Variation of the detergent-binding capacity and phospholipid content of membrane proteins when purified in different detergents.

Purified membrane proteins are ternary complexes consisting of protein, lipid, and detergent. Information about the amounts of detergent and endogenous phospholipid molecules bound to purified membrane proteins is largely lacking. In this systematic study, three model membrane proteins of different oligomeric states were purified in nine different detergents at commonly used concentrations and characterized biochemically and biophysically. Detergent-binding capacities and phospholipid contents of the model proteins were determined and compared. The insights on ternary complexes obtained from the experimental results, when put into a general context, are summarized as follows. 1), The amount of detergent and 2) the amount of endogenous phospholipids bound to purified membrane proteins are dependent on the size of the hydrophobic lipid-accessible protein surface areas and the physicochemical properties of the detergents used. 3), The size of the detergent and lipid belt surrounding the hydrophobic lipid-accessible surface of purified membrane proteins can be tuned by the appropriate choice of detergent. 4), The detergents n-nonyl-β-D-glucopyranoside and Cymal-5 have exceptional delipidating effects on ternary complexes. 5), The types of endogenous phospholipids bound to membrane proteins can vary depending on the detergent used for solubilization and purification. 6), Furthermore, we demonstrate that size-exclusion chromatography can be a suitable method for estimating the molecular mass of ternary complexes. The findings presented suggest a strategy to control and tune the numbers of detergent and endogenous phospholipid molecules bound to membrane proteins. These two parameters are potentially important for the successul crystallization of membrane proteins for structure determination by crystallographic approaches.

Cholesterol metabolism, transport, and hepatic regulation in dairy cows during transition and early lactation

The transition from the nonlactating to the lactating state represents a critical period for dairy cow lipid metabolism because body reserves have to be mobilized to meet the increasing energy requirements for the initiation of milk production. The purpose of this study was to provide a comprehensive overview on cholesterol homeostasis in transition dairy cows by assessing in parallel plasma, milk, and hepatic tissue for key factors of cholesterol metabolism, transport, and regulation. Blood samples and liver biopsies were taken from 50 multiparous Holstein dairy cows in wk 3 antepartum (a.p.), wk 1 postpartum (p.p.), wk 4 p.p., and wk 14 p.p. Milk sampling was performed in wk 1, 4, and 14 p.p. Blood and milk lipid concentrations [triglycerides (TG), cholesterol, and lipoproteins], enzyme activities (phospholipid transfer protein and lecithin:cholesterol acyltransferase) were analyzed using enzymatic assays. Hepatic gene expression patterns of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGC) synthase 1 (HMGCS1) and HMGC reductase (HMGCR), sterol regulatory element-binding factor (SREBF)-1 and -2, microsomal triglyceride transfer protein (MTTP), ATP-binding cassette transporter (ABC) A1 and ABCG1, liver X receptor (LXR) α and peroxisome proliferator activated receptor (PPAR) α and γ were measured using quantitative RT-PCR. Plasma TG, cholesterol, and lipoprotein concentrations decreased from wk 3 a.p. to a minimum in wk 1 p.p., and then gradually increased until wk 14 p.p. Compared with wk 4 p.p., phospholipid transfer protein activity was increased in wk 1 p.p., whereas lecithin:cholesterol acyltransferase activity was lowest at this period. Total cholesterol concentration and mass, and cholesterol concentration in the milk fat fraction decreased from wk 1 p.p. to wk 4 p.p. Both total and milk fat cholesterol concentration were decreased in wk 4 p.p. compared with wk 1 and 14 p.p. The mRNA abundance of genes involved in cholesterol synthesis (SREBF-2, HMGCS1, and HMGCR) markedly increased from wk 3 a.p. to wk 1 p.p., whereas SREBF-1 was downregulated. The expression of ABCA1 increased from wk 3 a.p. to wk 1 p.p., whereas ABCG1 was increased in wk 14 p.p. compared with other time points. In conclusion, hepatic expression of genes involved in the biosynthesis of cholesterol as well as the ABCA1 transporter were upregulated at the onset of lactation, whereas plasma concentrations of total cholesterol, phospholipids, lipoprotein-cholesterol, and TG were at a minimum. Thus, at the gene expression level, the liver seems to react to the increased demand for cholesterol after parturition. Whether the low plasma cholesterol and TG levels are due to impaired hepatic export mechanisms or reflect an enhanced transfer of these compounds into the milk to provide essential nutrients for the newborn remains to be elucidated.

Metabolomics reveals herbivore-induced metabolites of resistance and susceptibility in maize leaves and roots

Plants respond to herbivory by reprogramming their metabolism. Most research in this context has focused on locally induced compounds that function as toxins or feeding deterrents. We developed an ultra-high-pressure liquid chromatography time-of-flight mass spectrometry (UHPLC-TOF-MS)-based metabolomics approach to evaluate local and systemic herbivore-induced changes in maize leaves, sap, roots and root exudates without any prior assumptions about their function. Thirty-two differentially regulated compounds were identified from Spodoptera littoralis-infested maize seedlings and isolated for structure assignment by microflow nuclear magnetic resonance (CapNMR). Nine compounds were quantified by a high throughput direct nano-infusion tandem mass spectrometry/mass spectrometry (MS/MS) method. Leaf infestation led to a marked local increase of 1,3-benzoxazin-4-ones, phospholipids, N-hydroxycinnamoyltyramines, azealic acid and tryptophan. Only few changes were found in the root metabolome, but 1,3-benzoxazin-4-ones increased in the vascular sap and root exudates. The role of N-hydroxycinnamoyltyramines in plant–herbivore interactions is unknown, and we therefore tested the effect of the dominating p-coumaroyltyramine on S. littoralis. Unexpectedly, p-coumaroyltyramine was metabolized by the larvae and increased larval growth, possibly by providing additional nitrogen to the insect. Taken together, this study illustrates that herbivore attack leads to the induction of metabolites that can have contrasting effects on herbivore resistance in the leaves and roots.

Evolution of the spermadhesin gene family

Spermadhesins belong to a novel family of secretory proteins of the male genital tract. They are major proteins of the seminal plasma and have been found peripherally associated to the sperm surface. So far, they have only been detected in ungulates, specifically in pig, cattle, and horse, respectively. Spermadhesins form a subgroup of the superfamily of proteins with a CUB-domain that has been found in a variety of developmentally regulated proteins. The structure and function of the spermadhesins have been investigated in the pig. They are multifunctional proteins showing a range of ligand-binding abilities, e.g. to carbohydrates, phospholipids, and protease inhibitors, suggesting that they may be involved in different steps of fertilization. We report here the genomic organization of the porcine spermadhesin gene cluster as well as a detailed comparative analysis with respect to other mammalian species. The porcine spermadhesin genes are located on SSC 14q28-q29 in a region syntenic to HSA 10q26. The pig contains five closely linked spermadhesin genes, whereas only two spermadhesin genes are present in the cattle genome. Inactive copies of spermadhesin genes are still detectable in the human, chimp, and dog genome while the corresponding region was lost from the rodent genomes of mouse and rat. Within the pig, the five spermadhesin genes contain both highly diverged and highly conserved regions. Interestingly, the pattern of divergence does not correlate with the position of the exons. Evolutionary analyses suggest that the pattern of diversity is shaped by ancestral variation, recombination, and new mutations.

Phosphatidylethanolamine and phosphatidylcholine biosynthesis by the Kennedy pathway occurs at different sites in Trypanosoma brucei

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are among the most abundant phospholipids in biological membranes. In many eukaryotes, the CDP-ethanolamine and CDP-choline branches of the Kennedy pathway represent major and often essential routes for the production of PE and PC, with ethanolamine and choline/ethanolamine phosphotransferases (EPT and CEPT, respectively) catalysing the last reactions in the respective pathways. Although the site of PE and PC synthesis is commonly known to be the endoplasmic reticulum (ER), detailed information on the localization of the different phosphotransferases is lacking. In the unicellular parasite, Trypanosoma brucei, both branches of the Kennedy pathway are essential for cell growth in culture. We have previously reported that T. brucei EPT (TbEPT) catalyses the production of ether-type PE molecular species while T. brucei CEPT (TbCEPT) synthesizes diacyl-type PE and PC molecular species. We now show that the two enzymes localize to different sub-compartments of the ER. By expressing a series of tagged forms of the two enzymes in T. brucei parasites, in combination with sub-cellular fractionation and enzyme activity measurements, TbEPT was found exclusively in the perinuclear ER, a distinct area located close to but distinct from the nuclear membrane. In contrast, TbCEPT was detected in the bulk ER.

Response of the cholesterol metabolism to a negative energy balance in dairy cows depends on the lactational stage.

The response of cholesterol metabolism to a negative energy balance (NEB) induced by feed restriction for 3 weeks starting at 100 days in milk (DIM) compared to the physiologically occurring NEB in week 1 postpartum (p.p.) was investigated in 50 dairy cows (25 control (CON) and 25 feed-restricted (RES)). Blood samples, liver biopsies and milk samples were taken in week 1 p.p., and in weeks 0 and 3 of feed restriction. Plasma concentrations of total cholesterol (C), phospholipids (PL), triglycerides (TAG), very low density lipoprotein-cholesterol (VLDL-C) and low density lipoprotein-cholesterol (LDL-C) increased in RES cows from week 0 to 3 during feed restriction and were higher in week 3 compared to CON cows. In contrast, during the physiologically occurring NEB in week 1 p.p., C, PL, TAG and lipoprotein concentrations were at a minimum. Plasma phospholipid transfer protein (PLTP) and lecithin:cholesterol acyltransferase (LCAT) activities did not differ between week 0 and 3 for both groups, whereas during NEB in week 1 p.p. PLTP activity was increased and LCAT activity was decreased. Milk C concentration was not affected by feed restriction in both groups, whereas milk C mass was decreased in week 3 for RES cows. In comparison, C concentration and mass in milk were elevated in week 1 p.p. Hepatic mRNA abundance of sterol regulatory element-binding factor-2 (SREBF-2), 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), and ATP-binding cassette transporter (ABCA1) were similar in CON and RES cows during feed restriction, but were upregulated during NEB in week 1 p.p. compared to the non-lactating stage without a NEB. In conclusion, cholesterol metabolism in dairy cows is affected by nutrient and energy deficiency depending on the stage of lactation.

Trypanosoma brucei Bloodstream Forms Depend upon Uptake of myo-Inositol for Golgi Complex Phosphatidylinositol Synthesis and Normal Cell Growth

myo-Inositol is a building block for all inositol-containing phospholipids in eukaryotes. It can be synthesized de novo from glucose-6-phosphate in the cytosol and endoplasmic reticulum. Alternatively, it can be taken up from the environment via Na(+)- or H(+)-linked myo-inositol transporters. While Na(+)-coupled myo-inositol transporters are found exclusively in the plasma membrane, H(+)-linked myo-inositol transporters are detected in intracellular organelles. In Trypanosoma brucei, the causative agent of human African sleeping sickness, myo-inositol metabolism is compartmentalized. De novo-synthesized myo-inositol is used for glycosylphosphatidylinositol production in the endoplasmic reticulum, whereas the myo-inositol taken up from the environment is used for bulk phosphatidylinositol synthesis in the Golgi complex. We now provide evidence that the Golgi complex-localized T. brucei H(+)-linked myo-inositol transporter (TbHMIT) is essential in bloodstream-form T. brucei. Downregulation of TbHMIT expression by RNA interference blocked phosphatidylinositol production and inhibited growth of parasites in culture. Characterization of the transporter in a heterologous expression system demonstrated a remarkable selectivity of TbHMIT for myo-inositol. It tolerates only a single modification on the inositol ring, such as the removal of a hydroxyl group or the inversion of stereochemistry at a single hydroxyl group relative to myo-inositol.

INVESTIGATIONS ON THE INTRACELLULAR LOCALIZATION OF PHOSPHATIDYLSERINE SYNTHASE FROM ESCHERICHIA COLI

Phosphatidylserine synthase catalyzes the committed step in the synthesis of the major lipid of Escherichia coli, phosphatidylethanolamine, and may be involved in regulating the balance of the zwitterionic and anionic phospholipids in the membrane. Unlike the other enzymes involved in the biosynthesis of phospholipids in E. coli, phosphatidylserine synthase is not membrane associated but seems to have a high affinity for the ribosomal fraction of cells broken by various methods. Investigations on the enzyme in cell free extracts using glycerol gradient centrifugation revealed that the binding of the synthase to ribosomes may be prevented by the presence of highly basic compounds such as spermidine and by the presence of detergent-lipid substrate micelles under assay conditions. Thus phosphatidylserine synthase may not be ribosome associated under physiological conditions but associated with its membrane bound substrate (Louie and Dowhan (1980) J. Biol. Chem. 255, 1124).^ In addition homogeneous enzyme shows many of the properties of a membrane associated protein. It binds nonionic detergent such as Triton X-100, which is also required during purification of the enzyme. Optimal catalytic activity is also dependent on micelle or surface bound substrate. Phosphatidylserine synthase has been synthesized in vitro using a coupled transcription-translation system dependent on the presence of the cloned structural gene. The translation product was found to preferentially associate with the ribosomal fraction even in the presence of added E. coli membranes. Preferential membrane binding could be induced if the membranes were supplemented with the lipid substrate CDP-diacylglycerol. Similar effects were obtained with the acidic lipids phosphatidylglycerol and cardiolipin. On the other hand the zwitterionic lipid phosphatidylethanolamine and the lipid product phosphatidylserine did not cause any detectable membrane association. These results are consistent with the enzyme recognizing membrane bound substrate (Carman and Dowhan (1979) J. Biol. Chem. 254, 8391) and with the lipid charge influencing membrane interaction.^ Phosphatidylserine synthase is at a branch point in lipid metabolism, which may determine the distribution of the zwitterionic and anionic phospholipids in the membrane. The results obtained here indicate phosphatidylserine synthase may play a significant role in membrane lipid biosynthesis by maintaining charge balance of the E. coli membrane. In determining the localization of phosphatidylserine synthase in vitro one may have a better understanding of its function and control in vivo and may also have a better understanding of its role in membrane assembly.^

Novel mechanisms for PKC family enzymes to regulate PI3K signaling pathway

Phosphatidylinositol 3-kinase (PI3K) generates membrane phospholipids that serve as second messengers to recruit signaling proteins to plasma membrane consequently regulating cell growth and survival. PI3K is a heterodimer consisting of a catalytic p110 subunit and a regulatory p85 subunit. Association of the p85 with other signal proteins is critical for induced PI3K activation. Activated PI3K, in turn, leads to signal flows through a variety of PI3K effectors including PDK1, AKT, GSK3, BAD, p70 S6K and NFκB. The PI3K pathway is under regulation by multiple signal proteins representing cross-talk between different signaling cascades. In this study, we have evaluated the role of protein kinase C family kinases on signaling through PI3K at multiple levels. Firstly, we observed that the action of PKC specific inhibitors like Ro-31-8220 and GF109203X was associated with an increased AKT phosphorylation and activity, suggesting that PKC kinases might play a negative role in the regulation of PI3K pathway. Then, we demonstrated the stimulation of AKT by PKC inhibition was dependent on functional PI3K enzyme and able to be transmitted to the AKT effector p70 S6K. Furthermore, we showed an inducible physical association between the PKCζ isotype and AKT, which was accompanied by an attenuated AKT activity. However, a kinase-dead form of PKC failed to affect AKT. In the second part of our research we revealed the ability of a different PKC family member, PKCδ to bind to the p85 subunit of PI3K in response to oxidative stress, a process requiring the activity of src tyrosine kinases. The interaction was demonstrated to be a direct and specific contact between the carboxyl terminal SH2 domain of p85 and tyrosine phosphorylated PKCδ. Several different types of agonists were capable to induce this association including tyrosine kinases and phorbol esters with PKCδ tyrosine phosphorylation being integral components. Finally, the PKCδ-PI3K complex was related to a reduction in the AKT phosphorylation induced by src. A kinase-deficient mutant of PKCδ was equally able to inhibit AKT signal as the wild type, indicative of a process independent of PKCδ catalytic activity. Altogether, our data illustrate different PKC isoforms regulating PI3K pathway at multiple levels, suggesting a mechanism to control signal flows through PI3K for normal cell activities. Although further investigation is required for full understanding of the regulatory mechanism, we propose that complex formation of signal proteins in PI3K pathway and specific PKC isoforms plays important role in their functional linkage. ^

Function of cardiolipin in mitochondria of Saccharomyces cerevisiae

Cardiolipin and its precursor phosphatidylglycerol, phospholipids found uniquely in membranes engaged in oxidative phosphorylation, play important roles in multimeric complexes of the energy transducing system (ETS) associated with the inner mitochondrial membrane. A combined molecular genetic and biochemical approach was used to more precisely define the role of cardiolipin in cell processes. ^ Strains of yeast Saccharomyces cerevisiae unable to synthesize cardiolipin because of the crd1Δ allele (encodes cardiolipin synthase) with different phenotypes were analyzed to determine which phenotypes are due to lack of cardiolipin. We concluded that many of the severe phenotypes ascribed to cells lacking cardiolipin, particularly when grown at 37°C, are because of the synergistic interaction of the crd1Δ mutation with the reduced expression of the PET56 gene which encodes a component essential for the formation of functional mitochondrial ribosomes. We also demonstrate that much of the reduced mitochondrial function in crd1Δ is because of reduced expression of ETS components at elevated temperature. ^ A crd1Δ mutant of S. cerevisiae has less severe physiological changes than strains lacking both phosphatidylglycerol and cardiolipin due to an increased level of phosphatidylglycerol, which might partially substitute for the cardiolipin-requiring functions. By varying the level of cardiolipin, we were able to correlate phenotypes in a dose-dependent manner with the level of cardiolipin to support more strongly an involvement of cardiolipin in a particular cellular process. There is almost complete lack of a supercomplex composed of cytochrome bc1 complex (complex III) and cytochrome c oxidase (complex IV) in extracts of cardiolipin-lacking mitochondria when compared to wild type cells and the level of supercomplex varies in proportion to the cardiolipin levels. Reduced cardiolipin levels also compromise the growth properties of yeast in a dose-dependent manner suggesting that the loss in growth efficiency is related to a role of cardiolipin that cannot be replaced by phosphatidylglycerol. An independent kinetic approach was performed to compare organization of the respiratory chain in wild-type and cardiolipin-lacking mitochondria. Cardiolipin-lacking mitochondria display kinetic properties for electron transfer between complexes III and IV via cytochrome c consistent with cytochrome c being a freely diffusible carrier, confirming complexes III and IV exist as individual complexes and not associated into a supercomplex in cardiolipin-lacking mitochondria. ^

The LPS O -antigen is required for Myxococcus xanthus social motility and multicellular development

The gliding bacterium Myxococcus xanthus aggregates to form spore-filled fruiting bodies when starved at high density. All of the identified M. xanthus lipopolysaccharide (LPS) O-antigen biosynthesis mutants exhibit defective motility and fruiting-body development. To determine the cause of these phenotypes, the cell-surface properties of the LPS O-antigen mutants were compared to wild-type cells. The binding characteristics of wild-type and LPS O-antigen-defective strains to cationic resin indicate that the mutant cell surfaces are more electronegative. Antibiotic sensitivity and hexadecane adhesion assays indicate that the wild-type M. xanthus cell surface is hydrophobic, supporting the idea that phospholipids are present in the outer leaflet of the outer membrane. The absence of the LPS O-antigen appears to expose charges associated with phospholipids and LPS core/lipid A, resulting in a dramatic alteration of the cell-surface organization and charge. These differences may affect the interaction of the LPS O-antigen mutants with their substratum and neighboring cells, leading to defects in social and single-cell gliding motility and thus, deficiencies in fruiting body formation. ^ The LPS O-antigen biosynthetic mutations also bypass the requirement of 4521 gene expression for the cell-density signal, A signal. The 4521 gene is overexpressed in these mutants. This 4521 overexpression is dependent on the sensor kinase SasS. Co-development with wild-type cells, or the addition of crude polysaccharides or membrane vesicles restores the ability of LPS O-antigen mutants to form fruiting bodies and lowers 4521 developmental gene expression to wild-type levels. Wild-type vesicles may attach or incorporate into the outer membrane of the mutants that lack LPS O-antigen, restoring a wild-type periplasmic status and allowing for normal levels of 4521 activity and fruiting body formation. We propose that the LPS composition and the configuration of the outer membrane are important elements for the complex behavioral response of M. xanthus fruiting body development. ^

Studies of the microbial diversity on analyzed samples from from Peru Margin, Equatorial Pacific, Hydrate Ridge, and Juan de Fuca Ridge

Marine sediments harbor an enormous quantity of microorganisms, including a multitude of novel species. The habitable zone of the marine sediment column begins at the sediment-water interface and probably extends to depths of several thousands of meters. Studies of the microbial diversity in this ecosystem have mostly relied on molecular biological techniques. We used a complementary method - analysis of intact polar membrane lipids - to characterize the in-situ microbial community in sediments covering a wide range of environmental conditions from Peru Margin, Equatorial Pacific, Hydrate Ridge, and Juan de Fuca Ridge. Bacterial and eukaryotic phospholipids were only detected in surface sediments from the Peru Margin. In contrast, deeply buried sediments, independent of their geographic location, were dominated by archaeal diether and tetraether lipids with various polar head groups and core lipids. We compared ring distributions of archaeal tetraether lipids derived from polar glycosidic precursors with those that are present as core lipids. The distributions of these related compound pools were distinct, suggestive of different archaeal sources, i.e., the polar compounds derive from sedimentary communities and the core lipids are fossil remnants from planktonic communities with possible admixtures of decayed sedimentary archaea. This in-situ production of distinct archaeal lipid populations potentially affects applications of the TEX86 paleotemperature proxy as demonstrated by offsets in reconstructed temperatures between both pools. We evaluated how varying cell and lipid stabilities will influence the sedimentary pool by using a box-model. The results are consistent with (i) a requirement of continuous inputs of freshly synthesized lipids in subsurface sediments for explaining the observed distribution of intact polar lipids, and (ii) decreasing lipid inputs with increasing burial depth.

(Figures 2,3) Lipid composition of liver and muscles of the daubed shanny (Leptoclinus maculatus), Kongsfjorden

A comparative study on the lipid composition of the liver and muscles has been performed in daubed shanny caught in summer (July) in Arctic waters at three different sites (biotopes) along the north-western coast of Spitsbergen. In marine organisms living at high latitudes, lipids play an especially important role, primarily as reserve substances and as a factor influencing adaptation to severe environmental conditions. Since the ecology of daubed shanny is poorly known, the data obtained may be considered novel.

Composition of organic matter from samples of massive sulfides, bottom sediments, and seawater collected within the Logachev and Broken Spur hydrothermal fields, Mid-Atlantic Ridge

Lipid components of hydrothermal deposits from the unusual field at 14°45'N MAR and from the typical field at 29°N MAR were studied. For the first time mixed nature of organic matter (OM) from hydrothermal sulfide deposits was established with use of biochemical, gas chromatographic, and molecular methods of studies. In composition of OM lipids of phytoplankton, those of chemosynthesis bacteria and non-biogenic synthesis lipids were determined. Specific conditions of localization of sulfide deposits originated from ''black smokers'' (reducing conditions, absence of free oxygen, presence of reduced sulfur preventing OM from decomposition) let biogenic material, including bacterial one, be preserved in sulfide deposits. The hydrothermal system at 14°45'N MAR is characterized by geological, geochemical and thermodynamic conditions allowing abiogenic synthesis of methane and petroleum hydrocarbons. For sulfide deposits at 29°N and other active hydrothermal fields known at MAR, abiogenic synthesis of hydrocarbons occurs in lower scales.