Some isoforms of secretory phospholipase A 2 (sPLA 2 ) distinguish between healthy and damaged or apoptotic cells. This distinction reflects differences in membrane physical properties. Because various sPLA 2 isoforms respond differently to properties of artificial membranes such as surface charge, they should also behave differently as these properties evolve during a dynamic physiological process such as apoptosis. To test this idea, S49 lymphoma cell death was induced by glucocorticoid (6 -48 h) or calcium ionophore. Rates of membrane hydrolysis catalyzed by various concentrations of snake venom and human groups IIa, V, and X sPLA 2 were compared after each treatment condition. The data were analyzed using a model that evaluates the adsorption of enzyme to the membrane surface and subsequent binding of substrate to the active site. Results were compared temporally to changes in membrane biophysics and composition. Under control conditions, membrane hydrolysis was confined to the few unhealthy cells present in each sample. Increased hydrolysis during apoptosis and necrosis appeared to reflect substrate access to adsorbed enzyme for the snake venom and group X isoforms corresponding to weakened lipid-lipid interactions in the membrane. In contrast, apoptosis promoted initial adsorption of human groups V and IIa concurrent with phosphatidylserine exposure on the membrane surface. However, this observation was inadequate to explain the behavior of the groups V and IIa enzymes toward necrotic cells where hydrolysis was reduced or absent. Thus, a combination of changes in cell membrane properties during apoptosis and necrosis capacitates the cell for hydrolysis differently by each isoform.During programmed cell death, or apoptosis, a variety of changes occur in the plasma membrane of the cell. These include morphological alterations that emerge late in the process such as blebbing and increased permeability of the membrane. Earlier in the process, several more subtle membrane changes occur. The best studied is a loss of the normal asymmetrical transmembrane distribution of phospholipid species. Consequently, anionic lipids like phosphatidylserine, which are typically confined to the inner leaflet of the membrane, become exposed on the outer surface (1). In addition, studies with fluorescent membrane probes have revealed possible increases in fluidity and/or the spacing between lipid molecules that may precede or coincide with the loss of membrane asymmetry, depending on the cell type and mode of apoptosis (2-9). Recently, a latent increase in the order of membrane lipids has also been reported (9).A potential consequence of these events during apoptosis is enzymatic attack of the cell membrane by secretory phospholipase A 2 (sPLA 2 ).2 Ordinarily, healthy cells resist hydrolysis, but during apoptosis they become vulnerable to destruction by the enzyme (9 -11). Studies with snake venom phospholipase A 2 have identified possible ways by which this phenomenon relates to membrane physical properties (8,9,12). Prelimina...

Long chain acyl-CoA synthetases (ACSL) activate fatty acids for utilization by numerous metabolic pathways. Of the five mammalian ACSL isozymes known, ACSL5 is the only one located on mitochondria and thought to be involved in apoptosis. Fatty acids up-regulate ACSL5 and increase apoptosis susceptibility in human hepatocytes, thus, we hypothesize that ACLS5 is a promoting factor in hepatocellular lipoapoptosis. To investigate this mechanism, we have used immunochemical techniques and RNA interference as well as liquid chromatography, tandem mass spectrometry (LC-MS/MS). Fatty acid uptake led to up-regulation of ACSL5 expression and enzymatic activity in primary hepatocytes, HepG2 cells and steatotic liver. Over-expression of ACSL5 decreased HepG2 cell viability and increased susceptibility to TRAIL and TNFa, whereas knock down of ACSL5 reduced apoptosis susceptibility in fatty-acid treated HepG2 cells. Apoptosis sensitisation was accompanied by enhanced caspase-3/7 activity, but was not associated with up-regulation of DR4, DR5 or TNF-R1. By applying lipidomic techniques, we determined the effect of ACSL5 on the cellular amounts and subspecies of fatty acyl-CoAs as well as on sphingolipids, the downstream metabolites that are known to be important regulators of cell death and survival. High ACSL5 activity in HepG2 cells increased synthesis of long-chain acyl-CoAs by 50%, and enhanced ceramide and sphingomyelin levels by 2 to 3 fold. These results indicate that steatosis-induced upregulation of ACSL5 increased apoptosis susceptibility in human hepatocytes and that alterations in sphingolipid metabolism might contribute to ACSL5-mediated apoptotic effects. We propose that ACSL5 could play a role in promoting fatty acid-induced lipoapoptosis in hepatocytes as an important mechanism in fatty liver-related disorders.

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The proteins of the Bcl-2 family are key regulators of apoptosis, but their molecular mechanisms remains controversial. Two important aspects that center the debate involve the interaction network between the pro-and antiapoptotic family members and the role of their translocation to the mitochondrial outer membrane (MOM) during apoptosis. We have used FCCS to examine quantitatively the dynamic interactions of Bid and tBid with Bcl-xLDCt in solution and in lipid membranes. We found that only the active form tBid binds to Bcl-xLDCt and that the membrane strongly promotes binding between them. Importantly for drug design, a BH3 peptide from Bid disrupts the tBid/Bcl-xL complex in solution but not in lipid bilayers. Our findings convincingly suggest that the most relevant interaction between tBid and Bcl-xL happens in the membrane and reveal its significance as an additional regulatory stage for MOM permeabilization.