Lactobacillus gasseri K7 modulates the blood cell transcriptome of conventional mice infected with Escherichia coli O157:H7.

AIMS: As the immune cells underlying the intestinal barrier sense luminal microbial signals, blood cell transcriptomics may identify subclinical changes triggered by gut bacteria that may otherwise not be detected. We have therefore investigated how Lactobacillus gasseri K7 and enterohemorrhagic Escherichia coli O157:H7 modulate the blood cell transcriptome of mice possessing an intact microbiota. METHODS AND RESULTS: We have analysed the transcriptome of five groups of C57BL/6J mice: (i) control, (ii) inoculated with a single dose of E. coli, (iii) inoculated during 2 weeks with Lact. gasseri, (iv) co-inoculated with E. coli and Lact. gasseri, (v) inoculated with Lact. gasseri prior to E. coli infection. The transcriptome could distinguish between the five treatment groups. Gene characteristics of bacterial infection, in particular inflammation, were upregulated in the mice inoculated with E. coli. Lact. gasseri had only mild effects on the transcriptome but modified the gene expression induced by E. coli. CONCLUSIONS: The transcriptome differentiates mice inoculated orally with E. coli, Lact. gasseri and combinations of these two strains. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that the blood cell transcriptome can be used as a source of biomarkers to monitor the impact of probiotics in subclinical models of infectious disease.

Validation of biomarkers of food intake-critical assessment of candidate biomarkers.

Biomarkers of food intake (BFIs) are a promising tool for limiting misclassification in nutrition research where more subjective dietary assessment instruments are used. They may also be used to assess compliance to dietary guidelines or to a dietary intervention. Biomarkers therefore hold promise for direct and objective measurement of food intake. However, the number of comprehensively validated biomarkers of food intake is limited to just a few. Many new candidate biomarkers emerge from metabolic profiling studies and from advances in food chemistry. Furthermore, candidate food intake biomarkers may also be identified based on extensive literature reviews such as described in the guidelines for Biomarker of Food Intake Reviews (BFIRev). To systematically and critically assess the validity of candidate biomarkers of food intake, it is necessary to outline and streamline an optimal and reproducible validation process. A consensus-based procedure was used to provide and evaluate a set of the most important criteria for systematic validation of BFIs. As a result, a validation procedure was developed including eight criteria, plausibility, dose-response, time-response, robustness, reliability, stability, analytical performance, and inter-laboratory reproducibility. The validation has a dual purpose: (1) to estimate the current level of validation of candidate biomarkers of food intake based on an objective and systematic approach and (2) to pinpoint which additional studies are needed to provide full validation of each candidate biomarker of food intake. This position paper on biomarker of food intake validation outlines the second step of the BFIRev procedure but may also be used as such for validation of new candidate biomarkers identified, e.g., in food metabolomic studies.

Localization of the N-terminal methionine of rat liver cytochrome P-450 in the lumen of the endoplasmic reticulum.

Recent cumulative evidence suggests that liver microsomal cytochrome P-450 (P-450) is exposed to the cytosol with the exception of the N-terminal peptide (amino acid residues 1 to 21), or two peptides (residues 1 to 60). We tested the localization of the N-terminal methionine residue of P-450IIB1 of rat liver microsomes in the natural membrane with the site-specific reagent fluorescein isothiocyanate. The N-terminus of isolated P-450 was stoichiometrically modified in solution with fluorescein isothiocyanate. In intact microsomes, the N-terminus was not modified but became accessible to the reagent when the membrane was dissolved with Triton X-100. Our results indicate that the N-terminus faces the lumen of the endoplasmic reticulum, and we propose that P-450 spans the membrane only once with amino acid residues 1 to 21.

Unmyristoylated MARCKS-related protein (MRP) binds to supported planar phosphatidylcholine membranes.

We have recently shown that unmyristoylated MARCKS-related protein (MRP) does not bind to neutral phospholipid vesicles, unless negatively charged phospholipids are present. Similar behaviour has also been reported for MARCKS itself. Here we have compared the binding of MRP to neutral and negatively charged supported planar lipid bilayer membranes (SPLM) using two-mode waveguide spectroscopy. We find appreciable binding of unmyristoylated MRP to neutral SPLM. We propose that hydrophobic residues in the effector domain constitute an additional factor capable of mediating MRP-membrane interaction.

Chemical modification of rat liver microsomal cytochrome P-450: study of enzymic properties and membrane topology.

Isolated rat liver cytochrome P-450IIB1 was alkylated and acetylated at primary amino groups, and the position of the modified amino acids in the protein was identified. Alkylation of up to nine amino groups did not disturb the interaction of reconstituted P-450 and NADPH-cytochrome-P-450 reductase in a way that hydroxylation of benzphetamine was altered, whereas deethylation of 7-ethoxycoumarin was gradually reduced in parallel with impaired 7-ethoxycoumarin binding. Acetylation of four lysine residues completely inhibited binding and metabolism of 7-ethoxycoumarin but not of benzphetamine. These results suggest the presence of different substrate binding sites on P-450. Exhaustive proteolysis of modified P-450 in proteoliposomes liberated all but the N-terminal modified peptide and 85 to 90% of the cytochrome's mass from intact proteoliposomes. These findings further support our previously proposed model of P-450 topology (Vergères, G., Winterhalter, K.H. and Richter, C. (1989) Biochemistry 28, 3650-3655), in which P-450 is anchored to the membrane with the N-terminal peptide only, the N-terminal methionine facing the lumenal interior.

Cytochrome b5, its functions, structure and membrane topology.

The first part of the present communication reviews recent advances in our understanding of the known physiological functions of cytochrome b5. In addition, one section is devoted to a description of a recently discovered function of cytochrome b5, namely its involvement in the synthesis of the oncofetal antigen N-glycolylneuraminic acid. The second part of the article summarizes site-directed mutagenesis studies, primarily conducted in the author's laboratory, in both the catalytic heme-binding and membrane-binding domain of cytochrome b5. These studies have shown that: 1) the membrane binding domain of cytochrome b5 spans the bilayer; 2) cytochrome b5 lacking 19 COOH-terminal amino acids does not bind to membrane bilayers; and 3) specific amino acids in the membrane binding domain have been mutated and shown not to be essential for the function of cytochrome b5 with its redox partners.

Myristoylation-dependent N-terminal cleavage of the myristoylated alanine-rich C kinase substrate (MARCKS) by cellular extracts.

The myristoylated alanine-rich C kinase substrate (MARCKS) has been proposed to regulate the plasticity of the actin cytoskeleton at its site of attachment to membranes. In macrophages, MARCKS is implicated in various cellular events including motility, adhesion and phagocytosis. In this report we show that macrophage extracts contain a protease which specifically cleaves human MARCKS, expressed in a cell-free system or in E. coli, between Lys-6 and Thr-7. Cleavage of MARCKS decreases its affinity for macrophage membranes by ca. one order of magnitude, highlighting the contribution of the myristoyl moiety of MARCKS to membrane binding. Importantly, cleavage requires myristoylation of MARCKS. Furthermore, MARCKS-related protein (MRP), the second member of the MARCKS family, is not digested. Since Thr-7 is lacking in MRP this suggests that Thr-7 at the P1 position is important for the recognition of lipid-modified substrates. A different product is observed when MARCKS is incubated with a calf brain cytosolic extract. This product can be remyristoylated in the presence of myristoyl-CoA and N-myristoyl transferase, demonstrating that cycles of myristoylation/demyristoylation of MARCKS can be achieved in vitro. Although the physiological relevance of these enzymes still needs to be demonstrated, our results reveal the presence of a new class of cleaving enzymes recognizing lipid-modified protein substrates.

Microsomal cytochrome P-450: substrate binding, membrane interactions, and topology.

The microsomal monoxygenase system is of paramount importance for the metabolism of endogenous substrates and xenobiotics. It is capable of detoxifying many compounds, but also activates procarcinogens to carcinogens. Cytochrome P-450 is the terminal enzyme of the monoxygenase system. In this article we briefly review current knowledge of the nature of its active site, its interaction with the membrane, and its topology in the membrane. In contrast to previous proposals there is now strong evidence that cytochrome P-450 spans the membrane with only one short segment. Analysis of tryptophan fluorescence gives further evidence that most of the protein's mass protrudes from the membrane into the cytosolic space.

Expression of cytochrome b5 in yeast and characterization of mutants of the membrane-anchoring domain.

The soluble and membrane-bound forms of the synthetic rat cytochrome b5 gene have been expressed in Saccharomyces cerevisiae. In order to examine the topology and function of the COOH-terminal membrane binding domain of cytochrome b5, mutants have been constructed, expressed, purified, and partially characterized. Pro-115 is located in the middle of the putative alpha-helical membrane-anchoring domain of cytochrome b5 and has been hypothesized to give rise to either a hairpin-like loop or approximately equal to 26 degrees kink in the helix, depending on whether it exists, respectively, in the cis or trans configuration. The Pro-115----Ala mutant, which is expected to have a straight transmembrane helix, inserted normally into the endoplasmic reticulum and exhibited wild type levels of activity in yeast microsomes and in vitro in the cytochrome P-450 mixed function oxidation system. Since a hairpin structure does not appear to be essential, it is likely that the membrane binding domain of cytochrome b5 spans the membrane. Characterization of the truncated cytochrome b5 molecule, Pro-115----Stop, lacking 19 amino acids at the COOH terminus indicates that the distal part of the membrane binding domain of cytochrome b5 is necessary for in vivo binding to the endoplasmic reticulum and for functioning with its membrane-associated electron transfer partners. Replacement of Ser-104 to Met-125, the putative membrane-anchoring domain of cytochrome b5, with 22 leucine residues results in a protein which targets to the endoplasmic reticulum but the extent of its reduction is only 50% of that of the wild type in yeast microsomes. In vitro, the polyleucine mutant is unable to support substrate oxidation by cytochrome P-450. The mutation of Ala-131 and Glu-132, amino acids flanking the transmembrane domain, to lysines resulted in a protein with normal membrane topology and function.

The carboxyl terminus of the membrane-binding domain of cytochrome b5 spans the bilayer of the endoplasmic reticulum.

Preliminary studies (Vergères, G., and Waskell, L. (1992) J. Biol. Chem. 267, 12583-12591) have suggested that the carboxyl-terminal membrane-binding domain of cytochrome b5 traverses the membrane and that the carboxyl terminus is in the lumen of the endoplasmic reticulum. In order to confirm and extend these studies, additional experiments were conducted. The gene coding for rat cytochrome b5 was transcribed and the resulting mRNA was translated in vitro in a rabbit reticulocyte lysate in the presence of microsomes. The binding and topology of cytochrome b5 were investigated by treating microsomes containing the newly incorporated cytochrome b5 with carboxypeptidase Y and trypsin. Our studies indicate that cytochrome b5 is inserted both co- and post-translationally into microsomes in a topology in which the membrane-binding domain spans the bilayer with its COOH terminus in the lumen. Cytochrome b5 is also incorporated into microsomes pretreated with trypsin in a topology indistinguishable from the one resulting from the insertion of the protein into untreated microsomes, reconfirming that cytochrome b5 does not use the signal recognition particle-dependent translocation machinery. Our results do not allow a distinction to be made between a spontaneous insertion mode or some other trypsin-resistant receptor-mediated mechanism. A role for Pro115 in the middle of the membrane-binding domain of cytochrome b5 was also examined by mutating it to an alanine and subsequently characterizing the ability of the mutant protein to be incorporated into membranes. The mutant protein inserted more slowly in vitro into microsomes as well as into pure lipid bilayers by a factor of 2 to 3.

Binding of MARCKS (myristoylated alanine-rich C kinase substrate)-related protein (MRP) to vesicular phospholipid membranes.

The myristoylated alanine-rich C kinase substrate (MARCKS) protein family has two known members, MARCKS itself and MARCKS-related protein (MRP, also called MacMARCKS or F52). They are essential for brain development and are believed to regulate the structure of the actin cytoskeleton at the plasma membrane. Hence membrane binding is central to their function. MARCKS has been quite extensively characterized; MRP much less so. Despite the fact that MRP is only two thirds the size of MARCKS, it has hitherto been assumed that the two proteins have similar properties. Here we make a detailed study, including the effects of myristoylation, lipid composition, calmodulin and phosphorylation of the binding of MRP to phospholipid vesicles. We show that both the N-terminal myristoyl moiety and the central effector domain mediate binding. MRP behaves like MARCKS in the presence of neutral phospholipids. In contrast to MARCKS, however, the incorporation of 20% of negatively-charged phospholipids only marginally increases the affinity of myristoylated MRP. Co-operativity between the myristoyl moiety and the effector domain of MRP is weak and the protein has a significantly lower affinity for these vesicles compared with MARCKS. Furthermore, calmodulin or phosphorylation of the effector domain by the catalytic subunit of protein kinase C do not significantly decrease the binding of myristoylated MRP to negatively-charged phospholipid vesicles. Our results show that the mechanisms regulating the interactions of MARCKS and MRP with phospholipid vesicles are, at least quantitatively, different. In agreement with cellular studies, we therefore propose that MARCKS and MRP have different subcellular localization and, consequently, different functions.

Nonelectrostatic contributions to the binding of MARCKS-related protein to lipid bilayers.

The association of various protein constructs of MARCKS-related protein (MRP) lacking the myristoyl moiety or the basic effector domain (ED) or both to neutral and acidic supported planar phospholipid bilayer membranes has been monitored using two-mode optical waveguide spectroscopy. The importance of the myristoyl moiety for interaction with both neutral and acidic membranes is demonstrated but unmyristoylated MRP still binds appreciably to neutral membranes, albeit less than to acidic membranes. Only when both the myristoyl moiety and the ED are excised does the interaction fall to zero in the case of the acidic membranes, with very small residual binding still detectable in the presence of neutral membranes. These results point to the importance of hydrophobic interactions apart from those associated with the myristoyl moiety in the association of MRP with membranes. The ED is well endowed with hydrophobic as well as with basic residues, and the former are chiefly responsible for binding unmyristoylated MRP to neutral membranes: The very small residual attraction between MRP lacking both the myristoyl moiety and the ED is completely outweighed by electrostatic repulsion between the net acidic MRP and the acidic lipid head groups.

Regulation of the binding of myristoylated alanine-rich C kinase substrate (MARCKS) related protein to lipid bilayer membranes by calmodulin.

The effector domain (ED) of MARCKS proteins can associate with calmodulin (CaM) as well as with phospholipids. It is not clear, however, whether a complex between MARCKS proteins and CaM can form at the surface of phospholipid membranes or whether CaM and membranes compete for ED binding. Using two-mode waveguide spectroscopy, we have investigated how CaM regulates the association of MARCKS-related protein (MRP) with planar supported phospholipid bilayer membranes. Bringing a solution containing CaM into contact with membranes on which MRP had previously been deposited results in low-affinity binding of CaM to MRP. A preformed, high-affinity CaM MRP complex in the aqueous phase binds much more slowly than pure MRP to membranes. Similar observations were made when a peptide corresponding to the ED of MRP was used instead of MRP. Hence CaM cannot form a stable complex with MRP once the latter is bound at the membrane surface. CaM can, however, strongly retard the association of MRP with lipid membranes. The most likely interpretation of these results is that CaM and the phospholipid membrane share the same binding region at the ED and that the ED is forced by membrane binding to adopt a conformation unfavorable for CaM binding.

The myristoyl moiety of myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein is embedded in the membrane.

Members of the myristoylated alanine-rich protein kinase C substrate (MARCKS) family are involved in several cellular processes such as secretion, motility, mitosis, and transformation. In addition to their ability to bind calmodulin and to cross-link actin filaments, reversible binding to the plasma membrane is most certainly an important component of the so far unknown functions of these proteins. We have therefore investigated the binding of murine MARCKS-related protein (MRP) to lipid vesicles. The partition coefficient, Kp, describing the affinity of myristoylated MRP for acidic lipid vesicles (20% phosphatidylserine, 80% phosphatidylcholine) is 5-8 x 10(3) M-1, which is only 2-4 times larger than the partition coefficient for the unmyristoylated protein. Interestingly, the affinity of MRP for acidic lipid membranes is 20-30-fold smaller than reported for murine MARCKS (Kim, J., Shishido, T., Jiang, X., Aderem, A. A., and McLaughlin, S. (1994) J. Biol. Chem. 269, 28214-28219). Since only a marginal binding could be observed with neutral phosphatidylcholine vesicles, we propose that electrostatic interactions are the major determinant of the binding of MRP to pure lipid membranes. Although the myristoyl moiety does not contribute drastically to the binding of MRP to vesicles, photolabeling experiments with a photoreactive phospholipid probe show that the fatty acid is embedded in the bilayer. The same membrane topology was found for bovine brain MARCKS. Since the relatively low affinity of MRP for vesicles is insufficient to account for a stable anchoring of the protein to cellular membranes, insertion of the myristoyl moiety into the bilayer might favor the interaction of MRP with additional factors required for the binding of the protein to intracellular membranes.

Kinetics of the reduction of cytochrome b5 with mutations in its membrane-binding domain.

In an attempt to understand which amino acids in the membrane anchor of cytochrome b5 might be determinants of its ability to support the cytochrome P450-catalyzed oxidation of selected substrates, the synthetic rat cytochrome b5 gene has been mutated by site-directed mutagenesis. The mutant proteins have been expressed in Saccharomyces cerevisiae, purified and assayed for their ability to support the cytochrome P450-catalyzed metabolism of the cytochrome b5 requiring substrate methoxyflurane (G. Vergères and L. Waskell, 1992, J. Biol. Chem. 267, 12583-12591). The rate of reduction of the cytochromes b5 by cytochrome P450 reductase has been examined by stopped-flow spectrophotometry to determine whether an altered rate of reduction of cytochrome b5 could explain the observed activity of cytochrome b5 in the purified reconstituted mixed-function oxidase system. A mutant in which the 22-amino-acid membrane anchor was replaced by a sequence of 22 leucines was unable to support methoxyflurane metabolism in the reconstituted system and was reduced by cytochrome P450 reductase at a rate (k = 4.5 x 10(-3) s-1) slow enough to explain this finding. Comparison of the rate of reduction of this mutant cytochrome b5 in 0.025% Tergitol and 40 microM dilauroylphosphatidylcholine suggests that this slow rate of reduction may be explained partially by aggregation of the polyleucine protein. The Pro115Stop mutant protein, which has been truncated by 19 amino acids in its COOH terminus resulting in a protein with one-half of the putative membrane anchor, supports methoxyflurane oxidation at 12-20% of the rate of the wild type protein. In addition it is reduced by cytochrome P450 reductase at a rate which should be capable of supporting a normal rate of production formation. The fact that the Pro115Stop mutant can be reduced at a rate capable of supporting a normal rate of methoxyflurane oxidation but in fact only supports methoxyflurane oxidation at 30% of the normal rate suggests that the mutant protein is deficient in its interactions with cytochrome P450. The mutant proteins, Pro115Ala and Ala116Pro, behaved essentially as did the wild type protein demonstrating that the presence (Pro115Ala) or absence (Ala116Pro) of an alpha helix in the middle of the putative membrane-binding domain of cytochrome b5 was not a determinant of the interaction of cytochrome b5 with cytochrome P450 reductase and cytochrome P450.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of the effector domain of MARCKS and MARCKS-related protein with lipid membranes revealed by electric potential measurements.

We have investigated the binding of the effector domains of myristoylated alanine-rich C kinase substrate (MARCKS) and of MARCKS-related protein (MRP) to lipid model membranes. For membrane systems we used lipid monolayers on a Langmuir trough and black lipid membranes (BLM). The binding of the peptides was detected by monitoring changes in the boundary potential of the lipid membranes. The vibrating plate technique (VPT) and the method of inner field compensation (IFC) were used for the monolayer and for the BLM, respectively. We could show that the effector domain of MARCKS binds to acidic lipid membranes mainly via electrostatic interactions and to zwitterionic lipid membranes via hydrophobic interactions. Isobaric measurements on lipid monolayers revealed that binding of both effector domains is accompanied by partial insertion of the peptides into the membrane. Adsorption and insertion of the peptides could be followed simultaneously by the VPT and by recording the increase in area of the lipid monolayer, respectively. No temporal delay could be observed between adsorption and insertion of the peptides, demonstrating that adsorption is the rate-limiting step and that insertion is faster than the time resolution of the experiments, i.e., a few seconds. Both the IFC and the VPT did not show any significant difference between the behaviors of the effector domains of MARCKS and MRP. With the IFC we show that calcium can regulate the translocation of the MARCKS effector peptide between the membrane and calmodulin (CaM) in the bulk. Our results indicate, that the IFC and VPT are suitable qualitatively, and to a certain extent quantitatively, as membrane binding assays.

Identification of the membrane anchor of microsomal rat liver cytochrome P-450.

Cytochrome P450IIB1 isolated from rat liver microsomes was incorporated into phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (10:5:1 w/w) liposomes. Trypsinolysis of proteoliposomes and sequencing of the membrane-bound domains revealed that only one peptide, comprising amino acid residues 1-21, spans the membrane. Modification of the N-terminal methionine by membrane-impermeable fluorescein isothiocyanate occurred with the protein in solution but not in proteoliposomes. We conclude that in proteoliposomes cytochrome P-450 spans the membrane only with amino acid residues 1-21, the N-terminal methionine facing the lumen.

MARCKS-related protein binds to actin without significantly affecting actin polymerization or network structure. Myristoylated alanine-rich C kinase substrate.

Actinis a 42-kDa protein which, due to its ability to polymerize into filaments (F-actin), is one of the major constituents of the cytoskeleton. It has been proposed that MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) proteins play an important role in regulating the structure and mechanical properties of the actin cytoskeleton by cross-linking actin filaments. We have recently reported that peptides corresponding to the effector domain of MARCKS proteins promote actin polymerization and cause massive bundling of actin filaments. We now investigate the effect of MARCKS-related protein, a 20-kDa member of the MARCKS family, on both filament structure and the kinetics of actin polymerization in vitro. Our experiments document that MRP binds to F-actin with micromolar affinity and that the myristoyl chain at the N-terminus of MRP is not required for this interaction. In marked contrast to the effector peptide, binding of MRP is not accompanied by an acceleration of actin polymerization kinetics, and we also could not reliably observe an actin cross-linking activity of MRP.

Membrane binding of MARCKS-related protein studied by tryptophan fluorescence spectroscopy.

MARCKS-related protein (MRP) is a peripheral membrane protein whose binding to membranes is mediated by the N-terminal myristoyl moiety and a central, highly basic effector domain. MRP mediates cross-talk between protein kinase C and calmodulin and is thought to link the actin cytoskeleton to the plasma membrane. Since MRP contains no tryptophan residues, we mutated a phenylalanine in the effector domain to tryptophan (MRP F93W) and used fluorescence spectroscopy to monitor binding of the protein to phospholipid vesicles. We report in detail the evaluation procedure necessary to extract quantitative information from the raw data. The spectra of MRP F93W obtained in the presence of increasing amounts of lipid crossed at an isosbestic point, indicating a simple transition between two states: free and membrane-bound protein. The change in fluorescence toward values typical of a more hydrophobic environment was used to quantify membrane binding. The partition coefficient agreed well with values obtained previously by other methods. To study the interaction of the N-terminus of MRP with membranes, a tryptophan residue was also introduced at position 4 (MRP S4W). Our data suggest that only the myristoylated N-terminus interacted with liposomes. These results demonstrate the versatility of site-directed incorporation of tryptophan residues to study protein-membrane interactions.