Rate-limiting steps in the cytidine pathway for the synthesis of phosphatidylcholine and phosphatidylethanolamine.
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ABSTRACT: An analysis of the available data on the cytidine pathway for the synthesis of phosphatidylcholine and phosphatidylethanolamine, by the logic derived from the theoretical principles of metabolic regulation, shows that the first two reactions catalysed by choline (ethanolamine) kinase and phosphocholine (phosphoethanolamine) cytidylyltransferase are rate-limiting, whereas the phosphocholine (phosphoethanolamine) transferase step is near equilibrium in rat liver.
Project description:The nucleus contains a network of tubular invaginations of the nuclear envelope (NE), termed the nucleoplasmic reticulum (NR), implicated in transport, gene expression, and calcium homeostasis. Here, we show that proliferation of the NR, measured by the frequency of NE invaginations and tubules, is regulated by CTP:phosphocholine cytidylyltransferase-alpha (CCTalpha), the nuclear and rate-limiting enzyme in the CDP-choline pathway for phosphatidylcholine (PtdCho) synthesis. In Chinese hamster ovary (CHO)-K1 cells, fatty acids triggered activation and translocation of CCTalpha onto intranuclear tubules characteristic of the NR. This was accompanied by a twofold increase in NR tubules quantified by immunostaining for lamin A/C or the NE. CHO MT58 cells expressing a temperature-sensitive CCTalpha allele displayed reduced PtdCho synthesis and CCTalpha expression and minimal proliferation of the NR in response to oleate compared with CHO MT58 cells stably expressing CCTalpha. Expression of CCTalpha mutants in CHO58 cells revealed that both enzyme activity and membrane binding promoted NR proliferation. In support of a direct role for membrane binding in NR tubule formation, recombinant CCTalpha caused the deformation of liposomes into tubules in vitro. This demonstrates that a key nuclear enzyme in PtdCho synthesis coordinates lipid synthesis and membrane deformation to promote formation of a dynamic nuclear-cytoplasmic interface.
Project description:Methionine adenosyltransferase 1A (MAT1A) and glycine N-methyltransferase (GNMT) are the primary genes involved in hepatic S-adenosylmethionine (SAMe) synthesis and degradation, respectively. Mat1a ablation in mice induces a decrease in hepatic SAMe, activation of lipogenesis, inhibition of triglyceride (TG) release, and steatosis. Gnmt-deficient mice, despite showing a large increase in hepatic SAMe, also develop steatosis. We hypothesized that as an adaptive response to hepatic SAMe accumulation, phosphatidylcholine (PC) synthesis by way of the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway is stimulated in Gnmt(-/-) mice. We also propose that the excess PC thus generated is catabolized, leading to TG synthesis and steatosis by way of diglyceride (DG) generation. We observed that Gnmt(-/-) mice present with normal hepatic lipogenesis and increased TG release. We also observed that the flux from PE to PC is stimulated in the liver of Gnmt(-/-) mice and that this results in a reduction in PE content and a marked increase in DG and TG. Conversely, reduction of hepatic SAMe following the administration of a methionine-deficient diet reverted the flux from PE to PC of Gnmt(-/-) mice to that of wildtype animals and normalized DG and TG content preventing the development of steatosis. Gnmt(-/-) mice with an additional deletion of perilipin2, the predominant lipid droplet protein, maintain high SAMe levels, with a concurrent increased flux from PE to PC, but do not develop liver steatosis.These findings indicate that excess SAMe reroutes PE towards PC and TG synthesis and lipid sequestration.
Project description:Deep sequencing now provides detailed snapshots of ribosome occupancy on mRNAs. We leverage these data to parameterize a computational model of translation, keeping track of every ribosome, tRNA, and mRNA molecule in a yeast cell. We determine the parameter regimes in which fast initiation or high codon bias in a transgene increases protein yield and infer the initiation rates of endogenous Saccharomyces cerevisiae genes, which vary by several orders of magnitude and correlate with 5' mRNA folding energies. Our model recapitulates the previously reported 5'-to-3' ramp of decreasing ribosome densities, although our analysis shows that this ramp is caused by rapid initiation of short genes rather than slow codons at the start of transcripts. We conclude that protein production in healthy yeast cells is typically limited by the availability of free ribosomes, whereas protein production under periods of stress can sometimes be rescued by reducing initiation or elongation rates.
Project description:Nascent RNA-sequencing tracks transcription at nucleotide resolution. The genomic distribution of engaged transcription complexes, in turn, uncovers functional genomic regions. Here, we provide analytical steps to (1) identify transcribed regulatory elements de novo genome-wide, (2) quantify engaged transcription complexes at enhancers, promoter-proximal regions, divergent transcripts, gene bodies, and termination windows, and (3) measure distribution of transcription machineries and regulatory proteins across functional genomic regions. This protocol tracks engaged transcription complexes across functional genomic regions demonstrated in human K562 erythroleukemia cells. For complete details on the use and execution of this protocol, please refer to Vihervaara et al. (2021).
Project description: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.
Project description:Amino-oxyacetate (carboxymethoxylamine) was found to inhibit protein labelling in isolated liver cells. A similar degree of inhibition (about 70%) was observed of basal and substrate-stimulated rates of protein labelling, ruling out an action on the cellular energy state. Its effect does not seem to be related either to a perturbation of the reduction state of the NAD system or to rate changes in the gluconeogenic pathway. The following observations indicate that amino-oxyacetate inhibits protein labelling by limiting aspartate supply. Amino-oxyacetate was ineffective in a postmitochondrial supernatant under non-limiting amino acid supply conditions. The aspartate cellular content decreases in the presence of amino-oxyacetate, although most other amino acids tend to accumulate. L-Cycloserine was unable to decrease aspartate content and was ineffective in decreasing protein labelling. The inhibitory action of amino-oxyacetate was specifically reversed by incubating cells with amino acids that increase the cellular content of aspartate.
Project description:BackgroundPlasmalogens are either phosphatidylcholine (PC P) or phosphatidylethanolamine (PE P) glycerophospholipids containing a vinyl ether moiety in sn-1-position and an esterified fatty acid in sn-2 position. Multiple functions have been proposed, including reservoir of precursors for inflammatory mediators, modulation of membrane fluidity, and anti-oxidative properties. They could therefore play a role under conditions of metabolic stress. Especially enzymatically modified LDL (eLDL) and oxidatively modified LDL (oxLDL) represent modifications of LDL that are taken up by macrophages in atherosclerotic plaques. The aim of this study was to analyze plasmalogen related effects of eLDL and oxLDL in human monocyte derived macrophages, as well as the effects of HDL3 mediated deloading.MethodsElutriated monocytes from nine healthy donors were differentiated in vitro for four days. Macrophages were then loaded with native LDL, eLDL and oxLDL for 24h and subsequently deloaded with HDL3 for another 24h. Lipidomic and transcriptomic profiles were obtained.ResultsLoading of macrophages with eLDL and oxLDL led to a transient but strong elevation of lysophosphatidylcholine (LPC) most likely through direct uptake. Only eLDL induced increased levels of total PC, presumably through an induction of PC synthesis. On the other hand treatment with oxLDL led to a significant increase in PC P. Analysis of individual lipid species showed lipoprotein and saturation specific effects for LPC, PC P and PE P species. Membrane fluidity was decreased by the large amount of FC contained in the lipoproteins, as indicated by a lower PC to FC ratio after lipoprotein loading. In contrast the observed changes in the saturated to mono-unsaturated fatty acid (SFA to MUFA) and saturated to poly-unsaturated fatty acid (SFA to PUFA) ratios in PE P could represent a cellular reaction to counteract this effect by producing more fluid membranes. Transcriptomic analysis showed considerable differences between eLDL and oxLDL treated macrophages. As a common feature of both lipoproteins we detected a strong downregulation of pathways for endogenous lipid synthesis as well as for exogenous lipid uptake. Deloading with HDL3 had only minor effects on total lipid class as well as on individual lipid species levels, most of the time not reaching significance. Interestingly treatment with HDL3 had no effect on membrane fluidity under these conditions, although incubation with HDL3 was partially able to counteract the oxLDL induced transcriptomic effects. To investigate the functional effect of lipoprotein treatment on macrophage polarization we performed surface marker flow cytometry. Under our experimental conditions oxLDL was able to partially shift the surface marker pattern towards a pro-inflammatory M1-like phenotype. This is consistent with the consumption of arachidonic acid containing PE P species in oxLDL treated cells, presumably for the synthesis of inflammatory mediators.SummaryOur findings provide novel data on the lipoprotein induced, lipidomic and transcriptomic changes in macrophages. This can help us better understand the development of metabolic, inflammatory diseases as well as improve our background knowledge on lipid biomarkers in serum.
Project description:The ability to predict the mechanisms and the associated rate constants of protein-ligand unbinding is of great practical importance in drug design. In this work we demonstrate how a recently introduced metadynamics-based approach allows exploration of the unbinding pathways, estimation of the rates, and determination of the rate-limiting steps in the paradigmatic case of the trypsin-benzamidine system. Protein, ligand, and solvent are described with full atomic resolution. Using metadynamics, multiple unbinding trajectories that start with the ligand in the crystallographic binding pose and end with the ligand in the fully solvated state are generated. The unbinding rate k off is computed from the mean residence time of the ligand. Using our previously computed binding affinity we also obtain the binding rate k on. Both rates are in agreement with reported experimental values. We uncover the complex pathways of unbinding trajectories and describe the critical rate-limiting steps with unprecedented detail. Our findings illuminate the role played by the coupling between subtle protein backbone fluctuations and the solvation by water molecules that enter the binding pocket and assist in the breaking of the shielded hydrogen bonds. We expect our approach to be useful in calculating rates for general protein-ligand systems and a valid support for drug design.
Project description:Cell-to-cell variability in cellular components generates cell-to-cell diversity in RNA and protein production dynamics. As these components are inherited, this should also cause lineage-to-lineage variability in these dynamics. We conjectured that these effects on transcription are promoter initiation kinetics dependent. To test this, first we used stochastic models to predict that variability in the numbers of molecules involved in upstream processes, such as the intake of inducers from the environment, acts only as a transient source of variability in RNA production numbers, while variability in the numbers of a molecular species controlling transcription of an active promoter acts as a constant source. Next, from single-cell, single-RNA level time-lapse microscopy of independent lineages of Escherichia coli cells, we demonstrate the existence of lineage-to-lineage variability in gene activation times and mean RNA production rates, and that these variabilities differ between promoters and inducers used. Finally, we provide evidence that this can be explained by differences in the kinetics of the rate-limiting steps in transcription between promoters and induction schemes. We conclude that cell-to-cell and consequent lineage-to-lineage variability in RNA and protein numbers are both promoter sequence-dependent and subject to regulation.