Calcium inhibits diacylglycerol uptake by serum albumin

AbstractSerum albumin is an abundant protein in blood plasma, that is well-known for its ability to transport hydrophobic biomolecules and drugs. Recent hypotheses propose that serum albumin plays a role in the regulation of lipid metabolism in addition to its lipid transport properties. The present work explores the capacity of bovine serum albumin (BSA) to extract diacylglycerols (DAG) from phospholipid bilayers, and the inhibition of such interaction by divalent cations. Quantitative measurements using radioactive DAG and morphological evidence derived from giant unilamellar vesicles examined by confocal microscopy provide concurrent results. BSA extracts DAG from vesicles consisting of phosphatidylinositol/DAG. Long, saturated DAG species are incorporated more readily than the shorter-chain or unsaturated ones. Divalent cations hinder DAG uptake by BSA. For Ca2+, the concentration causing half-maximal inhibition is ≈10 μM; 90% inhibition is caused by 100 μM Ca2+. Sr2+ requires concentrations one order of magnitude higher, while Mg2+ has virtually no effect. As an example on how DAG uptake by BSA, and its inhibition by Ca2+, could play a regulating role in lipid metabolism, a PI-specific phospholipase C has been assayed in the presence of BSA and/or Ca2+. BSA activates the enzyme by removing the end-product DAG, but the activation is reverted by Ca2+ that inhibits DAG uptake

The Binding of Aβ42 Peptide Monomers to Sphingomyelin/Cholesterol/Ganglioside Bilayers Assayed by Density Gradient Ultracentrifugation

The binding of Aβ42 peptide monomers to sphingomyelin/cholesterol (1:1 mol ratio) bilayers containing 5 mol% gangliosides (either GM1, or GT1b, or a mixture of brain gangliosides) has been assayed by density gradient ultracentrifugation. This procedure provides a direct method for measuring vesicle-bound peptides after non-bound fraction separation. This centrifugation technique has rarely been used in this context previously. The results show that gangliosides increase by about two-fold the amount of Aβ42 bound to sphingomyelin/cholesterol vesicles. Complementary studies of the same systems using thioflavin T fluorescence, Langmuir monolayers or infrared spectroscopy confirm the ganglioside-dependent increased binding. Furthermore these studies reveal that gangliosides facilitate the aggregation of Aβ42 giving rise to more extended β-sheets. Thus, gangliosides have both a quantitative and a qualitative effect on the binding of Aβ42 to sphingomyelin/cholesterol bilayers.This work was supported in part by grants from the Spanish Ministry of Economy (grant FEDER MINECO PGC2018-099857-B-I00) and the Basque Government (grants No. IT1264-19 and IT1270-19)

Enzymatic Determination of Diglyceride Using an Iridium Nano-Particle Based Single Use, Disposable Biosensor

A single use, disposable iridium-nano particle contained biosensor had been developed for the determination of diglyceride (DG). In this study hydrogen peroxide, formed through the enzymatic breakdown of DG via lipase, glycerol kinase and glycerol 3-phosphate oxidase, was electrochemically oxidized at an applied potential of +0.5 V versus the Ag/AgCl reference electrode. The oxidation current was then used to quantify the diglyceride concentration. Optimum enzyme concentrations and the surfactant loading used were established for successful sensor response. Good linear performance was observed over a DG concentration range of 0 to 25 μM in phosphate buffer and bovine serum media

The Mechanochemistry of Endocytosis

An integrated theoretical model reveals how the chemical and the mechanical aspects of endocytosis are coordinated coherently in yeast cells, driving progression through the endocytic pathway and ensuring efficient vesicle scission in vivo

Pb(II) Induces Scramblase Activation and Ceramide-Domain Generation in Red Blood Cells

The mechanisms of Pb(II) toxicity have been studied in human red blood cells using confocal microscopy, immunolabeling, fluorescence-activated cell sorting and atomic force microscopy. The process follows a sequence of events, starting with calcium entry, followed by potassium release, morphological change, generation of ceramide, lipid flip-flop and finally cell lysis. Clotrimazole blocks potassium channels and the whole process is inhibited. Immunolabeling reveals the generation of ceramide-enriched domains linked to a cell morphological change, while the use of a neutral sphingomyelinase inhibitor greatly delays the process after the morphological change, and lipid flip-flop is significantly reduced. These facts point to three major checkpoints in the process: first the upstream exchange of calcium and potassium, then ceramide domain formation, and finally the downstream scramblase activation necessary for cell lysis. In addition, partial non-cytotoxic cholesterol depletion of red blood cells accelerates the process as the morphological change occurs faster. Cholesterol could have a role in modulating the properties of the ceramide-enriched domains. This work is relevant in the context of cell death, heavy metal toxicity and sphingolipid signaling.AGA was a predoctoral student supported by the Basque Government and later by the University of the Basque Country (UPV/EHU). This work was also supported in part by grants from the Spanish Government (FEDER/MINECO BFU 2015-66306-P to F.M.G. and A.A.) and the Basque Government (IT849-13 to F.M.G. and IT838-13 to A.A.), and by the Swiss National Science Foundation

Membrane fluidity matters: Hyperthermia from the aspects of lipids and membranes

Hyperthermia is a promising treatment modality for cancer in combination both with radio- and chemotherapy. In spite of its great therapeutic potential, the underlying molecular mechanisms still remain to be clarified. Due to lipid imbalances and 'membrane defects' most of the tumour cells possess elevated membrane fluidity. However, further increasing membrane fluidity to sensitise to chemo-or radiotherapy could have some other effects. In fact, hyperfluidisation of cell membrane induced by membrane fluidiser initiates a stress response as the heat shock protein response, which may modulate positively or negatively apoptotic cell death. Overviewing some recent findings based on a technology allowing direct imaging of lipid rafts in live cells and lipidomics, novel aspects of the intimate relationship between the 'membrane stress' of tumour cells and the cellular heat shock response will be highlighted. Our findings lend support to both the importance of membrane remodelling and the release of lipid signals initiating stress protein response, which can operate in tandem to control the extent of the ultimate cellular thermosensitivity. Overall, we suggest that the fluidity variable of membranes should be used as an independent factor for predicting the efficacy of combinational cancer therapies