Interdomain communication in the phosphatidylcholine regulatory enzyme, CCTα, relies on a modular αE helix.

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.

Remodeling of the interdomain allosteric linker upon membrane binding of CCTα pulls its active site close to the membrane surface.

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 αEC/J) using MD simulations, cross-linking, mutagenesis, and solvent accessibility suggested that membrane binding of domain M promotes remodeling of the αEC/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 αEC/J can interact with lipids independently of the M domain. We observed strong FRET between engineered tryptophans in the αEC/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.

Metabolism of a synthetic compared with a natural therapeutic pulmonary surfactant in adult mice.

Secreted pulmonary surfactant phosphatidylcholine (PC) has a complex intra-alveolar metabolism that involves uptake and recycling by alveolar type II epithelial cells, catabolism by alveolar macrophages, and loss up the bronchial tree. We compared the in vivo metabolism of animal-derived poractant alfa (Curosurf) and a synthetic surfactant (CHF5633) in adult male C57BL/6 mice. The mice were dosed intranasally with either surfactant (80 mg/kg body weight) containing universally 13C-labeled dipalmitoyl PC (DPPC) as a tracer. The loss of [U13C]DPPC from bronchoalveolar lavage and lung parenchyma, together with the incorporation of 13C-hydrolysis fragments into new PC molecular species, was monitored by electrospray ionization tandem mass spectrometry. The catabolism of CHF5633 was considerably delayed compared with poractant alfa, the hydrolysis products of which were cleared more rapidly. There was no selective resynthesis of DPPC and, strikingly, acyl remodeling resulted in preferential synthesis of polyunsaturated PC species. In conclusion, both surfactants were metabolized by similar pathways, but the slower catabolism of CHF5633 resulted in longer residence time in the airways and enhanced recycling of its hydrolysis products into new PC species.

A choline-releasing glycerophosphodiesterase essential for phosphatidylcholine biosynthesis and blood stage development in the malaria parasite.

The malaria parasite Plasmodium falciparum synthesizes significant amounts of phospholipids to meet the demands of replication within red blood cells. De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway is essential, requiring choline that is primarily sourced from host serum lysophosphatidylcholine (lysoPC). LysoPC also acts as an environmental sensor to regulate parasite sexual differentiation. Despite these critical roles for host lysoPC, the enzyme(s) involved in its breakdown to free choline for PC synthesis are unknown. Here, we show that a parasite glycerophosphodiesterase (PfGDPD) is indispensable for blood stage parasite proliferation. Exogenous choline rescues growth of PfGDPD-null parasites, directly linking PfGDPD function to choline incorporation. Genetic ablation of PfGDPD reduces choline uptake from lysoPC, resulting in depletion of several PC species in the parasite, whilst purified PfGDPD releases choline from glycerophosphocholine in vitro. Our results identify PfGDPD as a choline-releasing glycerophosphodiesterase that mediates a critical step in PC biosynthesis and parasite survival.

Malaria kills over half a million people every year worldwide. A single-celled parasite called Plasmodium falciparum is responsible for the most lethal form of the disease. This malaria-causing agent is carried by mosquitos which transmit the parasite to humans through their bite. Once in the bloodstream, the parasite enters red blood cells and starts to replicate so it can go on to infect other cells. Like our cells, P. falciparum is surrounded by a membrane, and further membranes surround a number of its internal compartments. To make these protective coats, the parasite has to gather a nutrient called choline to form an important building block in the membrane. The parasite gets most of its choline by absorbing and digesting a molecule known as lysoPC found in the bloodstream of its host. However, it was unclear precisely how the parasite achieves this. To address this question, Ramaprasad, Burda et al. used genetic and metabolomic approaches to study how P. falciparum breaks down lysoPC. The experiments found that mutant parasites that are unable to make an enzyme called GDPD were able to infect red blood cells, but failed to grow properly once inside the cells. The mutant parasites took up less choline and, as a result, also made fewer membrane building blocks. The team were able to rescue the mutant parasites by supplying them with large quantities of choline, which allowed them to resume growing. Taken together, the findings of Ramaprasad, Burda et al. suggest that P. falciparum uses GDPD to extract choline from lysoPC when it is living in red blood cells. More and more P. falciparum parasites are becoming resistant to many of the drugs currently being used to treat malaria. One solution is to develop new therapies that target different molecules in the parasite. Since it performs such a vital role, GDPD may have the potential to be a future drug target.

C1 Metabolism Inhibition and Nitrogen Deprivation Trigger Triacylglycerol Accumulation in Arabidopsis thaliana Cell Cultures and Highlight a Role of NPC in Phosphatidylcholine-to-Triacylglycerol Pathway.

Triacylglycerol (TAG) accumulation often occurs in growth limiting conditions such as nutrient deprivations. We analyzed and compared the lipid contents of Arabidopsis cells grown under two conditions that inhibited growth as a way to study interactions between membrane and storage lipids. In order to inhibit C1 metabolism, the first condition utilized methotrexate (MTX), a drug that inhibits methyl transfer reactions and potentially reduces Pi-choline synthesis, the polar head of phosphatidylcholine (PC). MTX-treated cells displayed a 10- to 15-fold increase in TAG compared to that found in control cells. This corresponded to a net increase of lipids as the total amount of membrane glycerolipids was minimally affected. Under this condition, PC homeostasis appeared tightly regulated and not strictly dependent on the rate of Pi-choline synthesis. The second condition we investigated involved nitrogen deprivation. Here, we observed a 40-fold increase of TAG. In these cells, the overall lipid content remained unchanged, but membrane lipids decreased by a factor of two suggesting a reduction of the membrane network and a rerouting of membrane lipids to storage lipids. Under all conditions, fatty acid (FA) analyses showed that the FA composition of TAG was comparable to that in PC, but different from that in acyl-CoA, suggesting that TAG accumulation involved PC-derived DAG moieties. In agreement, analyses by qPCR of genes coding for TAG synthesis showed a strong increase of non-specific phospholipase C (NPC) expressions, and experiments using labeled (fluorescent) PC indicated higher rates of PC-to-TAG conversion under both situations. These results highlight a role for NPC in plant cell oil production.

Fluorescent fatty acid analogs as a tool to study development of the picornavirus replication organelles.

Genome replication of positive strand RNA viruses of eukaryotes is universally associated with specialized membranous structures referred to as replication organelles. Accumulating evidence show that new membrane synthesis is important for the development of the replication organelles of diverse picornaviruses and likely for other positive strand RNA viruses as well. The hydrophobic part of the structural phospholipid molecules defining the barrier properties of biological membranes consists of two long chain fatty acid moieties attached to the glycerol backbone. Fluorescent long chain fatty acid analogs represent a very convenient tool to monitor membrane synthesis in infected cells offering significant advantages over conventional radioactively labeled compounds. Bodipy-containing fatty acid analogs are readily imported from the extracellular media and utilized in lipid synthesis by cellular machinery. The strong fluorescence of the Bodipy group allows monitoring the molecules in situ by fluorescent microscopy as well as provides an opportunity for quantitative assessment of fatty acid import in a multi-well plate format. Moreover lipids with incorporated fluorescent fatty acid chain can be resolved by thin layer chromatography and easily identified using conventional UV imaging systems thus providing a simple and convenient way of monitoring the perturbation of the lipid synthesis pathways upon infection.