Highly luminescent and multi-sensing aggregates co-assembled from Eu-containing polyoxometalate and an enzyme-responsive surfactant in water.

Hybrid co-assembly of polyoxometalates (POMs) with cationic organic matrices offers a preferable way to greatly enhance POM functionality as well as processability. Thus, multi-stimulus responsive supramolecular materials based on lanthanide-containing POMs with improved luminescence may be fabricated from appropriate components through this convenient strategy. Herein, we reported that the co-assembly of Na9(EuW10O36)·32H2O (EuW10) and a commercially available cationic surfactant, myristoylcholine chloride (Myr), in water could produce enhanced red-emitting luminescent aggregates, with their photophysical properties highly dependent on the molar ratio (R) between Myr and EuW10. The R of 36 was finally selected owing to the displayed superior luminescence intensity and good aggregate stability. The Myr/EuW10 hybrids induced by electrostatic and hydrophobic forces presented practically as multilamellar spheres with diameters varying from 80 to 300 nm. Compared to an aqueous solution of EuW10 nanoclusters, a 12-fold increase in absolute luminescence quantum yield (∼23.3%) was observed for the hybrid spheres, which was ascribed to the efficient shielding of water molecules. An unusual aggregation arrangement mechanism and the excellent photophysical properties of these aggregates were thoroughly investigated. Both the enzyme substrate character of Myr and the sensitive coordination structure of EuW10 to the surrounding environment made Myr/EuW10 aggregates exhibit multi-stimulus responsiveness to enzymes, pH, and transition metal ions, thus providing potential applications in fluorescence sensing, targeted-release, and optoelectronics.

Modification of lipid A structure and activity by the introduction of palmitoyltransferase gene to the acyltransferase-knockout mutant of Escherichia coli.

Lauroyltransferase gene (lpxL), Myristoyltransferase gene (lpxM) and palmitoyltransferase gene (crcA) of Escherichia coli BL21 were independently disrupted by the insertional mutations. The knockout mutant of two transferase genes (lpxL and crcA) produced lipid A with no lauric or palmitic acids and only a little amount of myristic acid. The mutant was susceptible to polymyxin B, but showed comparable growth with the wild-type strain at 30°C. The palmitoyltransferase gene from E. coli (crcA) or Salmonella (pagP) was amplified by PCR, cloned in pUC119, and transferred into the double-knockout mutant by transformation. The transformant contained palmitic acid in the lipid A, and recovered resistance to polymyxin B. Mass spectrometric analysis revealed that palmitic acid was linked to the hydroxyl group of 3-hydroxymyristic acid at C-2 position of proximal (reducing-end) glucosamine. LPS from the double-knockout mutant showed reduced IL-6-inducing activity to macrophage-like line cells compared to that of the wild-type strain, and the activity was only slightly restored by the introduction of palmitic acid to the lipid A. These results suggested that the introduction of one palmitic acid was enough to recover the integrity of the outer membrane, but not enough for the stimulation of macrophages.

The Structure of the Lipid A from the Halophilic Bacterium Spiribacter salinus M19-40T.

The study of the adaptation mechanisms that allow microorganisms to live and proliferate in an extreme habitat is a growing research field. Directly exposed to the external environment, lipopolysaccharides (LPS) from Gram-negative bacteria are of great appeal as they can present particular structural features that may aid the understanding of the adaptation processes. Moreover, through being involved in modulating the mammalian immune system response in a structure-dependent fashion, the elucidation of the LPS structure can also be seen as a fundamental step from a biomedical point of view. In this paper, the lipid A structure of the LPS from Spiribacter salinus M19-40T, a halophilic gamma-proteobacteria, was characterized through chemical analyses and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. This revealed a mixture of mono- and bisphosphorylated penta- to tri-acylated species with the uncommon 2 + 3 symmetry and bearing an unusual 3-oxotetradecaonic acid.

Branched phospholipids render lipid vesicles more susceptible to membrane-active peptides.

Iso- and anteiso-branched lipids are abundant in the cytoplasmic membranes of bacteria. Their function is assumed to be similar to that of unsaturated lipids in other organisms - to maintain the membrane in a fluid state. However, the presence of terminally branched membrane lipids is likely to impact other membrane properties as well. For instance, lipid acyl chain structure has been shown to influence the activity of antimicrobial peptides. Moreover, the development of resistance to antimicrobial agents in Staphylococcus aureus is accompanied by a shift in the fatty acid composition toward a higher fraction of anteiso-branched lipids. Little is known about how branched lipids and the location of the branch point affect the activity of membrane-active peptides. We hypothesized that bilayers containing lipids with low phase transition temperatures would tend to exclude peptides and be less susceptible to peptide-induced perturbation than those made from higher temperature melting lipids. To test this hypothesis, we synthesized a series of asymmetric phospholipids that only differ in the type of fatty acid esterified at the sn-2 position of the lipid glycerol backbone. We tested the influence of acyl chain structure on peptide activity by measuring the kinetics of release from dye-encapsulated lipid vesicles made from these synthetic lipids. The results were compared to those obtained using vesicles made from S. aureus and Staphylococcus sciuri membrane lipid extracts. Anteiso-branched phospholipids, which melt at very low temperatures, produced lipid vesicles that were only slightly less susceptible to peptide-induced dye release than those made from the iso-branched isomer. However, liposomes made from bacterial phospholipid extracts were generally much more resistant to peptide-induced perturbation than those made from any of the synthetic lipids. The results suggest that the increase in the fraction of anteiso-branched fatty acids in antibiotic-resistant strains of S. aureus is unlikely to be the sole factor responsible for the observed increased antibiotic resistance. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

G protein-membrane interactions I: Gαi1 myristoyl and palmitoyl modifications in protein-lipid interactions and its implications in membrane microdomain localization.

G proteins are fundamental elements in signal transduction involved in key cell responses, and their interactions with cell membrane lipids are critical events whose nature is not fully understood. Here, we have studied how the presence of myristic and palmitic acid moieties affects the interaction of the Gαi1 protein with model and biological membranes. For this purpose, we quantified the binding of purified Gαi1 protein and Gαi1 protein acylation mutants to model membranes, with lipid compositions that resemble different membrane microdomains. We observed that myristic and palmitic acids not only act as membrane anchors but also regulate Gαi1 subunit interaction with lipids characteristics of certain membrane microdomains. Thus, when the Gαi1 subunit contains both fatty acids it prefers raft-like lamellar membranes, with a high sphingomyelin and cholesterol content and little phosphatidylserine and phosphatidylethanolamine. By contrast, the myristoylated and non-palmitoylated Gαi1 subunit prefers other types of ordered lipid microdomains with higher phosphatidylserine content. These results in part explain the mobility of Gαi1 protein upon reversible palmitoylation to meet one or another type of signaling protein partner. These results also serve as an example of how membrane lipid alterations can change membrane signaling or how membrane lipid therapy can regulate the cell's physiology.

Β-hydroxymyristic acid as a chemical marker to detect endotoxins in dialysis water.

An analytical chemical method has been developed for determination of ß-hydroxymyristic acid (ß-HMA), a component of lipopolysaccharides (LPSs/endotoxins) in dialysis water. In our investigation, the ß-HMA component was used as a chemical marker for endotoxin presence in dialysis water because it is available in the molecular subunit (lipid A) and responsible for toxicity. It is the most abundant saturated fatty acid in that subunit. The developed method is based on fluorescence derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ). A high-performance liquid chromatographic separation of the ß-HMA derivative was achieved using an octadecyl silica column in gradient elution. A wide dynamic range of ß-HMA was tested and a calibration curve was constructed with accuracy of 90% and variability of less than 10%. The limits of detection and quantification obtained were 2 and 5µM, respectively. The developed method was applied to detect endotoxins in dialysis water by alkaline hydrolysis of LPS using NaOH (0.25M) at 60°C for 2h. After hydrolysis, free acid was detected as its NBD-PZ derivative using high-performance liquid chromatography/mass spectrometry (HPLC/MS). Good recovery rates ranging from 98 to 105% were obtained for ß-HMA in dialysis water.

Polylactide/Poly(ω-hydroxytetradecanoic acid) Reactive Blending: A Green Renewable Approach to Improving Polylactide Properties.

A green manufacturing technique, reactive extrusion (REx), was employed to improve the mechanical properties of polylactide (PLA). To achieve this goal, a fully biosourced PLA based polymer blend was conceived by incorporating small quantities of poly(ω-hydroxytetradecanoic acid) (PC14). PLA/PC14 blends were compatibilized by transesterification reactions promoted by 200 ppm titanium tetrabutoxide (Ti(OBu)4) during REx. REx for 15 min at 150 rpm and 200 °C resulted in enhanced blend mechanical properties while minimizing losses in PLA molecular weight. SEM analysis of the resulting compatibilized phase-separated blends showed good adhesion between dispersed PC14 phases within the continuous PLA phase. Direct evidence for in situ synthesis of PLA-b-PC14 copolymers was obtained by HMBC and HSQC NMR experiments. The size of the dispersed phase was tuned by the screw speed to "tailor" the blend morphology. In the presence of 200 ppm Ti(OBu)4, inclusion of only 5% PC14 increased the elongation at break of PLA from 3 to 140% with only a slight decrease in the tensile modulus (3200 to 2900 MPa). Furthermore, PLA's impact strength was increased by 2.4× that of neat PLA for 20% PC14 blends prepared by REx. Blends of PLA and PC14 are expected to expand the potential uses of PLA-based materials.

Structure of the bacterial deacetylase LpxC bound to the nucleotide reaction product reveals mechanisms of oxyanion stabilization and proton transfer.

The emergence of antibiotic-resistant strains of pathogenic bacteria is an increasing threat to global health that underscores an urgent need for an expanded antibacterial armamentarium. Gram-negative bacteria, such as Escherichia coli, have become increasingly important clinical pathogens with limited treatment options. This is due in part to their lipopolysaccharide (LPS) outer membrane components, which dually serve as endotoxins while also protecting Gram-negative bacteria from antibiotic entry. The LpxC enzyme catalyzes the committed step of LPS biosynthesis, making LpxC a promising target for new antibacterials. Here, we present the first structure of an LpxC enzyme in complex with the deacetylation reaction product, UDP-(3-O-(R-3-hydroxymyristoyl))-glucosamine. These studies provide valuable insight into recognition of substrates and products by LpxC and a platform for structure-guided drug discovery of broad spectrum Gram-negative antibiotics.

Oil-in-water microemulsion droplets of TDMAO/decane interconnected by the telechelic C18-EO150-C18: clustering and network formation.

The effect of a doubly hydrophobically end-capped water soluble polymer (C18-PEO150-C18) on the properties of an oil-in-water (O/W) droplet microemulsion (R ∼ 2.85 nm) has been studied as a function of the amount of added telechelic polymer. Macroscopically one observes a substantial increase of viscosity once a concentration of ∼5 hydrophobic stickers per droplet is surpassed and effective cross-linking of the droplets takes place. SANS measurements show that the size of the individual droplets is not affected by the polymer addition but it induces attractive interactions at low concentration and repulsive ones at high polymer content. Measurements of the diffusion coefficient by DLS and FCS show increasing sizes at low polymer addition that can be attributed to the formation of clusters of microemulsion droplets interconnected by the polymer. At higher polymer content the network formation leads to an additional slow relaxation mode in DLS that can be related to the rheological behaviour, while the self-diffusion observed in FCS attains a lower plateau value, i.e., the microemulsion droplets remain effectively fixed within the network. The combination of SANS, DLS, and FCS allows us to derive a self-consistent picture of the evolution of structure and dynamics of the mixed system microemulsion/telechelic polymer as a function of the polymer content, which is not only relevant for controlling the macroscopic rheological properties but also with respect to the internal dynamics as it is, for instance, relevant for the release and transport of active agents.

Characterization of low-energy excited states in the native state ensemble of non-myristoylated and myristoylated neuronal calcium sensor-1.

Information on the low-energy excited states of a given protein is important as this controls the structural adaptability and various biological functions of proteins such as co-operativity, response towards various external perturbations. In this article, we characterized individual residues in both non-myristoylated (non-myr) and myristoylated (myr) neuronal calcium sensor-1 (NCS-1) that access alternate states by measuring nonlinear temperature dependence of the backbone amide-proton (¹H(N)) chemical shifts. We found that ~20% of the residues in the protein access alternative conformations in non-myr case, which increases to ~28% for myr NCS-1. These residues are spread over the entire polypeptide stretch and include the edges of α-helices and ß-strands, flexible loop regions, and the Ca²(+)-binding loops. Besides, residues responsible for the absence of Ca²(+)-myristoyl switch are also found accessing alternative states. The C-terminal domain is more populated with these residues compared to its N-terminal counterpart. Individual EF-hands in NCS-1 show significantly different number of alternate states. This observation prompts us to conclude that this may lead to differences in their individual conformational flexibility and has implications on the functionality. Theoretical simulations reveal that these low-energy excited states are within an energy band of 2-4 kcal/mol with respect to the native state.

HIV-1 Nef membrane association depends on charge, curvature, composition and sequence.

Nef-mediated internalization of T-cell receptor molecules from the surface of an infected cell is required for the pathogenicity of HIV and disease progression to AIDS. This function depends on the N-terminal myristoylation of Nef, a lipid modification that targets the protein to membranes. We have analyzed how specific membrane properties and sequence motifs within Nef determine this interaction. Using time-resolved techniques we find that the association with membranes is a biphasic process with a fast rate for an electrostatic-driven protein-liposome interaction and a slow rate for the formation of an amphipathic helix. The rate of myristate insertion into liposomes depends on membrane curvature, while changes in the lipid composition with respect to phosphoinositides, cholesterol or sphingomyelin did not significantly alter the interaction. Moreover, Nef binding to membranes requires negatively charged liposomes, and mutations of basic and hydrophobic residues strongly diminished the association and changed the binding kinetics differently.

[Study on prescription of self-microemulsifying drug delivery system of mangiferin phospholipid complex].

OBJECTIVE: To study formulation of self-microemulsifying drug delivery system (SMEDDS) of mangiferin phospholipid complex and improve dissolution and bioavailability of mangiferin. METHODS: Ternary phase diagram was applied to optimize the prescription of self-microemulsifying drug delivery system of mangiferin phospholipid complex, and the best recipe was selected by comprehensive evaluation of the speed of microemulsifying, microemulsion size and electric potential. RESULTS: The optimum formulation of SMEDDS was composed of IPM-Cremphor EL35-labrasol = 2 : 4.8 : 3.2. CONCLUSION: Self-microemulsifying drug delivery system of mangiferin phospholipid complex can effectively improve the dissolution of Mangiferin.

Polymers from fatty acids: poly(ω-hydroxyl tetradecanoic acid) synthesis and physico-mechanical studies.

This Article describes the synthesis and physicomechanical properties of bioplastics prepared from methyl ω-hydroxytetradecanoic acid (Me-ω-OHC14), a new monomer available by a fermentation process using an engineered Candida tropicalis strain. Melt-condensation experiments were conducted using titanium tetraisopropoxide (Ti[OiPr](4)) as a catalyst in a two-stage polymerization (2 h at 200 °C under N(2), 4 h at 220 °C under 0.1 mmHg). Poly(ω-hydroxytetradecanoate), P(ω-OHC14), M(w), determined by SEC-MALLS, increased from 53K to 110K as the Ti(OiPr)(4) concentration increased from 50 to 300 ppm. By varying the polymerization conditions (catalyst concentration, reaction time, second-stage reaction temperature) a series of P(ω-OHC14) samples were prepared with M(w) values from 53K to 140K. The synthesized polyesters with M(w) ranging from 53K to 140K were subjected to characterization by DSC, TGA, DMTA, and tensile testing. Influences of P(ω-OHC14) molecular weight, melting point, and enthalpies of melting/crystallization on material tensile properties were explored. Cold-drawing tensile tests at room temperature for P(ω-OHC14) with M(w) 53K-78K showed a brittle-to-ductile transition. In contrast, P(ω-OHC14) with M(w) 53K undergoes brittle fracture. Increasing P(ω-OHC14) M(w) above 78K resulted in a strain-hardening phenomena and tough properties with elongation at break ~700% and true tensile strength of ~50 MPa. Comparisons between high density polyethylene and P(ω-OHC14) mechanical and thermal properties as a function of their respective molecular weights are discussed.

Structural features of lipid A of Rickettsia typhi.

Lipid A isolated from the Rickettsia typhi lipopolysaccharide (LPS) was investigated for its composition and structure using chemical analyses, gas chromatography-mass spectrometry (GC-MS), and electrospray ionization (ESI) combined with the tandem mass spectrometry (MS/MS). Our studies revealed a noticeable compositional and structural heterogeneity of lipid A with respect to the content of phosphate groups and the degree of acylation. It appeared that at least two molecular species were present in lipid A. The major species represented the hexaacyl lipid A consisting of the ß-(1--> 6)-linked D-glucosamine (GlcN) disaccharide backbone carrying two phosphate groups. One of them was linked to the glycosidic hydroxyl group of the reducing GlcN I and the other was ester linked to the O-4´ position of the non-reducing GlcN II. The primary fatty acids consisted of two 3-hydroxytetradecanoic [C14:0(3-OH)] and two 3-hydroxyhexadecanoic [C16:0(3-OH)] acids. The former were ester- and the latter amide-linked to both GlcN. Two secondary fatty acids were represented by the octadecanoic (C18:0) and hexadecanoic (C16:0) acids that were ester-linked at the N-2´ and O-3´ positions, respectively. In the minor lipid A species, ester-linked C18:0 was substituted by C16:0 at the C16:0(3-OH) of GlcN II. The R. typhi lipid A resembles structurally the classical forms of enterobacterial lipids A with high endotoxicity.

Importance of the long-chain fatty acid beta-hydroxylating cytochrome P450 enzyme YbdT for lipopeptide biosynthesis in Bacillus subtilis strain OKB105.

Bacillus species produce extracellular, surface-active lipopeptides such as surfactin that have wide applications in industry and medicine. The steps involved in the synthesis of 3-hydroxyacyl-coenzyme A (CoA) substrates needed for surfactin biosynthesis are not understood. Cell-free extracts of Bacillus subtilis strain OKB105 synthesized lipopeptide biosurfactants in presence of l-amino acids, myristic acid, coenzyme A, ATP, and H(2)O(2), which suggested that 3-hydroxylation occurs prior to CoA ligation of the long chain fatty acids (LCFAs). We hypothesized that YbdT, a cytochrome P450 enzyme known to beta-hydroxylate LCFAs, functions to form 3-hydroxy fatty acids for lipopeptide biosynthesis. An in-frame mutation of ybdT was constructed and the resulting mutant strain (NHY1) produced predominantly non-hydroxylated lipopeptide with diminished biosurfactant and beta-hemolytic activities. Mass spectrometry showed that 95.6% of the fatty acids in the NHY1 biosurfactant were non-hydroxylated compared to only ∼61% in the OKB105 biosurfactant. Cell-free extracts of the NHY1 synthesized surfactin containing 3-hydroxymyristic acid from 3-hydroxymyristoyl-CoA at a specific activity similar to that of the wild type (17 ± 2 versus 17.4 ± 6 ng biosurfactant min(-1)·ng·protein(-1), respectively). These results showed that the mutation did not affect any function needed to synthesize surfactin once the 3-hydroxyacyl-CoA substrate was formed and that YbdT functions to supply 3-hydroxy fatty acid for surfactin biosynthesis. The fact that YbdT is a peroxidase could explain why biosurfactant production is rarely observed in anaerobically grown Bacillus species. Manipulation of LCFA specificity of YbdT could provide a new route to produce biosurfactants with activities tailored to specific functions.

Biosynthesis of monomers for plastics from renewable oils.

Omega-hydroxyfatty acids are excellent monomers for synthesizing a unique family of polyethylene-like biobased plastics. However, ω-hydroxyfatty acids are difficult and expensive to prepare by traditional organic synthesis, precluding their use in commodity materials. Here we report the engineering of a strain of the diploid yeast Candida tropicalis to produce commercially viable yields of ω-hydroxyfatty acids. To develop the strain we identified and eliminated 16 genes encoding 6 cytochrome P450s, 4 fatty alcohol oxidases, and 6 alcohol dehydrogenases from the C. tropicalis genome. We also show that fatty acids with different chain lengths and degrees of unsaturation can be more efficiently oxidized by expressing different P450s within this strain background. Biocatalysis using engineered C. tropicalis is thus a potentially attractive biocatalytic platform for producing commodity chemicals from renewable resources.

Profiling of myristoylation in Toxoplasma gondii reveals an N-myristoylated protein important for host cell penetration.

N-myristoylation is a ubiquitous class of protein lipidation across eukaryotes and N-myristoyl transferase (NMT) has been proposed as an attractive drug target in several pathogens. Myristoylation often primes for subsequent palmitoylation and stable membrane attachment, however, growing evidence suggests additional regulatory roles for myristoylation on proteins. Here we describe the myristoylated proteome of Toxoplasma gondii using chemoproteomic methods and show that a small-molecule NMT inhibitor developed against related Plasmodium spp. is also functional in Toxoplasma. We identify myristoylation on a transmembrane protein, the microneme protein 7 (MIC7), which enters the secretory pathway in an unconventional fashion with the myristoylated N-terminus facing the lumen of the micronemes. MIC7 and its myristoylation play a crucial role in the initial steps of invasion, likely during the interaction with and penetration of the host cell. Myristoylation of secreted eukaryotic proteins represents a substantial expansion of the functional repertoire of this co-translational modification.

A microscopic parasite known as Toxoplasma gondii infects around 30% of the human population. Most infections remain asymptomatic, but in people with a compromised immune system, developing fetuses and people infected with particular virulent strains of the parasite, infection can be fatal. T. gondii is closely related to other parasites that also infect humans, including the one that causes malaria. These parasites have complex lifecycles that involve successive rounds of invading the cells of their hosts, growing and then exiting these cells. Signaling proteins found at specific locations within parasite cells regulate the ability of the parasites to interact with and invade host cells. Sometimes these signaling proteins are attached to membranes using lipid anchors, for example through a molecule called myristic acid. An enzyme called NMT can attach myristic acid to one end of its target proteins. The myristic acid tag can influence the ability of target proteins to bind to other proteins, or to membranes. Previous studies have found that drugs that inhibit the NMT enzyme prevent the malaria parasite from successfully invading and growing inside host cells. The NMT enzyme from T. gondii is very similar to that of the malaria parasite. Broncel et al. have shown that the drug developed against P. falciparum also inhibits the ability of T. gondii to grow. These findings suggest that drugs against the NMT enzyme may be useful to treat diseases caused by T. gondii and other closely-related parasites. Broncel et al. also identified 65 proteins in T. gondii that contain a myristic acid tag using an approach called proteomics. One of the unexpected 'myristoylated' proteins identified in the experiments is known as MIC7. This protein was found to be transported onto the surface of T. gondii parasites and is required in its myristoylated form for the parasite to successfully invade host cells. This was surprising as myristoylated proteins are generally thought to not enter the pathway that brings proteins to the outside of cell. These findings suggest that myristic acid on proteins that are secreted can facilitate interactions between cells, maybe by inserting the myristic acid into the cell membrane.

Requirement of the acyl-CoA carrier ACBD6 in myristoylation of proteins: Activation by ligand binding and protein interaction.

Glycine N-myristoylation is an essential acylation modification modulating the functions, stability, and membrane association of diverse cytosolic proteins in human cells. Myristoyl-CoA is the 14-carbon acyl donor of the acyltransferase reaction. Acyl-CoAs of a chain length compatible with the binding site of the N-myristoyltransferase enzymes (NMT) are competitive inhibitors, and the mechanism protecting these enzymes from unwanted acyl-CoA species requires the acyl-CoA binding protein ACBD6. The acyl-CoA binding domain (ACB) and the ankyrin-repeat motifs (ANK) of ACBD6 can perform their functions independently. Interaction of ANK with human NMT2 was necessary and sufficient to provide protection. Fusion of the ANK module to the acyl-CoA binding protein ACBD1 was sufficient to confer the NMT-stimulatory property of ACBD6 to the chimera. The ACB domain is dispensable and sequestration of the competitor was not the basis for NMT2 protection. Acyl-CoAs bound to ACB modulate the function of the ANK module and act as positive effector of the allosteric activation of the enzyme. The functional relevance of homozygous mutations in ACBD6 gene, which have not been associated with a disease so far, is presented. Skin-derived fibroblasts of two unrelated individuals with neurodevelopmental disorder and carrying loss of function mutations in the ACBD6 gene were deficient in protein N-myristoylation. These cells were sensitive to substrate analog competing for myristoyl-CoA binding to NMT. These findings account for the requirement of an ANK-containing acyl-CoA binding protein in the cellular mechanism protecting the NMT enzymes and establish that in human cells, ACBD6 supports the N-myristoylation of proteins.

Crystal structure and acyl chain selectivity of Escherichia coli LpxD, the N-acyltransferase of lipid A biosynthesis.

LpxD catalyzes the third step of lipid A biosynthesis, the R-3-hydroxyacyl-ACP-dependent N-acylation of UDP-3-O-(acyl)-alpha-D-glucosamine, and is a target for new antibiotic development. Here we report the 2.6 A crystal structure of the Escherichia coli LpxD homotrimer (EcLpxD). As is the case in Chlamydia trachomatis LpxD (CtLxpD), each EcLpxD chain consists of an N-terminal uridine-binding region, a left-handed parallel beta-helix (LbetaH), and a C-terminal alpha-helical domain. The backbones of the LbetaH domains of the two enzymes are similar, as are the positions of key active site residues. The N-terminal nucleotide binding domains are oriented differently relative to the LbetaH regions, but are similar when overlaid on each other. The orientation of the EcLpxD tripeptide (residues 303-305), connecting the distal end of the LbetaH and the proximal end of the C-terminal helical domains, differs from its counterpart in CtLpxD (residues 311-312); this results in a 120 degrees rotation of the C-terminal domain relative to the LbetaH region in EcLpxD versus CtLpxD. M290 of EcLpxD appears to cap the distal end of a hydrophobic cleft that binds the acyl chain of the R-3-hydroxyacyl-ACP donor substrate. Under standard assay conditions, wild-type EcLpxD prefers R,S-3-hydroxymyristoyl-ACP over R,S-3-hydroxypalmitoyl-ACP by a factor of 3, whereas the M290A mutant has the opposite selectivity. Both wild-type and M290A EcLpxD rescue the conditional lethality of E. coli RL25, a temperature-sensitive strain harboring point mutations in lpxD. Complementation with wild-type EcLpxD restores normal lipid A containing only N-linked hydroxymyristate to RL25 at 42 degrees C, as judged by mass spectrometry, whereas the M290A mutant generates multiple lipid A species containing one or two longer hydroxy fatty acids in place of the usual R-3-hydroxymyristate at positions 2 and 2'.

Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog.

Myristoylation is the attachment of the 14-carbon fatty acid myristate to the N-terminal glycine residue of proteins. Typically a co-translational modification, myristoylation of proapoptotic cysteinyl-aspartyl proteases (caspase)-cleaved Bid and PAK2 was also shown to occur post-translationally and is essential for their proper localization and proapoptotic function. Progress in the identification and characterization of myristoylated proteins has been impeded by the long exposure times required to monitor incorporation of radioactive myristate into proteins (typically 1-3 months). Consequently, we developed a nonradioactive detection methodology in which a bio-orthogonal azidomyristate analog is specifically incorporated co- or post-translationally into proteins at N-terminal glycines, chemoselectively ligated to tagged triarylphosphines and detected by Western blotting with short exposure times (seconds to minutes). This represents over a million-fold signal amplification in comparison to using radioactive labeling methods. Using rational prediction analysis to recognize putative internal myristoylation sites in caspase-cleaved proteins combined with our nonradioactive chemical detection method, we identify 5 new post-translationally myristoylatable proteins (PKC epsilon, CD-IC2, Bap31, MST3, and the catalytic subunit of glutamate cysteine ligase). We also demonstrate that 15 proteins undergo post-translational myristoylation in apoptotic Jurkat T cells. This suggests that post-translational myristoylation of caspase-cleaved proteins represents a novel mechanism widely used to regulate cell death.