Qualitative characterization of the rat liver mitochondrial lipidome using LC–MS profiling and high energy collisional dissociation (HCD) all ion fragmentation

Lipids play multiple roles essential for proper mitochondrial function, from their involvement in membrane structure and fluidity, cellular energy storage, and signaling. Lipids are also major targets for reactive species, and their peroxidation byproducts themselves mediate further damage. Thousands of lipid species, from multiple classes and categories, are involved in these processes, suggesting lipid quantitative and structural analysis can help provide a better understanding of mitochondrial physiological status. Due to the diversity of lipids that contribute to and reflect mitochondrial function, analytical methods should ideally cover a wide range of lipid classes, and yield both quantitative and structural information. We developed a high resolution LC–MS method that is able to monitor the major lipid classes found in biospecimens (i.e. biofluids, cells and tissues) with relative quantitation in an efficient, sensitive, and robust manner while also characterizing individual lipid side-chains, by all ion high energy collisional dissociation fragmentation and chromatographic alignment. This method was used to profile the liver mitochondrial lipids from 192 rats undergoing a dietary macronutrient study in which changes in mitochondria function are related to changes in the major fat and glycemic index component of each diet. A total of 381 unique lipids, spanning 5 of the major LIPID MAPS defined categories, including fatty acyls, glycerophospholipids, glycerolipids, sphingolipids and prenols, were identified in mitochondria using the non-targeted LC–MS analysis in both positive and negative mode. The intention of this report is to show the breadth of this non-targeted LC–MS profiling method with regards to its ability to profile, identify and characterize the mitochondrial lipidome and the details of this will be discussed.     

Qualitative characterization of the rat liver mitochondrial lipidome using LC–MS profiling and high energy collisional dissociation (HCD) all ion fragmentation

Lipids play multiple roles essential for proper mitochondrial function, from their involvement in membrane structure and fluidity, cellular energy storage, and signaling. Lipids are also major targets for reactive species, and their peroxidation byproducts themselves mediate further damage. Thousands of lipid species, from multiple classes and categories, are involved in these processes, suggesting lipid quantitative and structural analysis can help provide a better understanding of mitochondrial physiological status. Due to the diversity of lipids that contribute to and reflect mitochondrial function, analytical methods should ideally cover a wide range of lipid classes, and yield both quantitative and structural information. We developed a high resolution LC–MS method that is able to monitor the major lipid classes found in biospecimens (i.e. biofluids, cells and tissues) with relative quantitation in an efficient, sensitive, and robust manner while also characterizing individual lipid side-chains, by all ion high energy collisional dissociation fragmentation and chromatographic alignment. This method was used to profile the liver mitochondrial lipids from 192 rats undergoing a dietary macronutrient study in which changes in mitochondria function are related to changes in the major fat and glycemic index component of each diet. A total of 381 unique lipids, spanning 5 of the major LIPID MAPS defined categories, including fatty acyls, glycerophospholipids, glycerolipids, sphingolipids and prenols, were identified in mitochondria using the non-targeted LC–MS analysis in both positive and negative mode. The intention of this report is to show the breadth of this non-targeted LC–MS profiling method with regards to its ability to profile, identify and characterize the mitochondrial lipidome and the details of this will be discussed.

     

Lipid particles/droplets of the yeast Saccharomyces cerevisiae revisited: lipidome meets proteome

In the yeast Saccharomyces cerevisiae as in other eukaryotes non-polar lipids are a reservoir of energy and building blocks for membrane lipid synthesis. The yeast non-polar lipids, triacylglycerols (TG) and steryl esters (SE) are stored in so-called lipid particles/droplets (LP) as biologically inert form of fatty acids and sterols. To understand LP structure and function in more detail we investigated the molecular equipment of this compartment making use of mass spectrometric analysis of lipids (TG, SE, phospholipids) and proteins. We addressed the question whether or not lipid and protein composition of LP influence each other and performed analyses of LP from cells grown on two different carbon sources, glucose and oleate. Growth of cells on oleate caused dramatic cellular changes including accumulation of TG at the expense of SE, enhanced the amount of glycerophospholipids and strongly increased the degree of unsaturation in all lipid classes. Most interestingly, oleate as a carbon source led to adaptation of the LP proteome resulting in the appearance of several novel LP proteins. Localization of these new LP proteins was confirmed by cell fractionation. Proteomes of LP variants from cells grown on glucose or oleate, respectively, were compared and are discussed with emphasis on the different groups of proteins detected through this analysis. In summary, we demonstrate flexibility of the yeast LP lipidome and proteome and the ability of LP to adapt to environmental changes.     

Effects of omega-3 and -6 fatty acids on Mycobacterium tuberculosis in macrophages and in mice

We recently showed that treatment of macrophages prior to Mycobacterium tuberculosis infection with the pro-inflammatory omega-6 lipid, arachidonic acid (AA) enhanced bacterial killing whereas the anti-inflammatory, omega-3 lipid eicosapentaenoic acid (EPA) stimulated bacterial growth. Here we tested if these effects were depending on when lipids were added to macrophages: before or during Mycobacterium smegmatis or M. tuberculosis infection. Collectively, our data suggested that a high omega-6 diet might be beneficial against mycobacteriosis, while a high omega-3 diet might be detrimental. AA also stimulated TNF-alpha secretion in M. tuberculosis-infected macrophages whereas EPA inhibited this process. AA strongly activated the MAP kinase p38 in uninfected cells but M. tuberculosis infected cells blocked the ability of AA to activate p38; AA-dependent killing is therefore independent of p38. We therefore tested diets enriched in omega-3 and omega-6 lipids on a mouse model of tuberculosis. In contrast to the in vitro results, the omega-6 tended to increase survival of M. tuberculosis in mice, while omega-3- tended to increase pathogen killing. Overall our results together with those previously reported in the literature suggest that it is almost impossible to predict, at the whole organism level, if a diet enriched in omega-3 or -6 will be beneficial or detrimental to intracellular pathogens.     

Identification of arylamine N -acetyltransferase inhibitors as an approach towards novel anti-tuberculars

New anti-tubercular drugs and drug targets are urgently needed to reduce the time for treatment and also to identify agents that will be effective against Mycobacterium tuberculosis persisting intracellularly. Mycobacteria have a unique cell wall. Deletion of the gene for arylamine N-acetyltransferase (NAT) decreases mycobacterial cell wall lipids, particularly the distinctive mycolates, and also increases antibiotic susceptibility and killing within macrophage of Mycobacterium bovis BCG. The nat gene and its associated gene cluster are almost identical in sequence in M. bovis BCG and M. tuberculosis. The gene cluster is essential for intracellular survival of mycobacteria. We have therefore used pure NAT protein for high-throughput screening to identify several classes of small molecules that inhibit NAT activity. Here, we characterize one class of such molecules— triazoles—in relation to its effects on the target enzyme and on both M. bovis BCG and M. tuberculosis. The most potent triazole mimics the effects of deletion of the nat gene on growth, lipid disruption and intracellular survival. We also present the structure-activity relationship between NAT inhibition and effects on mycobacterial growth, and use ligand-protein analysis to give further insight into the structure-activity relationships. We conclude that screening a chemical library with NAT protein yields compounds that have high potential as anti-tubercular agents and that the inhibitors will allow further exploration of the biochemical pathway in which NAT is involved.     

Tracking microdomain dynamics in cell membranes

Studies of the diffusion of proteins and lipids in the plasma membrane of cells have long pointed to the presence of membrane domains. A major challenge in the field of membrane biology has been to characterize the various cellular structures and mechanisms that impede free diffusion in cell membranes and determine the consequences that membrane compartmentalization has on cellular biology. In this review, we will provide a brief summary of the classes of domains that have been characterized to date, focusing on recent efforts to identify the properties of lipid rafts in cells through measurements of protein and lipid diffusion.     

Lipids in the structure,folding, and function of the KcsA K+ channel

Lipid molecules surround an ion channel in its native environment of cellular membranes. The importance of the lipid bilayer and the role of lipid protein interactions in ion channel structure and function are not well understood. Here we demonstrate that the bacterial potassium channel KcsA binds a negatively charged lipid molecule. We have defined the potential binding site of the lipid molecule on KcsA by X-ray crystallographic analysis of a complex of KcsA with a monoclonal antibody Fab fragment. We also demonstrate that lipids are required for the in vitro refolding of the KcsA tetramer from the unfolded monomeric state. The correct refolding of the KcsA tetramer requires lipids, but it is not dependent on negatively charged lipids as refolding takes place in the absence of such lipids. We confirm that the presence of negatively charged lipids is required for ion conduction through the KcsA potassium channel, suggesting that the lipid bound to KcsA is important for ion channel function.     

A targeted mass spectrometric analysis of phosphatidylinositol phosphate species

The development of a new mass spectrometric lipid profiling methodology permits the identification of cellular phosphatidylinositol monophosphate/phosphatidylinositol bisphosphate/phosphatidylinositol trisphosphate (PIP/PIP2/PIP3) species that includes the fatty acyl composition. Using electrospray ionization mass spectrometry, we were able to resolve and identify 28 PIP and PIP2 compounds as well as 8 PIP3 compounds from RAW 264.7 or primary murine macrophage cell extracts. Analysis of PIP profiles after agonist stimulation of cells revealed the generation of differential PIP3 species and permitted us to propose a novel means for regulation and specificity in signaling through PIP3. This is the first reported identification of intact, cellular PIP3 by mass spectral analysis. The ability to analyze the fatty acyl chain composition of signaling lipids initiates new venues for investigation of the processes by which specific polyphosphoinositide species mediate.     

Neutral-red reaction is related to virulence and cell wall methyl-branched lipids in Mycobacterium tuberculosis

Searching for virulence marking tests for Mycobacterium tuberculosis, Dubos and Middlebrook reported in 1948 that in an alkaline aqueous solution of neutral-red, the cells of the virulent H37Rv M. tuberculosis strain fixed the dye and became red in color, whereas the cells of the avirulent H37Ra M. tuberculosis strain remained unstained. In the 1950 and 1960s, fresh isolates of M. tuberculosis were tested for this neutral-red cytochemical reaction and it was reported that they were neutral-red positive, whereas other mycobacteria of diverse environmental origins that were non-pathogenic for guinea pigs were neutral-red negative. However, neutral-red has not really been proven to be a virulence marker. To test if virulence is in fact correlated to neutral-red, we studied a clinical isolate of M. tuberculosis that was originally neutral-red positive but, after more than 1 year passing through culture mediums, turned neutral-red negative. We found that, in comparison to the original neutral-red positive strain, this neutral-red negative variant was attenuated in two murine models of experimental tuberculosis. Lipid analysis showed that this neutral-red negative natural mutant lost the capacity to synthesize pthiocerol dimycocerosates, a cell wall methyl-branched lipid that has been related to virulence in M. tuberculosis. We also studied the neutral-red of different gene-targeted M. tuberculosis mutants unable to produce pthiocerol dimycocerosates or other cell wall methyl-branched lipids such as sulfolipids, and polyacyltrehaloses. We found a negative neutral-red reaction in mutants that were deficient in more than one type of methyl-branched lipids. We conclude that neutral-red is indeed a marker of virulence and it indicates important perturbations in the external surface of M. tuberculosis cells.     

The ins and outs of endoplasmic reticulum‐controlled lipid biosynthesis

Endoplasmic reticulum (ER)‐localized enzymes synthesize the vast majority of cellular lipids. The ER therefore has a major influence on cellular lipid biomass and balances the production of different lipid categories, classes, and species. Signals from outside and inside the cell are directed to ER‐localized enzymes, and lipid enzyme activities are defined by the integration of internal, homeostatic, and external information. This allows ER‐localized lipid synthesis to provide the cell with membrane lipids for growth, proliferation, and differentiation‐based changes in morphology and structure, and to maintain membrane homeostasis across the cell. ER enzymes also respond to physiological signals to drive carbohydrates and nutritionally derived lipids into energy‐storing triglycerides. In this review, we highlight some key regulatory mechanisms that control ER‐localized enzyme activities in animal cells. We also discuss how they act in concert to maintain cellular lipid homeostasis, as well as how their dysregulation contributes to human disease.     

Cold adaptation in the Antarctic Archaeon Methanococcoides burtonii involves membrane lipid unsaturation

Direct analysis of membrane lipids by liquid chromatography-electrospray mass spectrometry was used to demonstrate the role of unsaturation in ether lipids in the adaptation of Methanococcoides burtonii to low temperature. A proteomics approach using two-dimensional liquid chromatography-mass spectrometry was used to identify enzymes involved in lipid biosynthesis, and a pathway for lipid biosynthesis was reconstructed from the M. burtonii draft genome sequence. The major phospholipids were archaeol phosphatidylglycerol, archaeol phosphatidylinositol, hydroxyarchaeol phosphatidylglycerol, and hydroxyarchaeol phosphatidylinositol. All phospholipid classes contained a series of unsaturated analogues, with the degree of unsaturation dependent on phospholipid class. The proportion of unsaturated lipids from cells grown at 4 degrees C was significantly higher than for cells grown at 23 degrees C. 3-Hydroxy-3-methylglutaryl coenzyme A synthase, farnesyl diphosphate synthase, and geranylgeranyl diphosphate synthase were identified in the expressed proteome, and most genes involved in the mevalonate pathway and processes leading to the formation of phosphatidylinositol and phosphatidylglycerol were identified in the genome sequence. In addition, M. burtonii encodes CDP-inositol and CDP-glycerol transferases and a number of homologs of the plant geranylgeranyl reductase. It therefore appears that the unsaturation of lipids may be due to incomplete reduction of an archaeol precursor rather than to a desaturase mechanism. This study shows that cold adaptation in M. burtonii involves specific changes in membrane lipid unsaturation. It also demonstrates that global methods of analysis for lipids and proteomics linked to a draft genome sequence can be effectively combined to infer specific mechanisms of key biological processes.     

Probing lipid-protein interactions using lipid microarrays

Lipids are central to the regulation and control of several cellular functions. They form many of the important structural features of cells, and are critical members of cellular signal transduction pathways. Cellular dysfunction is often caused by errors in lipid signaling; therefore, the proteins that interact with, synthesize or metabolize the lipids are potential therapeutic targets. Characterizing the contingent of cellular lipids and their abundance and how this is associated with disease will facilitate understanding how to intervene to correct diseases caused by dysfunctional lipid signaling. Since lipid-signaling networks involve several classes of proteins it is essential to determine the identity and role of these proteins in order to understand the networks. These proteins may be receptors, effectors, transporters or enzymes. We present tools, specifically, a lipid microarray platform, to uncover lipid-binding effector proteins that function in lipid signaling pathways. Lipid microarrays will allow researchers to obtain a comparable fingerprint of the proteins from a cell or tissue that bind to lipids, and also enable the identification of functionally important lipid-binding proteins. By applying a systematic approach to the quantification of lipid-protein interactions, lipid microarrays will provide an integrated knowledge base for the human lipidome. These tools have the potential to identify and validate targets to improve personalized medicine and health.     

The virulence-associated two-component PhoP-PhoR system controls the biosynthesis of polyketide-derived lipids in Mycobacterium tuberculosis

Two-component regulatory signal transduction systems are important elements of the adaptative response of prokaryotes to a variety of environmental stimuli. Disruption of PhoP-PhoR in Mycobacterium tuberculosis dramatically attenuates virulence, implying that this system directly and/or indirectly coordinates the expression of important virulence factors whose identity remains to be established. Interestingly, in knockingout the PhoP-PhoR two-component system in M. tuberculosis Mt103, dramatic changes in the colonial morphology, cording properties, and reactivity of the mutant strain to the basic dye neutral red, all intrinsic properties of tubercle bacilli known to correlate with virulence, were noted. Because deficiencies in the ability of the mutant to form serpentine cords and stain with the dye are likely the results of alterations of its cell envelope composition, we undertook to analyze the lipid content of phoP and phoP-phoR mutants constructed in two different strains of M. tuberculosis. Our results indicate that PhoP coordinately and positively regulates the synthesis of methyl-branched fatty acid-containing acyltrehaloses known to be restricted to pathogenic species of the M. tuberculosis complex, namely diacyltrehaloses, polyacyltrehaloses, and sulfolipids. Evidence is also provided that PhoP but not PhoR is required for the production of these lipids. This work represents an important step toward the functional characterization of PhoP-PhoR and the understanding of complex lipid synthesis in M. tuberculosis.     

Functional analyses of mycobacterial lipoprotein diacylglyceryl transferase and comparative secretome analysis of a mycobacterial lgt mutant

Preprolipopoprotein diacylglyceryl transferase (Lgt) is the gating enzyme of lipoprotein biosynthesis, and it attaches a lipid structure to the N-terminal part of preprolipoproteins. Using Lgt from Escherichia coli in a BLASTp search, we identified the corresponding Lgt homologue in Mycobacterium tuberculosis and two homologous (MSMEG_3222 and MSMEG_5408) Lgt in Mycobacterium smegmatis. M. tuberculosis lgt was shown to be essential, but an M. smegmatis ΔMSMEG_3222 mutant could be generated. Using Triton X-114 phase separation and [(14)C]palmitic acid incorporation, we demonstrate that MSMEG_3222 is the major Lgt in M. smegmatis. Recombinant M. tuberculosis lipoproteins Mpt83 and LppX are shown to be localized in the cell envelope of parental M. smegmatis but were absent from the cell membrane and cell wall in the M. smegmatis ΔMSMEG_3222 strain. In a proteomic study, 106 proteins were identified and quantified in the secretome of wild-type M. smegmatis, including 20 lipoproteins. All lipoproteins were secreted at higher levels in the ΔMSMEG_3222 mutant. We identify the major Lgt in M. smegmatis, show that lipoproteins lacking the lipid anchor are secreted into the culture filtrate, and demonstrate that M. tuberculosis lgt is essential and thus a validated drug target.     

Do lipids shape the eukaryotic cell cycle?

Successful passage through the cell cycle presents a number of structural challenges to the cell. Inceptive studies carried out in the last five years have produced clear evidence of modulations in the lipid profile (sometimes referred to as the lipidome) of eukaryotes as a function of the cell cycle. This mounting body of evidence indicates that lipids play key roles in the structural transformations seen across the cycle. The accumulation of this evidence coincides with a revolution in our understanding of how lipid composition regulates a plethora of biological processes ranging from protein activity through to cellular signalling and membrane compartmentalisation. In this review, we discuss evidence from biological, chemical and physical studies of the lipid fraction across the cell cycle that demonstrate that lipids are well-developed cellular components at the heart of the biological machinery responsible for managing progress through the cell cycle. Furthermore, we discuss the mechanisms by which this careful control is exercised.     

Separation and quantification of chicken and bovine growth plate cartilage matrix vesicle lipids by high-performance liquid chromatography using evaporative light scattering detection

Matrix vesicles (MV) are lipid bilayer-enclosed nanoscale structures that initiate extracellular mineral formation in most vertebrate species. Little attention has been given to differences between species in membrane lipid composition or to how new mineral is formed in MV. To explore more precisely the lipids of MV isolated from avian and bovine species, we developed a new high-performance liquid chromatography (HPLC) method used in combination with evaporative light scattering detection (ELSD) to quantify their lipid composition. HPLC analyses were performed on a Lichrosorb silica column using separate binary gradient elution systems for analyzing polar and nonpolar lipids. Standard mixtures of both phospholipids and nonpolar lipids were used to prepare calibration curves for each lipid, which were analyzed mathematically by polynomial regression for accurate quantitation. This new methodology provides high-resolution separations and quantitation of both the polar and the nonpolar lipids typically present in biological membranes, including lyso- (monoacyl-) phospholipids. We have applied this method to quantitate the phospholipid and nonpolar lipid composition of MV isolated from chicken and bovine growth plate cartilage. We also compared the ability of these MV to induce mineral formation. While the ability to induce mineralization and the lipid composition were generally similar, some significant differences between MV from these two disparate species were seen.     

M. tuberculosis Rv2252 encodes a diacylglycerol kinase involved in the biosynthesis of phosphatidylinositol mannosides (PIMs)

Phosphorylated lipids play important roles in biological systems, not only as structural moieties but also as modulators of cellular function. Phospholipids of pathogenic bacteria are known to play roles both as membrane components and as factors that modulate the infectious process. Mycobacterium tuberculosis is, however, noteworthy in that it has an extremely diverse repertoire of biologically active phosphorylated lipids that, in the absence of a specialized protein translocation system, appear to constitute the main means of communication with the host. Many of these lipids are derived from phosphatidylinositol (PI) that is differentially processed to give rise to phosphatidylinositol mannosides (PIMs) or lipoarabinomannan. In preliminary studies on the lipid processing enzymes associated with the bacterial cell wall, a kinase activity was noted that gave rise to a novel lipid species released by the bacterium. It was determined that this kinase activity was encoded by the ORF Rv2252. Rv2252 demonstrates the capacity to phosphorylate various amphipathic lipids of host and bacterial origin, in particular a M. tuberculosis derived diacylglycerol. Targeted deletion of the rv2252 gene resulted in disruption of the production of certain higher order PIM species, suggesting a role for Rv2252 in the biosynthetic pathway of PI, a PIM precursor.