Phosphoethanolamine Research Articles

Substrate structure-activity relationship reveals a limited lipopolysaccharide chemotype range for intestinal alkaline phosphatase

Lipopolysaccharide (LPS) from the Gram-negative bacterial outer membrane potently activates the human innate immune system. LPS is recognized by the Toll-like receptor 4/myeloid differentiation factor-2 (TLR4/MD2) complex, leading to the release of pro-inflammatory cytokines. Alkaline phosphatase (AP) is currently being investigated as an anti-inflammatory agent for detoxifying LPS through dephosphorylating lipid A, thus providing a potential treatment for managing both acute (sepsis) and chronic (metabolic endotoxemia) pathologies wherein aberrant TLR4/MD2 activation has been implicated. Endogenous LPS preparations are chemically heterogeneous, and little is known regarding the LPS chemotype substrate range of AP. Here, we investigated the activity of AP on a panel of structurally defined LPS chemotypes isolated from Escherichia coli and demonstrate that calf intestinal AP (cIAP) has only minimal activity against unmodified enteric LPS chemotypes. Pi was only released from a subset of LPS chemotypes harboring spontaneously labile phosphoethanolamine (PEtN) modifications connected through phosphoanhydride bonds. We demonstrate that the spontaneously hydrolyzed O-phosphorylethanolamine is the actual substrate for AP. We found that the 1- and 4'-lipid A phosphate groups critical in TLR4/MD2 signaling become susceptible to hydrolysis only after de-O-acylation of ester linked primary acyl chains on lipid A. Furthermore, PEtN modifications on lipid A specifically enhanced hTLR4 agonist activity of underacylated LPS preparations. Computational binding models are proposed to explain the limitation of AP substrate specificity imposed by the acylation state of lipid A, and the mechanism of PEtN in enhancing hTLR4/MD2 signaling.

Discrimination of oral squamous cell carcinoma from oral lichen planus by salivary metabolomics.

This study was conducted to distinguish salivary metabolites in oral squamous cell carcinoma (OSCC) from those in oral lichen planus (OLP) to identify practical biomarkers for the discrimination of OSCC from OLP. Whole unstimulated saliva samples were collected from patients with OSCC (n=34) and OLP (n=26). Hydrophilic metabolites in the saliva samples were comprehensively analysed by capillary electrophoresis mass spectrometry. To evaluate the discrimination ability of a combination of multiple markers, a multiple logistic regression (MLR) model was developed to differentiate OSCC from OLP. Fourteen metabolites were found to be significantly different between the OSCC and OLP groups. Among them, indole-3-acetate and ethanolamine phosphate were used to develop the MLR model. The combination of these two metabolites showed a high area under the receiver operating characteristic curve (0.856, 95% confidential interval: 0.762-0.950; p<.001) for discriminating OSCC from OLP. We identified salivary metabolites for discerning between OSCC and OLP, which is clinically important for detecting the malignant transformation of OLP by both dentists and oral surgery specialists. Our candidate salivary metabolites show potential for non-invasive screening of OSCC versus OLP.

Effect of ionic liquid on sugar-aromatic separation selectivity by metal-organic framework NU-1000 in aqueous solution

Leveraging on previously observed selectivity in separating sugars from aromatics in aqueous solution via adsorption to metal-organic framework NU-1000, we investigate the effect of ionic liquid (IL) cosolvent on this separation. Four ILs (cholinium lysinate, cholinium phosphate, ethanolamine acetate, and ethanolamine phosphate) were investigated at concentrations of 0.4, 10, and 20 vol%. When treated with an aqueous IL mixture containing glucose, xylose, coumaric acid, and ferulic acid, highly selective sugar-aromatic separations were observed for NU-1000 at 0.4 vol% for both cholinium lysinate and ethanolamine acetate. In contrast, amorphous carbon under similar conditions lacked selectivity, adsorbing similar amounts of aromatics to NU-1000 while also adsorbing ≥96 mg gadsorbent−1 of sugars. These results parallel those previously observed in pure aqueous solution. At higher concentrations of IL, however, NU-1000 displayed ≥56 mg gadsorbent−1 of sugar uptakes for all ILs, with the largest sugar uptakes being observed for ethanolamine acetate. This mirrored results when treating NU-1000 with sugars and IL in aqueous solution, in the absence of aromatics, in a way that depended non-monotonically on the IL concentration. We implicate coadsorption of IL onto NU-1000 as the reason why selectivity in the presence of IL is lower than the selectivity in pure aqueous solution.

Oxidative Stress and Lymphocyte Alterations in Chronic Relapsing Experimental Allergic Encephalomyelitis in the Rat Hippocampus and Protective Effects of an Ethanolamine Phosphate Salt.

Chronic relapsing experimental allergic encephalomyelitis (CR-EAE) exhibits neuropathological and immunological dysfunctions similar to those found in multiple sclerosis (MS) and has been used as an animal model of MS. Inflammatory infiltrates and oxidative stress have been linked to the development of both diseases. Ethanolamine plasmalogen derivates have been shown to be powerful antioxidants and immunomodulators. Therefore, the objective of this study was to analyse inflammatory infiltrates, the state of the oxidative defences and the possible protective effects of calcium, magnesium and phosphate ethanolamine (EAP) in the CR-EAE rat hippocampus. To this aim, we evaluated, by immunohistochemistry, T cell infiltrates, Iba-1+ (a marker of activated microglia) immunoreactivity and TUNEL (+) cells. We also measured the protein levels and activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GP) and glutathione reductase (GR). In addition, reduced (GSH) and oxidized (GSSG) glutathione levels, lipid peroxidation and cholesterol as well as desmosterol content were determined. We found an increase in T cell infiltrates and Iba1+ immunoreactivity, lipid peroxidation, SOD, GP and GR activities as well as enhanced cholesterol levels and a decrease in CAT activity, GSH and desmosterol levels in the first and second attack in the CR-EAE rat hippocampus. Pretreatment of CR-EAE rats with EAP led to a delay in the onset of the clinical signs of the disease as well as a decrease in inflammatory infiltrates and alterations of the antioxidant defences in the hippocampus. Altogether, the present results suggest a protective role of EAP in the CR-EAE rat hippocampus.

Influence of Environmental Conditions on the Fusion of Cationic Liposomes with Living Mammalian Cells.

Lipid-based nanoparticles, also called vesicles or liposomes, can be used as carriers for drugs or many types of biological macromolecules, including DNA and proteins. Efficiency and speed of cargo delivery are especially high for carrier vesicles that fuse with the cellular plasma membrane. This occurs for lipid mixture containing equal amounts of the cationic lipid DOTAP and a neutral lipid with an additional few percents of an aromatic substance. The fusion ability of such particles depends on lipid composition with phosphoethanolamine (PE) lipids favoring fusion and phosphatidyl-choline (PC) lipids endocytosis. Here, we examined the effects of temperature, ionic strength, osmolality, and pH on fusion efficiency of cationic liposomes with Chinese hamster ovary (CHO) cells. The phase state of liposomes was analyzed by small angle neutron scattering (SANS). Our results showed that PC containing lipid membranes were organized in the lamellar phase. Here, fusion efficiency depended on buffer conditions and remained vanishingly small at physiological conditions. In contrast, SANS indicated the coexistence of very small (~50 nm) objects with larger, most likely lamellar structures for PE containing lipid particles. The fusion of such particles to cell membranes occurred with very high efficiency at all buffer conditions. We hypothesize that the altered phase state resulted in a highly reduced energetic barrier against fusion.

How To Build a Bone: PHOSPHO1, Biomineralization, and Beyond.

ABSTRACTSince its characterization two decades ago, the phosphatase PHOSPHO1 has been the subject of an increasing focus of research. This work has elucidated PHOSPHO1's central role in the biomineralization of bone and other hard tissues, but has also implicated the enzyme in other biological processes in health and disease. During mineralization PHOSPHO1 liberates inorganic phosphate (Pi) to be incorporated into the mineral phase through hydrolysis of its substrates phosphocholine (PCho) and phosphoethanolamine (PEA). Localization of PHOSPHO1 within matrix vesicles allows accumulation of Pi within a protected environment where mineral crystals may nucleate and subsequently invade the organic collagenous scaffold. Here, we examine the evidence for this process, first discussing the discovery and characterization of PHOSPHO1, before considering experimental evidence for its canonical role in matrix vesicle–mediated biomineralization. We also contemplate roles for PHOSPHO1 in disorders of dysregulated mineralization such as vascular calcification, along with emerging evidence of its activity in other systems including choline synthesis and homeostasis, and energy metabolism. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

1873-P: NAFLD and Insulin Resistance Is Associated with a Specific Signature in Membrane Phospholipid Metabolites in Adolescents with Polycystic Ovary Syndrome (PCOS)

Obese adolescents with PCOS are at high risk of nonalcoholic Fatty Liver Disease (NAFLD) which contributes to the risk of diabetes. Impaired hepatic energy metabolism is associated with diabetes and characterized by decreased inorganic phosphates (Pi) and ATP hepatic content. Membrane phospholipids measured by magnetic resonance spectroscopy (MRS) may reflect endoplasmic reticulum (ER) stress (phosphodiesters; PDE) or turnover/composition of membrane phospholipids: precursors (phosphomonoesters (PME): phosphoethanolamine (PE) and phosphocholine (PC)) and degradation products (PDE). It is not known if adolescents with PCOS and NAFLD have impaired hepatic energy metabolism or MRS signature associated with insulin sensitivity (Si). Obese adolescents with PCOS (n=25) underwent fasted hepatic 31phosphorus-MRS to measure ATP, Pi, PME and PDE relative to total phosphates (TP). Liver stiffness was measured with MR elastography and hepatic fat fraction (HFF) by MRI-proton density, with an HFF &amp;gt;5.5% classified as NAFLD. A 6h oral sugar tolerance test (OSTT, 25g fructose+75g glucose) was performed. Insulin sensitivity (Si) was calculated with the oral minimal model. 12 girls (BMI 36.4 ±7.3 kg/m2, age 16 (15-17)) had NAFLD and 13 did not (BMI 34.0±4.9 kg/m2, age 16 (14-17)). PME/TP were lower in girls with NAFLD (p=0.007) as well as the ratio of PME/PDE (p=0.002). A lower PE/TP was associated with higher liver stiffness (r= -0.55; p=0.009) and lower Si (r=0.57; p= 0.03). Total ATP/TP, y-ATP/TP and Pi/TP were similar between groups (p&amp;gt;0.15). Obese adolescents with PCOS and NAFLD have a distinct signature in hepatic 31P-MRS that is associated with Si suggesting alterations in membrane phospholipids. However, changes in energetics seen in adults with NAFLD are not present. Further work is needed to understand how these changes in hepatic phospholipids relates to insulin sensitivity and risk for diabetes. Disclosure A. Carreau: Advisory Panel; Self; Pfizer Inc. Y. Garcia Reyes: None. B. Newcomer: None. D.E. Befroy: None. M.S. Brown: None. M. Cree-Green: None. Funding National Institutes of Health (K23DK107871); Doris Duke Charitable Foundation; National Center for Advancing Translational Sciences (UL1TR002535); Diabetes Canada

MON-494 Infantile Hypophosphatasia Diagnosed in Adulthood: A Case Report

Background: Hypophosphatasia (HPP) is a result of loss of function mutations within ALPL gene that encodes for the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). It is characterized by low serum alkaline phosphatase (ALP) activity. To date, HPP was recognized in 500-600 individuals in the United States. Clinical presentation and severity is broad, which can be explained by variable mutations in the TNSALP gene. Elevation of serum pyridoxal-5’-phosphate (vitamin B6), elevation of urinary of phosphoethanolamine (PEA) and finding of a mutation in the ALPL gene support the diagnosis. Asfotase alfa is a recombinant human alkaline phsosphatase that was approved in 2015 for treatment of patients, including adults, with pediatric-onset HPP. Case report: A 56 year old physician presented with a long history of low serum ALP on routine laboratory testing since childhood. During infancy he was diagnosed with craniosynostosis. He had delayed motor milestones and a waddling gait. He was never able to run as a child and he attended high school in a wheelchair. He had severe periodontal disease, lost his first tooth at age 4 and continued to lose his teeth despite excellent dental hygiene care. He has an extensive bone fractures history. He had his first fracture at the elbow at age 6 after he was pulled up by his hands. He developed metatarsal fractures around that time after a fall. He had a metacarpal fracture as an adult with bumping of his hand against the wall. At age 12 he developed hip pain and uveitis. He was diagnosed with ankylosing spondylitis (AS) at age 16 year with a positive HLA B 27. His most recent fracture was at age 45 after he slipped in the shower and sustained a hip fracture. At that time he had a BMD that showed osteoporosis. A bisphosphonate was offered but the patient refused medication as he was worried of the side effects. He has a family history of musculoskeletal disorders. His late mother had a history of low ALP, dental problems, metatarsal fractures and osteopenia. His sister has low ALP and had a low impact radial fracture. His sister’s son who is 18 years old was found to have low serum ALP and was found to have a Z score of -1.5 on BMD. When the patient presented to our clinic, his serum ALP was 22 U/L (n 40-115) and vitamin B 6 was 253 ng/ml (n 2.1-21.7). Genetic testing revealed heterozygous variant in the ALPL gene c.1251T>G (p.Asn417Lys). The patient’s sister and her son were found to have similar mutation of the ALPL gene. The patient was started on treatment with Asfotase alfa. After three months of treatment, the patient reported increased strength and reduced fatigue. Conclusion: We present a rare diagnosis of a 56 year old gentleman who was diagnosed with infantile HPP as an adult. The disease manifested as craniosynostosis, recurrent fractures and periodontal disease. He was started on Asfotase alfa with improvement of symptoms. .

Manganese-Induced Substrate Promiscuity in the Reaction Catalyzed by Phosphoglutamine Cytidylyltransferase from Campylobacter jejuni.

The leading cause of bacterial gastroenteritis, Campylobacter jejuni, is a Gram-negative pathogen that contains a unique O-methyl phosphoramidate (MeOPN) on its capsular polysaccharide. Previously, MeOPN has been linked to the evasion of host immune responses and serum resistance. Despite the involvement of MeOPN in pathogenicity, the complete biosynthesis of this modification is unknown; however, the first four enzymatic steps have been elucidated. The second enzyme in this pathway, Cj1416, is a CTP/phosphoglutamine cytididylyltransferase that catalyzes the displacement of pyrophosphate from MgCTP by l-glutamine phosphate to form CDP-l-glutamine. Initially, Cj1416 was predicted to use phosphoramidate to form cytidine diphosphoramidate, but no activity was detected with MgATP as a substrate. However, in the presence of MnCTP, Cj1416 can directly catalyze the formation of cytidine diphosphoramidate from phosphoramidate and MnCTP. Here we characterize the manganese-induced promiscuity of Cj1416. In the presence of Mn2+, Cj1416 catalyzes the formation of 12 different reaction products using l-glutamine phosphate, phosphoramidate, methyl phosphate, methyl phosphonate, phosphate, arsenate, ethanolamine phosphate, glycerol-1-phosphate, glycerol-2-phosphate, serinol phosphate, l-serine phosphate, or 3-phospho-d-glycerate as the nucleophile to displace pyrophosphate from CTP.

Early detection of changes in phospholipid metabolism during neoadjuvant chemotherapy in breast cancer patients using phosphorus magnetic resonance spectroscopy at 7T.

The purpose of this work was to investigate whether noninvasive early detection (after the first cycle) of response to neoadjuvant chemotherapy (NAC) in breast cancer patients was possible. 31P‐MRSI at 7 T was used to determine different phosphor metabolites ratios and correlate this to pathological response. 31P‐MRSI was performed in 12 breast cancer patients treated with NAC. 31P spectra were fitted and aligned to the frequency of phosphoethanolamine (PE). Metabolic signal ratios for phosphomonoesters/phosphodiesters (PME/PDE), phosphocholine/glycerophosphatidylcholine (PC/GPtC), phosphoethanolamine/glycerophosphoethanolamine (PE/GPE) and phosphomonoesters/in‐organic phosphate (PME/Pi) were determined from spectral fitting of the individual spectra and the summed spectra before and after the first cycle of NAC. Metabolic ratios were subsequently related to pathological response. Additionally, the correlation between the measured metabolic ratios and Ki‐67 levels was determined using linear regression.Four patients had a pathological complete response after treatment, five patients a partial pathological response, and three patients did not respond to NAC. In the summed spectrum after the first cycle of NAC, PME/Pi and PME/PDE decreased by 18 and 13%, respectively. A subtle difference among the different response groups was observed in PME/PDE, where the nonresponders showed an increase and the partial and complete responders a decrease (P = 0.32). No significant changes in metabolic ratios were found. However, a significant association between PE/Pi and the Ki‐67 index was found (P = 0.03).We demonstrated that it is possible to detect subtle changes in 31P metabolites with a 7 T MR system after the first cycle of NAC treatment in breast cancer patients. Nonresponders showed different changes in metabolic ratios compared with partial and complete responders, in particular for PME/PDE; however, more patients need to be included to investigate its clinical value.

Fifty years of lyase and a moment of truth: sphingosine phosphate lyase from discovery to disease

Sphingosine phosphate lyase (SPL) is the final enzyme in the sphingolipid degradative pathway, catalyzing the irreversible cleavage of long-chain base phosphates (LCBPs) to yield a long-chain aldehyde and ethanolamine phosphate (EP). SPL guards the sole exit point of sphingolipid metabolism. Its inactivation causes product depletion and accumulation of upstream sphingolipid intermediates. The main substrate of the reaction, sphingosine-1-phosphate (S1P), is a bioactive lipid that controls immune-cell trafficking, angiogenesis, cell transformation, and other fundamental processes. The products of the SPL reaction contribute to phospholipid biosynthesis and programmed cell-death activation. The main features of SPL enzyme activity were first described in detail by Stoffel et al. in 1969. The first SPL-encoding gene was cloned from budding yeast in 1997. Reverse and forward genetic strategies led to the rapid identification of other genes in the pathway and their homologs in other species. Genetic manipulation of SPL-encoding genes in model organisms has revealed the contribution of sphingolipid metabolism to development, physiology, and host-pathogen interactions. In 2017, recessive mutations in the human SPL gene SGPL1 were identified as the cause of a novel inborn error of metabolism associated with nephrosis, endocrine defects, immunodeficiency, acanthosis, and neurological problems. We refer to this condition as SPL insufficiency syndrome (SPLIS). Here, we share our perspective on the 50-year history of SPL from discovery to disease, focusing on insights provided by model organisms regarding the pathophysiology of SPLIS and how SPLIS raises the possibility of a hidden role for sphingolipids in other disease conditions.

Free, unlinked glycosylphosphatidylinositols on mammalian cell surfaces revisited

Glycosylphosphatidylinositols (GPIs) are linked to many cell-surface proteins, anchor these proteins in the membrane, and are well characterized. However, GPIs that exist in the free form on the mammalian cell surface remain largely unexplored. To investigate free GPIs in cultured cell lines and mouse tissues, here we used the T5-4E10 mAb (T5 mAb), which recognizes unlinked GPIs having an N-acetylgalactosamine (GalNAc) side chain linked to the first mannose at the nonreducing terminus. We detected free GPIs bearing the GalNAc side chain on the surface of Neuro2a and CHO, but not of HEK293, K562, and C2C12 cells. Furthermore, free GPIs were present in mouse pons, medulla oblongata, spinal cord, testis, epididymis, and kidney. Using a panel of Chinese hamster ovary cells defective in both GPI-transamidase and GPI remodeling pathway, we demonstrate that free GPIs follow the same structural remodeling pathway during passage from the endoplasmic reticulum to the plasma membrane as do protein-linked GPI. Specifically, free GPIs underwent post-GPI attachment to protein 1 (PGAP1)-mediated inositol deacylation, PGAP5-mediated removal of the ethanolamine phosphate from the second mannose, and PGAP3- and PGAP2-mediated fatty acid remodeling. Moreover, T5 mAb recognized free GPIs even if the inositol-linked acyl chain or ethanolamine-phosphate side chain linked to the second mannose is not removed. In contrast, addition of a fourth mannose by phosphatidylinositol glycan anchor biosynthesis class Z (PIGZ) inhibited T5 mAb-mediated detection of free GPIs. Our results indicate that free GPIs are normal components of the plasma membrane in some tissues and further characterize free GPIs in mammalian cells.

Ex Vivo1H NMR study of pituitary adenomas to differentiate various immunohistochemical subtypes

Pituitary adenomas (PAs) are benign growths arising from epithelial cells in the adenohypophysis of the pituitary gland. To date, there has been no detailed metabolic characterization of PAs of various subtypes. In this study, we report nuclear magnetic resonance (NMR) based metabolomic analysis of surgically resected tumors from forty five pituitary tumor patients [gonadotropic (LH/FSH-secreting) = 17; prolactinomas (PRL-secreting) = 11, Cushing’s disease (ACTH-secreting) = 4, non-functional = 5, and mixed = 8] who underwent transsphenoidal selective adenomectomy. Compared to LH/FSH-secreting tumors, PRL-secreting tumors showed statistically significant decrease in the levels of N-acetylaspartate (NAA), myo-inositol (mI), scyllo-inositol (sI), glycine, taurine, phosphoethanolamine (PE) and increase in the levels of glutamine. When compared with LH/FSH-secreting tumors, ACTH-secreting tumors showed statistically significant decrease in the levels of sI, glycine, PE and increase in the levels of aspartate. Although lipid extracts of PAs showed the presence of many common lipid molecules, only glycerophosphoethanolamine (GPE) showed statistically significant decrease in PRL, ACTH and non-functional subtypes when compared to LH/FSH-secreting tumors. Changes observed in these metabolite concentrations among various subtypes of PAs reflect metabolic heterogeneity in these tumors and may pave the way towards the development of metabolic markers to distinguish various immunohistochemical subtypes of PAs.

Comprehensive proteomic and metabolomic profiling of mcr-1-mediated colistin resistance in Escherichia coli

Spread of the mcr-1 gene in human and veterinary medicine has jeopardised the use of polymyxins, last-resort antibiotics against life-threatening multidrug-resistant Gram-negative bacteria. As a lipid-modifying gene, whether mcr-1 causes proteomic and metabolomic changes in bacteria and affects the corresponding metabolic pathway is largely unknown. In this study, label-free quantitative proteomics and untargeted metabolomics were used to profile comprehensive proteome and metabolome characteristics of mcr-1-mediated colistin-resistant and -susceptible Escherichia coli in order to gain further insight into the colistin resistance mechanism. Large sets of differentially expressed proteins (DEPs) and metabolites were identified that contributed to mcr-1-mediated antimicrobial resistance, predominantly in different growth conditions with and without colistin. mcr-1 caused downregulated expression of most proteins in order to adapt to drug pressure. Pathway analysis showed that metabolic processes were significantly affected, mainly related to glycerophospholipid metabolism, thiamine metabolism and lipopolysaccharide (LPS) biosynthesis. The substrate phosphoethanolamine (PEA) for mcr-1 to mediate colistin resistance was accumulated in colistin-resistant E. coli. Notably, mcr-1 not only caused PEA modification of the bacterial cell membrane lipid A but also affected the biosynthesis and transport of lipoprotein in colistin resistance by disturbing the expression of efflux pump proteins involved in the cationic antimicrobial peptide (CAMP) resistance pathway. Overall, disturbed glycerophospholipid metabolism and LPS biosynthesis as well as accumulation of the substrate PEA was closely related with mcr-1-mediated colistin resistance. These findings could provide further valuable information to inhibit colistin resistance by blocking this metabolic process.

Lipid-Dependent Alternating Access Mechanism of a Bacterial Multidrug ABC Exporter.

By undergoing conformational changes, active membrane transporters alternate between an inward-facing (IF) and an outward-facing (OF) state to transport their substrates across cellular membrane. The conformational landscape of membrane transporters, however, could be influenced by their environment, and the dependence of the alternating access mechanism on the lipid composition has not been understood at the molecular level. We have performed an extensive set of microsecond-level all-atom molecular dynamics (MD) simulations on bacterial ATP binding cassette (ABC) exporter Sav1866 in six different phosphocholine (PC) and phosphoethanolamine (PE) lipid membrane environments. This study mainly focuses on the energetically downhill OF-to-IF conformational transition of Sav1866 upon the ATP hydrolysis. We observe that the transporter undergoes large-scale conformational changes in the PE environment, particularly in the POPE lipids, resulting in an IF-occluded conformation, a transition that does not occur when the transporter is embedded in any of the PC lipid bilayers. We propose that the PE lipids facilitate the closing of the protein on the periplasmic side due to their highly polar headgroups that mediate the interaction of the two transmembrane (TM) bundles by a network of lipid–lipid and lipid–protein hydrogen bonds. POPE lipids in particular facilitate the closure of periplasmic gate by promoting a hinge formation in TM helices and an interbundle salt bridge formation. This study explains how the alternating access mechanism and the flippase activity in ABC exporters could be lipid-dependent.

Structural characterization of phosphoethanolamine-modified lipid A from probiotic Escherichia coli strain Nissle 1917.

Gut microbiota, a complex microbial community inhabiting human or animal intestines recently regarded as an endocrine organ, has a significant impact on human health. Probiotics can modulate gut microbiota and the gut environment by releasing a range of bioactive compounds. Escherichia coli (E. coli) strain Nissle 1917 (EcN), a Gram-negative bacterial strain, has been used to treat gastrointestinal (GI) disorders (i.e., inflammatory bowel disease, diarrhea, ulcerative colitis, and so on). However, endotoxicity of lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria in the gut, is known to have a strong influence on gut inflammation and maintenance of gut homeostasis. Therefore, characterizing the chemical structure of lipid A which determines the toxicity of LPS is needed to understand nonpathogenic colonization and commensalism properties of EcN in the gut more precisely. In the present study, MALDI multiple-stage mass spectrometry analysis of lipid A extracted from EcN demonstrates that hexaacylated lipid A (m/z 1919.19) contains a glucosamine disaccharide backbone, a myristate, a laurate, four 3-hydroxylmyristates, two phosphates, and phosphoethanolamine (PEA). PEA modification of lipid A is known to contribute to cationic antimicrobial peptide (CAMP) resistance of Gram-negative bacteria. To confirm the role of PEA in CAMP resistance of EcN, minimum inhibitory concentrations (MICs) of polymyxin B and colistin were determined using a wild-type strain and a mutant strain with deletion of eptA gene encoding PEA transferase. Our results confirmed that MICs of polymyxin B and colistin for the wild-type were twice as high as those for the mutant. These results indicate that EcN can more efficiently colonize the intestine through PEA-mediated tolerance despite the presence of CAMPs in human gut such as human defensins. Thus, EcN can be used to help treat and prevent many GI disorders.

Structural and mechanistic insights into polymyxin resistance mediated by EptC originating from Escherichia coli.

Gram-negative bacteria defend against the toxicity of polymyxins by modifying their outer membrane lipopolysaccharide (LPS). This modification mainly occurs through the addition of cationic molecules such as phosphoethanolamine (PEA). EcEptC is a PEA transferase from Escherichia coli (E. coli). However, unlike its homologs CjEptC (Campylobacter jejuni) and MCR-1, EcEptC is unable to mediate polymyxin resistance when overexpressed in E. coli. Here, we report crystal structures of the C-terminal putative catalytic domain (EcEptCΔN, 205-577 aa) of EcEptC in apo and Zn2+ -bound states at 2.10 and 2.60 Å, respectively. EcEptCΔN is arranged into an α-β-α fold and equipped with the zinc ion in a conserved mode. Coupled with isothermal titration calorimetry (ITC) data, we provide insights into the mechanism by which EcEptC recognizes Zn2+ . Furthermore, structure comparison analysis indicated that disulfide bonds, which play a key role in polymyxin resistance, were absent in EcEptCΔN. Supported by structural and biochemical evidence, we reveal mechanistic implications for disulfide bonds in PEA transferase-mediated polymyxin resistance. Significantly, because the structural effects exhibited by disulfide bonds are absent in EcEptC, it is impossible for this protein to participate in polymyxin resistance in E. coli. DATABASE: Structural data are available in the PDB under the accession numbers 6A82 and 6A83. ENZYME: EC 2.7.8.43.

Fluorescent ZnS Quantum Dots-Phosphoethanolamine Nanoconjugates for Bioimaging Live Cells in Cancer Research.

Many human diseases, including metabolic, immune, and central nervous system disorders, as well as several types of cancers, are the consequence of an important alteration in lipid-related metabolic biomolecules. Although recognized that one of the most important metabolic hallmarks of cancer cells is deregulation of lipid metabolism, the multiple complex signaling pathways are poorly understood yet. Thus, in this research, novel nanoconjugates made of ZnS quantum dots (QDs) were directly synthesized in aqueous media using phosphoethanolamine (PEA) as the capping ligand, which is an important biomolecule naturally present in cells for de novo biosynthesis of fatty acids and phospholipids involved in the cell structure (e.g., membrane), differentiation, and cancer growth. These QD–PEA bio-nanoconjugates were characterized by spectroscopical and morphological techniques. The results demonstrated that fluorescent ZnS nanocrystalline QDs were produced with uniform spherical morphology and estimated sizes of 3.3 ± 0.6 nm. These nanoconjugates indicated core–shell colloidal nanostructures (ZnS QD–PEA) with the hydrodynamic diameter (HD) of 26.0 ± 3.5 nm and ζ-potential centered at −30.0 ± 4.5 mV. The cell viability response using mitochondrial activity assay in vitroconfirmed no cytotoxicity at several concentrations of PEA (biomolecule) and the ZnS–PEA nanoconjugates. Moreover, these nanoconjugates effectively behaved as fluorescent nanomarkers for tracking the endocytic pathways of cancer cells using confocal laser scanning microscopy bioimaging. Hence, these results proved that biofunctionalized ZnS–PEA nanoprobes offer prospective tools for cellular bioimaging with encouraging forecast for future applications as active fluorescent biomarker conjugates in metabolic-related cancer research.

Loss of Phosphoethanolamine N-Methyltransferases Abolishes Phosphatidylcholine Synthesis and Is Lethal.

Plants use several pathways to synthesize phosphatidylcholine (PC), the major phospholipid of eukaryotic cells. PC has important structural and signaling roles. One pathway plants use for synthesis is the phospho-base methylation pathway, which forms the head-group phosphocholine through the triple methylation of phosphoethanolamine (PEA) catalyzed by phosphoethanolamine N-methyltransferases (PEAMTs). Our understanding of that pathway and its physiological importance remains limited. We recently reported that disruption of Arabidopsis thaliana PEAMT1/NMT1 and PEAMT3/NMT3 induces severe PC deficiency leading to dwarfism and impaired development. However, the double nmt1 nmt3 knock-out mutant is viable. Here, we show that this is enabled by residual PEAMT activity through a third family member, NMT2. The triple nmt1 nmt2 nmt3 knock-out mutant cannot synthesize PC from PEA and is lethal. This shows that, unlike mammals and yeast, Arabidopsis cannot form PC from phosphatidyl ethanolamine (PE), and demonstrates that methylation of PEA is the sole, and vital, entry point to PC synthesis. We further show that Arabidopsis has evolved an expanded family of four nonredundant PEAMTs through gene duplication and alternate use of the NMT2 promoter. NMT2 encodes two PEAMT variants, which greatly differ in their ability to perform the initial phospho-base methylation of PEA. Five amino acids at the N terminus of PEAMTs are shown to each be critical for the catalysis of that step committing to PC synthesis. As a whole, these findings open new avenues for enzymatic engineering and the exploration of ways to better tune phosphocholine and PC synthesis to environmental conditions for improved plant performance.

Effect of the redox reactivity of vanadium ions enhanced by phosphorylethanolamine based catalyst on the performance of vanadium redox flow battery

Phosphorylethanolamine (PHOS) doped carbon nanotube (CNT) is newly suggested as a catalyst for enhancing the redox reactions of vanadium ions and vanadium redox flow battery (VRFB) performance. For synthesizing the catalyst, CNT is linked to the phosphate group of the PHOS, forming phosphate functionalized carbon nanotube (POH-CNT). Its catalytic activity is then compared with those of pure CNT and carboxylic acid functionalized CNT (CA-CNT) catalysts. Regarding the redox reactivity of vanadium ions, POH-CNT exhibits superior catalytic activity and reaction reversibility for VO2+/VO2+ reaction to CNT and CA-CNT because of (i) the chelation ability of phosphate group, (ii) its low electron delocalization capability and (iii) its high acid dissociation constant. By the role as chelating agent of phosphate group, the density of active sites for reaction of VO2+ and VO2+ ions in POH-CNT increase. Also, the low electron delocalization and high acid dissociation constant induce effective adsorption and desorption of VO2+ and VO2+ ions. Such effects facilitate the VO2+/VO2+ reaction. In contrast, regarding V2+/V3+ reaction, POH-CNT shows equivalent reactivity to CA-CNT because phosphate and carboxyl groups form similar active sites for the V2+/V3+ reaction. The above results are supported by calculating kinetic parameters, such as charge transfer resistance and diffusion coefficient, while chemical bonding of the catalysts is examined by XPS. Finally, as the POH-CNT is used as catalyst for positive electrode prompting the VO2+/VO2+ reaction, performance of VRFB single cell is improved.