Karel W. A. Wirtz

Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress

Reactive oxygen species are required for cell proliferation but can also induce apoptosis. In proliferating cells this paradox is solved by the activation of protein kinase B (PKB; also known as c-Akt), which protects cells from apoptosis. By contrast, it is unknown how quiescent cells that lack PKB activity are protected against cell death induced by reactive oxygen species. Here we show that the PKB-regulated Forkhead transcription factor FOXO3a (also known as FKHR-L1) protects quiescent cells from oxidative stress by directly increasing their quantities of manganese superoxide dismutase (MnSOD) messenger RNA and protein. This increase in protection from reactive oxygen species antagonizes apoptosis caused by glucose deprivation. In quiescent cells that lack the protective mechanism of PKB-mediated signalling, an alternative mechanism is induced as a consequence of PKB inactivity. This mechanism entails the activation of Forkhead transcription factors, the transcriptional activation of MnSOD and the subsequent reduction of reactive oxygen species. Increased resistance to oxidative stress is associated with longevity. The model of Forkhead involvement in regulating longevity stems from genetic analysis in Caenorhabditis elegans, and we conclude that this model also extends to mammalian systems.

Transfer of phospholipids between membrane

A variety of techniques, both biochemical and physical, have shed light upon the structure of biological membranes. A consensus of opinion has developed by which the membrane is thought to consist of a phospholipid bilayer interspersed with proteins. Excellent reviews on this subject have recently been published. It is interesting to note that the current membrane model accentuates the fluidity of the membrane. Phospholipids undergo a rapid lateral diffusion within the two monolayers of the bilayer. Membrane proteins are also thought to diffuse freely in the lipid matrix. Which phospholipids and proteins in the membrane are in this state of motion, and how the movement of phospholipids and proteins is interrelated, are the subjects of intensive research.

The human MDR3 P-glycoprotein promotes translocation of phosphatidylcholine through the plasma membrane of fibroblasts from transgenic mice.

The mouse mdr2 P‐glycoprotein (P‐gp) and its human MDR3 homologue are present in high concentrations in the canalicular membrane of hepatocytes. Mice lacking this protein are unable to secrete phosphatidylcholine (PC) into bile, suggesting that this P‐gp is a PC translocator. We have tested this in fibroblasts from transgenic mice expressing the MDR3 gene under a vimentin promoter. Transgenic and control fibroblasts were incubated with [14C]choline to label PC. When the labeled cells were incubated with a PC transfer protein and acceptor liposomes, transfer of radioactive PC was enhanced in transgenic cells relative to the wild type controls. We conclude that the MDR3 P‐glycoprotein is able to promote the transfer of PC from the inner to the outer leaflet of the plasma membrane, supporting the idea that this protein functions as a PC flippase.

Some properties of phosphatidylcholine exchange protein purified from beef liver

A phospholipid exchange protein has been purified 2680-fold from beef liver. The assay of the exchange activity of the protein was based on the transfer of [14C]phosphatidylcholine from microsomes labeled with [14C]phosphatidylcholine to liposomes. The homogeneity of the protein has been established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoelectrophoresis and isoelectric focusing. The protein has a molecular weight of approximately 22000 and an isoelectric point of 5.8. The amino acid composition has been determined. The protein contains one disulfide bridge and has glutamic acid as the N-terminal amino acid. Phospholipid, tentatively identified as phosphatidylcholine, was found to be present in the protein preparation. The protein stimulated specifically the exchange of phosphatidylcholine between mitochondria and microsomes from rat liver.

FRET microscopy demonstrates molecular association of non‐specific lipid transfer protein (nsL‐TP) with fatty acid oxidation enzymes in peroxisomes

The fate of fluorescently labeled pre‐nsL‐TP (Cy3‐pre‐nsL‐TP) microinjected into BALB/c 3T3 fibroblasts was investigated by confocal laser scanning microscopy. The protein exhibited a distinct punctate fluorescence pattern and colocalized to a high degree with the immunofluorescence pattern for the peroxisomal enzyme acyl‐CoA oxidase. Proteolytic removal of the C‐terminal leucine of the putative peroxisomal targeting sequence (AKL) resulted in a diffuse cytosolic fluorescence. These results indicate that microinjected Cy3‐pre‐nsL‐TP is targeted to peroxisomes. The association of nsL‐TP with peroxisomal enzymes was investigated in cells by measuring fluorescence resonance energy transfer (FRET) between the microinjected Cy3‐pre‐nsL‐TP and Cy5‐labeled antibodies against the peroxisomal enzymes acyl‐CoA oxidase, 3‐ketoacyl‐CoA thiolase, bifunctional enzyme, PMP70 and catalase. The technique of photobleaching digital imaging microscopy (pbDIM), used to quantitate the FRET efficiency on a pixel‐by‐pixel basis, revealed a specific association of nsL‐TP with acyl‐CoA oxidase, 3‐ketoacyl‐CoA thiolase and bifunctional enzyme in the peroxisomes. These observations were corroborated by subjecting a peroxisomal matrix protein fraction to affinity chromatography on Sepharose‐immobilized pre‐nsL‐TP. Acyl‐CoA oxidase was retained. These studies provide strong evidence for a role of nsL‐TP in the regulation of peroxisomal fatty acid β‐oxidation, e.g. by facilitating the presentation of substrates and/or stabilization of the enzymes.

RATIO-FLUORESCENCE MICROSCOPY OF LIPID OXIDATION IN LIVING CELLS USING C11-BODIPY581/591

A ratio‐fluorescence assay was developed for on‐line localization and quantification of lipid oxidation in living cells. The assay explores the oxidative sensitivity of C11‐BODIPY581/591. Upon oxidation, the fluorescence of this fluorophore shifts from red to green. The probe incorporates readily into cellular membranes and is about twice as sensitive to oxidation as arachidonic acid. Using confocal microscopy, the cumene hydroperoxide‐induced oxidation of C11‐BODIPY581/591 was visualized at the sub‐cellular level in rat‐1 fibroblasts. Pre‐loading of the cells with tocopherol retarded this oxidation. The data demonstrate that C11‐BODIPY581/591 is a valuable tool to quantify lipid oxidation and anti‐oxidant efficacy in single cells.

Phosphatidylcholine exchange protein from beef liver.

The exchange of phosphatidyicholine was investigated between two separate monolayers and between monolayer and liposomes. The phosphatidylcholine exchange protein from beef liver acted in those systems as a carrier of phosphatidyicholine.

Peroxisomes in human fibroblasts have a basic pH.

eroxisomes are single-membrane-bound organelles found in nearly all eukaryotic cells. These organelles have a central function in lipid metabolism, including the beta-oxidation of verylong-chain and branched-chain fatty acids and the biosynthesis of ether phospholipids and cholesterol. Another characteristic of peroxisomes is their ability to degrade hydrogen peroxide by catalase. A deficiency in one or more peroxisomal enzymes has been linked to at least 20 (often lethal) disorders, showing the key role of this organelle in normal functioning of the human body. Peroxisomes are fragile structures that easily lose their integrity upon isolation. This poses a serious problem for studying these organelles in vitro and explains why our knowledge about the properties of the peroxisomal membrane, including the change in pH across it, is limited. In vivo, peroxisomes have been shown to be closed structures that are impermeable to NADH and NADPH, implying the existence of NADP redox shuttles. Here we study the pH in peroxisomes by targeting a pH-sensitive fluorescent reporter group to these organelles in living fibroblasts. We attained specific targeting by conjugating the fluorophore to a membrane-permeable peptide that contains a type-I peroxisome-targeting sequence (PTS1; amino-acid sequence AKL). Using this peptide probe, we establish that peroxisomes of human fibroblasts have a pH of 8.2 ± 0.3. Fibroblasts from RCDP (rhizomelic form of chondrodysplasia punctata) type 1 patients with severe mutations in the PEX7 protein, which result in an isolated defect in peroxisomal import of proteins with a PTS2 sequence, are still capable of importing the probe into peroxisomes, but have a pH of 6.5 ± 0.3. We covalently linked the pH-sensitive (5and 6-)carboxySNAFL-2 moiety to the PTS1-containing heptapeptide acetylCKGGAKL-COOH at the lysine near the amino terminus. This peptide probe (SNAFL-2–PTS1) was rapidly taken up into the cells and a punctate pattern of fluorescence was found, indicative of a peroxisomal localization. To confirm that these structures are indeed peroxisomes, we used a fixable analogue (BODIPY–PTS1) in co-localization studies with Cy5-labelled antibodies against different peroxisomal proteins (Fig. 1a–c). The probe was targeted only towards the peroxisomes (Fig. 1b). Further evidence that the probe was incorporated into peroxisomes came from studies of human fibroblasts with defects in peroxisomal import of PTS1bearing proteins. This probe was not incorporated into peroxiP

ISOLATION OF A PROTEIN FROM BEEF LIVER WHICH SPECIFICALLY STIMULATES THE EXCHANGE OF PHOSPHATIDYLCHOLINE

1. 1. The soluble protein fraction from beef liver contains a protein which stimulates phospholipid exchange. The activity of this protein has been assayed by determining the stimulation of phosphatidylcholine exchange either between 32P-labeled mitochondria and microsomes or between mitochondria labeled with [14C]phosphatidylcholine and microsomes. 2. 2. The phospholipid exchange protein from beef liver has been purified 310-fold with an overall recovery of activity of 15 %. (NH4)2SO4 precipitation, ion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Sephadex G-75 and Sephadex G-50 were used. 3. 3. The isolated phospholipid exchange protein stimulated specifically the exchange of phosphatidylcholine between mitochondria and microsomes. This specificity was also apparent in the phospholipid exchange between microsomes and liposomes consisting of egg yolk phosphatidylcholine and between mitochondria and liposomes consisting of egg yolk phosphatidylcholine (70 mole %) and rat liver phosphatidylethanolamine (30 mole %).

The protein-mediated net transfer of phosphatidylinositol in model systems

The phospholipid monolayer technique has been used to study the transfer activity of the phospholipid exchange protein from beef brain. In measuring the transfer between a monolayer consisting of equimolar amounts of phosphatidylcholine and phosphatidylinositol and liposomes consisting of 98 mol% phosphatidylcholine and 2 mol% phosphatidylinositol, the beef brain protein demonstrates an 8-fold higher transfer activity for phosphatidylinositol than for phosphatidylcholine. Under similar conditions the phosphatidylcholine exchange protein from beef liver showed a great preference for phosphatidylcholine. Phosphatidylcholine liposomes devoid of phosphatidylinositol still functioned as receptors of phosphatidylinositol when the beef brain exchange protein was present. This indicates that this protein can catalyse a net transfer of phosphatidylinopsitol. Binding of both phosphatidylinositol and phosphatidylcholine to the beef brain protein was shown.