters, and membrane-bound enzymes. Marked alterations in neural membrane glycerophospholipid composition have been reported to occur in neurological disorders. These alterations result in changes in membrane fluidity and permeability. These processes along with the accumulation of lipid peroxides and compromised energy metabolism may be responsible for the neurodegeneration observed in neurological disorders.