Spontaneous Intermembrane Transfer of Various Cholesterol-Derived Hydroperoxide Species:  Kinetic Studies with Model Membranes and Cells

Vila, Andrew; Korytowski, Witold; Girotti, Albert W.

doi:10.1021/bi011408r

Cited by 43 publications

(74 citation statements)

References 50 publications

(131 reference statements)

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“…The PBS was Chelex-treated to remove redox metal ions; DFO, an iron chelator/redox inhibitor (43), was used for added protection against any possible CL oxidation or CLOOH turnover. A small amount of [ 14 C]Ch (2 mol %, ϳ0.1 Ci/ml) was periodically included in SUV preparations as an internal standard, its transfer properties being well established (29). Stock LUVs consisted of 1.0 mM POPC in PBS, 0.1 mM DFO (pH 7.4).…”

Section: Methodsmentioning

confidence: 99%

“…POPC/CL (8:2, mol/mol) donors were also used to compare the responses to CLOOH and parental CL. Complete reaction systems included recombinant nsLTP, which is known to facilitate intermembrane transfer of LOOHs as well as unoxidized lipids (29,30). For CLOOH-bearing SUV donors, the sensitivity of LUV acceptors to permeabilization by tBid in the presence of Ca 2ϩ was found to be strongly enhanced after an 18-h co-incubation with nsLTP (Fig.…”

Section: Comparative Effects Of CL and Clooh In Sensitizing Luvs To Pmentioning

confidence: 99%

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Permeabilization of the Mitochondrial Outer Membrane by Bax/Truncated Bid (tBid) Proteins as Sensitized by Cardiolipin Hydroperoxide Translocation

Korytowski

Basova

Piłat

et al. 2011

Journal of Biological Chemistry

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Cytochrome c (cyt c) release upon oxidation of cardiolipin (CL) in the mitochondrial inner membrane (IM) under oxidative stress occurs early in the intrinsic apoptotic pathway. We postulated that CL oxidation mobilizes not only cyt c but also CL itself in the form of hydroperoxide (CLOOH) species. Relatively hydrophilic CLOOHs could assist in apoptotic signaling by translocating to the outer membrane (OM), thus promoting recruitment of the pro-apoptotic proteins truncated Bid (tBid) and Bax for generation of cyt c-traversable pores. Initial testing of these possibilities showed that CLOOH-containing liposomes were permeabilized more readily by tBid plus Ca 2؉ than CL-containing counterparts. Moreover, CLOOH translocated more rapidly from IM-mimetic to OM-mimetic liposomes than CL and permitted more extensive OM permeabilization. We found that tBid bound more avidly to CLOOH-containing membranes than to CL counterparts, and binding increased with increasing CLOOH content. Permeabilization of CLOOH-containing liposomes in the presence of tBid could be triggered by monomeric Bax, consistent with tBid/Bax cooperation in pore formation. Using CL-null mitochondria from a yeast mutant, we found that tBid binding and cyt c release were dramatically enhanced by transfer acquisition of CLOOH. Additionally, we observed a pre-apoptotic IM-to-OM transfer of oxidized CL in cardiomyocytes treated with the Complex III blocker, antimycin A. These findings provide new mechanistic insights into the role of CL oxidation in the intrinsic pathway of oxidative apoptosis.Cytochrome c (cyt c) 2 dissociation from the mitochondrial inner membrane (IM), movement into the intermembrane space, and then release into cytosol is recognized as a key early event in the intrinsic (mitochondrion-initiated) pathway of oxidative stress-induced apoptosis (1-3). How this occurs is still not completely understood, but accumulating evidence suggests that oxidative modification of cardiolipin (CL), e.g. conversion to hydroperoxide species (CLOOHs), plays an important role (4 -7). CL (diphosphatidylglycerol) is located primarily in the mitochondrial IM of normal eukaryotic cells, where it interacts with and supports the functions of cyt c, Complex III (cytochromes b and c 1 ), and Complex IV (cytochrome c oxidase) (8). Unlike most other phospholipids in which only the sn-2 fatty acyl group is unsaturated, both sn-1 and sn-2 groups of natural CL are typically unsaturated. In mammalian heart, for example, tetra-linoleoyl CL composes 75-80% of overall CL molecular species (9). Consequently, CL oxidizability and -OOH content per average oxidized molecule are typically much higher than for more conventional phospholipids such as those in the phosphatidylcholine and phosphatidylethanolamine families. Model studies with bovine heart CL in thin film or liposomal form have shown that its normally strong interaction with some fraction of IM cyt c is progressively weakened with increasing -OOH content (4), suggesting possible involvement in pro-apoptotic cyt c releas...

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Section: Methodsmentioning

confidence: 99%

Section: Comparative Effects Of CL and Clooh In Sensitizing Luvs To Pmentioning

confidence: 99%

Permeabilization of the Mitochondrial Outer Membrane by Bax/Truncated Bid (tBid) Proteins as Sensitized by Cardiolipin Hydroperoxide Translocation

Korytowski

Basova

Piłat

et al. 2011

Journal of Biological Chemistry

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“…The transfer activities of recombinant SCP-2 and bSCP-2 were determined by measuring the kinetics of [ 14 C]cholesterol translocation from small unilamellar vesicles (50 nm SUVs) to red cell ghost membranes [14,27,28]. Departure from SUV donors is the rate limiting step in the transfer process [27].…”

Section: Transfer Assaymentioning

confidence: 99%

“…Departure from SUV donors is the rate limiting step in the transfer process [27]. Stock SUVs consisting of 1.0 mM egg PC, 0.8 mM [ 14 C]cholesterol (~1 μCi/ml), and 0.02 mM DCP in bulk suspension were prepared by lipid extrusion, as described [27,28]. Unsealed ghosts were prepared by hypotonic lysis of human erythrocytes [27].…”

Section: Transfer Assaymentioning

confidence: 99%

“…Assay mixtures (2.0 ml starting volume) typically contained 0.6 mM total ghost lipid, 0.06 mM total SUV lipid, and 5-10 μM SCP-2 or bSCP-2 in PBS/0.1 mM EDTA/0.1 mM DFO (pH 7.4). The chelators were included to minimize any metal ion-catalyzed lipid or protein oxidation [14,27,28]. At various times during transfer incubation at 37 °C, a 0.1 ml sample was removed, mixed with ice-cold PBS/EDTA/DFO, and centrifuged (16000 × g, 10 min), after which 0.1 ml of the SUV-containing supernatant was analyzed by scintillation counting.…”

Section: Transfer Assaymentioning

confidence: 99%

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Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

Kernstock

Girotti

2007

Analytical Biochemistry

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A new approach for analyzing lipid-lipid transfer protein interactions is described. The transfer protein is genetically engineered for expression with a C-terminal biotinylated peptide extension (AviTag ® ). This allows protein anchoring to a streptavidin-coated chip for surface plasmon resonance (SPR)-based assessment of lipid binding. Sterol carrier protein-2 (SCP-2), involved in the intracellular trafficking of cholesterol, fatty acids and other lipids, was selected as the prototype. Biotinylated SCP-2 (bSCP-2) was expressed in E. coli, purified to homogeneity by mutated streptavidin (SoftLink ® ) affinity chromatography, and confirmed by mass spectrometry to contain one biotin group at the expected position. Intermembrane [ 14 C]cholesterol transfer was strongly enhanced by bSCP-2, demonstrating that it was functional. Using bSCP-2 immobilized on a Biacore streptavidin chip, we determined on-and off-rate constants along with equilibrium dissociation constants for the following analytes: oleic acid, linoleic acid, cholesterol, and fluorophore (NBD)-derivatized cholesterol. The dissociation constant for NBD-cholesterol was similar to that determined by fluorescence titration for SCP-2 in solution, thus validating the SPR approach. This method can be readily adapted to other transfer proteins and has several advantages over existing techniques for measuring lipid binding, including (i) ability to study lipids in their natural states, i.e. without relatively large reporter groups; and (ii) ability to measure on-and off-rate constants as well as equilibrium constants.

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Generation and Reactivity of Lipid Hydroperoxides in Biological Systems

Girotti

Korytowski

2013

Patai's Chemistry of Functional Groups

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Numerous reviews have been published about the structural/functional damage caused by peroxidation of unsaturated lipids (phospholipids, glycolipids, cholesterol) in model membranes, cellular membranes, and lipoproteins. Less attention has been given to hydroperoxide species (LOOHs), which arise as intermediates of free radical‐mediated (chain) peroxidation or by direct ene‐addition of singlet molecular oxygen. This chapter focuses on nonenzymatic LOOH formation and on LOOH reactivity in biological systems, including cell membranes and lipoproteins. Specialized techniques for analyzing these processes are described. Two competing LOOH reaction pathways are discussed in detail: (i) iron‐catalyzed one‐electron reduction to oxyl radicals (LO • ), which expand chain peroxidation, and (ii) enzyme‐catalyzed two‐electron reduction to alcohols (LOH), which inhibits further chain propagation. In addition to these competing fates, LOOHs can undergo spontaneous or protein‐assisted translocation between membranes, lipoproteins, and membranes and lipoproteins, a more recently recognized phenomenon. Intracellular LOOH transfer could result in damage/toxicity enhancement or abatement, depending on prooxidant vs. antioxidant availability. We also discuss a novel, but still poorly understood response to LOOH trafficking, namely, activation of redox signaling pathways. We postulate that LOOHs, unlike the better known signaling molecule, H 2 O 2 , will be targeted to relatively hydrophobic “sensor” sites and that these sites are distinct from those targeted by H 2 O 2 . Thus, the authors' intent in preparing this chapter was to discuss not only the well‐established chemistry of LOOH generation and turnover, but also LOOH involvement in signal transduction, the importance of which in the field of redox signaling is only beginning to be appreciated.

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Spontaneous Intermembrane Transfer of Various Cholesterol-Derived Hydroperoxide Species: Kinetic Studies with Model Membranes and Cells

Cited by 43 publications

References 50 publications

Permeabilization of the Mitochondrial Outer Membrane by Bax/Truncated Bid (tBid) Proteins as Sensitized by Cardiolipin Hydroperoxide Translocation

Permeabilization of the Mitochondrial Outer Membrane by Bax/Truncated Bid (tBid) Proteins as Sensitized by Cardiolipin Hydroperoxide Translocation

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

Generation and Reactivity of Lipid Hydroperoxides in Biological Systems

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