A functional analysis regarding solution detection of antibodies versus SARSCoV2 nucleoprotein

Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to oxaloacetate). It is also known that the histone acetyltransferase p300 can induce PCK1 acetylation in cells, but whether that is a direct or indirect function was not known. Here we initially set out to determine whether p300 can acetylate directly PCK1 in vitro. We report that p300 weakly acetylates PCK1, but surprisingly, using several techniques including protein crystallization, mass spectrometry, isothermal titration calorimetry (ITC), saturation-transfer difference nuclear magnetic resonance (STD-NMR) and molecular docking, we found that PCK1 is also able to acetylate itself using acetyl-CoA independently of p300. This reaction yielded an acetylated recombinant PCK1 with a 3-fold decrease in kcat without changes in Km for all substrates. Acetylation stoichiometry was determined for 14 residues, including residues lining the active site. Structural and kinetic analyses determined that site-directed acetylation of K244, located inside the active site, altered this site and rendered the enzyme inactive. Additionally, we found that acetyl-CoA binding to the active site is specific and metal dependent. Our findings provide direct evidence for acetyl-CoA binding and chemically reacting with the active site of PCK1 and suggest a newly discovered regulatory mechanism of PCK1 during metabolic stress.Eukaryotic initiation factor 2B (eIF2B) serves as a vital control point within protein synthesis and regulates translation initiation in response to cellular stress. Mutations within eIF2B result in the fatal disease, leukoencephalopathy with vanishing white matter (VWM). Previous biochemical studies on VWM mutations have illustrated that changes in the activity of eIF2B poorly correlates with disease severity. This suggests that there may be additional characteristics of eIF2B contributing to VWM pathogenesis. Here, we investigated whether the localisation of eIF2B to eIF2B bodies was integral for function and whether this localisation could provide insight into the pathogenesis of VWM. We demonstrate that the regulatory subunit, eIF2Bα, is required for the assembly of eIF2B bodies in yeast and that loss of eIF2B bodies correlates with an inability of cells to regulate eIF2B activity. Mutational analysis of eIF2Bα showed that missense mutations which disrupt the regulation of eIF2B similarly disrupt the assembly of eIF2B bodies. In contrast, when eIF2Bα mutations which impact the catalytic activity of eIF2B were analysed, eIF2B bodies were absent and instead eIF2B localised to small foci, termed microfoci. FRAP analysis highlighted that within these microfoci, eIF2 shuttles more slowly indicating that formation of eIF2B bodies correlates with full eIF2B activity. When eIF2Bα VWM mutations were analysed a diverse impact on localisation was observed, which did not seem to correlate with eIF2B activity. These findings provide key insights into how the eIF2B body assembles and suggest that the body is a fundamental part of the translational regulation via eIF2α phosphorylation.The death of photoreceptor cells in dry age-related macular degeneration (AMD) and autosomal recessive Stargardt disease (STGD1) is closely associated with disruption in all-trans-retinal (atRAL) clearance in neural retina. In this study, we reveal that the overload of atRAL leads to photoreceptor degeneration through activating ferroptosis, a nonapoptotic form of cell death. Ferroptosis of photoreceptor cells induced by atRAL resulted from increased ferrous ion (Fe2+), elevated ACSL4 expression, system Xc‾ inhibition and mitochondrial destruction. Fe2+ overload, tripeptide glutathione (GSH) depletion and damaged mitochondria in photoreceptor cells exposed to atRAL provoked reactive oxygen species (ROS) production, which, together with ACSL4 activation, promoted lipid peroxidation and thereby evoked ferroptotic cell death. Moreover, exposure of photoreceptor cells to atRAL activated COX2, a well-accepted biomarker for ferroptosis onset. In addition to GSH supplement, inhibiting either Fe2+ by deferoxamine mesylate salt (DFO) or lipid peroxidation with ferrostatin-1 (Fer-1) protected photoreceptor cells from ferroptosis caused by atRAL. Abca4-/-Rdh8-/- mice exhibiting defects in atRAL clearance is an animal model for dry AMD and STGD1. We observed that ferroptosis was indeed present in neural retina of Abca4-/-Rdh8-/- mice after light exposure. More importantly, photoreceptor atrophy and ferroptosis in light-exposed Abca4-/-Rdh8-/- mice were effectively alleviated by intraperitoneally injected Fer-1, a selective inhibitor of ferroptosis. Our study suggests that ferroptosis is one of the important pathways of photoreceptor cell death in retinopathies arising from excess atRAL accumulation, and should be pursued as a novel target for protection against dry AMD and STGD1.The carnitine/organic cation transporter novel 2 (OCTN2) is responsible for the cellular uptake of carnitine in most tissues. Ziprasidone Being a transmembrane protein OCTN2 must interact with the surrounding lipid microenvironment to function. Among the main lipid species that constitutes eukaryotic cells, cholesterol level is highly dynamic under a number of physio-pathological conditions. This work describes how plasma membrane cholesterol modulates OCTN2 transport of L-carnitine in human embryonic kidney 293 cells overexpressing OCTN2 (OCTN2-HEK293) and in proteoliposomes harboring human OCTN2. We manipulated the cholesterol content of intact cells, assessed by thin layer chromatography, through short exposures to empty and/or cholesterol-saturated methyl-β-cyclodextrin (mβcd), whereas free cholesterol was used to enrich reconstituted proteoliposomes. We measured OCTN2 transport using [3H]L-carnitine, and expression levels and localization by surface biotinylation and western blotting. A 20-minute preincubation with mβcd reduced the cellular cholesterol content and inhibited L-carnitine influx by 50% in comparison to controls. Analogously, the insertion of cholesterol in OCTN2-proteoliposomes stimulated L-carnitine uptake in a dose-dependent manner. Carnitine uptake in cells incubated with empty mβcd and cholesterol-saturated mβcd to preserve cholesterol content was comparable to controls, suggesting that the mβcd effect on OCTN2 was cholesterol dependent. Cholesterol stimulated L-carnitine influx in cells by markedly increasing the affinity for L-carnitine and in proteoliposomes by significantly enhancing the affinity for Na+ and, in turn, the L-carnitine maximal transport capacity. Because of the antilipogenic and antioxidant features of L-carnitine, the stimulatory effect of cholesterol on L-carnitine uptake might represent a novel protective effect against lipid-induced toxicity and oxidative stress.