cytochrome-c-t and cholesterol-alpha-oxide

Cholesterol undergoes ozonolysis to afford a variety of oxysterol products, including cholesterol-5,6-epoxide (CholEp) and the isomeric aldehydes secosterol A (seco A) and secosterol B (seco B). These oxysterols display numerous important biological activities, including protein adduction; however, much remains to be learned about the identity of the reactive species and the range of proteins modified by these oxysterols. Here, we synthesized alkynyl derivatives of cholesterol-derived oxysterols and employed a straightforward detection method to establish secosterols A and B as the most protein-reactive of the oxysterols tested. Model adduction studies with an amino acid, peptides, and proteins provide evidence for the potential role of secosterol dehydration products in protein adduction. Hydrophobic separation methods-Folch extraction and solid phase extraction (SPE)-were successfully applied to enrich oxysterol-adducted peptide species, and LC-MS/MS analysis of a model peptide-seco adduct revealed a unique fragmentation pattern (neutral loss of 390 Da) for that species. Coupling a hydrophobic enrichment method with proteomic analysis utilizing characteristic fragmentation patterns facilitates the identification of secosterol-modified peptides and proteins in an adducted protein. More broadly, these improved enrichment methods may give insight into the role of oxysterols and ozone exposure in the pathogenesis of a variety of diseases, including atherosclerosis, Alzheimer's disease, Parkinson's disease, and asthma.

Topics: Aldehydes; Amino Acid Sequence; Biotin; Cholesterol; Chromatography, High Pressure Liquid; Click Chemistry; Cytochromes c; Humans; Hydrophobic and Hydrophilic Interactions; Isomerism; Molecular Sequence Data; Ozone; Peptides; Proteins; Serum Albumin; Solid Phase Extraction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Streptavidin

The induction of apoptosis in cells of the arterial wall is a critical event in the development of atheroma. 7beta-Hydroxycholesterol (7beta-OH) and cholesterol-5beta,6beta-epoxide (beta-epoxide) are components of oxLDL and have previously been shown to be potent inducers of apoptosis. However, the exact mechanisms through which these oxysterols induce apoptosis remains to be fully elucidated. The specific interaction of the Fas death receptor with Fas ligand (FasL) initiates a caspase cascade culminating in apoptosis. The purpose of the present study was to determine the involvement of Fas signalling in 7beta-OH-and beta-epoxide-induced apoptosis. To this end we employed the Fas/FasL antagonist, Kp7-6, and examined the effect of Fas inhibition on oxysterol-induced cell death in U937 cells. Fas levels were increased following 24 h exposure to 30 micro M 7beta-OH while treatment with 30 micro M beta-epoxide had no effect. Kp7-6 reduced the Fas content of 7beta-OH-treated cells to control levels and partially protected against 7beta-OH-induced apoptosis. This coincided with a decrease in cytochrome c release along with a reduction in caspase-3 and caspase-8 activity. Our data implicate Fas signalling in the apoptotic pathway induced by 7beta-OH and also highlight differences between apoptosis induced by 7beta-OH and beta-epoxide.

Topics: Apoptosis; Caspases; Cholesterol; Cytochromes c; Enzyme-Linked Immunosorbent Assay; fas Receptor; Humans; Hydroxycholesterols; Monocytes; Signal Transduction; U937 Cells

Oxysterols are oxygenated derivatives of cholesterol that may be formed endogenously or absorbed from the diet. Significant amounts of oxysterols have frequently been identified in foods of animal origin, in particular highly processed foods. To date, oxysterols have been shown to possess diverse biological activities; however, recent attention has focused on their potential role in the development of atherosclerosis. Oxysterols have been reported to induce apoptosis in cells of the arterial wall, a primary process in the development of atheroma. The aim of the present study was to identify the role of the mitochondria in the apoptotic pathways induced by the oxysterols 7beta-hydroxycholesterol (7beta-OH) and cholesterol-5beta,6beta-epoxide (beta-epoxide) in U937 cells. To this end, we investigated the effects of these oxysterols on mitochondrial membrane potential, caspase-8 activity, the mitochondrial permeability transition pore and cytochrome c release. 7beta-OH-induced apoptosis was associated with a loss in mitochondrial membrane potential after 2 h, accompanied by cytochrome c release from the mitochondria into the cytosol after 16 h. Pre-treatment with a range of inhibitors of the mitochondrial permeability transition pore protected against 7beta-OH-induced cell death. In contrast, beta-epoxide induced a slight increase in caspase-8 activity but had no effect on mitochondrial membrane potential or cytochrome c release. The present results confirm that 7beta-OH-induced apoptosis occurs via the mitochondrial pathway and highlights differences in the apoptotic pathways induced by 7beta-OH and beta-epoxide in U937 cells.

Topics: Apoptosis; Caspase 8; Caspases; Cholesterol; Cytochromes c; Enzyme Activation; Humans; Hydroxycholesterols; Ion Channels; Membrane Potentials; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Monocytes; U937 Cells