Kinetic Evaluation of Cell Membrane Hydrolysis during Apoptosis by Human Isoforms of Secretory Phospholipase A2

Abstract

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.

Phospholipases A2 and neural membrane dynamics: implications for Alzheimer’s disease

“…Secretory PLA 2 is involved in neuroinflammation Secretory PLA 2 s comprises a superfamily of more than 10 different isoforms of low molecular mass (13-19 kDa) proteins that are widely present in mammalian cells, body fluids, and in secretagogues and venom (Murakami et al 2010). Many isoforms are associated with dynamic physiological and pathological processes including inflammation and apoptosis (Olson et al 2010). In the central nervous system, sPLA 2 -IIA has been shown to play a role in mediating several important physiological processes, including the regulation of neurotransmitter release, neuritogenesis, and neuronal apoptosis ).…”

Investigation into the Role of Phosphatidylserine in Modifying the Susceptibility of Human Lymphocytes to Secretory Phospholipase A2 using Cells Deficient in the Expression of Scramblase.

“…This sensitivity is important because it prevents damage to the membranes of normal healthy mammalian cells while allowing efficient catalysis of bacterial membranes and those of damaged or dying cells [2][3][4][5][6]. In particular, the membranes of several types of cultured leukocytes have been reported to convert from being resistant to becoming susceptible to the action of the enzyme during apoptosis and other forms of biochemical cell death [4,5,[7][8][9][10].…”

“…The nature of the biophysical changes in the cell membrane that trigger catalysis by sPLA 2 during cell death is not yet established. Nevertheless, important clues have been identified from biophysical studies of both artificial bilayers and living cells [6][7][8][10][11][12][13][14][15][16][17][18]. Two prominent candidates have emerged: 1) a reduction in the strength of interactions among adjacent phospholipids resulting in increased lipid mobility and greater interlipid spacing and 2) the presence of anionic lipids on the membrane surface [6,10,14,19].…”

“…Nevertheless, important clues have been identified from biophysical studies of both artificial bilayers and living cells [6][7][8][10][11][12][13][14][15][16][17][18]. Two prominent candidates have emerged: 1) a reduction in the strength of interactions among adjacent phospholipids resulting in increased lipid mobility and greater interlipid spacing and 2) the presence of anionic lipids on the membrane surface [6,10,14,19]. The hypothesis is that reduced interactions among neighboring phospholipids allow substrates to migrate into the active site of adsorbed sPLA 2 , while negative charge facilitates initial adsorption of the enzyme to the membrane surface [11,18,[20][21][22].…”

“…However, during apoptosis and other forms of cell death, PS is translocated to the outer leaflet [23][24][25]. Consequently, the exposed PS functions as a marker for processes that eliminate the dying cell such as phagocytosis [24,[26][27][28] and, perhaps, hydrolysis by sPLA 2 [10,18,19]. Arguments for the importance of lipid-neighbor interactions and of surface PS are accumulating, although the existing evidence from cells depends on temporal correlations rather than direct experimental manipulations [6][7][8]10,14].…”