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Fatty acids stored in triacylglycerol-rich lipid droplets are assembled with a surface monolayer composed primarily of phosphatidylcholine (PC). Fatty acids stimulate PC synthesis by translocating CTP:phosphocholine cytidylyltransferase (CCT) α to the inner nuclear membrane, nuclear lipid droplets (nLD) and lipid associated promyelocytic leukemia (PML) structures (LAPS). Huh7 cells were used to identify how CCTα translocation onto these nuclear structures are regulated by fatty acids and phosphorylation of its serine-rich P-domain. Oleate treatment of Huh7 cells increased nLDs and LAPS that became progressively enriched in CCTα. In cells expressing the phosphatidic acid phosphatase Lipin1α or 1β, the expanded pool of nLDs and LAPS had a proportional increase in associated CCTα. In contrast, palmitate induced few nLDs and LAPS and inhibited the oleate-dependent translocation of CCTα without affecting total nLDs. Phospho-memetic or phospho-null mutations in the P-domain revealed that a 70% phosphorylation threshold, rather than site-specific phosphorylation, regulated CCTα association with nLDs and LAPS. In vitro candidate kinase and inhibitor studies in Huh7 cells identified cyclin-dependent kinase (CDK) 1 and 2 as putative P-domain kinases. In conclusion, CCTα translocation onto nLDs and LAPS is dependent on available surface area and fatty acid composition, as well as threshold phosphorylation of the P-domain potentially involving CDKs.

Phosphatidylcholine has essential functions in many eukaryotic cells, and its de novo biosynthesis is rate-limited by cytidine triphosphate:phosphocholine cytidylyltransferase (CCT). Although the biological and biochemical functions of CCT have been reported in mammals and several plants, this key enzyme has yet to be examined at a genome-wide level. As such, certain fundamental questions remain unanswered, including the evolutionary history, genetic and functional relationships, and structural variations among CCTs in the green lineage. In the current study, in-depth phylogenetic analysis, as well as the conservation and diversification in CCT gene structure and motif patterns, indicated that CCTs exist broadly in chlorophytes, bryophytes, lycophytes, monilophytes, gymnosperms, early-diverging angiosperms, monocots, and eudicots, and form eight relatively conserved clades. To further explore the potential function of selection pressure, we conducted extensive selection pressure analysis with a representative CCT gene, CCT1 from the model plant Arabidopsis thaliana (AthCCT1), and identified two positive selection sites, L59 and Q156. Site-directed mutagenesis and in vitro enzyme assays demonstrated that these positively selected sites were indeed important for the activity and substrate affinity of AthCCT1, and subsequent 3D structure analyses explained the potential biochemical mechanism. Taken together, our results unraveled the evolution and diversity of CCTs in the green lineage, as well as their association with the enzyme's biochemical and structural properties, and expanded our understanding of this important enzyme at the genome-wide level., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The two branches of the Kennedy pathways (CDP-choline and CDP-ethanolamine) are the predominant pathways responsible for the synthesis of the most abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively, in mammalian membranes. Recently, hereditary diseases associated with single gene mutations in the Kennedy pathways have been identified. Interestingly, genetic diseases within the same pathway vary greatly, ranging from muscular dystrophy to spastic paraplegia to a childhood blinding disorder to bone deformations. Indeed, different point mutations in the same gene (PCYT1; CCTα) result in at least three distinct diseases. In this review, we will summarize and review the genetic diseases associated with mutations in genes of the Kennedy pathway for phospholipid synthesis. These single-gene disorders provide insight, indeed direct genotype-phenotype relationships, into the biological functions of specific enzymes of the Kennedy pathway. We discuss potential mechanisms of how mutations within the same pathway can cause disparate disease., (Copyright © 2020 © 2020 Tavasoli et al. Published by Elsevier Inc. All rights reserved.)

The phospholipid biosynthesis of the malaria parasite, Plasmodium falciparum is a key process for its survival and its inhibition is a validated antimalarial therapeutic approach. The second and rate-limiting step of the de novo phosphatidylcholine biosynthesis is catalysed by CTP: phosphocholine cytidylyltransferase (PfCCT), which has a key regulatory function within the pathway. Here, we investigate the functional impact of the key structural differences and their respective role in the structurally unique pseudo-heterodimer PfCCT protein in a heterologous cellular context using the thermosensitive CCT-mutant CHO-MT58 cell line. We found that a Plasmodium-specific lysine-rich insertion within the catalytic domain of PfCCT acts as a nuclear localization signal and its deletion decreases the nuclear propensity of the protein in the model cell line. We further showed that the putative membrane-binding domain also affected the nuclear localization of the protein. Moreover, activation of phosphatidylcholine biosynthesis by phospholipase C treatment induces the partial nuclear-to-cytoplasmic translocation of PfCCT. We additionally investigated the cellular function of several PfCCT truncated constructs in a CHO-MT58 based rescue assay. In absence of the endogenous CCT activity we observed that truncated constructs lacking the lysine-rich insertion, or the membrane-binding domain provided similar cell survival ratio as the full length PfCCT protein.

While most of the articles in this issue review the workings of integral membrane enzymes, in this review, we describe the catalytic mechanism of an enzyme that contains a soluble catalytic domain but appears to catalyze its reaction on the membrane surface, anchored and assisted by a separate regulatory amphipathic helical domain and inter-domain linker. Membrane partitioning of CTP: phosphocholine cytidylyltransferase (CCT), a key regulatory enzyme of phosphatidylcholine metabolism, is regulated chiefly by changes in membrane phospholipid composition, and boosts the enzyme's catalytic efficiency >200-fold. Catalytic enhancement by membrane binding involves the displacement of an auto-inhibitory helix from the active site entrance-way and promotion of a new conformational ensemble for the inter-domain, allosteric linker that has an active role in the catalytic cycle. We describe the evidence for close contact between membrane lipid, a compact allosteric linker, and the CCT active site, and discuss potential ways that this interaction enhances catalysis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

CTP:phosphocholine cytidylyltransferase-alpha (CCTα) and CCTβ catalyze the rate-limiting step in phosphatidylcholine (PC) biosynthesis. CCTα is activated by association of its α-helical M-domain with nuclear membranes, which is negatively regulated by phosphorylation of the adjacent P-domain. To understand how phosphorylation regulates CCT activity, we developed phosphosite-specific antibodies for pS319 and pY359+pS362 at the N- and C-termini of the P-domain, respectively. Oleate treatment of cultured cells triggered CCTα translocation to the nuclear envelope (NE) and nuclear lipid droplets (nLDs) and rapid dephosphorylation of pS319. Removal of oleate led to dissociation of CCTα from the NE and increased phosphorylation of S319. Choline depletion of cells also caused CCTα translocation to the NE and S319 dephosphorylation. In contrast, Y359 and S362 were constitutively phosphorylated during oleate addition and removal, and CCTα-pY359+pS362 translocated to the NE and nLDs of oleate-treated cells. Mutagenesis revealed that phosphorylation of S319 is regulated independently of Y359+S362, and that CCTα-S315D+S319D was defective in localization to the NE. We conclude that the P-domain undergoes negative charge polarization due to dephosphorylation of S319 and possibly other proline-directed sites and retention of Y359 and S362 phosphorylation, and that dephosphorylation of S319 and S315 is involved in CCTα recruitment to nuclear membranes.

De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway involves highly endergonic biochemical reactions that must be fine-tuned with energy homeostasis. Previous studies have shown that CTP:phosphocholine cytidylyltransferase (CCT) is an important regulatory enzyme in this pathway and that its activity can be controlled at both transcriptional and posttranslational levels. Here we identified an important additional mechanism regulating plant CCT1 activity. Comparative analysis revealed that Arabidopsis CCT1 (AtCCT1) contains catalytic and membrane-binding domains that are homologous to those of rat CCT1. In contrast, the C-terminal phosphorylation domain important for stringent regulation of rat CCT1 was apparently missing in AtCCT1. Instead, we found that AtCCT1 contains a putative consensus site (Ser-187) for modification by sucrose nonfermenting 1-related protein kinase 1 (SnRK1 or KIN10/SnRK1.1), involved in energy homeostasis. Phos-tag SDS-PAGE coupled with MS analysis disclosed that SnRK1 indeed phosphorylates AtCCT1 at Ser-187, and we found that AtCCT1 phosphorylation substantially reduces its activity by as much as 70%. An S187A variant exhibited decreased activity, indicating the importance of Ser-187 in catalysis, and this variant was less susceptible to SnRK1-mediated inhibition. Protein truncation and liposome binding studies indicated that SnRK1-mediated AtCCT1 phosphorylation directly affects the catalytic domain rather than interfering with phosphatidate-mediated AtCCT1 activation. Overexpression of the AtCCT1 catalytic domain in Nicotiana benthamiana leaves increased PC content, and SnRK1 co-expression reduced this effect. Taken together, our results suggest that SnRK1 mediates the phosphorylation and concomitant inhibition of AtCCT1, revealing an additional mode of regulation for this key enzyme in plant PC biosynthesis., (© 2019 Caldo et al.)

CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme in phosphatidylcholine (PC) synthesis, is an amphitropic enzyme that regulates PC homeostasis. Recent work has suggested that CCTα activation by binding to a PC-deficient membrane involves conformational transitions in a helix pair (αE) that, along with a short linker of unknown structure (J segment), bridges the catalytic domains of the CCTα dimer to the membrane-binding (M) domains. In the soluble, inactive form, the αE helices are constrained into unbroken helices by contacts with two auto-inhibitory (AI) helices from domain M. In the active, membrane-bound form, the AI helices are displaced and engage the membrane. Molecular dynamics simulations have suggested that AI displacement is associated with hinge-like bending in the middle of the αE, positioning its C terminus closer to the active site. Here, we show that CCTα activation by membrane binding is sensitive to mutations in the αE and J segments, especially within or proximal to the αE hinge. Substituting Tyr-213 within this hinge with smaller uncharged amino acids that could destabilize interactions between the αE helices increased both constitutive and lipid-dependent activities, supporting a link between αE helix bending and stimulation of CCT activity. The solvent accessibilities of Tyr-213 and Tyr-216 suggested that these tyrosines move to new partially buried environments upon membrane binding of CCT, consistent with a folded αE/J structure. These data suggest that signal transduction through the modular αE helix pair relies on shifts in its conformational ensemble that are controlled by the AI helices and their displacement upon membrane binding., (© 2019 Taneva et al.)

The rate-limiting step in the biosynthesis of the major membrane phospholipid, phosphatidylcholine, is catalyzed by CTP:phosphocholine cytidylyltransferase (CCT), which is regulated by reversible membrane binding of a long amphipathic helix (domain M). The M domain communicates with the catalytic domain via a conserved ∼20-residue linker, essential for lipid activation of CCT. Previous analysis of this region (denoted as the αE C /J) using MD simulations, cross-linking, mutagenesis, and solvent accessibility suggested that membrane binding of domain M promotes remodeling of the αE C /J into a more compact structure that is required for enzyme activation. Here, using tryptophan fluorescence quenching, we show that the allosteric linker lies superficially on the membrane surface. Analyses with truncated CCTs show that the αE C /J can interact with lipids independently of the M domain. We observed strong FRET between engineered tryptophans in the αE C /J and vesicles containing dansyl-phosphatidylethanolamine that depended on the native J sequence. These data are incompatible with the extended conformation of the αE helix observed in the previously determined crystal structure of inactive CCT but support a bent αE helix conformation stabilized by J segment interactions. Our results suggest that the membrane-adsorbed, folded allosteric linker may partially cover the active site cleft and pull it close to the membrane surface, where cytidyl transfer can occur efficiently in a relatively anhydrous environment., (© 2019 Knowles et al.)

CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in phosphatidylcholine (PC) synthesis and is activated by binding to PC-deficient membranes. Mutations in the gene encoding CCTα ( PCYT1A ) cause three distinct pathologies in humans: lipodystrophy, spondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD), and isolated retinal dystrophy. Previous analyses showed that for some disease-linked PCYT1A variants steady state levels of CCTα and PC synthesis were reduced in patient fibroblasts, but other variants impaired PC synthesis with little effect on CCT levels. To explore the impact on CCT stability and function we expressed WT and mutant CCTs in COS-1 cells, which have very low endogenous CCT. Over-expression of two missense variants in the catalytic domain (V142M and P150A) generated aggregated enzymes that could not be refolded after solubilization by denaturation. Other mutations in the catalytic core that generated CCTs with reduced solubility could be purified. Five variants destabilized the catalytic domain-fold as assessed by lower transition temperatures for unfolding, and three of these manifested defects in substrate K m values. A mutation (R223S) in a signal-transducing linker between the catalytic and membrane-binding domains also impaired enzyme kinetics. E280del, a single amino acid deletion in the autoinhibitory helix increased the constitutive (lipid-independent) enzyme activity ∼4-fold. This helix also participates in membrane binding, and surprisingly E280del enhanced the enzyme's response to anionic lipid vesicles ∼4-fold. These in vitro analyses on purified mutant CCTs will complement future measurements of their impact on PC synthesis in cultured cells and in tissues with a stringent requirement for CCTα., (© 2019 Cornell et al.)

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.

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., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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, Lys 122 The impacts of the AI helix on active-site dynamics and positioning of Lys 122 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 Lys 122 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 Lys 122 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., (© 2018 Ramezanpour et al.)

The methylerythritol phosphate (MEP) pathway is an essential metabolic pathway found in malaria parasites, but absent in mammals, making it a highly attractive target for the discovery of novel and selective antimalarial therapies. Using high-throughput screening, we have identified 2-phenyl benzo[d]isothiazol-3(2H)-ones as species-selective inhibitors of Plasmodium spp. 2-C-methyl- D -erythritol-4-phosphate cytidyltransferase (IspD), the third catalytic enzyme of the MEP pathway. 2-Phenyl benzo[d]isothiazol-3(2H)-ones display nanomolar inhibitory activity against P. falciparum and P. vivax IspD and prevent the growth of P. falciparum in culture, with EC 50 values below 400 nM. In silico modeling, along with enzymatic, genetic and crystallographic studies, have established a mechanism-of-action involving initial non-covalent recognition of inhibitors at the IspD binding site, followed by disulfide bond formation through attack of an active site cysteine residue on the benzo[d]isothiazol-3(2H)-one core. The species-selective inhibitory activity of these small molecules against Plasmodium spp. IspD and cultured parasites suggests they have potential as lead compounds in the pursuit of novel drugs to treat malaria.

The amphipathic helical (AH) membrane binding motif is recognized as a major device for lipid compositional sensing. We explore the function and mechanism of sensing by the lipid biosynthetic enzyme, CTP:phosphocholine cytidylyltransferase (CCT). As the regulatory enzyme in phosphatidylcholine (PC) synthesis, CCT contributes to membrane PC homeostasis. CCT directly binds and inserts into the surface of bilayers that are deficient in PC and therefore enriched in lipids that enhance surface charge and/or create lipid packing voids. These two membrane physical properties induce the folding of the CCT M domain into a ≥60 residue AH. Membrane binding activates catalysis by a mechanism that has been partially deciphered. We review the evidence for CCT compositional sensing, and the membrane and protein determinants for lipid selective membrane-interactions. We consider the factors that promote the binding of CCT isoforms to the membranes of the ER, nuclear envelope, or lipid droplets, but exclude CCT from other organelles and the plasma membrane. The CCT sensing mechanism is compared with several other proteins that use an AH motif for membrane compositional sensing. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A unique, unsolved O-mannosyl glycan on α-dystroglycan is essential for its interaction with protein ligands in the extracellular matrix. Defective O-mannosylation leads to a group of muscular dystrophies, called dystroglycanopathies. Mutations in isoprenoid synthase domain containing (ISPD) represent the second most common cause of these disorders, however, its molecular function remains uncharacterized. The human ISPD (hISPD) crystal structure showed a canonical N-terminal cytidyltransferase domain linked to a C-terminal domain that is absent in cytidyltransferase homologs. Functional studies demonstrated cytosolic localization of hISPD, and cytidyltransferase activity toward pentose phosphates, including ribulose 5-phosphate, ribose 5-phosphate, and ribitol 5-phosphate. Identity of the CDP sugars was confirmed by liquid chromatography quadrupole time-of-flight mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy. Our combined results indicate that hISPD is a cytidyltransferase, suggesting the presence of a novel human nucleotide sugar essential for functional α-dystroglycan O-mannosylation in muscle and brain. Thereby, ISPD deficiency can be added to the growing list of tertiary dystroglycanopathies., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.

Cation-π interactions to cognate ligands in enzymes have key roles in ligand binding and enzymatic catalysis. We have deciphered the key functional role of both charged and aromatic residues within the choline binding subsite of CTP:phosphocholine cytidylyltransferase and choline kinase from Plasmodium falciparum. Comparison of quaternary ammonium binding site structures revealed a general composite aromatic box pattern of enzyme recognition sites, well distinguished from the aromatic box recognition site of receptors., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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.

The unique steps in the synthesis of an unusual osmolyte in hyperthermophiles, di-myo-inositol-1,1'-phosphate (DIP), involve the production of CDP-inositol and its condensation with an inositol-1-phosphate molecule to form phosphorylated DIP. While many organisms fuse both activities into a single enzyme, the two are separate in Thermotoga maritima. The crystal structure of the T. maritima inositol-1-phosphate cytidylyltransferase, which as a soluble protein may transiently associate with its membrane-embedded partner phospho-DIP synthase (P-DIPS), has now been obtained. The structure shows a conserved motif of sugar nucleotide transferases (COG1213) with a structurally reinforced C-terminal Cys bonded to the core of the protein. A bound arsenosugar identifies the location of the active site for inositol 1-phosphate. Based on homologous structures from several species and the identification of the crucial conserved aspartate residue, a catalytic mechanism for this enzyme is proposed as well as a mode for its association with P-DIPS. This structure imposes constraints on the mode of association, communication and temperature activation of two separate enzymes in T. maritima. For the first time, a working model for the membrane-bound P-DIPS unit has been constructed. This sheds light on the functioning of the phosphatidylserine and phosphatidylinositol synthases involved in many physiological processes that are homologous to P-DIPS. This work provides fresh insights into the synthesis of the unusual thermoprotective compound DIP in hyperthermophiles.

The enzyme CTP:phosphocholine cytidylyltransferase (CCT) is essential in the lipid biosynthesis of Plasmodia (Haemosporida), presenting a promising antimalarial target. Here, we identified two independent gene duplication events of CCT within Apicomplexa and characterized a truncated construct of Plasmodium falciparum CCT that forms a dimer resembling the molecular architecture of CCT enzymes from other sources. Based on biophysical and enzyme kinetics methods, our data show that the CDP-choline product of the CCT enzymatic reaction binds to the enzyme considerably stronger than either substrate (CTP or choline phosphate). Interestingly, in the presence of Mg²⁺ , considered to be a cofactor of the enzyme, the binding of the CTP substrate is attenuated by a factor of 5. The weaker binding of CTP:Mg²⁺ , similarly to the related enzyme family of aminoacyl tRNA synthetases, suggests that, with lack of Mg²⁺ , positively charged side chain(s) of CCT may contribute to CTP accommodation. Thermodynamic investigations by isothermal titration calorimetry and fluorescent spectroscopy studies indicate that accommodation of the choline phosphate moiety in the CCT active site is different when it appears on its own as one of the substrates or when it is linked to the CDP-choline product. A tryptophan residue within the active site is identified as a useful internal fluorescence sensor of enzyme-ligand binding. Results indicate that the catalytic mechanism of Plasmodium falciparum CCT may involve conformational changes affecting the choline subsite of the enzyme., (© 2013 FEBS.)

CTP:phosphocholine cytidylyltransferase (CCT), the regulatory enzyme in the synthesis of phosphatidylcholine, is activated by binding membranes using a lipid-induced amphipathic helix (domain M). Domain M functions to silence catalysis when CCT is not membrane engaged. The silencing mechanism is unknown. We used photo-cross-linking and mass spectrometry to identify contacts between domain M and other CCT domains in its soluble form. Each of four sites in domain M forged cross-links to the same set of peptides that flank the active site and overlap at helix αE at the base of the active site. These cross-links were broken in the presence of activating lipid vesicles. Mutagenesis of domain M revealed that multiple hydrophobic residues within a putative auto-inhibitory (AI) motif contribute to the contact with helix αE and silencing. Helix αE was confirmed as the docking site for domain M by deuterium exchange analysis. We compared the dynamics and fold stability of CCT domains by site-directed fluorescence anisotropy and urea denaturation. The results suggest a bipartite structure for domain M: a disordered N-terminal portion and an ordered C-terminal AI motif with an unfolding transition identical with that of helix αE. Reduction in hydrophobicity of the AI motif decreased its order and fold stability, as did deletion of the catalytic domain. These results support a model in which catalytic silencing is mediated by the docking of an amphipathic AI motif onto the amphipathic helices αE. An unstructured leash linking αE with the AI motif may facilitate both the silencing contact and its membrane-triggered disruption., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

CTP:phosphocholine cytidylyltransferase (CCT), an amphitropic enzyme that regulates phosphatidylcholine synthesis, is composed of a catalytic head domain and a regulatory tail. The tail region has dual functions as a regulator of membrane binding/enzyme activation and as an inhibitor of catalysis in the unbound form of the enzyme, suggesting conformational plasticity. These functions are well conserved in CCTs across diverse phyla, although the sequences of the tail regions are not. CCT regulatory tails of diverse origins are composed of a long membrane lipid-inducible amphipathic helix (m-AH) followed by a highly disordered segment, reminiscent of the Parkinson disease-linked protein, α-synuclein, which we show shares a novel sequence motif with vertebrate CCTs. To unravel features required for silencing, we created chimeric enzymes by fusing the catalytic domain of rat CCTα to the regulatory tail of CCTs from Drosophila, Caenorhabditis elegans, or Saccharomyces cerevisiae or to α-synuclein. Only the tail domains of the two invertebrate CCTs were competent for both suppression of catalytic activity and for activation by lipid vesicles. Thus, both silencing and activating functions of the m-AH can tolerate significant changes in length and sequence. We identified a highly amphipathic 22-residue segment in the m-AH with features conserved among animal CCTs but not yeast CCT or α-synuclein. Deletion of this segment from rat CCT increased the lipid-independent V(max) by 10-fold, equivalent to the effect of deleting the entire tail, and severely weakened membrane binding affinity. However, membrane binding was required for additional increases in catalytic efficiency. Thus, full activation of CCT may require not only loss of a silencing conformation in the m-AH but a gain of an activating conformation, promoted by membrane binding.

CTP:phosphocholine cytidylyltransferase (CCT) is an amphitropic protein regulating phosphatidylcholine synthesis. Lipid-induced folding of its amphipathic helical (AH) membrane-binding domain activates the enzyme. In this study we examined the membrane deforming property of CCT in vitro by monitoring conversion of vesicles to tubules, using transmission electron microscopy. Vesicle tubulation was proportional to the membrane density of CCT and proceeded either as growth from a pre-formed surface bud, or as a global transformation of roughly spherical vesicles into progressively thinner tubules. The tubulation pathway depended on the lipid compositional heterogeneity of the vesicles, with heterogeneous mixtures supporting the bud-extension pathway. Co-existence of vesicles alongside thick and thin tubules suggested that CCT can discriminate between flat membrane surfaces and those with emerging curvature, binding preferentially to the latter. Thin tubules had a limiting diameter of ~12nm, likely representing bilayer cylinders with a very high density of 1 CCT/50 lipids. The AH segment was necessary and sufficient for tubulation. AH regions from diverse CCT sources, including C. elegans, had tubulation activity that correlated with α-helical length. The AH motifs in CCT and the Parkinson's-related protein, α-synuclein, have similar features, however the CCT AH was more effective in its membrane remodeling function. That CCT can deform vesicles of physiologically relevant composition suggests that CCT binding to membranes may initiate deformations required for organelle morphogenesis and at the same time stimulate synthesis of the PC required for the development of these regions., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Archaeoglobus fulgidus, a hyperthermophilic archaeon, accumulates di-myo-inositol phosphate (DIP) in response to heat stress. Recently, the pathway for biosynthesis of DIP has been elucidated in this organism and involves a bifunctional enzyme that contains two domains: CTP:inositol-1-phosphate cytidylyltransferase (IPCT) as a soluble domain and di-myo-inositol-1,3'-phosphate-1-phosphate synthase (DIPPS) as a membrane domain. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis of the IPCT domain from A. fulgidus in the apo form are reported. The crystals diffracted to 2.4 Å resolution using a synchrotron source and belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 154.7, b = 83.9, c = 127.7 Å.

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in eukaryotic membranes and its biosynthetic pathway is generally controlled by CTP:Phosphocholine Cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCT is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum (ER) translocation; however, most of the enzyme is intranuclearly located. Here we demonstrate that CCTα is concentrated in the nucleoplasm of MDCK cells. Confocal immunofluorescence revealed that extracellular hypertonicity shifted the diffuse intranuclear distribution of the enzyme to intranuclear domains in a foci pattern. One population of CCTα foci colocalised and interacted with lamin A/C speckles, which also contained the pre-mRNA processing factor SC-35, and was resistant to detergent and salt extraction. The lamin A/C silencing allowed us to visualise a second more labile population of CCTα foci that consisted of lamin A/C-independent foci non-resistant to extraction. We demonstrated that CCTα translocation is not restricted to its redistribution from the nucleus to the ER and that intranuclear redistribution must thus be considered. We suggest that the intranuclear organelle distribution of CCTα is a novel mechanism for the regulation of enzyme activity., (Copyright © 2010 Elsevier B.V. All rights reserved.)

CTP:phosphocholine cytidylyltransferase (CCT), critical for phosphatidylcholine biosynthesis, is activated by translocation to the membrane surface. The lipid activation region of Caenorhabditis elegans CCT is between residues 246 and 266 of the 347 amino acid polypeptide, a region proposed to form an amphipathic alpha helix. When leucine 246, tryptophan 249, isoleucine 256, isoleucine 257, or phenylalanine 260, on the hydrophobic face of the helix, were changed individually to serine low activity was observed in the absence of lipid vesicles, similar to wild-type CCT, while lipid stimulated activity was reduced compared to wild-type CCT. Mutational analysis of phenylalanine 260 implicated this residue as a contributor to auto-inhibition of CCT while mutation of L246, W249, I256, and I257 simultaneously to serine resulted in significantly higher activity in the absence of lipid vesicles and an enzyme that was not lipid activated. These results support a concerted mechanism of lipid activation that requires multiple residues on the hydrophobic face of the putative amphipathic alpha helix.

CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in the synthesis of phosphatidylcholine, the most abundant phospholipid in eukaryotic cell membranes. The CCT-catalyzed transfer of a cytidylyl group from CTP to phosphocholine to form CDP-choline is regulated by a membrane lipid-dependent mechanism imparted by its C-terminal membrane binding domain. We present the first analysis of a crystal structure of a eukaryotic CCT. A deletion construct of rat CCTalpha spanning residues 1-236 (CCT236) lacks the regulatory domain and as a result displays constitutive activity. The 2.2-A structure reveals a CCT236 homodimer in complex with the reaction product, CDP-choline. Each chain is composed of a complete catalytic domain with an intimately associated N-terminal extension, which together with the catalytic domain contributes to the dimer interface. Although the CCT236 structure reveals elements involved in binding cytidine that are conserved with other members of the cytidylyltransferase superfamily, it also features nonconserved active site residues, His-168 and Tyr-173, that make key interactions with the beta-phosphate of CDP-choline. Mutagenesis and kinetic analyses confirmed their role in phosphocholine binding and catalysis. These results demonstrate structural and mechanistic differences in a broadly conserved protein fold across the cytidylyltransferase family. Comparison of the CCT236 structure with those of other nucleotidyltransferases provides evidence for substrate-induced active site loop movements and a disorder-to-order transition of a loop element in the catalytic mechanism.

Monoubiquitination aids in the nuclear export and entrance of proteins into the lysosomal degradative pathway, although the mechanisms are unknown. Cytidylyltransferase (CCTalpha) is a proteolytically sensitive lipogenic enzyme containing an NH(2)-terminal nuclear localization signal (NLS). We show here that CCTalpha is monoubiquitinated at a molecular site (K(57)) juxtaposed near its NLS, resulting in disruption of its interaction with importin-alpha, nuclear exclusion, and subsequent degradation within the lysosome. Cellular expression of a CCTalpha-ubiquitin fusion protein that mimics the monoubiquitinated enzyme resulted in cytoplasmic retention. A CCTalpha K(57R) mutant exhibited an extended half-life, was retained in the nucleus, and displayed proteolytic resistance. Importantly, by using CCTalpha-ubiquitin hybrid constructs that vary in the intermolecular distance between ubiquitin and the NLS, we show that CCTalpha monoubiquitination masks its NLS, resulting in cytoplasmic retention. These results unravel a unique molecular mechanism whereby monoubiquitination governs the trafficking and life span of a critical regulatory enzyme in vivo.

CTP:phosphocholine cytidylyltransferase alpha (CCTalpha), the rate-limiting enzyme in the CDP-choline pathway for phosphatidylcholine (PtdCho) synthesis, is activated by translocation to nuclear membranes. However, CCTalpha is cytoplasmic in cells with increased capacity for PtdCho synthesis and following acute activation, suggesting that nuclear export is linked to activation. The objective of this study was to identify which CCTalpha domains were involved in nuclear export in response to the lipid activators farnesol (FOH) and oleate. Imaging of CCT-green fluorescent protein (GFP) mutants expressed in CCTalpha-deficient CHO58 cells showed that FOH-mediated translocation to nuclear membranes and export to the cytoplasm required the membrane binding amphipathic helix (domain M). Nuclear export was reduced by a mutation that mimics constitutive phosphorylation of the CCT phosphorylation (P) domain. However, domain M alone was sufficient to promote translocation to the nuclear envelope and export of a nuclear-localized GFP construct in FOH- or oleate-treated CHO58 cells. In the context of acute activation with lipid mediators, nuclear export of CCT-GFP mutants correlated with in vitro activity but not PtdCho synthesis. This study describes a nuclear export pathway that is dependent on membrane interaction of an amphipathic helix, thus linking lipid-dependent activation to the nuclear/cytoplasmic distribution of CCTalpha.

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the conversion of phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline for the eventual synthesis of phosphatidylcholine (PC). The enzyme is regulated by reversible association with cellular membranes, with the rate of catalysis increasing following membrane association. Two isoforms of CCT appear to be present in higher eukaryotes, including Drosophila melanogaster, which contains the tandem genes Cct1 and Cct2. Before this study, the CCT1 isoform had not been characterized and the cellular location of each enzyme was unknown. In this investigation, the cDNA encoding the CCT1 isoform from D. melanogaster has been cloned and the recombinant enzyme purified and characterized to determine catalytic properties and the effect of lipid vesicles on activity. CCT1 exhibited a V max of 23904 nmol of CDP-choline min (-1) mg (-1) and apparent K m values for phosphocholine and CTP of 2.29 and 1.21 mM, respectively, in the presence of 20 muM PC/oleate vesicles. Cytidylyltransferases require a divalent cation for catalysis, and the cation preference of CCT1 was found to be as follows: Mg (2+) > Mn (2+) = Co (2+) > Ca (2+) = Ni (2+) > Zn (2+). The activity of the enzyme is stimulated by a variety of lipids, including phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, diphosphatidylglycerol, and the fatty acid oleate. Phosphatidylethanolamine and phosphatidic acid, however, did not have a significant effect on CCT1 activity. The cellular location of both CCT1 and CCT2 isoforms was elucidated by expressing green fluorescent fusion proteins in cultured D. melanogaster Schneider 2 cells. CCT1 was identified as the nuclear isoform, while CCT2 is cytoplasmic.

CTP:phosphocholine cytidylyltransferase (CCT), a rate-limiting enzyme in phosphatidylcholine synthesis, is regulated by reversible membrane interactions mediated by an amphipathic helical domain (M) that binds selectively to anionic lipids. CCT is a dimer; thus the functional unit has two M domains. To probe the functional contribution of each domain M we prepared a CCT heterodimer composed of one full-length subunit paired with a CCT subunit truncated before domain M that was also catalytically dead. We compared this heterodimer to the full-length homodimer with respect to activation by anionic vesicles, vesicle binding affinities, and promotion of vesicle aggregation. Surprisingly for all three functions the dimer with just one domain M behaved similarly to the dimer with two M domains. Full activation of the wild-type subunit was not impaired by loss of one domain M in its partner. Membrane binding affinities were the same for dimers with one versus two M domains, suggesting that the two M domains of the dimer do not engage a single bilayer simultaneously. Vesicle cross-bridging was also unhindered by loss of one domain M, suggesting that another motif couples with domain M for cross-bridging anionic membranes. Mutagenesis revealed that the positively charged nuclear localization signal sequence constitutes that second motif for membrane cross-bridging. We propose that the two M domains of the CCT dimer engage a single bilayer via an alternating binding mechanism. The tethering function involves the cooperation of domain M and the nuclear localization signal sequence, each engaging separate membranes. Membrane binding of a single M domain is sufficient to fully activate the enzymatic activity of the CCT dimer while sustaining the low affinity, reversible membrane interaction required for regulation of CCT activity.

The nucleoplasmic reticulum (NR), a nuclear membrane network implicated in signaling and transport, is formed by the biosynthetic and membrane curvature-inducing properties of the rate-limiting enzyme in phosphatidylcholine synthesis, CTP:phosphocholine cytidylyltransferase (CCT) alpha. The NR is formed by invagination of the nuclear envelope and has an underlying lamina that may contribute to membrane tubule formation or stability. In this study we investigated the role of lamins A and B in NR formation in response to expression and activation of endogenous and fluorescent protein-tagged CCTalpha. Similarly to endogenous CCTalpha, CCT-green fluorescent protein (GFP) reversibly translocated to nuclear tubules projecting from the NE in response to oleate, a lipid promoter of CCT membrane binding. Coexpression and RNA interference experiments revealed that both CCTalpha and lamin A and B were necessary for NR proliferation. Expression of CCT-GFP mutants with compromised membrane-binding affinity produced fewer nuclear tubules, indicating that the membrane-binding function of CCTalpha promotes the expansion of the NR. Proliferation of atypical bundles of nuclear membrane tubules by a CCTalpha mutant that constitutively associated with membranes revealed that expansion of the double-bilayer NR requires the coordinated assembly of an underlying lamin scaffold and induction of membrane curvature by CCTalpha.

CTP:phosphocholine cytidylyltransferase (CCTalpha) is a proteolytically sensitive enzyme essential for production of phosphatidylcholine, the major phospholipid of animal cell membranes. The molecular signals that govern CCTalpha protein stability are unknown. An NH(2)-terminal PEST sequence within CCTalpha did not serve as a degradation signal for the proteinase, calpain. Calmodulin (CaM) stabilized CCTalpha from calpain proteolysis. Adenoviral gene transfer of CaM in cells protected CCTalpha, whereas CaM small interfering RNA accentuated CCTalpha degradation by calpains. CaM bound CCTalpha as revealed by fluorescence resonance energy transfer and two-hybrid analysis. Mapping and site-directed mutagenesis of CCTalpha uncovered a motif (LQERVDKVK) harboring a vital recognition site, Gln(243), whereby CaM directly binds to the enzyme. Mutagenesis of CCTalpha Gln(243) not only resulted in loss of CaM binding but also led to complete calpain resistance in vitro and in vivo. Thus, calpains and CaM both access CCTalpha using a structurally similar molecular signature that profoundly affects CCTalpha levels. These data suggest that CaM, by antagonizing calpain, serves as a novel binding partner for CCTalpha that stabilizes the enzyme under proinflammatory stress.

CTP:phosphocholine cytidylyltransferase (CCT) is a multi-domain enzyme that regulates phosphatidylcholine synthesis. It converts to an active form upon binding cell membranes, and interdomain dissociations have been hypothesized to accompany this process. To identify these interdomain and membrane interactions, the tertiary structures of three forms of CCTalpha were probed by monitoring accessibility to proteases. Time-limited digestion with chymotrypsin or arginine C of soluble CCTalpha (CCT(sol)), phospholipid vesicle-bound CCT (CCT(mem)), and a soluble constitutively active CCT truncated at amino acid 236 generated complex mixtures of peptides that were resolved and identified by gel electrophoresis/immunoblotting and by matrix-assisted laser desorption/ionization-mass spectrometry, with or without coupling to capillary liquid chromatography. Identification of cleavage sites enabled assembly of peptide bond accessibility maps for each CCT form. Our results reveal a approximately 80-residue core within the catalytic domain (domain C) as the most inaccessible region in all three forms and the C-terminal phosphorylation domain as the most accessible. Membrane binding has little effect on the protease accessibility of these domains. To map the protease sites onto the catalytic domain, its three-dimensional structure was modeled from the atomic coordinates of glycerol-phosphate cytidylyltransferase (Protein Data Bank code 1COZ). The protease inaccessibility of most sites in domain C could be explained by burial or location within secondary structural elements. The accessibility of the N-terminal domain (domain N) was enhanced upon membrane binding. Residues Phe(234)-Leu(303) were inaccessible in CCT(mem), suggesting burial in the membrane. Surprisingly, residues Leu(274)-Leu(303) of this domain were also inaccessible in CCT(sol). We propose that this region is buried by interdomain contacts with domain N in CCT(sol). Membrane binding and burial of domain M in the lipid bilayer may disrupt this interaction, leading to increased exposure of sites in domain N.

Homeostasis of phosphatidylcholine (PC) is regulated by the opposing actions between CTP:phosphocholine cytidylyltransferase (CT) and the group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)). We investigated this process during the cell cycle. PC mass doubles during late G(1) and early S phase when its rate of catabolism is lowest. We show that iPLA(2) activity is cell cycle-dependent with peak activity during G(2)/M and late S phase. iPLA(2) activity declines during G(1) and is lowest at the G(1)/S transition and early S phase. The accumulation of PC correlates with decreased iPLA(2) activity, suggesting that regulation of this enzyme contributes to phospholipid accumulation. The levels of 80 kDa iPLA(2) protein do not change and thus cannot account for changes in enzyme activity. Reverse transcriptase and real-time PCR experiments show that splice variant iPLA(2) mRNAs are preferentially expressed during G(2)/M. Immunoblot analyses with an antibody directed against the N terminus of iPLA(2) revealed a approximately 50 kDa protein that is of appropriate size to be the truncated protein encoded by the ankyrin-iPLA(2)-1 splice variant mRNA. The levels of truncated iPLA(2) protein were high in cells in late G(1) and S phase cells that had low iPLA(2) activity and low in G(2)/M cells that had high iPLA(2) activity. The truncated protein co-immunoprecipitated with full-length iPLA(2), indicating a physical interaction between the two proteins. Together, these data suggest that truncated iPLA(2) proteins associate with active iPLA(2) and down-regulate its activity during G(1). This down-regulation may contribute to phospholipid accumulation during the cell cycle.

CTP:phosphocholine cytidylyltransferase (CT) is the key regulatory enzyme in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. Hepatic cells express both an alpha and a beta2 isoform of CT and can also synthesize phosphatidylcholine via the sequential methylation of phosphatidylethanolamine catalyzed by phosphatidylethanolamine N-methyltransferase. To ascertain the functional importance of CTalpha, we created a mouse in which the hepatic CTalpha gene was specifically inactivated by the Cre/LoxP procedure. In CTalpha knockout mice, hepatic CT activity (due to residual CTbeta2 activity as well as activity in nonhepatic cells) was 15% of normal, whereas phosphatidylethanolamine N-methyltransferase activity was elevated 2-fold compared with controls. Lipid analyses of the liver indicated that female knockout mice had reduced phosphatidylcholine levels and accumulated triacylglycerols. The plasma phosphatidylcholine concentration was reduced in the CTalpha knockout (independent of gender), as were levels of high density lipoproteins (cholesterol and apoAI) and very low density lipoproteins (triacylglycerols and apoB100). Experiments in which mice were injected with Triton WR1339 indicated that apoB secretion was decreased in hepatic-specific CTalpha knockout mice compared with controls. These results suggest an important role for hepatic CTalpha in regulating both hepatic and systemic lipid and lipoprotein metabolism.

CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme that controls phosphatidylcholine synthesis, is regulated by reversible interactions with membranes containing anionic lipids. Previous work demonstrated that CCT is a homodimer. In this work we show that the structure of the dimer interface is altered upon encountering membranes that activate CCT. Chemical cross-linking reactions were established which captured intradimeric interactions but not random CCT dimer collisions. The efficiency of capturing covalent cross-links with four different reagents was diminished markedly upon presentation of activating anionic lipid vesicles but not zwitterionic vesicles. Experiments were conducted to show that the anionic vesicles did not interfere with the chemistry of the cross-linking reactions and did not sequester available cysteine sites on CCT for reaction with the cysteine-directed cross-linking reagent. Thus, the loss of cross-linking efficiency suggested that contact sites at the dimer interface had increased distance or reduced flexibility upon binding of CCT to membranes. The regions of the enzyme involved in dimerization were mapped using three approaches: 1) limited proteolysis followed by cross-linking of fragments, 2) yeast two-hybrid analysis of interactions between select domains, and 3) disulfide bonding potential of CCTs with individual cysteine to serine substitutions for the seven native cysteines. We found that the N-terminal domain (amino acids 1-72) is an important participant in forming the dimer interface, in addition to the catalytic domain (amino acids 73-236). We mapped the intersubunit disulfide bond to the cystine 37 pair in domain N and showed that this disulfide is sensitive to anionic vesicles, implicating this specific region in the membrane-sensitive dimer interface.

The genomes of Treponema denticola and Treponema pallidum contain a gene, licCA, which is predicted to encode a fusion protein containing choline kinase and CTP:phosphocholine cytidylyltransferase activities. Because both organisms have been reported to contain phosphatidylcholine, this raises the possibility that they use a CDP-choline pathway for the biosynthesis of phosphatidylcholine. This report shows that phosphatidylcholine is a major phospholipid in T. denticola, accounting for 35-40% of total phospholipid. This organism readily incorporated [14C]choline into phosphatidylcholine, indicating the presence of a choline-dependent biosynthetic pathway. The licCA gene was cloned, and recombinant LicCA had choline kinase and CTP:phosphocholine cytidylyltransferase activity. The licCA gene was disrupted in T. denticola by erythromycin cassette mutagenesis, resulting in a viable mutant. This disruption completely blocked incorporation of either [14C]choline or 32Pi into phosphatidylcholine. The rate of production of another phospholipid in T. denticola, phosphatidylethanolamine, was elevated considerably in the licCA mutant, suggesting that the elevated level of this lipid compensated for the loss of phosphatidylcholine in the membranes. Thus it appears that T. denticola does contain a licCA-dependent CDP-choline pathway for phosphatidylcholine biosynthesis.

Charged droplet processing methodology, that utilizes electrodynamic levitation technology to control the trajectories of picoliter volume charged droplets and deliver them to a target plate at atmospheric pressure, has been developed. Termed wall-less sample preparation (WaSP), this methodology offers several features that could prove beneficial to the preparation of sample spots from separation column effluents for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. These features include solute pre-concentration factors of 10(1) to 10(3) due to volatile solvent evaporation prior to droplet deposition onto the target plate, high spatial accuracy of the deposition position of each processed droplet (+/-5 microm), and the ability to prepare sample spots as small as 20 microm in diameter from a single droplet. Here a new mode of operation of this methodology is described and used as an offline post-column pre-concentrating interface between capillary liquid chromatography (capLC) and a target plate for offline MALDI-MS. Using a fraction from the capLC separation of peptides produced by the proteolytic digestion of the protein cytidine 5'-triphosphate:phosphocholine cytidylyltransferase, MALDI sample spots were prepared using the dried-droplet method, direct piezoelectric droplet dispensing, and the processing of piezo-dispensed droplets by WaSP. The sample spot morphology was investigated using light microscopy, and peptide ion abundances produced by MALDI were measured using time-of-flight (TOF) MS. The advantages of developing an online capLC/WaSP interface with MALDI-MS in the future are discussed along with some of the challenges that may be encountered in such an endeavor., (Copyright (c) 2004 John Wiley & Sons, Ltd.)

Patterning of the Drosophila egg requires cooperation between the germline cells and surrounding somatic follicle cells. In order to identify genes involved in follicle cell patterning, we analyzed enhancer trap lines expressed in specific subsets of follicle cells. Through this analysis, we have identified tandem Drosophila genes homologous to CTP: phosphocholine cytidylyltransferase (CCT), the second of three enzymes in the CDP-choline pathway, which is used to synthesize phosphatidylcholine. Drosophila Cct1 is expressed at high levels in three specific subsets of follicle cells, and this expression is regulated, at least in part, by the TGF-beta and Egfr signaling pathways. Mutations in Cct1 result in a number of defects, including a loss of germline stem cell maintenance, mispositioning of the oocyte, and a shortened operculum, suggesting that Cct1 plays multiple roles during oogenesis. In addition, Cct1 mutants display a novel branched ovariole phenotype, demonstrating a requirement for this gene during ovarian morphogenesis. These data provide the first evidence for a specific role for CCT, and thus for phosphatidylcholine, in patterning during development.

Phospholipid biosynthesis plays a key role in malarial infection and is regulated by CCT (CTP:phosphocholine cytidylyltransferase). This enzyme belongs to the group of amphitropic proteins which are regulated by reversible membrane interaction. To assess the role of the putative membrane-binding domain of Plasmodium falciparum CCT (PfCCT), we synthesized three peptides, K21, V20 and K54 corresponding to residues 274-294, 308-327 and 274-327 of PfCCT respectively. Conformational behaviour of the peptides, their ability to bind to liposomes and to destabilize lipid bilayers, and their insertion properties were investigated by different biophysical techniques. The intercalation mechanisms of the peptides were refined further by using surface-pressure measurements on various monolayers at the air/water interface. In the present study, we show that the three studied peptides are able to bind to anionic and neutral phospholipids, and that they present an alpha-helical conformation upon lipid binding. Peptides V20 and the full-length K54 intercalate their hydrophobic parts into an anionic bilayer and, to a lesser extent, a neutral one for V20. Peptide K21 interacts only superficially with both types of phospholipid vesicles. Adsorption experiments performed at the air/water interface revealed that peptide K54 is strongly surface-active in the absence of lipid. Peptide V20 presents an atypical behaviour in the presence of phosphatidylserine. Whatever the initial surface pressure of a phosphatidylserine film, peptide V20 and phosphatidylserine entities seem linked together in a special organization involving electrostatic and hydrophobic interactions. We showed that PfCCT presents different lipid-dependence properties from other studied CCTs. Although the lipid-binding domain seems to be located in the C-terminal region of the enzyme, as with the mammalian counterpart, the membrane anchorage, which plays a key role in the enzyme regulation, is driven by two alpha-helices, which behave differently from one another.

CTP:phosphocholine cytidylyltranferase (CCT) regulates phosphatidylcholine (PC) biosynthesis. Its activity is controlled by reversible interactions with membrane lipids, mediated by an internal segment referred to as domain M. Although domain M peptides adopt an amphipathic alpha-helical structure when membrane bound, the structure of this domain in the context of the whole enzyme in the lipid-free and lipid-bound state is unknown. Here we derive lipid-induced secondary structural changes in CCTalpha using circular dichroism and three deconvolution programs. The analysis of two fragments, CCT236 (CCT1-236, housing the catalytic domain) and a synthetic domain M peptide (CCT237-293) aided the assignment of structural change to specific domains. To carry out this study, we developed a micellar lipid activating system that would avoid generation of CCT-induced lipid vesicle aggregates that interfere with the CD analysis. Lysophosphatidylcholine/phosphatidylglycerol (LPC/PG) mixed micelles supported full activation of CCT and caused an increase in the alpha-helix content of full-length CCT from 25 to 41%, at the expense of all other conformations. LPC/PG also induced an increase in alpha-helix content of the domain M peptide from 7 to 85% at the expense of all other conformers. This lipid system did not significantly affect the secondary structure of CCT236, nor did it affect the proteolytic fragmentation pattern of this region within full-length CCT, suggesting that the region containing the catalytic domain changes very little upon membrane activation of CCT. Our data suggest that lipids trigger a conformational switch in domain M from a mixed structure to an alpha-helix, thus creating a hydrophobic face for membrane insertion. Our results negate the idea that domain M is entirely helical in both the soluble and membrane-bound forms of CCT.

We investigated effects of pro-atherogenic oxidized lipoproteins on phosphatidylcholine (PtdCho) biosynthesis in murine lung epithelial cells (MLE-12). Cells surface-bound, internalized, and degraded oxidized low density lipoproteins (Ox-LDL). Ox-LDL significantly reduced [3H]choline incorporation into PtdCho in cells by selectively inhibiting the activity of the rate-regulatory enzyme, CTP:phosphocholine cytdylyltransferase (CCT). Ox-LDL coordinately increased the cellular turnover of CCTalpha protein as determined by [35S]methionine pulse-chase studies by inducing the calcium-activated proteinase, calpain. Forced expression of calpain or exposure of cells to the calcium ionophore, A23187, increased CCTalpha degradation, whereas overexpression of the endogenous calpain inhibitor, calpastatin, attenuated Ox-LDL-induced CCTalpha degradation. The effects of Ox-LDL on CCTalpha breakdown were attenuated in calpain-deficient cells. In vitro calpain digestion of CCTalpha isolated from cells transfected with truncated or internal deletion mutants indicated multiple cleavage sites within the CCTalpha primary structure, leading to the generation of a 26-kDa (p26) fragment. Calpain hydrolysis of purified CCTalpha generated p26, which upon NH2-terminal sequencing localized a calpain attack site within the CCTalpha amino terminus. Expression of a CCTalpha mutant where the amino-terminal cleavage site and a putative carboxyl-terminal hydrolysis region were modified resulted in an enzyme that was significantly less sensitive to proteolytic cleavage and restored the ability of cells to synthesize surfactant PtdCho after Ox-LDL treatment. Thus, these results provide a critical link between proatherogenic lipoproteins and their metabolic target, CCTalpha, resulting in impaired surfactant metabolism.

CTP:phosphocholine cytidylyltransferase (CCT) is a key regulatory enzyme in phosphatidylcholine (PtdCho) biosynthesis, and in mammals, there are two distinct genes that encode enzymes that catalyze this reaction. This work defines the structures of both the murine CCT genes (Pcyt1a and Pcyt1b) and identifies a new CCT protein, CCTbeta3, with a unique amino terminus that arises from an alternate initiation exon. CCTalpha is expressed in all tissues, and is most abundant in liver, kidney and heart. A second CCTalpha transcript is described that initiates from a separate untranslated exon that is most highly expressed in testis. The CCTbeta isoforms are most highly expressed in brain and reproductive tissues. CCTbeta3 is not expressed in embryonic brain tissues, but is a significant transcript in the adult. These data suggest unique roles for the CCT protein isoforms in the differential regulation of PtdCho biosynthesis in specific tissues.

CTP:phosphocholine cytidylyltransferase alpha (CCTalpha) contains a central region that functions as a catalytic domain, converting phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline for the subsequent synthesis of phosphatidylcholine. We have investigated the catalytic role of lysine 122 and arginine 196 of rat CCTalpha using site-directed mutagenesis and a baculovirus expression system. Arginine 196 is part of the highly conserved RTEGIST motif, while lysine 122 has not previously been identified by protein sequence alignment as a candidate catalytic amino acid. Removing the side chain of lysine 122 compromises the catalytic ability of CCTalpha, decreasing the apparent V(max) value in mutant enzymes Lys122Ala and Lys122Arg to 0.30 and 0.09% of the wild-type value, respectively. The decrease in V(max) is accompanied by dramatic 471- and 80-fold increases in the apparent K(m) value for phosphocholine but no greater than 3-fold increases in the apparent Hill constant (K*) value for CTP. Mutation of arginine 196 to lysine results in an enzyme that retains 24% of the wild-type V(max) value with a modest 5-fold increase in the K(m) value for phosphocholine. However, the Arg196Lys mutant enzyme exhibits a 23-fold increase in the K* value for CTP. These data suggest lysine 122 and arginine 196 of rat CTP:phosphocholine cytidylyltransferase are functionally important amino acids, perhaps at or near the active site involved in forming contacts with the substrates phosphocholine and CTP, respectively.

Amphitropic proteins are regulated by reversible membrane interaction. Anionic phospholipids generally promote membrane binding of such proteins via electrostatics between the negatively charged lipid headgroups and clusters of basic groups on the proteins. In this study of one amphitropic protein, a cytidylyltransferase (CT) that regulates phosphatidylcholine synthesis, we found that substitution of lysines to glutamine along both interfacial strips of the membrane-binding amphipathic helix eliminated electrostatic binding. Unexpectedly, three glutamates also participate in the selectivity for anionic membrane surfaces. These glutamates become protonated in the low pH milieu at the surface of anionic, but not zwitterionic membranes, increasing protein positive charge and hydrophobicity. The binding and insertion into lipid vesicles of a synthetic peptide containing the three glutamates was pH-dependent with an apparent pK(a) that varied with anionic lipid content. Glutamate to glutamine substitution eliminated the pH dependence of the membrane interaction, and reduced anionic membrane selectivity of both the peptide and the whole CT enzyme examined in cells. Thus anionic lipids, working via surface-localized pH effects, can promote membrane binding by modifying protein charge and hydrophobicity, and this novel mechanism contributes to the membrane selectivity of CT in vivo.

Pneumococcal LicC is a member of the nucleoside triphosphate transferase superfamily and catalyzes the transfer of a cytidine monophosphate from CTP to phosphocholine to form CDP-choline. The structures of apo-LicC and the LicC-CDP-choline-Mg(2+) ternary complex were determined, and the comparison of these structures reveals a significant conformational change driven by the multivalent coordination of Mg(2+). The key event is breaking the Glu(216)-Arg(129) salt bridge, which triggers the coalescence of four individual beta-strands into two extended beta-sheets. These movements reorient the side chains of Trp(136) and Tyr(190) for the optimal binding and alignment of the phosphocholine moiety. Consistent with these conformational changes, LicC operates via a compulsory ordered kinetic mechanism. The structures explain the substrate specificity of LicC for CTP and phosphocholine and implicate a direct role for Mg(2+) in aligning phosphocholine for in-line nucleophilic attack and stabilizing the negative charge that develops in the pentacoordinate transition state. These results provide a structural basis for assigning a specific role for magnesium in the catalytic mechanism of pneumococcal LicC.

Streptococcus pneumoniae is a member of a small group of bacteria that display phosphocholine on the cell surface, covalently attached to the sugar groups of teichoic acid and lipoteichoic acid. The putative pathway for this phosphocholine decoration is, in its first two enzymes, functionally similar to the CDP-choline pathway used for phosphatidylcholine biosynthesis in eukaryotes. We show that the licC gene encodes a functional CTP:phosphocholine cytidylyltransferase (CCT). The enzyme has been expressed and purified to homogeneity. Assay conditions were optimized, particularly with respect to linearity with time, pH, Mg(2+), and ammonium sulfate concentration. The pure enzyme has K(M) values of 890+/-240 microM for CTP, and 390+/-170 microM for phosphocholine. The k(cat) is 17.5+/-4.0 s(-1). S. pneumoniae CTP:phosphocholine cytidylyltransferase (SpCCT) is specific for CTP or dCTP as the nucleotide substrate. SpCCT is strongly inhibited by Ca(2+). The IC(50) values for recombinant and native SpCCT are 0.32+/-0.04 and 0.27+/-0.03 mM respectively. The enzyme is also inhibited by all other tested divalent cations, including Mg(2+) at high concentrations. The cloning and expression of this enzyme sets the stage for design of inhibitors as possible antipneumococcal drugs.