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Esposti MDLipids, cardiolipin and apoptosis: a greasy licence to kill. Cell Death Differ. 9: 234-236
Abstract
Cell death and differentiation is a monthly research journal focused on the exciting field of programmed cell death and apoptosis. It provides a single accessible source of information for both scientists and clinicians, keeping them up-to-date with advances in the field. It encompasses programmed cell death, cell death induced by toxic agents, differentiation and the interrelation of these with cell proliferation.
... The activation of the apoptotic pathway leads to changes in the molar content of several mitochondrial lipids, including cardiolipin, that have been linked to apoptotic regulation [53,54,[58][59][60][61][62], summarized in Fig. S1. To gain insight into the regulatory role of cardiolipin we measured the protonation-dependent insertion of Bcl-xL ΔTM into membranes containing increasing concentrations of cardiolipin (Fig. 3a). ...
... Several studies have suggested that mitochondrial membrane lipids play a role in the action of the Bcl-2 proteins and therefore in the regulation of apoptosis [2,23,25,[58][59][60][61][62][74][75][76][77][78][79][80]. The most prominent example is that of cardiolipin, an anionic phospholipid specific to mitochondria [81,82], which is strongly linked to apoptotic regulation [58][59][60][61][62]. ...
... Several studies have suggested that mitochondrial membrane lipids play a role in the action of the Bcl-2 proteins and therefore in the regulation of apoptosis [2,23,25,[58][59][60][61][62][74][75][76][77][78][79][80]. The most prominent example is that of cardiolipin, an anionic phospholipid specific to mitochondria [81,82], which is strongly linked to apoptotic regulation [58][59][60][61][62]. The exact mechanism behind the role of lipids on the modulation of apoptosis as a whole, however, remains unclear. ...
Bcl-xL is a member of the Bcl-2 family of apoptotic regulators, responsible for inhibiting the permeabilization of the mitochondrial outer membrane, and a promising anti-cancer target. Bcl-xL exists in the following conformations, each believed to play a role in the inhibition of apoptosis: (a)a soluble folded conformation, (b)a membrane-anchored (by its C-terminal α8 helix)form, which retains the same fold as in solution and (c)refolded membrane-inserted conformations, for which no structural data are available. Previous studies established that in the cell Bcl-xL exists in a dynamic equilibrium between soluble and membranous states, however, no direct evidence exists in support of either anchored or inserted conformation of the membranous state in vivo. In this in vitro study, we employed a combination of fluorescence and EPR spectroscopy to characterize structural features of the bilayer-inserted conformation of Bcl-xL and the lipid modulation of its membrane insertion transition. Our results indicate that the core hydrophobic helix α6 inserts into the bilayer without adopting a transmembrane orientation. This insertion disrupts the packing of Bcl-xL and releases the regulatory N-terminal BH4 domain (α1)from the rest of the protein structure. Our data demonstrate that both insertion and refolding of Bcl-xL are modulated by lipid composition, which brings the apparent pK a of insertion to the threshold of physiological pH. We hypothesize that conformational rearrangements associated with the bilayer insertion of Bcl-xL result in its switching to a so-called non-canonical mode of apoptotic inhibition. Presented results suggest that the alteration in lipid composition before and during apoptosis can serve as an additional factor regulating the permeabilization of the mitochondrial outer membrane.
... At the level used (40 nM), tBid alone had no significant effect on CLOOH-enriched mitochondria (not shown), whereas Bax alone (100 nM) caused some net cyt c release but much less than when combined with tBid (Fig. 6). Western analyses also showed that YZD5 mitochondria that had been transfer-enriched in CLOOH retained much more tBid after washing than non-CLOOH controls (Fig. 6), in agreement with previous evidence that some form of CL is required for synergistic pro-apoptotic binding of tBid and Bax to the mitochondrial OM (15)(16)(17)(18)(19)(20). ...
... Pre-oligomerized Bax can permeabilize the OM in the absence of CL and tBid (57,58), but this appears to be physiologically irrelevant vis à vis apoptosis. Substantial evidence from various laboratories suggests that CL or some metabolite thereof is presented at the OM surface in early response to a stress signal (15)(16)(17)(18)(19)(20). This facilitates binding of cytosolic tBid, which serves as a nexus for Bax binding and oligomerization, giving transmembrane megapores through which cyt c, Smac/DIABLO, AIF, and other apoptogenic proteins can pass (18 -21). ...
... Some of this could be accounted for by formation of CLOOHs and other oxidation products. Various hydrolyzed species, including monolyso-CL and dilyso-CL have also been identified (20,28), consistent with phospholipids being more susceptible to phospholipase action after being oxidized (61). Another highly relevant property of CL is its tendency, like cyt c, to become mobilized under oxidative pressure and to translocate to other membrane sites. ...
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 Ca2+ 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.
... Whether or not caspases are engaged, dying cells generally show a proliferation of intracellular membranes, which implies major changes in the metabolism and traffic of intracellular membranes and their lipid constituents [3]. These changes impact on the mutual relationship between organelles, especially mitochondria, endoplasmic reticulum and the endolysosomal compartment, which are intimately connected by membrane contacts and lipid traffic. ...
... However, this simple concept needs rectification, since other players and reactions have now been shown to connect death receptors to mitochondria. For instance, full-length Bid also moves to mitochondria [3,5]. In addition, caspase-independent changes occur in the mitochondrial outer membranes that contribute to a 'priming' for the action of (t)Bid and other pro-apoptotic proteins [6,7]. ...
... A seminal paper [9] showed that tBid preferentially associates with liposomes containing CL levels comparable with those present in mitochondria. We then found that reversible binding to phospholipids and lysolipids enables Bid to transport CL metabolites between donor and acceptor membranes [3,5,7]. Interestingly, tBid is more effective than full-length Bid and displays an increased affinity towards MCL (monolysocardiolipin) [7]. ...
The role of mitochondria in cell death has been increasingly appreciated in the last few years and is now well established in a variety of cellular systems. At present we know that the involvement of mitochondria is regulated by proteins of the Bcl-2 (B-cell lymphocytic-leukaemia proto-oncogene 2) family, which biochemically act by altering the properties of mitochondrial membranes to facilitate the release of apoptogenic proteins like cytochrome c and Smac/Diablo that, once released into the cytosol, are crucial for activating the caspase cascade of cell degradation. The precise mechanism of the pro-apoptotic action is not fully understood yet, but could be clarified in the near future. Instrumental to this clarification is the emerging evidence that CL (cardiolipin), an unusual membrane lipid that is predominantly present in mitochondria, is required for the action of major pro-apoptotic proteins like Bid and Bax. New results obtained in myeloid cells further sustain this possibility and suggest that Bid may be involved in the metabolic cycle of CL re-modelling. In agreement with this postulate, preliminary results indicate a down-regulation of Bid in parallel to the genetic deficiency in CL re-modelling that is associated with a rare human disease, 'Barth Syndrome'. Intriguingly, this disease is characterized by neutropenia, suggesting a link between myeloid differentiation and cell death (and myeloid lymphoma pathogenesis too). I will project current results and trends towards future investigations on the involvement of CL and mitochondrial membranes in myeloid differentiation, cell death and disease.
... [1][2][3][4] In addition, apoptosis has been reported to induce changes in the remodelling of membrane lipids. [5][6][7][8] Physiologically, phosphatidylcholine (PC) and sphingomyelin (SM) are almost exclusively located in the outer leaflet of the plasma membrane, while phosphatidylserine (PS) and 70% of phosphatidylethanolamine (PE) are located in the inner leaflet of the plasma membrane. 9 Apoptosisinduced changes in the intracellular location of phospholipids are well documented for PS, which is translocated to the outer leaflet of the plasma membrane as a consequence of downregulation of the ATP-dependent aminophospholipid translocase and an activation of a nonspecific lipid scramblase. ...
... [5][6][7] The negatively charged lipid cardiolipin (CL), which is normally confined to the mitochondrial inner membrane, 10 has also been shown to undergo alterations in its traffic and remodelling during apoptosis. 8,11,12 In particular, it has been reported that CL moves to the outer leaflet of inner mitochondrial membrane in apoptotic cells 11 and, in another cell system, becomes exposed onto the cell surface. 12 Changes in CL distribution appeared to occur prior or concomitantly to membrane exposure of PS, but after the onset of an overproduction of reactive oxygen species (ROS). ...
... 11 The importance of lipid traffic in apoptosis is highlighted by the observation that Bid, a proapoptotic protein of the Bcl-2 family that is involved in death receptor-mediated death in many cell types, [13][14][15][16] displays lipid transfer activity between endoplasmic reticulum (ER) and mitochondria. 17 The preferential interaction with negatively charged phospholipids such as phosphatidylglycerol (PG) and its derivative CL 17,18 has suggested that Bid may be involved in the metabolic cycle of CL. 8 We have evaluated this possibility in light of our previous observation that CL becomes exposed on the surface of cells undergoing apoptosis, 12 which has been documented also using a-CL antibodies selected from patients with the antiphospholipid antibody syndrome (APS). 19 In particular, we studied how Bid, which is required for mediating mitochondrial membrane damage during Fas-induced apoptosis, 15,16 might be involved in the intracellular transport of CL (as well as its metabolites) and potentially account for its relocation onto the plasma membrane of Fas-stimulated cells. ...
We previously reported that during death receptor-mediated apoptosis, cardiolipin (CL) relocates to the cell surface, where it reacts with autoantibodies from antiphospholipid syndrome sera. Here, we analysed the intracellular distribution of CL and its metabolites during the early phase of cell death signalling triggered by Fas stimulation in U937 cells and mouse liver. We found a redistribution of mitochondrial CL to the cell surface by using confocal microscopy and flow cytometry. Mass spectrometry revealed that CL and its metabolites relocated from mitochondria to other intracellular organelles during apoptosis, with a conversion into non-mitochondrial lipids. Concomitantly, cytosolic Bid relocated to the light membranes comprised in fraction P100, including the plasma membrane and associated vesicular systems. A direct Bid-CL interaction was demonstrated by the observation that CL and monolysoCL coimmunoprecipitated with Bid especially after Fas stimulation, suggesting a dynamic interaction of the protein with CL and its metabolites.
... Cardiolipin or diphosphatidylglycerol, as it is traditionally called in microbiology literature, is a dimeric phospholipid that provided one of the first hallmarks for the bacterial origin of mitochondria (14,37). In eukaryotic cells, it is synthesized in mitochondria and remodeled with the participation of extramitochondrial enzymes but normally resides in the inner mitochondrial membrane (38,39). In prokaryotes, cardiolipin and its various derivatives are constituents of the cytoplasmic membrane, often fulfilling essential roles in viability (40). ...
... Cardiolipin or diphosphatidylglycerol is a typical membrane lipid of prokaryotes that is present almost exclusively in the inner membrane of mitochondria (14,(37)(38)(39)(40)(41)(42). In this work, we have tackled the complicated issue of the evolutionary pathways of cardiolipin biosynthesis and metabolism (14,37,40). ...
We breathe at the molecular level when mitochondria in our cells consume oxygen to extract energy from nutrients. Mitochondria are characteristic cellular organelles that derive from aerobic bacteria and carry out oxidative phosphorylation and other key metabolic pathways in eukaryotic cells. The precise bacterial origin of mitochondria and, consequently, the ancestry of the aerobic metabolism of our cells remain controversial despite the vast genomic information that is now available. Here, we use multiple approaches to define the most likely living relatives of the ancestral bacteria from which mitochondria originated. These bacteria live in marine environments and exhibit the highest frequency of aerobic traits and genes for the metabolism of fundamental lipids that are present in the membranes of eukaryotes, sphingolipids, and cardiolipin.
... CL is one of the most unsaturated lipids in the body, and the degree of unsaturation is maintained by means of constant remodeling process (44). Phospholipase A2 (Pla2) is a key enzyme catalyzes the hydrolysis of immature CL to lysoCL, which is involved in CL remodeling (25,26,28). ...
... Phospholipase A2 (Pla2) is a key enzyme catalyzes the hydrolysis of immature CL to lysoCL, which is involved in CL remodeling (25,26,28). In the remodeling process of CL, lysocardiolipin acyltransferase 1 (LCLAT1), also known as ALCAT1 or LYCAT, catalyzes the acylation of lysoCL to produce CL (27,28,44). In the present study, upregulation of Pla2 and reduction of LCLAT1 expression in the kidney of db/db mice may lead to the accumulation of lyso-CL in the db/db mice. ...
Exposure to chronic hyperglycemia due to diabetes mellitus can lead to the development and progression of diabetic kidney disease (DKD). We have recently reported that reduced superoxide production is associated with mitochondrial dysfunction in the kidneys of mouse models of type 1 DKD. We also demonstrated that humans with DKD have significantly reduced levels of mitochondrion-derived metabolites in their urine. Here, we examined renal superoxide production in a type 2 diabetes animal model, the db/db mouse, and the role of a mitochondrial-protectant, MTP-131 (also called elamipretide, SS-31, or Bendavia) in restoring renal superoxide production and ameliorating DKD. We found that18-week-old db/db mice have reduced renal and cardiac superoxide levels, as measured by dihydroethidium oxidation, and increased levels of albuminuria, mesangial matrix accumulation, and urinary hydrogen peroxide (H 2 O 2 ). Administration of MTP-131 significantly inhibited the increases in albuminuria, urinary H 2 O 2 , and mesangial matrix accumulation in the db/db mice, and fully preserved levels of renal superoxide production in these mice. MTP-131 also reduced total renal lysocardiolipin (lysoCL) and major lysoCL subspecies, and preserved lysocardiolipin acyltransferase 1 (LCLAT1) expression in the db/db mice. These results indicate that in type 2 diabetes, DKD is associated with reduced renal and cardiac superoxide levels and that MTP-131 protects against DKD and preserves physiological superoxide levels possibly by regulating cardiolipin remodeling.
... Recent studies showed that lipid second messengers play important roles in initiating apoptosis (36)(37)(38)(39)(40)(41)(42). To investigate if ApoL6 interacted with lipids/fatty acids during its induction of mitochondria-mediated apoptosis and if the BH3 domain in ApoL6 affected binding of lipid/fatty acids of ApoL6, we immunoprecipitated lipoprotein complex containing ApoL6.V5 using agarose-conjugated anti-V5 antibody from both induced and control DLD-1.ApoL6.V5 cells. ...
... It has been suggested, moreover, that apolipoproteins are also involved in the intracellular lipid transport for the supply of lipid components to the membranes of organelles, a critical step in the maintenance of membrane fluidity and integrity (30,31). Importantly, recent studies have shown that lipid messengers, such as ceramide, sphingolipids, ganglioside GD3, and oxidized cholesterol, play important roles in inducing matrix metalloproteinase and initiating apoptosis (36)(37)(38)(39)(40)(41)(42)(43). However, how those lipid messengers are transported to particular intracellular sites (e.g., mitochondria, endoplasmic reticulum, and lysosome) and what mechanisms they employ to induce apoptosis remain largely unknown. ...
... 8,13 This observation, despite contrasting results, 15 has increased current awareness of the role of cardiolipin in the mechanism of membrane permeabilisation by proapoptotic Bcl-2 proteins. 1,16,17 However, other evidence has shown that mitochondrial cardiolipin decreases during apoptosis induced by a variety of stimuli, [18][19][20] thus creating a paradox. If Bid and Bax, which are predominantly cytosolic proteins, require cardiolipin for their action on mitochondria, 1,13,17 how can they relocate to apoptotic mitochondria, which contain reduced levels of the same lipid? ...
... [18][19][20] Cardiolipin degradation with simultaneous MCL accumulation suggests that apoptosis signalling induces alteration in the remodelling cycle of mitochondrial membranes. 11,16 Of note, MCL resides in both membranes of mitochondria and is involved in remodelling nascent cardiolipin into its mature species. 21,22,30 Apoptosis mediated by death receptors has been shown to enhance the remodelling of cellular lipids, 35 in part due to early activation of phosphatidylcholine-specific phospholipase C (PC-PLC 36 ). ...
Recent evidence indicates that the mitochondrial lipid cardiolipin may be instrumental in the proapoptotic action of Bcl-2 family proteins on mitochondrial membranes, leading to the release of apoptogenic factors. However, contrasting evidence indicates that progressive loss of cardiolipin occurs during apoptosis. Here we show that Bid, a crucial proapoptotic protein that integrates the action of other Bcl-2 family members, exhibits discrete specificity for metabolites of cardiolipin, especially monolysocardiolipin (MCL). MCL, normally present in the remodelling of mitochondrial lipids, progressively increases in mitochondria during Fas-mediated apoptosis as a by-product of cardiolipin degradation, and also enhances Bid binding to membranes. MCL may thus play a crucial role in connecting lipid metabolism, relocation of Bid to mitochondria and integrated action of Bcl-2 proteins on mitochondrial membranes. We propose that Bid interaction with MCL 'primes' the mitochondrial outer membrane via segregation of lipid domains, facilitating membrane discontinuity and leakage of apoptogenic factors.
... CL is required for the reconstituted activity of a number of key mitochondrial enzymes involved in cellular energy metabolism and may be the 'glue' that holds the respiratory chain together [3,4]. Given the importance of loss of CL [5,6], and accumulation of its immediate metabolic product MLCL (monolysocardiolipin) [7], in mitochondria-mediated apoptosis, it is possible that rapid CL synthesis and/or its resynthesis from MLCL is required in response to proapoptotic stimuli-mediated CL degradation to restore cellular homoeostasis and prevent the entry of cells into apoptosis. Hence, increased CL biosynthesis and resynthesis may be a protective mechanism against apoptosis [8,9]. ...
... We show that CL de novo biosynthesis is increased in hepatocytes during liver regeneration and that an increase in CL resynthesis coincides with this increase in de novo CL biosynthesis. [1][2][3][4][5][6][7][8][9][10][11][12][13][14] . MLCL [a mixture of 1 (1-acyl-sn-glycerol-3-phosphoryl)-3 (1 ,2 -diacyl-sn-glycerol-3-phosphoryl)glycerol and 1 (1,2-diacyl-sn-glycerol-3-phosphoryl)-3 (1 -acyl-sn-glycerol-3phosphoryl)glycerol], produced by phospholipase A 2 hydrolysis of bovine heart CL, was obtained from Avanti Polar Lipids (Alabaster, AL, U.S.A.). ...
CL (cardiolipin) is a major mitochondrial membrane phospholipid important for the regulation of mitochondrial function. We examined CL de novo biosynthesis and its resynthesis in isolated rat liver hepatocytes prepared 48 h subsequent to two-thirds PHx (partial hepatectomy). The pool size of CL and its de novo biosynthesis from [1,3-(3)H]glycerol were increased 3.3-fold (P<0.05) and 3.1-fold (P<0.05) respectively in hepatocytes prepared from PHx rats compared with sham-operated controls. The reason for the increased CL biosynthesis was a 65% increase (P<0.05) in enzymic activity in PGP-S (phosphatidylglycerolphosphate synthase), a key enzyme in de novo CL biosynthesis. The increase in PGP-S activity was due to a 3-fold increase (P<0.05) of hepatic PGP-S mRNA expression. The increase in de novo CL biosynthesis and pool size corresponded to a 2.3-fold increase (P<0.05) in the amount of [1-14C]linoleic acid incorporated into CL of hepatocytes prepared from PHx rats compared with sham-operated controls, indicating an increase in CL resynthesis. The activity of MLCL-AT (monolysocardiolipin acyltransferase), a rate-limiting enzyme of CL resynthesis, was increased by 43% (P<0.05) in hepatocytes prepared from PHx rats compared with sham-operated controls; this result would explain the increase in [1-14C]linoleic acid incorporation into CL. The increase in MLCL-AT activity was due to an increase in hepatic MLCL-AT protein expression. The results show that CL de novo biosynthesis and its resynthesis are increased during liver regeneration.
... Recent studies showed that lipid second messengers play important roles in initiating apoptosis (36)(37)(38)(39)(40)(41)(42). To investigate if ApoL6 interacted with lipids/fatty acids during its induction of mitochondria-mediated apoptosis and if the BH3 domain in ApoL6 affected binding of lipid/fatty acids of ApoL6, we immunoprecipitated lipoprotein complex containing ApoL6.V5 using agarose-conjugated anti-V5 antibody from both induced and control DLD-1.ApoL6.V5 cells. ...
... It has been suggested, moreover, that apolipoproteins are also involved in the intracellular lipid transport for the supply of lipid components to the membranes of organelles, a critical step in the maintenance of membrane fluidity and integrity (30,31). Importantly, recent studies have shown that lipid messengers, such as ceramide, sphingolipids, ganglioside GD3, and oxidized cholesterol, play important roles in inducing matrix metalloproteinase and initiating apoptosis (36)(37)(38)(39)(40)(41)(42)(43). However, how those lipid messengers are transported to particular intracellular sites (e.g., mitochondria, endoplasmic reticulum, and lysosome) and what mechanisms they employ to induce apoptosis remain largely unknown. ...
Cancer cells frequently possess defects in the genetic and biochemical pathways of apoptosis. Members of the Bcl-2 family play pivotal roles in regulating apoptosis and possess at least one of four Bcl-2 homology (BH) domains, designated BH1 to BH4. The BH3 domain is the only one conserved in proapoptotic BH3-only proteins and plays an important role in protein-protein interactions in apoptosis by regulating homodimerization and heterodimerization of the Bcl-2 family members. To date, 10 BH3-only proapoptotic proteins have been identified and characterized in the human genome. The completion of the Human Genome Project and the availability of various public databases and sequence analysis algorithms allowed us to use the bioinformatic database-mining approach to identify one novel BH3-only protein, apolipoprotein L6 (ApoL6). The full-length cDNA of ApoL6 was identified, cloned, and functionally expressed in p53-null colorectal cancer cells (DLD-1). We found that overexpression of wild-type ApoL6 induced mitochondria-mediated apoptosis in DLD-1 cells characterized by release of cytochrome c and Smac/DIABLO from mitochondria and activation of caspase-9, whereas ApoL6 BH3 domain deletion allele did not. In addition, overexpression of ApoL6 also induced activation of caspase-8. Furthermore, we showed that adenovirus harboring the full-length cDNA of ApoL6 induced marked apoptosis in a variety of cancer cell types, and ApoL6 recruited and interacted with lipid/fatty acid components during the induction of apoptosis. To our knowledge, this is the first example that intracellular overproduction of an apolipoprotein induces marked apoptosis.
... β2GPI was further suggested to interact with the mitochondrial phospholipid, cardiolipin (CL). CL externalization during apoptosis may lead to the expose of CL to β2GPI 39,40 . The Cys281-Cys288 region of β2GPI was identified as the interaction site 41 . ...
Beta‐2‐glycoprotein I (β2GPI) is the major antigen for the antiphospholipid antibodies in the antiphospholipid syndrome. The exposed epitope in domain I of β2GPI can be recognized by the anti‐β2GPI antibody. Here, we prepared the anionic di‐oleoyl‐phosphatidylserine (DOPS) and cardiolipin (CL) liposomes to interact with the β2GPI. The conformational changes of β2GPI upon binding with the liposomes were analyzed using hydrogen/deuterium exchange mass spectrometry (HDXMS). The exchange level of sequence 21‐27 significantly increased after β2GPI had interacted with DOPS. This change indicated a reduced interaction between domain I and domain V, inferring to a protrusion of the sequence 21‐27 from the ring conformation. After β2GPI had interacted with CL for 30 min, the exchange levels in 4 of the 5 domains increased significantly. The deuteration levels of sequence 1‐20, 21‐27, 196‐205, 273‐279 and 297‐306 increased, suggesting that these regions had become more exposed, and the domain I was no longer in contact with domain V. The increasing deuteration levels in sequence 70‐86, 153‐162, 191‐198, 196‐205 and 273‐279 indicated β2GPI undergoing conformational changes to expose these inner regions, suggesting a structural transition. Overall, DOPS and CL induced minor conformational changes of β2GPI at sequence 21‐27 and forms an intermediate conformation after 10‐min of interaction. After a complete protein‐lipid interaction, high negatively charged CL membrane induced a major conformation transition of β2GPI.
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... Interestingly, unsaturated fatty acid-containing PC lipids were up-regulated in the hippocampus of female rats following postnatal DEHP treatment. The carbon-carbon double bonds in unsaturated PC lipids amplify the oxidization potential thereby increasing the vulnerability to apoptotic cell death [57,58]. The overexpression of anti-apoptotic protein bcl-2 in HL-60 cells increased saturated and decreased unsaturated fatty acids in phospholipids (which include PCs) providing protection against oxidation and promoting cell survival [58]. ...
Slight changes in the abundance of certain lipid species in the brain may drastically alter normal neurodevelopment via membrane stability, cell signalling, and cell survival. Previous findings have demonstrated that postnatal exposure to di (2-ethylhexyl) phthalate (DEHP) disrupts normal axonal and neural development in the hippocampus. The goal of the current study was to determine whether postnatal exposure to DEHP alters the lipid profile in the hippocampus during postnatal development. Systemic treatment with 10 mg/kg DEHP during postnatal development led to elevated levels of phosphatidylcholine and sphingomyelin in the hippocampus of female rats. There was no effect of DEHP exposure on the overall abundance of phosphatidylcholine or sphingomyelin in male rats or of lysophosphatidylcholine in male or female rats. Individual analyses of each identified lipid species revealed 10 phosphatidylcholine and six sphingomyelin lipids in DEHP-treated females and a single lysophosphatidylcholine in DEHP-treated males with a two-fold or higher increase in relative abundance. Our results are congruent with previous work that found that postnatal exposure to DEHP had a near-selective detrimental effect on hippocampal development in males but not females. Together, results suggest a neuroprotective effect of these elevated lipid species in females.
... Several evidences showed that CL becomes exposed on the plasma membrane of cells undergoing apoptosis induced by death receptors, like CD95/Fas and tumor necrosis factoralpha (TNF-) [14,64,87]. Translocation onto the cell surface implies a leakage of CL (and/or of its metabolites) from the normal remodelling cycle [88], probably as a consequence of an apoptosis-mediated increase of ER and secretory membranes. Interestingly, mass spectroscopy analysis has demonstrated an early degradation of mitochondrial CL into its immediate metabolite, monolysocardiolipin, during CD95/Fas-induced apoptosis [43]. ...
Apoptosis has been reported to induce changes in the remodelling of membrane lipids; after death receptor engagement, specific changes of lipid composition occur not only at the plasma membrane, but also in intracellular membranes. This paper focuses on one important aspect of apoptotic changes in cellular lipids, namely, the redistribution of the mitochondria-specific phospholipid, cardiolipin (CL). CL predominantly resides in the inner mitochondrial membrane, even if the rapid remodelling of its acyl chains and the subsequent degradation occur in other membrane organelles. After death receptor stimulation, CL appears to concentrate into mitochondrial “raft-like” microdomains at contact sites between inner and outer mitochondrial membranes, leading to local oligomerization of proapoptotic proteins, including Bid. Clustering of Bid in CL-enriched contacts sites is interconnected with pathways of CL remodelling that intersect membrane traffic routes dependent upon actin. In addition, CL association with cytoskeleton protein vimentin was observed. Such novel association also indicated that CL molecules may be expressed at the cell surface following apoptotic stimuli. This observation adds a novel implication of biomedical relevance. The association of CL with vimentin at the cell surface may represent a “new” target antigen in the context of the apoptotic origin of anti-vimentin/CL autoantibodies in Antiphospholipid Syndrome.
... VDAC controls the entry of calcium into mitochondria by IP3R-mediated calcium signals [67]. The release of a large amount of calcium through the IP3R and RyR can lead to: (1) an excessive entry of calcium into the matrix of mitochondria and induction of the mitochondrial permeability transition pore (MPTP) opening [68]; (2) activation of calpains, a family of calcium-dependent cysteine proteases, whose substrates are the pro-apoptotic proteins Bax and Bid [6,[69][70][71]; (3) alterations of calcium-dependent phospholipids scramblers, which alter membrane biology to promote apoptosis or necrosis [6,72,73]; (4) the activation of nitric oxide synthase (NOS), which exacerbates oxidative stress [6,74]; (5) activation of calcium-sensitive mitochondrial fission protein DRP1 implicated in Bax-induced release of cytochrome c from mitochondria. As referred before, it is believed that impairment of ER-mitochondria calcium coupling is tightly modulated by members of the Bcl2 family [6,58,[75][76][77][78]. ...
Neurodegenerative disorders such as Huntington's disease, amyotrophic lateral sclerosis and Parkinson's disease have in common the presence of protein aggregates in specific brain areas where significant neuronal loss is detected. In these pathologies, several evidences support a close correlation between neurodegeneration and endoplasmic reticulum (ER) stress, a condition that arises from ER lumen overload with misfolded proteins. Under these conditions, ER stress sensors initiate the unfolded protein response to restore normal ER function. If stress is too prolonged, or adaptive responses fail, apoptotic cell death ensues. Therefore, it was recently suggested that the manipulation of the ER unfolded protein response could be an effective strategy to avoid neuronal loss in neurodegenerative disorders. We will review the mechanisms underlying ER stress-associated neurodegeneration and discuss the possibility of ER as a therapeutic target.
... Mitochondrial membrane lipids influence a great deal of physiologic processes that grasp a wide range of relevant pathways. Mitochondrial membrane biogenesis [10,11], mitochondrial fusion and fission [12,13], apoptosis [14,15] and mitochondrial oxidative phosphorylation [16] are among the processes known to involve mitochondrial membrane lipids. Other than just the presence of specific lipids, membrane lipid dynamics and distribution were proposed to mediate mitochondrial membrane-protein interactions [17]. ...
The traditional view of mitochondria as cell powerhouses is a matter of common knowledge, but the overall view of these extraordinary organelles has been revolutionized in the last years. In fact, a large number of important and diverse processes take place at the mitochondrial level, which clearly surpass the energy production scope, intruding the critical fragile balance between cell life and death. The entangled biochemistry of mitochondrial membranes has been found to be dependent on specific lipid requirements, with cardiolipin holding a great part of the raised functional interest. Mitochondria contain a complex membrane system, based on a variety of lipids and exquisite asymmetries. Mitochondria lipid membrane composition depends on a tight interplay with the endoplasmic reticulum, from which some of the lipids present in the mitochondrial membranes have to be imported, at least in the form of precursors. Here, we review some external interventions resulting in alterations of mitochondrial lipid content, namely dietary interventions and genetic manipulation. Such manipulations of mitochondrial membrane lipid composition should result in physiological impact, given the importance of lipid-protein interactions within the mitochondrial membrane boundaries. We provide arguments for future experiments using the most modern chemical and biophysical approaches as well as computer simulation studies applied to appropriate biological membrane model systems, in order to identify the effects exerted by diet-induced lipid changes on membrane physical properties.
... These protein modifications can result in detrimental consequences, such as alteration of protein function, activation of immune and inflammatory responses, and activation of cell death pathways. 20,77 Oxidatively modified proteins and lipoproteins, including oxidized LDL, lipid peroxidation products, and nitrated tyrosines, have been identified in patients with coronary artery disease and in atherosclerotic animal models. 19,78,79 Mitochondrial antioxidant enzymes and ETC complexes could be potential targets of such abnormal protein modifications. ...
Mitochondrial-derived reactive oxygen species (mtROS) is one of the major sources of cellular ROS, and excessive mtROS is associated with atherosclerosis progression in both human and mouse models. This review aims to summarize the most recent studies showing the existence, the causes and pathological consequences of excessive mtROS in atherosclerosis. Despite numerous association and causation studies demonstrating the importance of mtROS in atherosclerosis progression, the failure of antioxidant therapy in human randomized clinical trials demands more definitive, cell-type specific investigations. Better mechanistic understanding of mtROS in atherosclerosis may lead to more effective therapeutic strategies.
... In addition to respiratory disfunction, S. cerevisae coq mutants present hypersensitivity to oxidized polyunsaturated fatty acids and elevated formation of lipid hydroperoxides [50]. Enhanced lipid peroxidation promoted by exacerbated ROS production also changes mitochondrial lipid composition, affecting mtDNA maintenance [3,7] and promoting apoptosis in human cells [51]. ROS overproduction was also detected in CoQ yeast mutants [31,39,47], and in cell lines obtained from patients with primary CoQ deficiencies [13]. ...
S. cerevisiae has three distinct inner mitochondrial membrane NADH dehydrogenases mediating the transfer of electrons from NADH to Coenzyme Q (CoQ): Nde1p, Nde2p and Ndi1p. The active site of Ndi1p faces the matrix side, while the enzymatic activities of Nde1p and Nde2p are restricted to the intermembrane space side, where they are responsible for cytosolic NADH oxidation. Here, we genetically manipulate yeast strains in order to alter the redox state of CoQ and NADH dehydrogenases to evaluate the consequences on mtDNA maintenance. Interestingly, nde1 deletion was protective for mtDNA in strains defective in CoQ function. Additionally, the absence of functional Nde1p promoted a decrement in the rate of hydrogen peroxide (H2O2) release in isolated mitochondria from different yeast strains. On the other hand, over-expression of the predominant NADH-dehydrogenase NDE1 elevated the rate of mtDNA loss and was toxic to coq10 and coq4 mutants. Increased CoQ synthesis through COQ8 over-expression also demonstrated that there is a correlation between CoQ respiratory function and mtDNA loss: supra-physiological CoQ levels were protective against mtDNA loss in the presence of oxidative imbalance generated by Nde1p excess or exogenous H2O2. Altogether, our results indicate that impairment in the oxidation of cytosolic NADH by Nde1p is deleterious toward mitochondrial biogenesis due to an increment in reactive oxygen species release.
... Unexpectedly the lipid-binding assay provides evidence for an interaction of P2X7R and P2X4R fusion proteins with cardiolipin. This specialized lipid, which is involved in apoptotic processes and discussed as a surface marker of apoptotic cells [55][56][57], is mainly found in mitochondria [58], but also detected in lysosomes and the endoplasmatic reticulum. A localization of P2X4R predominantly within intracellular compartments, especially in lysosomes, was demonstrated for cultured rat microglia, vascular endothelial cells and freshly isolated peritoneal macrophages [59]. ...
P2X4 and P2X7 receptors are ATP-gated ion channels that are co-expressed in alveolar epithelial type I cells. Both receptors are localized to the plasma membrane and partly associated with lipid rafts. Here we report on our study in an alveolar epithelial cell line of the molecular organization of P2X7R and P2X4R receptors and the effect of their knockdown. Native gel electrophoresis reveals three P2X7R complexes of approximately 430, approximately 580 and approximately 760 kDa. The latter two correspond exactly in size to signals of Cav-1, the structural protein of caveolae. Interestingly knockdown of P2rx7 affects protein levels, the intracellular distribution and the supramolecular organization of Cav-1 as well as of P2X4R, which is mainly detected in a complex of approximately 430 kDa. Our data suggest upregulation of P2X4R as a compensatory mechanism of P2X7R depletion.
... [19], providing specificity for targeting of tBid to mitochondria and regulating the oligomerization of Bax [20]. In particular, a preferential interaction of tBid with MCL has been observed [21], suggesting that this protein of the bcl family has lipid transfer activity between mitochondria and endoplasmic reticulum [22]. It has been proposed that Bid interaction with MCL ''primes" the mitochondrial outer membrane via segregation of lipid domains, facilitating membrane discontinuity and leakage of apoptogenic factors. ...
Cardiolipin (CL) has recently been shown to provide an anchor and an essential activating platform for caspase-8 on mitochondria. We hypothesize that these platforms may correspond to "raft-like" microdomains, which have demonstrated to be detectable on mitochondrial membrane of cells undergoing apoptosis. The role for CL in "raft-like" microdomains could be to anchor caspase-8 at contact sites between inner and outer membranes, facilitating its self-activation, Bid cleavage and apoptosis execution. The role played by "raft-like" microdomains in the apoptotic program could introduce a new task in the pathogenetic studies on human diseases associated with cardiolipin dismetabolism.
... It is also particularly rich in unsaturated fatty acids and, as a consequence, is highly susceptible to oxidative attack. The high degree of CL unsaturation is maintained by a constant remodeling process integrated between the mitochondria and the endoplasmic reticulum (Esposti, 2002;van der Berg et al., 1993;Hatch et al., 1993;Schlame and Rü stow, 1990). Changes in CL content have also been reported to trigger the release of Cyt c from mitochondria during the initiation of the apoptotic process, due to oxidative damage or to the alteration of its biosynthetic pathway (Petrosillo et al., 2001;Shidoji et al., 1999). ...
... Mitochondrial phospholipids compromise a significant proportion of the entire phospholipid content of most eukaryotic cells and possess diverse roles in the regulation of varied mitochondrial processes. CL (cardiolipin) or bis-(1,2-diacyl-sn-glycero-3-phospho)-1 ,3 -sn-glycerol is a key mitochondrial membrane phospholipid involved in ATP generation, mitochondrial-mediated apoptosis, diabetes and BTHS (Barth Syndrome), a rare and often fatal X-linked genetic disorder in young boys associated with cardiomyopathy [2][3][4][5][6][7][8][9][10][11]. CL modulates the activity of a number of key mitochondrial membrane enzymes involved in the electron transport chain including cytochrome c oxidase, carnitine palmitoyltransferase, creatine phosphokinase, pyruvate translocator, mono-, di-and tri-carboxylate carriers, glycerol-3-phosphate dehydrogenase, phosphate transporter, ATP/ADP translocase and ATP synthase (reviewed in [4,12]). ...
CL (cardiolipin) is a key phospholipid involved in ATP generation. Since progression through the cell cycle requires ATP we examined regulation of CL synthesis during S-phase in human cells and investigated whether CL or CL synthesis was required to support nucleotide synthesis in S-phase. HeLa cells were made quiescent by serum depletion for 24 h. Serum addition resulted in substantial stimulation of [methyl-(3)H]thymidine incorporation into cells compared with serum-starved cells by 8 h, confirming entry into the S-phase. CL mass was unaltered at 8 h, but increased 2-fold by 16 h post-serum addition compared with serum-starved cells. The reason for the increase in CL mass upon entry into S-phase was an increase in activity and expression of CL de novo biosynthetic and remodelling enzymes and this paralleled the increase in mitochondrial mass. CL de novo biosynthesis from D-[U-(14)C]glucose was elevated, and from [1,3-(3)H]glycerol reduced, upon serum addition to quiescent cells compared with controls and this was a result of differences in the selection of precursor pools at the level of uptake. Triascin C treatment inhibited CL synthesis from [1-(14)C]oleate but did not affect [methyl-(3)H]thymidine incorporation into HeLa cells upon serum addition to serum-starved cells. Barth Syndrome lymphoblasts, which exhibit reduced CL, showed similar [methyl-(3)H]thymidine incorporation into cells upon serum addition to serum-starved cells compared with cells from normal aged-matched controls. The results indicate that CL de novo biosynthesis is up-regulated via elevated activity and expression of CL biosynthetic genes and this accounted for the doubling of CL seen during S-phase; however, normal de novo CL biosynthesis or CL itself is not essential to support nucleotide synthesis during entry into S-phase of the human cell cycle.
... Although cytochrome C is perhaps the best characterised factor to be released from the mitochondria during apoptotis, several other released proteins have also been identified, including Smac/DIABLO, HtrA2, Endo G and AIF (apoptosis-inducing factor) (6). Furthermore, the mitochondrialspecific lipid, cardiolipin (26) which under normal situations couples to cytochrome C, is also released from the mitochondria during apoptosis and is another of the acidic phospholipids reported to modulate the PMCA activity (45). Binding and/or regulation of Ca 2+ transport proteins by mitochondrially released proteins is not an unprecedented phenomenon. ...
Impairment of the normal spatiotemporal pattern of intracellular Ca(2+) ([Ca(2+)](i)) signaling, and in particular, the transition to an irreversible "Ca(2+) overload" response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this Ca(2+) overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide (H(2)O(2)) evokes a Ca(2+) overload response and inhibition of plasma membrane Ca(2+)-ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938-C950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focusing on the role of the mitochondria. Using a [Ca(2+)](i) clearance assay in which mitochondrial Ca(2+) uptake was blocked with Ru-360, H(2)O(2) (50 microM-1 mM) markedly inhibited the PMCA activity. This H(2)O(2)-induced inhibition of the PMCA correlated with mitochondrial depolarization (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using Magnesium Green fluorescence). The H(2)O(2)-induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid. These data suggest that oxidant-induced opening of the mPTP and mitochondrial depolarization may lead to an inhibition of the PMCA that is independent of mitochondrial Ca(2+) handling and ATP depletion, and we speculate that this may involve the release of a mitochondrial factor. Such a phenomenon may be responsible for the Ca(2+) overload response, and for the transition between apoptotic and necrotic cell death thought to be important in many disease states.
... These are preferentially transferred by tBid to the mitochondria and may contribute to the loss of membrane integrity. 57,59 Mitochondrial proteins released during SR>apoptotic cell death Cytochrome c Cytochrome c was the first apoptogenic intermembrane protein identified as being released from mitochondria during apoptosis. Using a cell free system, Xiaodong Wang and colleagues showed that cytochrome c is required for the proteolytic activation of procaspase-3. ...
Mitochondria are 'life-essential' organelles for the production of metabolic energy in the form of ATP. Paradoxically mitochondria also play a key role in controlling the pathways that lead to cell death. This latter role of mitochondria is more than just a 'loss of function' resulting in an energy deficit but is an active process involving different mitochondrial proteins. Cytochrome c was the first characterised mitochondrial factor shown to be released from the mitochondrial intermembrane space and to be actively implicated in apoptotic cell death. Since then, other mitochondrial proteins, such as AIF, Smac/DIABLO, endonuclease G and Omi/HtrA2, were found to undergo release during apoptosis and have been implicated in various aspects of the cell death process. Members of the Bcl-2 protein family control the integrity and response of mitochondria to apoptotic signals. The molecular mechanism by which mitochondrial intermembrane space proteins are released and the regulation of mitochondrial homeostasis by Bcl-2 proteins is still elusive. This review summarises and evaluates the current knowledge concerning the complex role of released mitochondrial proteins in the apoptotic process.
... Finally, PG condenses with another CDP-DG molecule to form CL. CL is localized to mitochondria and is required for the reconstituted activity of a number of key mammalian mitochondrial enzymes involved in cellular energy metabolism, including cytochrome c oxidase (8), carnitine palmitoyltransferase (9), creatine phosphokinase (10), pyruvate translocator (11), tricarboxylate carrier (12), mitochondrial glycerol-3-phosphate dehydrogenase (13), phosphate transporter (14), ADP/ATP carrier (15), and the ATP synthase (16). In addition, several recent studies have implicated CL loss in the regulation of mitochondrial-mediated apoptosis (reviewed in Ref. 17). Thus, the appropriate content of CL is an important requirement for activation of enzymes involved in mitochondrial respiration and in the control of programmed cell death. ...
In mammalian tissues cardiolipin is rapidly remodeled by monolysocardiolipin acyltransferase subsequent to its de novo biosynthesis (Ma, B. J., Taylor, W. A, Dolinsky, V. W., and Hatch, G. M. (1999) J. Lipid Res. 40, 1837-1845). We report here the purification and characterization of a monolysocardiolipin acyltransferase activity from pig liver mitochondria. Monolysocardiolipin acyltransferase activity was purified over 1000-fold by butanol extraction, hydroxyapatite chromatography, and preparative SDS-PAGE. The purified 74-kDa protein catalyzed acylation of monolysocardiolipin to cardiolipin with [(14)C]linoleoyl coenzyme A. Photoaffinity labeling of the protein with 12-[(4-[(125)I]azidosalicyl)amino]dodecanoyl coenzyme A indicated coenzyme A was bound at its active site and photoaffinity cross-linking of 12-[(4-azidosalicyl)amino]dodecanoyl coenzyme A to the enzyme inhibited enzyme activity. Enzyme activity was optimum at pH 7.0, and the enzyme did not utilize other lysophospholipids as substrate. The purified enzyme was heat-labile and exhibited an isoelectric point of pH 5.4. To determine the enzymes kinetic mechanism the effect of varying concentrations of linoleoyl coenzyme A and monolysocardiolipin on initial velocity were determined. Double-reciprocal plots revealed parallel lines consistent with a ping pong kinetic mechanism. When the enzyme was incubated in the absence of monolysocardiolipin, coenzyme A was produced from linoleoyl coenzyme A at a rate consistent with the formation of an enzyme-linoleate intermediate. The true K(m) value for linoleoyl coenzyme A and true K(m) value for monolysocardiolipin were 100 and 44 microM, respectively. The calculated V(max) was 6802 pmol/min per mg of protein. A polyclonal antibody, raised in rabbits to the purified protein, cross-reacted with the protein in crude pig liver mitochondrial fractions. In liver mitochondria prepared from thyroxine-treated rats, the level of the protein was elevated compared with euthyroid controls indicating that expression of monolysocardiolipin acyltransferase is regulated by thyroid hormone. The study represents the first purification and characterization of a monolysocardiolipin acyltransferase activity from any organism.
... The specificity of the fatty acid pattern of CL can be the result of an active deacylation-reacylation cycle between CL and monolysoCL in rat liver mitochondria [46]. Interestingly, Bid has been shown to display lipid transfer activity [47], by interacting with CL and lysoCL [48]. It is noteworthy that CL has been observed to decrease in ischemia, in ageing and in apoptosis [35]. ...
Mitochondria are key players in fundamental processes such as energy production and adaptive responses to cellular stress, including apoptosis. Mitochondrial membranes may undergo permeabilization when perturbed by a number of intracellular stress mediators, and consequently may allow the release of intramitochondrial proteins. This event triggers and amplifies the cellular apoptotic program, provided sufficient energy is available. Mitochondrial membranes are therefore both targets and regulators of intracellular pathways controlling cell fate. Evidence is emerging that the integration and biological outcome of these pathways might be critically dependent on the unique lipid composition of mitochondrial membranes.
... An increased ability to remove damaged lipids could help to explain these data. There is some recent evidence that Bcl-2 family members affect membrane lipid metabolism [77]. ...
Dexamethasone-treated WEHI7.2 mouse thymoma cells readily undergo apoptosis. WEHI7.2 variants that overexpress catalase (CAT38) or Bcl-2 (Hb12) show a delay or lack of apoptosis, respectively, when treated with dexamethasone. This is accompanied by a delay or lack of cytochrome c release from the mitochondria suggesting that alterations in the signaling phase of apoptosis are responsible for the observed resistance. Because membranes are a rich source of signaling molecules, we have used 31P NMR spectroscopy to compare phospholipids and their metabolites in WEHI7.2, CAT38 and Hb12 cells after dexamethasone treatment. Increased lysophosphatidylcholine (lysoPtdC) content accompanied phosphatidylserine (PtdS) externalization in the WEHI7.2 cells. Both changes were delayed in CAT38 cells suggesting phosphatidylcholine (PtdC) metabolites may play a role in steroid-induced apoptotic signaling. The steroid-resistant Hb12 cells showed a dramatic increase in glycerophosphocholine (GPC) content, suggesting increased phospholipid turnover may contribute to the anti-apoptotic mechanism of Bcl-2.
... The mitochondrial permeability transition is promoted by direct oxidative damage to mitochondrial lipids, most notably cardiolipin (Petrosillo et al., 2003). This oxidative damage of mitochondrial lipids is largely independent of BCL-2 family function but does serve to synergize their pro-apoptotic effects (Esposti, 2002;Lehmann and Shatrov, 2002). We hypothesize that T. gondii is unable to suppress cytochrome c release triggered by mitochondrial damage resulting from exposure to high UV doses ( Fig. 3A) but enables infected cells to recover or establish a higher threshold for release (Fig. 3A). ...
Cells infected with the protozoan parasite Toxoplasma gondii are resistant to diverse apoptotic stimuli. In this study, we perform a detailed analysis of the manipulation of the mitochondrial arm of the apoptotic cascade by the parasite. Apoptosis was induced using irradiation with ultraviolet light (UV), and the kinetics of caspase activation, cytochrome c release and activation of the upstream signalling pathways were examined. The evidence clearly points to T. gondii targeting multiple steps in the transmission [inhibition of c-Jun N-terminal kinase (JNK) activation in response to UV], triggering (inhibition of cytochrome c release by affecting the balance of pro- and anti-apoptotic BCL-2 family members) and execution (inhibition of caspase 9 and caspase 3) phases of the apoptotic cascade. Interestingly, the multilevel pattern of inhibition that emerges suggests that the global inhibition of the mitochondrial arm of apoptosis is not likely to be contributed to by the small subset of mitochondria recruited to the T. gondii parasitophorous vacuole membrane.
... Cardiolipin (CL) makes up about 15–20% of mitochondrial phospholipids in eukaryotic cells (Hatch, 1996; Jakovcic et al., 1971). As an anionic phospholipid predominantly found in the mitochondrial inner membrane (Gallet et al., 1997; Hatch, 1996), CL plays an essential role in many critical mitochondrial functions, such as solute transport (Battelli et al., 1992; Hoffmann et al., 1994; Mende et al., 1983), protein and phospholipid import (Ardail et al., 1991; Chupin et al., 1995; Eilers et al., 1989; Endo et al., 1989; Shiao et al., 1995), oxidative phosphorylation (Eble et al., 1990; Fry and Green, 1981; Paradies et al., 1997; Petrosillo et al., 2003; Robinson, 1993) and mitochondriamediated apoptosis (Esposti, 2002; Kirkland et al., 2002; Kriska et al., 2005; McMillin and Dowhan, 2002; Nakagawa, 2004). Much of CL's importance to various mitochondrial processes can be ascribed to its interaction with individual proteins or complexes , which in turn require CL to maintain their structural or functional integrity. ...
The Saccharomyces cerevisiae cardiolipin (CL) synthase encoded by the CRD1 gene catalyses the synthesis of CL, which is localized to the inner mitochondrial membrane and plays an important role in mitochondrial function. To investigate how CRD1 expression is regulated, a lacZ reporter gene was placed under control of the CRD1 promoter and the 5'-untranslated region of its mRNA (P(CRD1)-lacZ). P(CRD1)-lacZ expression was 2.5 times higher in early stationary phase than in logarithmic phase for glucose grown cells. Non-fermentable growth resulted in a two-fold elevation in expression relative to glucose grown cells. A shift from glycerol to glucose rapidly repressed expression, whereas a shift from glucose to glycerol had the opposite effect. The derepression of P(CRD1)-lacZ expression by non-fermentable carbon sources was dependent on mitochondrial respiration. These results support a tight coordination between translation and transcription of the CRD1 gene, since similar effects by the above factors on CRD1 mRNA levels have been reported. In glucose-grown cells, P(CRD1)-lacZ expression was repressed 70% in a pgs1delta strain (lacks phosphatidylglycerol and CL) compared with wild-type and rho- cells and elevated 2.5-fold in crd1delta cells, which have increased phosphatidylglycerol levels, suggesting a role for phosphatidylglycerol in regulating CRD1 expression. Addition of inositol to the growth medium had no effect on expression. However, expression was elevated in an ino4delta mutant but not in ino2delta cells, suggesting multiple and separate functions for the inositol-responsive INO2/INO4 gene products, which normally function as a dimer in regulating gene function.
The occurrence of 3-methylglutaconic aciduria (3-MGA) is a well understood phenomenon in leucine oxidation and ketogenesis disorders (primary 3-MGAs). In contrast, its genesis in non-canonical (secondary) 3-MGAs, a growing-up group of disorders encompassing more than a dozen of inherited metabolic diseases, is a mystery still remaining unresolved for three decades. To puzzle out this anthologic problem of metabolism, three clues were considered: (i) the variety of disorders suggests a common cellular target at the cross-road of metabolic and signaling pathways, (ii) the response to leucine loading test only discriminative for primary but not secondary 3-MGAs suggests these latter are disorders of extramitochondrial HMG-CoA metabolism as also attested by their failure to increase 3-hydroxyisovalerate, a mitochondrial metabolite accumulating only in primary 3-MGAs, (iii) the peroxisome is an extramitochondrial site possessing its own pool and displaying metabolism of HMG-CoA, suggesting its possible involvement in producing extramitochondrial 3-methylglutaconate (3-MG). Following these clues provides a unifying common basis to non-canonical 3-MGAs: constitutive mitochondrial dysfunction induces AMPK activation which, by inhibiting early steps in cholesterol and fatty acid syntheses, pipelines cytoplasmic acetyl-CoA to peroxisomes where a rise in HMG-CoA followed by local dehydration and hydrolysis may lead to 3-MGA yield. Additional contributors are considered, notably for 3-MGAs associated with hyperammonemia, and to a lesser extent in CLPB deficiency. Metabolic and signaling itineraries followed by the proposed scenario are essentially sketched, being provided with compelling evidence from the literature coming in their support.
To study the cytostatic and some biological effects of aminoferrocene using mice with L1210 lymphoid leukemia.
Experiments were performed on BDF1 male mice (DBA/2, female × C57Bl/6, male) with transplantable L1210 lymphoid leukemia. Determination of antitumor activity of Benzyl-Fc Boron (Bn), it was injected intraperitoneally 6 times daily, starting on day 2 after L1210 leukemia cell transplantation. Doses of Bn such as 26; 260 and 2600 μg/kg were used. The determination of intracellular content of cardiolipin, thiols, reactive oxygen species (ROS) and also analysis of Annexin V positivity and mitochondrial transmembrane potential (JC-1 staining) were performed with use of flow cytometry. The levels of "free iron" complexes, transferrin active forms and the rate of NO generation were measured by EPR-specroscopy.
Six daily injections of Bn at a dose of 26 μg/kg resulted in an increased survival of mice with L1210 leukemia by 28% (p < 0.05). Bn led to an increase of apoptotic cells number and ROS amount in leukemia cells. Besides, Bn caused a decrease of cardiolipin and nonprotein thiol compounds content. The membrane electrochemical potential of cell mitochondria was decreased also after Bn administration. Studies using EPR-spectroscopy revealed a significant increase in a level of "free iron", content of transferrin active species and generation rate of NO by inducible NO-synthase in L1210 cells after aminoferrocene administration.
Our data indicate that Benzyl-Fc Boron can be promising candidate for realizing a new strategy of anticancer therapy with the use of ROS-inducing agents.
Mitochondrially-generated reactive oxygen species are involved in a myriad of signalling and damaging pathways in different tissues. Also, mitochondria are an important target of reactive oxygen and nitrogen species. Here we discuss basic mechanisms of mitochondrial oxidant generation and removal and the main factors affecting mitochondrial redox balance. We also discuss the interaction between mitochondrial reactive oxygen and nitrogen species, and the involvement of these oxidants in mitochondrial diseases, cancer, neurological, and cardiovascular disorders.
Living cells are known to exhibit great morphological, functional, spatial and temporal heterogeneity. Hence, the study of cells in a bulk, whether this bulk is homogenous or heterogeneous, does not provide sufficiently detailed or interpretable results. An advantageous approach would rather be a comprehensive study of cell biological activity in single isolated living cells. In this study, we present an imaging approach for studying pre-apoptotic and very early apoptotic events, during cell death induced by lysophosphatidylcholine (LPC) at the single cell level. The aim of this study is to investigate intracellular events, such as the mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) formation, before and immediately after LPC introduction to the lymphocytes at the level of individual cells. A new protocol of sequential staining was developed to study the relation between early apoptosis signs (PS externalization), MMP changes and intracellular ROS production rates at an individual Jurkat cell resolution. Simultaneous kinetic assessments of MMP, intracellular ROS levels and phosphatidylserine (PS) externalization were performed at a single cell resolution, using Optical LiveCell™ Array technology and image analysis. The parameters were measured and analyzed both before and during exposure to inducers in a Jurkat cell population, including three groups of single cells: spontaneous apoptotic cells, induced apoptotic cells and fully functional living cells. Exogenous LPC caused a heterogeneous intracellular response among Jurkat cells immediately after its introduction. Subgroups of cells with opposite changes of MMP and different kinetics of ROS increase, were revealed within the whole cell population. The subset of apoptosis-induced Jurkat cells, which became apoptotic within 3h after the LPC introduction, exhibited higher initial MMP compared to fully functional or spontaneous apoptotic cells. LPC-induced apoptosis was accompanied by a concomitant increase in intracellular ROS levels. In the present study, a method is described to assess the intracellular events in cells which were initially different in their physiological status. The individual T lymphocytes (Jurkat cells) in vitro have various susceptibilities to LPC effects at the very early stage of contact with the inducer. The apoptotic effect of LPC in individual Jurkat cells is associated with a relatively higher initial MMP before the introduction of the inducer and with a faster ROS formation within the affected cells. Such divergence may be significant in regulating the balance of lymphocyte subsets in pathological sites, either maintaining or preventing the inflammation components of atherosclerosis. We conclude that the presented approach provides the researcher, not only with the cell retaining methodology, but with opportunities to observe and find the distinctive cell subsets within the whole cell population as well, thus helping to define more exactly the role and importance of such sub-populations in physiological or pathological conditions.
Solution pH is believed to serve as an intricate regulatory switch in the induction of apoptosis central to embryonic development and cellular homeostasis. Herein, using an array of biophysical techniques, we provide evidence that acidic pH promotes the assembly of BclXL apoptotic repressor into a megadalton oligomer with a plume-like appearance and harboring structural features characteristic of a molten globule. Strikingly, our data reveal that pH tightly modulates not only oligomerization but also ligand binding and membrane insertion of BclXL in a highly subtle manner. Thus, while oligomerization and the accompanying molten globular content of BclXL is least favorable at pH 6, both of these structural features become more pronounced under acidic and alkaline conditions. However, membrane insertion of BclXL appears to be predominantly favored under acidic conditions. In a remarkable contrast, while ligand binding to BclXL optimally occurs at pH 6, it is diminished by an order of magnitude at lower and higher pH. This reciprocal relationship between BclXL oligomerization and ligand binding lends new insights into how pH modulates functional versatility of a key apoptotic regulator and strongly argues that the molten globule may serve as an intermediate primed for membrane insertion in response to apoptotic cues.
Cardiolipin (CL) is a mitochondria-specific phospholipid and is critical for maintaining the integrity of mitochondrial membrane and mitochondrial function. CL also plays an active role in mitochondria-dependent apoptosis by interacting with cytochrome c (cyt c), tBid and other important Bcl-2 proteins. The unique structure of CL with four linoleic acid side chains in the same molecule and its cellular location make it extremely susceptible to free radical oxidation by reactive oxygen species including free radicals derived from peroxidase activity of cyt c/CL complex, singlet oxygen and hydroxyl radical. The free radical oxidation products of CL have been emerged as important mediators in apoptosis. In this review, we summarize the free radical chemical mechanisms that lead to CL oxidation, recent development in detection of oxidation products of CL by mass spectrometry and the implication of CL oxidation in mitochondria-mediated apoptosis, mitochondrial dysfunction and human diseases.
The antiphospholipid syndrome (APS) is an autoimmune disease characterized by arterial and venous thrombosis, recurrent miscarriages or fetal loss, and circulating antiphospholipid antibodies (aPL). Enzyme-linked immunosorbent assays for anticardiolipin and anti-β2-glycoprotein I antibodies and clotting assays for the lupus anticoagulant are the tests recommended for detecting aPL. However, the aPL are a heterogeneous group of antibodies directed against anionic phospholipids but also toward phospholipid-binding plasma proteins or phospholipid-protein complexes. β2-glycoprotein I (β2GPI) is the playmaker antigen of APS, however during apoptosis, lysophospholipids can become exposed on the cell surface, and mainly through their interaction with β2GPI, they can become targets of aPL. Some CL metabolites are likely to escape from the remodeling cycle. This would account for the progressive loss of mitochondrial CL during apoptosis, as well as for the presence of CL and lyso-CL at the cell surface, where they can interact with β2GPI and become targets of aPL. Other recognized targets of aPL are represented by phosphatidylserine, lyso(bis)phosphatidic acid, Phosphatidylethanolamine, vimentin, and annexin A5. These molecules may allow improving the knowledge on the pathogenesis, and the early identification of APS. Although several studies have shown the presence of antibodies directed against other antigens in APS, their clinical relevance is still a matter of debate, and it needs to be confirmed with experimental data and longitudinal studies.
The plasma membrane Ca(2+)-ATPase (PMCA) is an ATP-driven pump that is critical for the maintenance of low resting [Ca(2+)](i) in all eukaryotic cells. Metabolic stress, either due to inhibition of mitochondrial or glycolytic metabolism, has the capacity to cause ATP depletion and thus inhibit PMCA activity. This has potentially fatal consequences, particularly for non-excitable cells in which the PMCA is the major Ca(2+) efflux pathway. This is because inhibition of the PMCA inevitably leads to cytosolic Ca(2+) overload and the consequent cell death. However, the relationship between metabolic stress, ATP depletion and inhibition of the PMCA is not as simple as one would have originally predicted. There is increasing evidence that metabolic stress can lead to the inhibition of PMCA activity independent of ATP or prior to substantial ATP depletion. In particular, there is evidence that the PMCA has its own glycolytic ATP supply that can fuel the PMCA in the face of impaired mitochondrial function. Moreover, membrane phospholipids, mitochondrial membrane potential, caspase/calpain cleavage and oxidative stress have all been implicated in metabolic stress-induced inhibition of the PMCA. The major focus of this review is to challenge the conventional view of ATP-dependent regulation of the PMCA and bring together some of the alternative or additional mechanisms by which metabolic stress impairs PMCA activity resulting in cytosolic Ca(2+) overload and cytotoxicity.
We examined if lipopolysaccharide (LPS) treatment of mice affected cardiolipin (CL) synthesis. Mice were injected i.p. with LPS, the liver harvested, and CL synthase (CLS) enzyme activity and its mRNA expression examined. Treatment of mice with LPS resulted in a 55% decrease (p < 0.01) in mRNA expression of murine CLS compared to controls, but CLS enzyme activity was unaltered. The pool size of liver CL and other phospholipids were unaltered by LPS treatment. A similar effect was observed in murine epidermal fat pad and in vitro in RAW mouse macrophages and in human HepG2 cells. LPS treatment of HepG2 cells transiently expressing a histidine-tagged human cardiolipin synthase-1 (hCLS1) reduced hCLS1 mRNA and newly synthesized CLS activity indicating that LPS inhibits production of newly synthesized hCLS1 via reduction in hCLS1 mRNA. The results clearly indicate that CLS mRNA levels cannot be correlated with CLS enzyme activity nor CL content in the LPS model of inflammation.
Biological membrane function, in part, depends upon the local regulation of lipid composition. The spatial heterogeneity of membrane lipids has been extensively explored in the context of cholesterol and phospholipid acyl-chain-dependent domain formation, but the effects of lipid head groups and soluble factors in lateral lipid organization are less clear. In this contribution, the effects of divalent calcium ions on domain formation in monolayers containing phosphatidylinositol 4,5-bisphosphate (PIP2), a polyanionic, multifunctional lipid of the cytosolic leaflet of the plasma bilayer, are reported. In binary monolayers of PIP2 mixed with zwitterionic lipids, calcium induced a rapid, PIP2-dependent surface pressure drop, with the concomitant formation of laterally segregated, PIP2-rich domains. The effect was dependent upon head-group multivalency, because lowered pH suppressed the surface-pressure effect and domain formation. In accordance with previous observations, inclusion of cholesterol in lipid mixtures induced coexistence of two liquid phases. Phase separation strongly segregated PIP2 to the cholesterol-poor phase, suggesting a role for cholesterol-dependent lipid demixing in regulating PIP2 localization and local concentration. Similar to binary mixtures, subphase calcium induced contraction of ternary cholesterol-containing monolayers; however, in these mixtures, calcium induced an unexpected, PIP2- and multivalency-dependent decrease in the miscibility phase transition surface pressure, resulting in rapid dissolution of the domains. This result emphasizes the likely critical role of subphase factors and lipid head-group specificity in the formation and stability of cholesterol-dependent domains in cellular plasma membranes.
Bid is an abundant pro-apoptotic protein of the Bcl-2 family that is crucial for death receptor-mediated apoptosis in many cell systems. Bid action has been proposed to involve the mitochondrial re-location of its truncated form, tBid, to facilitate the release of apoptogenic proteins like cytochrome c. However, the precise mechanism of (t)Bid action is unknown. To advance our knowledge, this review evaluates the basic steps of Bid activation--caspase cleavage, dissociation of tBid, and lipid-mediated mitochondrial relocation--and their structure-function aspects. Relevant current information is thoroughly examined to outline the problems that hamper our understanding of the possible roles of Bid in cell life and death, and suggest valuable directions for obtaining a clarification of its pro-apoptotic mechanism.
The mitochondrion has long been known both as a chemical powerplant and as a cellular compartment housing various biosynthetic pathways. However, studies on the function of mitochondria in apoptotic cell death have revealed a versatility and complexity of these organelles previously unsuspected. The mechanisms proposed for mitochondrial involvement in cell death are diverse and highly controversial. In one model, mitochondria are seen as passive containers that can be made to leak out cytotoxic proteins. In other scenarios, however, certain more or less familiar aspects of mitochondrial physiology, such as oxidative phosphorylation, generation of oxygen radicals, dynamic morphological rearrangements, calcium overload, and permeability transition, are proposed to play crucial roles. In this review, we examine a few promising mechanisms that have been gaining attention recently.
Fatty acids and other lipids have multiple roles in the cell, functioning as structural components, participating in intracellular signalling and serving as metabolic fuel. Various compounds that influence cellular lipid metabolism can reduce the growth of malignant cells, and dietary as well as pharmacological strategies for modulating lipid metabolism have therefore been suggested as possible approaches for cancer prevention and treatment. By chemically modifying fatty acids (e.g., butyrates, retinoids), new potential anticancer agents have been produced that possess increased metabolic stability and more specific and potent biological activity compared to the natural fatty acids. Possible therapeutic targets for such modified fatty acids include: i) Histone deacetylase; ii) nuclear hormone receptors (retinoid receptors), peroxisome proliferator-activated receptors; iii) cyclooxygenase-2; iv) intracellular signalling involving protein farnesylation and Ras activation; and v) various mitochondrial functions. Although several fatty acid derivatives have been thoroughly investigated in experimental models, clinical data on toxicity and pharmacological interactions are not available for the majority of these agents. However, several promising novel compounds are now being evaluated in preclinical and early clinical studies, and future research will hopefully reveal new formulations and therapy schedules that will improve the outcome of patients with malignant disorders.
Phospholipid scramblase 3 (PLS3) is a newly recognized member of a family of proteins responsible for phospholipid translocation between two lipid compartments. To study PLS3 function in mitochondria, we disrupted its conserved calcium-binding motif yielding an inactive mutant PLS3(F258V). Cells transfected with PLS3(F258V) exhibited reduced proliferative capacity. Mitochondrial analysis revealed that PLS3(F258V)-expressing cells have decreased mitochondrial mass shown by lower cytochrome c and cardiolipin (CL) content, poor mitochondrial respiration, and reduced oxygen consumption and intracellular ATP; whereas wild-type PLS3-transfected cells exhibit increased mitochondrial mass and enhanced respiration. Electron microscopic examination revealed that the mitochondria in PLS3(F258V)-expressing cells have densely packed cristae and are fewer in number and larger than those in control cells. The abnormal mitochondrial metabolism and structure in PLS3(F258V)-expressing cells were associated with decreased sensitivity to UV- and tBid-induced apoptosis and diminished translocation of CL to the mitochondrial outer membrane. In contrast, wild-type PLS3-transfected cells displayed increased sensitivity to apoptosis and enhanced CL translocation. These studies identify PLS3 as a critical regulator of mitochondrial structure and respiration, and CL transport in apoptosis.
In addition to their function as major energy-providing organelles of the cell, mitochondria accomplish a crucial role in apoptosis. The pro-apoptotic BH3-only members of the Bcl-2 family continuously sense the cellular integrity and well-being at various subcellular levels. If these sentinels are induced, released or activated, they converge on the release of mitochondrial intermembrane space proteins such as cytochrome c, the oxidoreductase AIF, endonuclease G, Smac/DIABLO and the serine protease Omi/HtrA2. We discuss how Bcl-2 family members integrate diverse survival and death signals and act as central regulators of apoptosis. Furthermore, we describe the current knowledge on the role of mitochondrial proteins in apoptotic cell death, discuss the molecular mechanisms of their release and the apoptotic role of mitochondria from a phylogenetic and immunological point of view.
Phospholipids are important structural and functional components of all biological membranes and define the compartmentation of organelles. Mitochondrial phospholipids comprise a significant proportion of the entire phospholipid content of most eukaroytic cells. In the heart, a tissue rich in mitochondria, the mitochondrial phospholipids provide for diverse roles in the regulation of various mitochondrial processes including apoptosis, electron transport, and mitochondrial lipid and protein import. It is well documented that alteration in the content and fatty acid composition of phospholipids within the heart is linked to alterations in myocardial electrical activity. In addition, reduction in the specific mitochondrial phospholipid cardiolipin is an underlying biochemical cause of Barth Syndrome, a rare and often fatal X-linked genetic disease that is associated with cardiomyopathy. Thus, maintenance of both the content and molecular composition of phospholipids synthesized within the mitochondria is essential for normal cardiac function. This review will focus on the function and regulation of the biosynthesis and resynthesis of mitochondrial phospholipids in the mammalian heart.
In this article we overview major aspects of membrane lipids in the complex area of cell death, comprising apoptosis and various forms of programmed cell death. We have focused here on glycerophospholipids, the major components of cellular membranes. In particular, we present a detailed appraisal of mitochondrial lipids that attract increasing interest in the field of cell death, while the knowledge of their re-modelling and traffic remains limited. It is hoped that this review will stimulate further studies by lipid experts to fully elucidate various aspects of membrane lipid homeostasis that are discussed here. These studies will undoubtedly reveal new and important connections with the established players of cell death and their action in promoting or blocking membrane alteration of mitochondria and other organelles. We conclude that the new dynamic era of cell death research will pave the way for a better understanding of the 'chemistry of apoptosis'.
The BCL-2 family proteins constitute a critical control point in apoptosis. BCL-2 family proteins display structural homology
to channel-forming bacterial toxins, such as colicins, transmembrane domain of diphtheria toxin, and the N-terminal domain
of δ-endotoxin. By analogy, it has been hypothesized the BCL-2 family proteins would unfold and insert into the lipid bilayer
upon membrane association. We applied the site-directed spin labeling method of electron paramagnetic resonance spectroscopy
to the pro-apoptotic member BID. Here we show that helices 6-8 maintain an α-helical conformation in membranes with a lipid
composition resembling mitochondrial outer membrane contact sites. However, unlike colicins and the transmembrane domain of
diphtheria toxin, these helices of BID are bound to the lipid bilayer without adopting a transmembrane orientation. Our study
presents a more detailed model for the reorganization of the structure of tBID on membranes.
Apoptosis is the best-characterized form of programmed cell death (PCD) and is of fundamental importance in tissue homeostasis. In mammalian systems, there are two major pathways that are involved in the initiation of apoptosis: the "extrinsic" death receptor pathway and the "intrinsic" mitochondrial pathway. Although these pathways act independently to initiate the death machinery in some cellular systems, in many cell types, including numerous tumor cells, there is delicate coordination and cross talk between the extrinsic and intrinsic pathways, which leads to the activation of the executioner caspase cascade. Additionally, there appears to be a fine balance between the caspase-mediated arm of death receptor signaling that engages mitochondria and the caspase-independent arm that promotes vacuole proliferation in many cells. Here, we review our current knowledge about the layers of complexity that are posed by the interactions between death receptor-induced pathways and how they influence mitochondria to regulate cellular life and death decisions.
Recent studies suggest a close relationship between cell metabolism and apoptosis. We have evaluated changes in lipid metabolism on permeabilized hepatocytes treated with truncated Bid (tBid) in the presence of caspase inhibitors and exogenous cytochrome c. The measurement of beta-oxidation flux by labeled palmitate demonstrates that tBid inhibits beta-oxidation, thereby resulting in the accumulation of palmitoyl-coenzyme A (CoA) and depletion of acetyl-carnitine and acylcarnitines, which is pathognomonic for inhibition of carnitine palmitoyltransferase-1 (CPT-1). We also show that tBid decreases CPT-1 activity by a mechanism independent of both malonyl-CoA, the key inhibitory molecule of CPT-1, and Bak and/or Bax, but dependent on cardiolipin decrease. Overexpression of Bcl-2, which is able to interact with CPT-1, counteracts the effects exerted by tBid on beta-oxidation. The unexpected role of tBid in the regulation of lipid beta-oxidation suggests a model in which tBid-induced metabolic decline leads to the accumulation of toxic lipid metabolites such as palmitoyl-CoA, which might become participants in the apoptotic pathway.
Since the aCL test was first described, several reports have described the heterogeneity of aPL, which binds to different anionic phospholipids, proteins, or to a phospholipid-protein complex. It has been recently reported that antiphospholipids (aPLs) from the sera of patients with the antiphospholipid syndrome (APS) are able to bind some newly identified antigens, the lyso(bis)phosphatidic acid (LBPA), lipid restricted to the late endosomes, and the sulfatides, acidic glycosphingolipids involved in the hemostatic process. Of interest, aLBPAs are present in the sera of a large number of patients with APS showing similar sensitivity and specificity compared to anti-beta(2) glycoprotein I antibodies (abeta(2)-GPIs) and close association with lupus anticoagulant. Moreover, beta(2)-GPI binds to sulfatides and the majority of the aPL reacting with cardiolipin-beta(2)-GPI complex also react with the sulfatide-beta(2)-GPI complex. Different mechanisms involved in the production of autoantibodies in autoimmune diseases have been proposed and, among them, apoptosis or programmed cell death seems to play a leading role. The relocation of CL and its metabolites during apoptosis may represent an in vivo trigger for the generation of aCL, and the higher reactivity of sera from APS patients to monolysocardiolipin, the immediate degradation product of mitochondrial CL validates this hypothesis. Finally, increasing evidence suggests that oxidative stress could be a pathogenic link between aPL and thrombosis, and antioxidant treatment may have some efficacy in preventing the clinical manifestations of this syndrome.
Reduction of mitochondrial cardiolipin (CL) levels has been postulated to compromise directly the function of several essential enzymes and processes of the mitochondria. There is limited genetic evidence for the critical roles with which CL and its precursor phosphatidylglycerol (PG) have been associated. A null allele of the PGS1 gene from Saccharomyces cerevisiae, which encodes the enzyme responsible for the synthesis of the CL precursor PG phosphate, was created in a yeast strain in which PGS1 expression is exogenously regulated by doxycycline. The addition of increasing concentrations of doxycycline to the growth medium causes a proportional decrease to undetectable levels of PGS1 transcript, PG phosphate synthase activity, and PG plus CL. The doubling time of this strain with increasing doxycycline increases to senescence in non-fermentable carbon sources or at high temperatures, conditions that do not support growth of the pgs1Delta strain. Doxycycline addition also causes mitochondrial abnormalities as observed by fluorescence microscopy. Products of four mitochondrial encoded genes (COX1, COX2, COX3, and COB) and one nuclear encoded gene (COX4) associated with the mitochondrial inner membrane are not present when PGS1 expression is fully repressed. No translation of these proteins can be detected in cells lacking the PGS1 gene product, although transcription and splicing appear unaffected. Protein import of other nuclear encoded proteins remains unaffected. The remaining proteins encoded by mitochondrial DNA are expressed and translated normally. Thus, the molecular basis for the lack of mitochondrial function in pgs1Delta cells is the failure to translate gene products essential to the electron transport chain.
Recent evidence supports the theory that mitochondrial homeostasis is the key regulatory step in apoptosis through the actions of members of the Bcl-2 family. Pro-apoptotic members of the family, such as Bax, Bad and Bid, can induce the loss of outer-membrane integrity with subsequent redistribution of pro-apoptotic proteins such as cytochrome c that are normally located in the intermembrane spaces of mitochondria. The anti-apoptotic members of the family, such as Bcl-2 and Bcl-XL, protect the integrity of the mitochondrion and prevent the release of death-inducing factors. Bid normally exists in an inactive state in the cytosol, but after cleavage by caspase 8, the carboxy-terminal portion (tBid) moves from cytosol to mitochondria, where it induces release of cytochrome c. Here we address the question of what mediates specific targeting of tBid to the mitochondria. We provide evidence that cardiolipin, which is present in mitochondrial membranes, mediates the targeting of tBid to mitochondria through a previously unknown three-helix domain in tBid. These findings implicate cardiolipin in the pathway for cytochrome c release.
Cellular levels of free arachidonic acid (AA) are controlled by a deacylation/reacylation cycle whereby the fatty acid is liberated by phospholipases and reincorporated by acyltransferases. We have found that the esterification of AA into membrane phospholipids is a Ca(2+)-independent process and that it is blocked up to 60-70% by a bromoenollactone (BEL) that is a selective inhibitor of a newly discovered Ca(2+)-independent phospholipase A2 (PLA2) in macrophages. The observed inhibition correlates with a decreased steady-state level of lysophospholipids as well as with the inhibition of the Ca(2+)-independent PLA2 activity in these cells. This inhibition is specific for the Ca(2+)-independent PLA2 in that neither group IV PLA2, group II PLA2, arachidonoyl-CoA synthetase, lysophospholipid:arachidonoyl-CoA acyltransferase, nor CoA-independent transacylase is affected by treatment with BEL. Moreover, two BEL analogs that are not inhibitors of the Ca(2+)-independent PLA2--namely a bromomethyl ketone and methyl-BEL--do not inhibit AA incorporation into phospholipids. Esterification of palmitic acid is only slightly affected by BEL, indicating that de novo synthetic pathways are not inhibited by BEL. Collectively, the data suggest that the Ca(2+)-independent PLA2 in P388D1 macrophages plays a major role in regulating the incorporation of AA into membrane phospholipids by providing the lysophospholipid acceptor employed in the acylation reaction.
Fas-mediated apoptosis of human leukemic U937 cells was accompanied by increased arachidonic acid (AA) and oleic acid release
from membrane glycerophospholipids, indicating phospholipase A2 (PLA2) activation. During apoptosis, type IV cytosolic PLA2 (cPLA2), a PLA2 isozyme with an apparent molecular mass of 110 kDa critical for stimulus-coupled AA release, was converted to a 78-kDa fragment
with concomitant loss of catalytic activity. Cleavage of cPLA2 correlated with increased caspase-3-like protease activity in apoptotic cells and was abrogated by a caspase-3 inhibitor.
A mutant cPLA2 protein in which Asp522 was replaced by Asn, which aligns with the consensus sequence of the caspase-3 cleavage site (DXXD↓X), was resistant to apo-ptosis-associated proteolysis. Moreover, a COOH-terminal deletion mutant of cPLA2 truncated at Asp522 comigrated with the 78-kDa fragment and exhibited no enzymatic activity. Thus, caspase-3-mediated cPLA2 cleavage eventually leads to destruction of a catalytic triad essential for cPLA2 activity, thereby terminating its AA-releasing function. In contrast, the activity of type VI Ca2+-independent PLA2 (iPLA2), a PLA2 isozyme implicated in phospholipid remodeling, remained intact during apoptosis. Inhibitors of iPLA2, but neither cPLA2 nor secretory PLA2 inhibitors, suppressed AA release markedly and, importantly, delayed cell death induced by Fas. Therefore, we conclude that
iPLA2-mediated fatty acid release is facilitated in Fas-stimulated cells and plays a modifying although not essential role in the
apoptotic cell death process.
Investigations into the capacity of the Bcl-2 protein to prevent apoptosis have targeted mitochondria as key sites of the preventative action accorded by Bcl-2 to cells. Using novel approaches with fluorescence probes and autofluorescence detection of endogenous NAD(P)H, we have examined the effects of expressing Bcl-2 in the Bcl-2 negative Burkitt's lymphoma cell line Daudi. We evaluated for the first time the effect of Bcl-2 expression on the intracellular distribution and production of hydrogen peroxide, under basal conditions and after treatment with apoptosis inducing agents, ceramide analogs and tumor necrosis factor (TNF)-alpha. Increased availability of mitochondrial NAD(P)H was detected in Bcl-2-expressing cells and was correlated with an increased constitutive mitochondrial production of hydrogen peroxide. Although production of hydrogen peroxide was increased by either C(6)-ceramide or TNF-alpha in Bcl-2 negative Daudi cells commensurate with the early phases of apoptosis, this increase did not occur in Bcl-2-expressing cells. Thus, Bcl-2 appears to allow cells to adapt to an increased state of oxidative stress, fortifying the cellular anti-oxidant defenses and counteracting the radical overproduction imposed by different cell death stimuli. Furthermore, we report altered cytological features of mitochondria during the early phases of apoptosis induced by C(6)-ceramide and TNF-alpha. In particular, mitochondria changed in appearance, clustering in the perinuclear region and Bcl-2 expression prevented these changes from occurring.
Anticardiolipin (anti-CL) antibodies, diagnostic for antiphospholipid antibody syndrome, are associated with increased risks of venous and arterial thrombosis. Because CL selectively enhances activated protein C/protein S-dependent anticoagulant activities in purified systems and because CL is not known to be a normal plasma component, we searched for CL in plasma. Plasma lipid extracts [chloroform/methanol (2:1, vol/vol)] were subjected to analyses by using TLC, analytical HPLC, and MS. A plasma lipid component was purified that was indistinguishable from reference CL (M:1448). When CL in 40 fasting plasma lipid extracts (20 males, 20 females) was quantitated by using HPLC, CL (mean +/- SD) was 14.9 +/- 3.7 microgram/ml (range 9.1 to 24.2) and CL was not correlated with phosphatidylserine (3.8 +/- 1.7 microgram/ml), phosphatidylethanolamine (64 +/- 20 microgram/ml), or choline-containing phospholipid (1,580 +/- 280 microgram/ml). Based on studies of fasting blood donors, CL (>/=94%) was recovered in very low density, low density, and high density lipoproteins (11 +/- 5.3%, 67 +/- 11.0%, and 17 +/- 10%, respectively), showing that the majority of plasma CL (67%) is in low density lipoprotein. Analysis of relative phospholipid contents of lipoproteins indicated that high density lipoprotein is selectively enriched in CL and phosphatidylethanolamine. These results shows that CL is a normal plasma component and suggest that the epitopes of antiphospholipid antibodies could include CL or oxidized CL in lipoproteins or in complexes with plasma proteins (e. g., beta(2)-glycoprotein I, prothrombin, protein C, or protein S) or with platelet or endothelial surface proteins.
Multigenic programs controlling susceptibility to apoptosis in response to ionizing radiation have not yet been defined. Here, using DNA microarrays, we show gene expression patterns in an apoptosis-sensitive and apoptosis-resistant murine B cell lymphoma model system both before and after irradiation. From the 11,000 genes interrogated by the arrays, two major patterns emerged. First, before radiation exposure the radioresistant LYar cells expressed significantly greater levels of message for several genes involved in regulating intracellular redox potential. Compared with LYas cells, LYar cells express 20- to 50-fold more mRNA for the tetraspanin CD53 and for fructose-1,6-bisphosphatase. Expression of both of these genes can lead to the increase of total cellular glutathione, which is the principle intracellular antioxidant and has been shown to inhibit many forms of apoptosis. A second pattern emerged after radiation, when the apoptosis-sensitive LYas cells induced rapid expression of a unique cluster of genes characterized by their involvement in mitochondrial electron transport. Some of these genes have been previously recognized as proapoptotic; however others, such as uncoupling protein 2, were not previously known to be apoptotic regulatory proteins. From these observations we propose that a multigenic program for sensitivity to apoptosis involves induction of transcripts for genes participating in mitochondrial uncoupling and loss of membrane potential. This program triggers mitochondrial release of apoptogenic factors and induces the "caspase cascade." Conversely, cells resistant to apoptosis down-regulate these biochemical pathways, while activating pathways for establishment and maintenance of high intracellular redox potential by means of elevated glutathione.
Cells determine the bilayer characteristics of different membranes by tightly controlling their lipid composition. Local changes in the physical properties of bilayers, in turn, allow membrane deformation, and facilitate vesicle budding and fusion. Moreover, specific lipids at specific locations recruit cytosolic proteins involved in structural functions or signal transduction. We describe here how the distribution of lipids is directed by proteins, and, conversely, how lipids influence the distribution and function of proteins.
Apoptosis has been identified recently as a component of many cardiac pathologies. However, the potential triggers of programmed cell death in the heart and the involvement of specific metabolic pathway(s) are less well characterized. Detachment of cytochrome c from the mitochondrial inner membrane is a necessary first step for cytochrome c release into the cytosol and initiation of apoptosis. The saturated long chain fatty acid, palmitate, induces apoptosis in rat neonatal cardiomyocytes and diminishes content of the mitochondrial anionic phospholipid, cardiolipin. These changes are accompanied by 1) acyl chain saturation of phosphatidic acid and phosphatidylglycerol, 2) large increases in the levels of these two phospholipids, and 3) a decline in cardiolipin synthesis. Although cardiolipin synthase activity is unchanged, saturated phosphatidylglycerol is a poor substrate for this enzyme. Under these conditions, decreased cardiolipin synthesis and release of cytochrome c are directly and significantly correlated. The results suggest that phosphatidylglycerol saturation and subsequent decreases in cardiolipin affect the association of cytochrome c with the inner mitochondrial membrane, directly influencing the pathway to cytochrome c release and subsequent apoptosis.
Lipoxygenases are a family of enzymes which dioxygenate unsaturated fatty acids, thus initiating lipoperoxidation of membranes and the synthesis of signaling molecules. Consequently, they induce structural and metabolic changes in the cell in a number of pathophysiological conditions. Recently, a pro-apoptotic effect of lipoxygenase, and of the hydroperoxides produced thereof, has been reported in different cells and tissues, leading to cell death. Anti-apoptotic effects of lipoxygenases have also been reported; however, this has often been based on the use of enzyme inhibitors. Here we review the characteristics of the lipoxygenase family and its involvement in the initiation of oxidative stress-induced apoptosis. Finally, we discuss the role of lipoxygenase activation in apoptosis of animal and plant cells, suggesting a common signal transduction pathway in cell death conserved through evolution of both kingdoms.
Virtually every cell in the body restricts phosphatidylserine (PS) to the inner leaflet of the plasma membrane by energy-dependent transport from the outer to the inner leaflet of the bilayer. Apoptotic cells of all types rapidly randomize the asymmetric distribution, bringing PS to the surface where it serves as a signal for phagocytosis. A myriad of phagocyte receptors have been implicated in the recognition of apoptotic cells, among them a PS receptor, yet few ligands other than PS have been identified on the apoptotic cell surface. Since apoptosis and the associated exposure of PS on the cell surface is probably over 600 million years old, it is not surprising that evolution has appropriated aspects of this process for specialized purposes such as blood coagulation, membrane fusion and erythrocyte differentiation. Failure to efficiently remove apoptotic cells may contribute to inflammatory responses and autoimmune diseases resulting from chronic, inappropriate exposure of PS.
Bid is an abundant proapoptotic protein of the Bcl-2 family that is crucial for the induction of death receptor-mediated apoptosis
in primary tissues such as liver. Bid action has been proposed to involve the relocation of its truncated form, tBid, to mitochondria
to facilitate the release of apoptogenic cytochrome c. The mechanism of Bid relocation to mitochondria was unclear. We report here novel biochemical evidence indicating that Bid
has lipid transfer activity between mitochondria and other intracellular membranes, thereby explaining its dynamic relocation
to mitochondria. First, physiological concentrations of phospholipids such as phosphatidic acid and phosphatidylgycerol induced
an accumulation of full-length Bid in mitochondria when incubated with light membranes enriched in endoplasmic reticulum.
Secondly, native and recombinant Bid, as well as tBid, displayed lipid transfer activity under the same conditions and at
the same nanomolar concentrations leading to mitochondrial relocation and release of cytochrome c. Thus, Bid is likely to be involved in the transport and recycling of mitochondrial phospholipids. We discuss how this new
role of Bid may relate to its proapoptotic action.
We investigate numerically the interaction of a stream of granular particles with a resting obstacle in two dimensions. For the case of high stream velocity we find that the force acting on the obstacle is proportional to the square of the stream velocity, the density and the obstacle size. This behaviour is equivalent to that of non-interacting hard spheres. For low stream velocity a gap between the obstacle and the incoming stream particles appears which is filled with granular gas of high temperature and low density. As soon as the gap appears the force does not depend on the square of velocity of the stream but the dependency obeys another law.
Liver mitochondrial cardiolipin (CL) is distinguished from other phospholipids by the presence of linoleoyl in almost all molecular species, and the biosynthesis of these species is not yet understood. The present study was carried out in order to test the hypothesis that the linoleoyl proportion of CL may be specifically enriched by a deacylation-reacylation cycle. Incorporation of [14C]glycerol 3-phosphate into the metabolites of the CL pathway was accompanied by formation of 14C-labelled monolyso- and dilyso-CL. Labelling of dilyso-CL was increased or decreased by stimulation or inhibition respectively of mitochondrial phospholipase A2. These data suggest a rapid deacylation of newly formed [14C]CL by phospholipase A2, whereas endogenous mitochondrial CL was very resistant to hydrolytic degradation. Unlike dilyso-CL, monolyso-CL could be reacylated by [14C]linoleoyl residues. [14C]Linoleoyl incorporation into CL was also observed when exogenous CL was added instead of monolyso-CL, thus indicating the concerted action of de- and re-acylation. Although 1-palmitoyl-2-[14C]linoleoyl-phosphatidylcholine was a suitable acyl donor under experimental conditions, the reaction was not a transacylation but required splitting of [14C]linoleic acid from phosphatidylcholine and formation of [14C]linoleoyl-CoA as an intermediate. The [14C]linoleoyl was mainly bound to the sn-2(2") position of CL, and a small portion (about 20%) to the sn-1(1") position. It is concluded that a cycle, comprising CL deacylation and monolyso-CL reacylation by linoleoyl-CoA, provides a potential mechanism for the remodelling of molecular species of newly formed CL.
Three mechanisms are commonly suggested for the movemerlt of lipids between intracellular organelles: transfer mediated by cytosolic lipid transfer proteins, vesicle-mediated transfer, and transfer via regions of membrane continuity between organelles. The mechanism of translocation of phosphatidylserine from its site of synthesis on the endoplasmic reticulum (and related membranes) to the site of phosphatidylserine decarboxylation in the mitochondria has been investigated. Several experiments indicate that a transfer mediated by soluble cytosolic phospholipid transfer proteins, or by vesicles, is unlikely. Rather, the most likely mode of import of newly-synthesized phosphatidylserine into mitochondria is contact between the endoplasmic reticulum and mitochondrial membranes. In support of this mechanism we have isolated an endoplasmic reticulum-like."mitochondria-associated membrane" fraction and shown that it has some, but not all, properties of the endoplasmic reticulum. The mitochondria-associated membranes are enriched in lipid biosynthetic enzymes, especially phosphatidylserine synthase. When either phosphatidylserine translocation to mitochondria is blocked (by ATP depletion), or phosphatidylserine decarboxylation is blocked (with hydroxylamine), newly-synthesized phosphatidylserine accumulates in the mitochondria-associated membrane but not in microsomes, suggesting that phosphatidylserine traverses the mitochondria-associated membrane on its route from the endoplasmic reticulum to mitochondria.
In vitro studies in purified plasma systems have suggested that triglyceride-rich lipoproteins such as chylomicrons, very low density lipoproteins, and their remnants promote activation of factor VII through activated factor XII (XIIa) and the intrinsic coagulation pathway. We specifically examined the roles of factors XII, XI, and IX in activation of factor VII during alimentary lipemia in vivo in humans and addressed the issue of whether generation of activated factor VII (VIIa) is accompanied by increased thrombin production. For this purpose XIIa, factor IX activation peptide (IXP), VIIa, prothrombin fragment 1 + 2 (F1 + 2), and thrombin-antithrombin complex (TAT) were determined in plasma samples taken before and 3, 6, and 9 hours after intake of a mixed meal type of oral fat load in 24 healthy men The VIIa response to fat intake was also determined in 7 patients with single coagulation-factor deficiency, of whom 2 were deficient in factor XII, 2 in factor XI, and 3 in factor IX. Postprandial activation of factors IX and VII occurred in the healthy individuals, whereas the plasma levels of XIIa did not change in response to the test meal. Of note, plasma concentrations of F1 + 2 were unaltered during alimentary lipemia, and TAT levels showed a small decrease (P < .05) in the 3-hour sample compared with the fasting level, indicating that thrombin generation is not stimulated in the postprandial state, despite the generation of activated factor IX (IXa) and VIIa. Factor VIIa increased in the postprandial period in the 2 factor XII-deficient patients who underwent the oral fat tolerance test but appeared to remain unchanged in the factor XI- and factor IX-deficient patients. Therefore, the current concept that activation of factor XII plays a pivotal role in initiating the sequence of events linking postprandial lipemia to activation of factor VII is contradicted by the present study. Whether activation of factor XI by triglyceride rich lipoproteins initiates these reactions needs to be demonstrated in future studies.
To demonstrate interaction between the mitochondrial membrane proteins CPT I and Bcl-2 the yeast two-hybrid system was used. Full-length CPT I was required for binding to Bcl-2. Direct protein interaction was confirmed in a GST binding assay and in coimmunoprecipitations using two different kinds of anti-Bcl-2 antibodies.
Cardiolipin is the principal polyglycerophospholipid found in the heart and most mammalian tissues. This phospholipid is the only phospholipid localized exclusively to the mitochondria of mammalian cells. Cardiolipin appears to be involved, either directly or indirectly, in the modulation of a number of cellular processes including the activation of mitochondrial enzymes and hence production of energy by oxidative phosphorylation. The regulatory properties which govern cardiolipin biosynthesis, its remodeling and trafficking are beginning to emerge. Studies in the isolated perfused rat heart and H9c2 cardiac myoblast cells have indicated that the rate-limiting step of cardiolipin biosynthesis, via the cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol pathway, is the conversion of phosphatidic acid and cytidine-5'-triphosphate to cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol. The cellular level of cytidine-5'-triphosphate appears to control the production of cardiolipin in H9c2 cells. The activities of the other enzymes of the cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol pathway of cardiolipin biosynthesis in the heart may be modulated by thyroid hormone and unsaturated fatty acids. In addition, extra-mitochondrial cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol and phosphatidylglycerol may be utilized for cardiolipin biosynthesis in the heart and permeabilized cells. Cardiolipin may be readily hydrolyzed by phospholipases and may be remodeled by a deacylation-reacylation pathway. Studies with a Chinese hamster lung fibroblast cell line CCL16-B2 have indicated that the remodeling of cardiolipin is markedly altered in the mitochondria of these cells and that this alteration in remodeling may be one of the underlying mechanisms for the mutation in oxidative energy production in these cells. Host cell cardiolipin may be trafficked from the mitochondria to an intracellular bacterial parasite Chlamydia trachomatis. The purpose of this review is to briefly discuss some of the more recent findings in cardiolipin metabolism in the heart and mammalian cells and to provide insight into their possible implications in the regulation of some cellular functions in mammalian tissues and cells.
A CHO-K1 cell mutant with a specific decrease in cellular phosphatidylethanolamine (PE) level was isolated as a variant resistant to Ro09-0198, a PE-directed antibiotic peptide. The mutant was defective in the phosphatidylserine (PS) decarboxylation pathway for PE formation, in which PS produced in the endoplasmic reticulum is transported to mitochondria and then decarboxylated by an inner mitochondrial membrane enzyme, PS decarboxylase. Neither PS formation nor PS decarboxylase activity was reduced in the mutant, implying that the mutant is defective in some step of PS transport. The transport processes of phospholipids between the outer and inner mitochondrial membrane were analyzed by use of isolated mitochondria and two fluorescence-labeled phospholipid analogs, 1-palmitoyl-2-[N-[6(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-PS (C6-NBD-PS) and C6-NBD-phosphatidylcholine (C6-NBD-PC). On incubation with the CHO-K1 mitochondria, C6-NBD-PS was readily decarboxylated to C6-NBD-PE, suggesting that the PS analog was partitioned into the outer leaflet of mitochondria and then translocated to the inner mitochondrial membrane. The rate of decarboxylation of C6-NBD-PS in the mutant mitochondria was reduced to approximately 40% of that in the CHO-K1 mitochondria. The quantity of phospholipid analogs translocated from the outer leaflet of mitochondria into inner mitochondrial membranes was further examined by selective extraction of the analogs from the outer leaflet of mitochondria. In the mutant mitochondria, the translocation of C6-NBD-PS was significantly reduced, whereas the translocation of C6-NBD-PC was not affected. These results indicate that the mutant is defective in PS transport between the outer and inner mitochondrial membrane and provide genetic evidence for the existence of a specific mechanism for intramitochondrial transport of PS.
Cytochrome c (cyt. c) is a proapoptotic factor that binds preferentially to cardiolipin (CL), a mitochondrial lipid, but not to cardiolipin hydroperoxide (CL-OOH). Cyt. c that had bound to CL liposomes was liberated on peroxidation of the liposomes by a radical. The generation of CL-OOH in mitochondria occurred before the release of cyt. c in rat basophile leukaemia (RBL)2H3 cells that had been induced to undergo apoptosis by exposure to hypoglycaemia with 2-deoxyglucose (2DG). The amount of cyt. c bound to CL prepared from the mitochondria of 2DG-treated cells was lower than that of untreated cells. The release of cyt. c was completely suppressed when the production of CL-OOH in mitochondria was inhibited by the overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx). The fluorescence from CL-labelling dye (10-N-nonyl Acridine Orange) decreased on the induction of apoptosis by 2DG. However, no decrease in fluorescence was observed in PHGPx-overexpressing cells. Cyt. c was released from mitochondria that had been isolated from control cells on peroxidation by t-butylhydroperoxide, but no similar liberation of cyt. c from mitochondria isolated from mitochondrial PHGPx-overexpressing cells was observed. These findings suggest that the generation of CL-OOH in mitochondria might be a primary event that triggers the release of cyt. c from mitochondria in the apoptotic process in which mitochondrial PHGPx participates as an anti-apoptotic factor by preventing the formation of CL-OOH.
Lipid peroxidation of membrane phospholipids can proceed both enzymatically via the mammalian 15-lipoxygenase-1 or the NADPH-cytochrome P-450 reductase system and non-enzymatically. In some cells, such as reticulocytes, this process is biologically programmed, whereas in the majority of biological systems lipid peroxidation is a deleterious process that has to be repaired via a deacylation-reacylation cycle of phospholipid metabolism. Several reports in the literature pinpoint a stimulation by lipid peroxidation of the activity of secretory phospholipase A(2)s (mainly pancreatic and snake venom enzymes) which was originally interpreted as a repair function. However, recent experiments from our laboratory have demonstrated that in mixtures of lipoxygenated and native phospholipids the former are not preferably cleaved by either secretory or cytosolic phospholipase A(2)s. We propose that the platelet activating factor (PAF) acetylhydrolases of type II, which cleave preferentially peroxidised or lipoxygenated phospholipids, are competent for the phospholipid repair, irrespective of their role in PAF metabolism. A corresponding role of Ca(2+)-independent phospholipase A(2), which has been proposed to be involved in phospholipid remodelling in biomembranes, has not been addressed so far. Direct and indirect 15-lipoxygenation of phospholipids in biomembranes modulates cell signalling by several ways. The stimulation of phospholipase A(2)-mediated arachidonic acid release may constitute an alternative route of the arachidonic acid cascade. Thus, 15-lipoxygenase-mediated oxygenation of membrane phospholipids and its interaction with phospholipase A(2)s may play a crucial role in the pathogenesis of diseases, such as bronchial asthma and atherosclerosis.
This study provides evidence that cardiolipin (CL) molecules are expressed on the surface of apoptotic cells and are recognized by antiphospholipid antibodies, purified from patients with the antiphospholipid antibody syndrome (APS). CL expression on cell surface was demonstrated by high performance thin layer chromatography analysis of phospholipids from plasma membrane purified fractions and by the positive staining with the CL-specific dye nonyl-acridine orange. This finding was complemented with the observation that aCL IgG purified from patients with APS bind to the surface of apoptotic cells. This staining shows a clustered distribution mostly localized on surface blebs. Interestingly, CL exposure on the cell surface preceded the DNA fragmentation, as shown by cytofluorimetric analysis. These findings demonstrate that exposure of CL molecules on the cell plasma membrane is an early event of the apoptotic cellular program that may represent an in vivo trigger for the generation of aCL.
Pro- and anti-apoptotic members of the Bcl-2 family control the permeability of the outer mitochondrial membrane. They could do this either by forming autonomous pores in the membrane or by collaborating with components of the permeability transition pore. Here we discuss why we favour the first of these possibilities.
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