Genetic screening reveals phospholipid metabolism as a key regulator of the biosynthesis of the redox-active lipid coenzyme Q.

Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases

Alpha-tocotrienol is the most abundant tocotrienol isomer circulated in plasma and lipoproteins after postprandial tocotrienol-rich vitamin E supplementation

<p>Abstract</p> <p>Background</p> <p>Tocotrienols (T3) and tocopherols (T), both members of the natural vitamin E family have unique biological functions in humans. T3 are detected in circulating human plasma and lipoproteins, although at concentrations significantly lower than α-tocopherol (α-T). T3, especially α-T3 is known to be neuropotective at nanomolar concentrations and this study evaluated the postprandial fate of T3 and α-T in plasma and lipoproteins.</p> <p>Methods</p> <p>Ten healthy volunteers (5 males and 5 females) were administered a single dose of vitamin E [526 mg palm tocotrienol-rich fraction (TRF) or 537 mg α-T] after 7-d pre-conditioning on a T3-free diet. Blood was sampled at baseline (fasted) and 2, 4, 5, 6, 8, and 24 h after supplementation. Concentrations of T and T3 isomers in plasma, triacylglycerol-rich particles (TRP), LDL, and HDL were measured at each postprandial interval.</p> <p>Results</p> <p>After TRF supplementation, plasma α-T3 and γ-T3 peaked at 5 h (α-T3: 4.74 ± 1.69 μM; γ-T3: 2.73 ± 1.27 μM). δ-T3 peaked earlier at 4 h (0.53 ± 0.25 μM). In contrast, α-T peaked at 6 h (30.13 ± 2.91 μM) and 8 h (37.80 ± 3.59 μM) following supplementation with TRF and α-T, respectively. α-T was the major vitamin E isomer detected in plasma, TRP, LDL, and HDL even after supplementation with TRF (composed of 70% T3). No T3 were detected during fasted states. T3 are detected postprandially only after TRF supplementation and concentrations were significantly lower than α-T.</p> <p>Conclusions</p> <p>Bio-discrimination between vitamin E isomers in humans reduces the rate of T3 absorption and affects their incorporation into lipoproteins. Although low absorption of T3 into circulation may impact some of their physiological functions in humans, T3 have biological functions well below concentration noted in this study.</p

Cryo-EM reveals distinct conformations of E. coli ATP synthase on exposure to ATP

ATP synthase produces the majority of cellular energy in most cells. We have previously reported cryo-EM maps of autoinhibited E. coli ATP synthase imaged without addition of nucleotide (Sobti et al. 2016), indicating that the subunit ε engages the α, β and γ subunits to lock the enzyme and prevent functional rotation. Here we present multiple cryo-EM reconstructions of the enzyme frozen after the addition of MgATP to identify the changes that occur when this ε inhibition is removed. The maps generated show that, after exposure to MgATP, E. coli ATP synthase adopts a different conformation with a catalytic subunit changing conformation substantially and the ε C-terminal domain transitioning via an intermediate \u27half-up\u27 state to a condensed \u27down\u27 state. This work provides direct evidence for unique conformational states that occur in E. coli ATP synthase when ATP binding prevents the ε C-terminal domain from entering the inhibitory \u27up\u27 state

Association between both lipid and protein oxidation and the risk of fatal or non-fatal coronary heart disease in a human population.

The role of oxidative damage in the aetiology of coronary disease remains controversial, as clinical trials investigating the effect of antioxidants have not generally been positive. In the present study, 227 coronary cases, identified from a cohort study, were matched, by age and gender, with 420 controls in a nested case-control design. Stored plasma samples were analysed for F2-isoprostanes by stable isotope dilution MS, and specifically oxidized forms of apoA-I (apolipoprotein A-I) by HPLC of HDL (high-density lipoprotein). Median values of F2-isoprostanes were higher in plasma samples that contained oxidized apoA-I compared with samples with undetectable oxidized apoA-I (1542 compared with 1165 pmol/l). F2-Isoprostanes were significantly correlated with variants of non-oxidized apoA-II (r=-0.15) and were associated with HDL-cholesterol (P&lt;0.0001). F2-Isoprostanes in cases (median, 1146 pmol/l) were not different from controls (1250 pmol/l); the odds ratio (95% confidence interval) for a 1 S.D. increase in F2-isoprostanes was 1.08 (0.91-1.29). Similarly, there was no independent association between the presence of oxidized apoA-I, detected in approx. 20% of the samples, and coronary risk. In conclusion, we found no evidence of associations between markers of lipid (F2-isoprostanes) and protein (oxidized apoA-I) oxidation and the risk of fatal or non-fatal coronary heart disease in a general population. This may be due to a true lack of association or insufficient power

Transition to 37°C reveals importance of NADPH in mitigating oxidative stress in stored RBCs.

The RBC storage lesion is a multiparametric response that occurs during storage at 4°C, but its impact on transfused patients remains unclear. In studies of the RBC storage lesion, the temperature transition from cold storage to normal body temperature that occurs during transfusion has received limited attention. We hypothesized that multiple deleterious events might occur in this period of increasing temperature. We show dramatic alterations in several properties of therapeutic blood units stored at 4°C after warming them to normal body temperature (37°C), as well as febrile temperature (40°C). In particular, the intracellular content and redox state of NADP(H) were directly affected by post-storage incubation at 37°C, as well as by pro-oxidant storage conditions. Modulation of the NADPH-producing pentose phosphate pathway, but not the prevention of hemoglobin autoxidation by conversion of oxyhemoglobin to carboxyhemoglobin, provided protection against storage-induced alterations in RBCs, demonstrating the central role of NADPH in mitigating increased susceptibility of stored RBCs to oxidative stress. We propose that assessing RBC oxidative status after restoration of body temperature constitutes a sensitive method for detecting storage-related alterations that has the potential to improve the quality of stored RBCs for transfusion