Cholinephosphate Cytidylyltransferase Research Articles

KDM2B regulates choline kinase expression and neuronal differentiation of neuroblastoma cells.

The process of neuronal differentiation is associated with neurite elongation and membrane biogenesis, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. During neuroblast differentiation, the transcription of two genes involved in PtdCho biosynthesis are stimulated: Chka gene for choline kinase (CK) alpha isoform and Pcyt1a gene for CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. Here we show that CKα is essential for neuronal differentiation. In addition, we demonstrated that KDM2B regulates CKα expression and, as a consequence, neuronal differentiation. This factor is up-regulated in the course of the neuroblasts proliferative and undifferentiated state and down-regulated during differentiation induced by retinoic acid (RA). During proliferation, KDM2B binds to the Box2 located in the Chka promoter repressing its transcription. Interestingly, KDM2B knockdown enhances the levels of CKα expression in neuroblast cells and induces neuronal differentiation even in the absence of RA. These results suggest that KDM2B is required for the appropriate regulation of CKα during neuronal differentiation and to the maintaining of the undifferentiated stage of neuroblast cells.

Rhodotorula glutinis as a living cell liposome to deliver polypeptide drugs in vivo

The potential advantages of recombinant microbes as oral drug carriers for curing diseases have attracted much attention. The use of recombinant oil microbes as living cell liposomes to carry polypeptide drugs may be an ideal polypeptide oral drug delivery system. GM4-ΔTS was constructed by LFH-PCR from Rhodotorula glutinis GM4, which was screened and preserved in our laboratory, and then transferred into choline-phosphate cytidylyltransferase (CCT), which is a rate-limiting enzyme for lecithin synthesis. The results showed that the CCT gene was highly expressed in the GM4-ΔTS strain and could significantly increase fatty acid and lecithin contents in GM4-ΔTS-PGK1-CCT. Moreover, insulin, H22-LP, and α-MSH were successfully introduced into cells in vitro, and the strain no longer proliferated in vivo, for safe and controllable polypeptide drug delivery. In vivo, normal mice were intragastrically administered with recombinant strains carrying insulin and α-MSH, and different levels of polypeptide drugs were detected in serum and tissue, respectively. Then, recombinant strains carrying insulin were administered to type II diabetes mellitus mice. The results showed that the strains could effectively reduce blood glucose levels in mice, which indicated that the recombinant strains could carry insulin into the body, and the drug effect was remarkable. Therefore, recombinant GM4-ΔTS-PGK1-CCT strains were successfully used as living cell liposomes to carry insulin, H22-LP, and α-MSH peptides into the body for the first time; additionally, these strains have enhanced safety, controllability, and efficacy.

Phosphatidylcholine synthesis regulates triglyceride storage and chylomicron secretion by Caco2 cells

Intracellular lipid droplets (LDs) supply fatty acids for energy, membrane biogenesis, and lipoprotein secretion. The surface monolayer of LDs is composed of phospholipids, primarily phosphatidylcholine (PC), that stabilize the neutral lipid core of triglyceride (TG). To determine the relationship between PC synthesis and TG storage and secretion in chylomicrons, we used a model of intestinal-derived human epithelial colorectal adenocarcinoma (Caco2) cells with knockout of PCYT1A, which encodes the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase (CCT)α in the CDP-choline pathway, that were treated with the fatty acid oleate. CRISPR/Cas9 knockout of CCTα in Caco2 cells (Caco2-KO cells) reduced PC synthesis by 50%. Compared with Caco2 cells, Caco2-KO cells exposed to oleate had fewer and larger LDs and greater TG accumulation as a result. The addition of exogenous lysophosphatidylcholine to Caco2-KO cells reversed the LD morphology defect. Caco2-KO cells, differentiated into epithelial monolayers, accumulated intracellular TG and had deficient TG and chylomicron-associated apoB48 secretion; apoB100 secretion was unaffected by CCTα knockout or oleate. Metabolic-labeling and LD imaging of Caco2-KO cells indicated preferential shuttling of de novo synthesized TG into larger LDs rather than into chylomicrons. Thus, reduced de novo PC synthesis in Caco2 cells enhances TG storage in large LDs and inhibits apoB48 chylomicron secretion.

Structural determinants of the catalytic mechanism of Plasmodium CCT, a key enzyme of malaria lipid biosynthesis

The development of the malaria parasite, Plasmodium falciparum, in the human erythrocyte, relies on phospholipid metabolism to fulfil the massive need for membrane biogenesis. Phosphatidylcholine (PC) is the most abundant phospholipid in Plasmodium membranes. PC biosynthesis is mainly ensured by the de novo Kennedy pathway that is considered as an antimalarial drug target. The CTP:phosphocholine cytidylyltransferase (CCT) catalyses the rate-limiting step of the Kennedy pathway. Here we report a series of structural snapshots of the PfCCT catalytic domain in its free, substrate- and product-complexed states that demonstrate the conformational changes during the catalytic mechanism. Structural data show the ligand-dependent conformational variations of a flexible lysine. Combined kinetic and ligand-binding analyses confirm the catalytic roles of this lysine and of two threonine residues of the helix αE. Finally, we assessed the variations in active site residues between Plasmodium and mammalian CCT which could be exploited for future antimalarial drug design.

An auto-inhibitory helix in CTP:phosphocholine cytidylyltransferase hijacks the catalytic residue and constrains a pliable, domain-bridging helix pair

The activity of CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme in phosphatidylcholine synthesis, is regulated by reversible interactions of a lipid-inducible amphipathic helix (domain M) with membrane phospholipids. When dissociated from membranes, a portion of the M domain functions as an auto-inhibitory (AI) element to suppress catalysis. The AI helix from each subunit binds to a pair of α helices (αE) that extend from the base of the catalytic dimer to create a four-helix bundle. The bound AI helices make intimate contact with loop L2, housing a key catalytic residue, Lys122 The impacts of the AI helix on active-site dynamics and positioning of Lys122 are unknown. Extensive MD simulations with and without the AI helix revealed that backbone carbonyl oxygens at the point of contact between the AI helix and loop L2 can entrap the Lys122 side chain, effectively competing with the substrate, CTP. In silico, removal of the AI helices dramatically increased αE dynamics at a predicted break in the middle of these helices, enabling them to splay apart and forge new contacts with loop L2. In vitro cross-linking confirmed the reorganization of the αE element upon membrane binding of the AI helix. Moreover, when αE bending was prevented by disulfide engineering, CCT activation by membrane binding was thwarted. These findings suggest a novel two-part auto-inhibitory mechanism for CCT involving capture of Lys122 and restraint of the pliable αE helices. We propose that membrane binding enables bending of the αE helices, bringing the active site closer to the membrane surface.

Rational Design of an Efficient Halotolerant Enzymatic System for In Vitro One-Pot Synthesis of Cytidine Diphosphate Choline.

Salt accumulation often impedes cytidine diphosphate choline (CDP-choline) in vitro biosynthetic process. In this work a halotolerant in vitro enzymatic system is developed to solve this problem. It applies a halotolerant choline-phosphate cytidylyltransferase (CCT) obtained from rational design instructed by a unique strategy, which refers to one of the features of naturally occurring halophilic enzymes. By increasing acidic residues on protein surface where is most variable with respect to amino acid in the sequence alignment with other CCT, the mutants are obtained. The mutants represent higher catalytic activities and IC50 values (inhibit activity by 50%) at high-salt concentrations. Furthermore, when the halotolerant CCT is applied to in vitro one-pot biosynthesis of CDP-choline, the maximum titer and productivity are 161 ± 3.5 mM and 6.2 ± 0.1 mM L-1 h-1 , respectively. When acetate concentration increases, it still keeps relatively high reaction rate and is 2.2-fold higher than process using wild-type CCT (3.87 mM L-1 h-1 comparing with 1.74 mM L-1 h-1 ). This halotolerant system has great potential for industrial use, and the rational design concept can be applied to modify other enzymes, addressing the salt accumulation problem in in vitro systems, and gives insight into resolving by-product inhibition during reaction.

A Combination of the Aerosolized PPAR-γ Agonist Pioglitazone and a Synthetic Surfactant Protein B Peptide Mimic Prevents Hyperoxia-Induced Neonatal Lung Injury in Rats

Background: Despite improvements in perinatal care, bronchopulmonary dysplasia (BPD) in extremely premature infants has not decreased. Postnatal surfactant therapy provides symptomatic relief from respiratory distress syndrome, but does not translate into a reduction in BPD. Therefore, the search for effective interventions to prevent BPD continues. Objectives: Since PPAR-γ agonists have been demonstrated to promote neonatal lung maturation and injury repair, we hypothesized that a formulation of a PPAR-γ agonist, pioglitazone (PGZ) and a synthetic lung surfactant (a surfactant protein B peptide mimic, B-YL) combined would stimulate lung maturation and block hyperoxia-induced neonatal lung injury more effectively than either modality alone. Methods: One-day-old Sprague-Dawley rat pups were administered PGZ + B-YL via nebulization every 24 h for up to 72 h. The pups were exposed to either 21 or 95% O₂, and then sacrificed. Their lungs were examined for markers of lung maturation (levels of PPAR-γ, SP-C and choline-phosphate cytidylyltransferase [CCT-α] and [³H]triolein uptake) and injury repair (bronchoalveolar lavage cell count and protein content, and levels of LEF-1, fibronectin, ALK5, and β-catenin) by Western blot analysis. Results: Markers of alveolar epithelial/mesenchymal maturation (PPAR-γ, SP-C, CCT-α, and triolein uptake) increased significantly in the PGZ + B-YL group, more than with either drug alone. Similarly, markers of hyperoxia-induced lung injury were blocked effectively with PGZ + B-YL treatment. Conclusions: Nebulized PPAR-γ agonist PGZ with a synthetic lung surfactant accelerates lung maturation and prevents neonatal hyperoxia-induced lung injury more than either modality alone, with the potential to provide more effective prevention of BPD.

Gene variants of the phosphatidylcholine synthesis pathway do not contribute to RDS inthe Chinese population.

To determine population-based prevalence and disease contribution of phosphatidylcholine synthetic pathway-associated gene variants in a native southern Chinese cohort. We used bloodspots from 2010 that were obtained from the Guangxi Neonatal Screening Center in Nannning China and included the Han (n=443) and Zhuang (n=313) ethnic groups. We sequenced the exons of cholinephosphate cytidylyltransferase (PCYT1B) lysophospholipid acyltransferase 1 (LPCAT1), and cholinephosphotransferase (CHPT1) genes, and analyzed both rare and common exonic variants. We obtained five mutations (G199D, A299V, G434C, Y490C, L312S) with eight alleles in the three candidate genes. The collapsed minor allele frequency for candidate genes was not significantly different between the Han and Zhuang populations (0.0045 vs. 0.0064, respectively, P=0.725). The combined Han and Zhuang pool collapsed carrier frequency of rare mutation allele was found to be 1.06%, which is much higher than previously reported for the Missouri population (0.1%). Further, we detected six exonic common variants (three in LPCAT1 and three in CHPT1), with three non-synonymous variants (F162S, F341L, M427K) among them. Two of the non-synonymous exonic variants (F341L, M427K) were not found in CHB; F341L was also not previously reported in exome sequencing project. The population-based frequency of mutations in the phosphatidylcholine synthesis pathway-associated genes PCYT1B LPCAT1, CHPT1 is low in southern Chinese newborns and there is no evidence of contribution to population-based disease burden of respiratory distress syndrome. As a population-based study of rare mutations and common variants, this work is valuable in directing future research.

Synaptic Membrane Synthesis in Rats Depends on Dietary Sufficiency of Vitamin C, Vitamin E, and Selenium: Relevance for Alzheimer's Disease.

Chronic consumption of a diet enriched with nutritional precursors of phospholipids, including uridine and the polyunsaturated fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), was shown previously to enhance levels of brain phospholipids and synaptic proteins in rodents. Vitamin C, vitamin E, and selenium may directly affect the breakdown or synthesis of membrane phospholipids. The present study investigated the necessity of antioxidants for the effectiveness of supplementation with uridine plus DHA and EPA (as fish oil) in rats. Rats were randomized to four treatment groups and received, for 6 weeks, one of four experimental diets, i.e., a diet low in antioxidants, a diet high in antioxidants, a diet low in antioxidants supplemented with DHA+EPA+uridine, or a diet high in antioxidants supplemented with DHA+EPA+uridine. On completion of dietary treatment, rats were sacrificed, and brain levels of phospholipids, synaptic proteins, and two enzymes involved in phospholipid synthesis (choline-phosphate cytidylyltransferase, PCYT1A, and choline/ethanolamine phosphotransferase, CEPT1) were analyzed. Levels of phospholipids, the pre- and post-synaptic proteins Synapsin-1 and PSD95, and the enzymes PCYT1A and CEPT1 were significantly enhanced by combined supplementation of DHA+EPA+uridine and antioxidants and not enhanced by supplementation of DHA+EPA+uridine with insufficient antioxidant levels. Our data suggest that dietary vitamin C, vitamin E, and selenium are essential for the phospholipid precursors’ effects on increasing levels of membrane phospholipids and synaptic proteins, the indirect indicators of synaptogenesis. Their concomitant supply may be relevant in Alzheimer’s disease patients, because the disease is characterized by synapse loss and lower plasma and brain levels of phospholipid precursors and antioxidants.

Effects of dietary phospholipids on growth performance and expression of key genes involved in phosphatidylcholine metabolism in larval and juvenile large yellow croaker, Larimichthys crocea

The objective of this study was to investigate the effects of dietary phospholipids (PLs) on survival rate, growth performance and expression of key genes related with phosphatidylcholine (PC) metabolism in larval and juvenile large yellow croaker, Larimichthys crocea. Three isonitrogenous and isolipidic diets with graded levels of PLs (2.53%, 6.32% and 12.7%) were randomly allocated to triplicate groups of larvae (initial body weight: 3.86±0.24mg) eight times daily for 30days. Five isonitrogenous and isolipidic diets with graded levels of PLs (1.32%, 3.05%, 5.86%, 7.4% and 9.63%) were randomly allocated to triplicate groups of juveniles (initial body weight: 7.36±0.33g) twice daily to apparent satiation for 60days. Results showed that survival rate and specific growth rate of larvae fed the diet with 2.53% PLs were significantly lower compared with those of larvae in other groups (P<0.05). However, specific growth rate and survival rate were not significantly different among dietary treatments in juveniles. No significant difference was observed in the transcript levels of key genes involved in PC de novo synthesis, including CTP: choline phosphate cytidylyltransferase and phosphatidylethanolamine N-methyltransferase, in both the whole larvae body and the livers of juveniles fed diets with different concentrations of PLs. Although no significant difference was found, the mRNA expression levels of 1, 2-diacylglycerol choline phosphotransferase increased with the increasing levels of dietary PLs in the livers of juveniles, with acyl-CoA: diacylglycerol acyltransferase 2 showing the opposite trend. These results confirmed that the beneficial effects of dietary PLs on survival and growth performance of large yellow croaker were restricted to fish larvae. Dietary PLs might reduce lipid deposition in the liver of juvenile large yellow croaker through utilizing more diacylglycerol for PC synthesis, rather than triglyceride synthesis. Statement of relevanceThe present study was conducted to investigate the effects of dietary phospholipids (PLs) on survival rate, growth performance and expression of key genes related to phosphatidylcholine (PC) biosynthesis in larval and juvenile large yellow croaker, (Larimichthys crocea). The results are reliable and of both theoretical and practical importance. For “theoretical importance”, our study provided the first evidence that dietary PLs might reduce lipid deposition in the liver of juvenile large yellow croaker through utilizing more diacylglycerol for PC synthesis, rather than triglyceride synthesis, which could contribute to better understanding the mechanisms underlying the discrepancy in requirements for phospholipids between larval and juvenile large yellow croaker. For “practical importance”, our study confirmed that the beneficial effects of dietary PLs on growth performance of large yellow croaker were restricted to larvae rather than juveniles (at least 7.36g) and these findings could be applied to reduce the supplementation of PLs in commercial feeds for large yellow croaker, which in turn might reduce the cost of the commercial feeds for large yellow croaker.The work described has not been submitted elsewhere for publication, in whole or in part, and all the authors listed have approved the manuscript that is enclosed. I have read and have abided by the statement of ethical standards for manuscripts submitted to Aquaculture.

Efficient multi-enzyme-catalyzed CDP-choline production driven by an ATP donor module.

Cytidine diphosphate choline (CDP-choline) has been applied for treating acute craniocerebral injury and allowing recovery of consciousness after brain surgery. In this study, an acetate kinase (ACK)/acetyl phosphate system was used to supply ATP and combined with Escherichia coli-overexpressed CMP kinase (CMK), NDP kinase (NDK), choline phosphate cytidylyltransferase (CCT), and choline kinase (CKI) to produce CDP-choline from CMP and choline chloride. Within 1h, 49mM CDP-choline was produced, for a molar yield of 89.9 and 68.4% based on CMP and choline chloride, respectively; the utilization efficiency of energy (UEE) was 79.5%. Acetyl phosphate, sodium acetate, and CTP inhibited the reaction when the concentration exceeded 18.5, 600, and 30mM, respectively. This inhibition could be overcome by controlling the rate of acetyl phosphate, CMP addition or using KOH instead of NaOH to regulate the pH in fed-batch transformation. After 24h, the maximum titer was 124.1±2.7mM, the productivity was 5.1±0.1mMl-1h-1, the molar yield to CMP and choline chloride were 83.8 and 63.7%, respectively, and the UEE was 58.2%. This high yield and productivity of CDP-choline through biocatalysis suggest future application at the industrial scale.

Participation of prostaglandin D2 in the mobilization of the nuclear-localized CTP:phosphocholine cytidylyltransferase alpha in renal epithelial cells

Phosphatidylcholine (PC) is the main constituent of mammalian cell membranes. Consequently, preservation of membrane PC content and composition — PC homeostasis — is crucial to maintain cellular life. PC biosynthetic pathway is generally controlled by CTP:phosphocholine cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCTα is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum redistribution. However, most of the enzyme is located inside the nuclei. Here, we demonstrate that CCTα is the most abundant isoform in renal collecting duct cells, and its redistribution is dependent on endogenous prostaglandins. Previously we have demonstrated that PC synthesis was inhibited by indomethacin (Indo) treatment, and this effect was reverted by exogenous PGD2. In this work we found that Indo induced CCTα distribution into intranuclear Lamin A/C foci. Exogenous PGD2 reverted this effect by inducing CCTα redistribution to nuclear envelope, suggesting that PGD2 maintains PC synthesis by CCTα mobilization. Interestingly, we found that the effect of PGD2 was dependent on ERK1/2 activation. In conclusion, our previous observations and the present results lead us to suggest that papillary cells possess the ability to maintain their structural integrity through the synthesis of their own survival molecule, PGD2, by modulating CCTα intracellular location.

Metformin Decouples Phospholipid Metabolism in Breast Cancer Cells.

IntroductionThe antidiabetic drug metformin, currently undergoing trials for cancer treatment, modulates lipid and glucose metabolism both crucial in phospholipid synthesis. Here the effect of treatment of breast tumour cells with metformin on phosphatidylcholine (PtdCho) metabolism which plays a key role in membrane synthesis and intracellular signalling has been examined.MethodsMDA-MB-468, BT474 and SKBr3 breast cancer cell lines were treated with metformin and [3H-methyl]choline and [14C(U)]glucose incorporation and lipid accumulation determined in the presence and absence of lipase inhibitors. Activities of choline kinase (CK), CTP:phosphocholine cytidylyl transferase (CCT) and PtdCho-phospholipase C (PLC) were also measured. [3H] Radiolabelled metabolites were determined using thin layer chromatography.ResultsMetformin-treated cells exhibited decreased formation of [3H]phosphocholine but increased accumulation of [3H]choline by PtdCho. CK and PLC activities were decreased and CCT activity increased by metformin-treatment. [14C] incorporation into fatty acids was decreased and into glycerol was increased in breast cancer cells treated with metformin incubated with [14C(U)]glucose.ConclusionThis is the first study to show that treatment of breast cancer cells with metformin induces profound changes in phospholipid metabolism.

Selective Targeting of Lipid Droplets by Proteins

Lipid droplets (LDs) are organelles that store neutral lipids in cells. LDs have a core of neutral lipids, including triacylglycerols and sterol esters, surrounded by a phospholipid monolayer. LDs can grow dramatically to store neutral lipids in its core, which requires a commensurate increase in the size of the LD membrane. CTP:phosphocholine cytidylyltransferase (CCT) plays a key role in regulating the size of LDs, as it the rate-limiting enzyme in phosphatidylcholine (PC) synthesis, which is a major component of the LD outer coat. CCT has been shown to have a binding preference for LD as opposed to bilayer membranes, although the cause of this preference is not known. Previous studies have shown that large hydrophobic packing defects are key for the binding of amphipathic helices (AH) to membranes. We have studied LDs and bilayer membranes by extensive long time scale molecular dynamics (MD) simulations. In our results, we observe differing properties between LD membranes and bilayer membranes, including an increase in the frequency of large defects in LD membranes. We also find, using μs-long MD simulations, that an increase in the size of hydrophobic lipid packing defects in LDs compared to bilayer membranes plays a large role in the specificity of CCT AH initial binding to membranes as compared to bilayer membranes. Moreover, we frequently observe detachment of CCT from bilayer membranes, suggesting the lack of large hydrophobic defects destabilizes this interaction sufficiently to make binding to bilayers thermodynamically less favorable.

Molecular Mechanism for the Thermo-Sensitive Phenotype of CHO-MT58 Cell Line Harbouring a Mutant CTP:Phosphocholine Cytidylyltransferase.

Control and elimination of malaria still represents a major public health challenge. Emerging parasite resistance to current therapies urges development of antimalarials with novel mechanism of action. Phospholipid biosynthesis of the Plasmodium parasite has been validated as promising candidate antimalarial target. The most prevalent de novo pathway for synthesis of phosphatidylcholine is the Kennedy pathway. Its regulatory and often also rate limiting step is catalyzed by CTP:phosphocholine cytidylyltransferase (CCT). The CHO-MT58 cell line expresses a mutant variant of CCT, and displays a thermo-sensitive phenotype. At non-permissive temperature (40°C), the endogenous CCT activity decreases dramatically, blocking membrane synthesis and ultimately leading to apoptosis. In the present study we investigated the impact of the analogous mutation in a catalytic domain construct of Plasmodium falciparum CCT in order to explore the underlying molecular mechanism that explains this phenotype. We used temperature dependent enzyme activity measurements and modeling to investigate the functionality of the mutant enzyme. Furthermore, MS measurements were performed to determine the oligomerization state of the protein, and MD simulations to assess the inter-subunit interactions in the dimer. Our results demonstrate that the R681H mutation does not directly influence enzyme catalytic activity. Instead, it provokes increased heat-sensitivity by destabilizing the CCT dimer. This can possibly explain the significance of the PfCCT pseudoheterodimer organization in ensuring proper enzymatic function. This also provide an explanation for the observed thermo-sensitive phenotype of CHO-MT58 cell line.

Phosphatidylcholine alteration identified using MALDI imaging MS in HBV-infected mouse livers and virus-mediated regeneration defects.

In this study, we investigated whether hepatitis B virus (HBV) causes the alteration of lipid metabolism and composition during acute infection and liver regeneration in a mouse model. The liver controls lipid biogenesis and bile acid homeostasis. Infection of HBV causes various liver diseases and impairs liver regeneration. As there are very few reports available in the literature on lipid alterations by HBV infection or HBV-mediated liver injury, we have analyzed phospholipids that have important roles in liver regeneration by using matrix-assisted laser desorption/ionization (MALDI)-imaging mass spectrometry (IMS) in the livers of HBV model mice. As a result, we identified different phosphatidylcholines (PCs) showing significant changes in their composition as well as cationized ion adduct formation in HBV-infected mouse livers which are associated with virus-mediated regeneration defects. To find the factor of altered PCs, the expression kinetics of enzymes was also examined that regulate PC biosynthesis during liver regeneration. It is noteworthy that the expression of choline-phosphate cytidylyltransferase A (PCYT1A) was significantly delayed in wild type HBV-expressing livers. Moreover, the amount of hepatic total PC was also significantly decreased in wt HBV-expressing mice. These results suggest that infection of HBV alters the composition of PCs which may involve in HBV-mediated regeneration defects and liver disease.

Evaluation of the inhibitory effects of miltefosine on the growth of Babesia and Theileria parasites

Miltefosine, a membrane-active synthetic ether-lipid analogue, has antiproliferative and antiparasitic effects. In this study, the inhibitory effects of miltefosine were evaluated against three Babesia species and Theileria equi in vitro and against Babesia microti in mice. The drug showed significant growth inhibition from an initial parasitemia of 1% for Babesia bovis, Babesia bigemina, Babesia caballi, and T. equi with IC50 values of 25, 10.2, 10.4, and 99μM, respectively. Complete inhibition was observed at 200μM of miltefosine on the third day of culture for the three Babesia species and 400μM on the fourth day for T. equi. Reverse-transcription PCR (RT-PCR) showed that miltefosine inhibited the transcription of choline-phosphate cytidylyltransferase in B. bovis. Miltefosine at a dose rate of 30mg/kg resulted in a 71.7% inhibition of B. microti growth in BALB/c mice. Miltefosine might be used for drug therapy in babesiosis.

The Curvature Sensitivity of a Membrane-Binding Amphipathic Helix Can Be Modulated by the Charge on a Flanking Region

Membrane-induced amphipathic helices (m-AH) can act as membrane curvature sensors by binding preferentially to hydrophobic lipid packing defects enriched in curved surfaces. Reliance on hydrophobicity and membrane curvature for binding is enhanced when electrostatic interactions are weak. We probed the role of modifying membrane and protein charge on the curvature sensing of two m-AH-containing proteins, CTP:phosphocholine cytidylyltransferase (CCT) and α-synuclein (α-syn). The m-AH domains in both proteins are flanked by disordered tails with multiple phosphoserines (CCT) or acidic residues (α-syn), which we mutated to glutamate or serine to modify protein charge. Analysis of binding to vesicles of varying curvature showed that increasing the negative charge of the tail region decreased the binding strength and augmented the curvature dependence, especially for CCT. We attribute this to charge repulsion. Conversely, increasing the membrane negative charge dampened the curvature dependence. Our data suggest that discrimination of curved versus flat membranes with high negative charge could be modulated by phosphorylation.

Choline kinase alpha expression during RA-induced neuronal differentiation: Role of C/EBPβ

Neuronal differentiation is a complex process characterized by a halt in proliferation and extension of neurites from the cell body. This process is accompanied by changes in gene expression that mediate the redirection leading to neurite formation and function. Acceleration of membrane phospholipids synthesis is associated with neurite elongation, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. The transcription of two genes in particular encoding key enzymes in the CDP–choline pathway for PtdCho biosynthesis are stimulated; the Chka gene for choline kinase (CK) alpha isoform and the Pcyt1a gene for the CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. We report that the stimulation of CKα expression during retinoic acid (RA) induced differentiation depends on a promoter region that contains two CCAAT/Enhancer-binding Protein-β (C/EBPβ) sites. We demonstrate that during neuronal differentiation of Neuro-2a cells, RA induces Chka expression by a mechanism that involves ERK1/2 activation which triggers C/EBPβ expression. Elevated levels of C/EBPβ bind to the Chka proximal promoter (Box1) inducing CKα expression. In addition we identified a downstream sequence named Box2 which together with Box1 is required for the promoter to reach the full induction. This is the first elucidation of the mechanism by which the expression of Chka is coordinately regulated during neuronal differentiation.

Structural Basis for Autoinhibition of CTP:Phosphocholine Cytidylyltransferase (CCT), the Regulatory Enzyme in Phosphatidylcholine Synthesis, by Its Membrane-binding Amphipathic Helix

CTP:phosphocholine cytidylyltransferase (CCT) interconverts between an inactive soluble and active membrane-bound form in response to changes in membrane lipid composition. Activation involves disruption of an inhibitory interaction between the αE helices at the base of the active site and an autoinhibitory (AI) segment in the regulatory M domain and membrane insertion of the M domain as an amphipathic helix. We show that in the CCT soluble form the AI segment functions to suppress kcat and elevate the Km for CTP. The crystal structure of a CCT dimer composed of the catalytic and AI segments reveals an AI-αE interaction as a cluster of four amphipathic helices (two αE and two AI helices) at the base of the active sites. This interaction corroborates mutagenesis implicating multiple hydrophobic residues within the AI segment that contribute to its silencing function. The AI-αE interaction directs the turn at the C-terminal end of the AI helix into backbone-to-backbone contact with a loop (L2) at the opening to the active site, which houses the key catalytic residue, lysine 122. Molecular dynamics simulations suggest that lysine 122 side-chain orientations are constrained by contacts with the AI helix-turn, which could obstruct its engagement with substrates. This work deciphers how the CCT regulatory amphipathic helix functions as a silencing device.