Lysophosphatidylcholine Acyltransferase 1 (LPCAT1) Specifically Interacts with Phospholipid Transfer Protein StarD10 to Facilitate Surfactant Phospholipid Trafficking in Alveolar Type II Cells

Pulmonary surfactant, a mixture of proteins and phospholipids, plays an important role in facilitating gas exchange by maintaining alveolar stability. Saturated phosphatidylcholine (SatPC), the major component of surfactant, is synthesized both de novo and by the remodeling of unsaturated phosphatidylcholine (PC) by lyso-PC acyltransferase 1 (LPCAT1). After synthesis in the endoplasmic reticulum, SatPC is routed to lamellar bodies (LBs) for storage prior to secretion. The mechanism by which SatPC is transported to LB is not understood. The specificity of LPCAT1 for lyso-PC as an acyl acceptor suggests that formation of SatPC via LPCAT1 reacylation is a final step in SatPC synthesis prior to transport. We hypothesized that LPCAT1 forms a transient complex with SatPC and specific phospholipid transport protein(s) to initiate trafficking of SatPC from the endoplasmic reticulum to the LB. Herein we have assessed the ability of different StarD proteins to interact with LPCAT1. We found that LPCAT1 interacts with StarD10, that this interaction is direct, and that amino acids 79–271 of LPCAT1 and the steroidogenic acute regulatory protein-related lipid transfer (START) domain of START domain-containing protein 10 (StarD10) are sufficient for this interaction. The role of StarD10 in trafficking of phospholipid to LB was confirmed by the observation that knockdown of StarD10 significantly reduced transport of phospholipid to LB. LPCAT1 also interacted with one isoform of StarD7 but showed no interaction with StarD2/PC transfer protein.     

De novo phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy

ABSTRACT

Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using ¹³C-labeled choline and ¹³C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues.     

A novel lysophosphatidylcholine acyl transferase activity is expressed by peroxiredoxin 6

The phospholipase A₂ (PLA₂) activity of peroxiredoxin (Prdx)6 has important physiological roles in the synthesis of lung surfactant and in the repair of peroxidized cell membranes. These functions require the activity of a lysophospholipid acyl transferase as a critical component of the phospholipid remodeling pathway. We now describe a lysophosphatidylcholine acyl transferase (LPCAT) activity for Prdx6 that showed a strong preference for lysophosphatidylcholine (LPC) as the head group and for palmitoyl CoA in the acylation reaction. The calculated kinetic constants for acylation were K_m 18 μM and V_max 30 nmol/min/mg protein; the V_max was increased 25-fold by phosphorylation of the protein while K_m was unchanged. Study of recombinant protein in vitro and in mouse pulmonary microvascular endothelial cells infected with a lentiviral vector construct indicated that amino acid D31 is crucial for LPCAT activity. A linear incorporation of labeled fatty acyl CoA into dipalmitoyl phosphatidylcholine (PC) indicated that LPC generated by Prdx6 PLA₂ activity remained bound to the enzyme for the reacylation reaction. Prdx6 is the first LPCAT enzyme with demonstrated cytoplasmic localization. Thus, Prdx6 is a complete enzyme comprising both PLA₂ and LPCAT activities for the remodeling pathway of PC synthesis or for repair of membrane lipid peroxidation.     

Characterization of human lysophospholipid acyltransferase 3

Esterifying lysophospholipids may serve a variety of functions, including phospholipid remodeling and limiting the abundance of bioactive lipids. Recently, a yeast enzyme, Lpt1p, that esterifies an array of lysophospholipids was identified. Described here is the characterization of a human homolog of LPT1 that we have called lysophosphatidylcholine acyltransferase 3 (LPCAT3). Expression of LPCAT3 in Sf9 insect cells conferred robust esterification of lysophosphatidylcholine in vitro. Kinetic analysis found apparent cooperativity with a saturated acyl-CoA having the lowest K_0.5 (5 μM), a monounsaturated acyl-CoA having the highest apparent V_max (759 nmol/min/mg), and two polyunsaturated acyl-CoAs showing intermediate values. Lysophosphatidylethanolamine and lysophosphatidylserine were also utilized as substrates. Electrospray ionization mass spectrometric analysis of phospholipids in Sf9 cells expressing LPCAT3 showed a relative increase in phosphatidylcholine containing saturated acyl chains and a decrease in phosphatidylcholine containing unsaturated acyl chains. Targeted reduction of LPCAT3 expression in HEK293 cells had essentially an opposite effect, resulting in decreased abundance of saturated phospholipid species and more unsaturated species. Reduced LPCAT3 expression resulted in more apoptosis and distinctly fewer lamellipodia, suggesting a necessary role for lysophospholipid esterification in maintaining cellular function and structure.     

Radioiodinated antibodies,a tool in studies on the presence and role of inactive enzyme forms: Regulation of chalcone synthase in parsley cell suspension cultures

A new technique, the quantitative determination of total enzyme concentrations by specific immunoprecipitation with purified, radioiodinated antibodies, was used to investigate the presence and possible roles of inactive enzyme in the regulation of chalcone synthase. Dark-grown cell suspension cultures from parsley (Petroselinum hortense) contained neither catalytically active nor detectable amounts of immunoprecipitable chalcone synthase. Irradiation induced large increases and subsequent decreases of both. Significant differences in the peak positions and in the half-lives of active and total chalcone synthase indicated that induced cells contained inactive as well as active enzyme forms. The presence of inactive enzyme could be explained by two different modes of regulation, (i) simultaneous de novo synthesis of active and inactive enzyme (“Simultaneous Model”), or (ii) de novo synthesis of active enzyme only, with sequential steps of inactivation and degradation (“Sequential Model”). Both models were compatible with experimental results, as analyzed mathematically by investigating the relations between curves for rate of enzyme synthesis, enzyme activity, total enzyme, and half-lives of active and total enzyme. However, the “Simultaneous Model” postulated that de novo synthesis of inactive enzyme represented always the vast majority of total enzyme synthesis, while the Sequential Model integrated inactive enzyme with facility in a sequence of irreversible inactivation and degradation of active enzyme. Experiments with repeated induction indicated that cells containing large amounts of inactive enzyme increased enzyme activity by de novo synthesis rather than by activation of preexisting inactive enzyme.     

Wogonin induces differentiation and neurite outgrowth of neural precursor cells

Currently, [³H]uridine is most often used to monitor rRNA synthesis in cultured cells. We show here that radiolabeled ribonucleoside triphosphates, such as [α-³³P]UTP, in culture medium were also incorporated efficiently not only into cells but also into de novo RNA, particularly rRNA. Using this method, we first revealed that endoplasmic reticulum (ER) stress inducers such as tunicamycin and thapsigargin suppressed de novo rRNA synthesis, and that PERK, but not IRE1α or ATF6, mediated the suppression. PERK is known to mediate the suppression of de novo protein synthesis via phosphorylation of eIF2α. Consistently, other translational inhibitors such as PSI, proteasomal inhibitor, and cycloheximide suppressed de novo rRNA synthesis. eIF2α knockdown also suppressed both de novo protein and rRNA syntheses. Furthermore, ER stress reduced cellular ATP levels, and the suppression of rRNA synthesis apparently mitigated their reduction. These observations provided a close link between ATP levels and suppression of de novo rRNA synthesis at ER stress, and we proposed a novel feedback mechanism, in which ATP levels were maintained via suppression of de novo rRNA synthesis in ATP-demanding stresses, such as ER stress.     

Phosphatidylinositol synthesis at the endoplasmic reticulum

Phosphatidylinositol (PI) is a minor phospholipid with a characteristic fatty acid profile; it is highly enriched in stearic acid at the sn-1 position and arachidonic acid at the sn-2 position. PI is phosphorylated into seven specific derivatives, and individual species are involved in a vast array of cellular functions including signalling, membrane traffic, ion channel regulation and actin dynamics. De novo PI synthesis takes place at the endoplasmic reticulum where phosphatidic acid (PA) is converted to PI in two enzymatic steps. PA is also produced at the plasma membrane during phospholipase C signalling, where hydrolysis of phosphatidylinositol (4,5) bisphosphate (PI(4,5)P₂) leads to the production of diacylglycerol which is rapidly phosphorylated to PA. This PA is transferred to the ER to be also recycled back to PI. For the synthesis of PI, CDP-diacylglycerol synthase (CDS) converts PA to the intermediate, CDP-DG, which is then used by PI synthase to make PI. The de novo synthesised PI undergoes remodelling to acquire its characteristic fatty acid profile, which is altered in p53-mutated cancer cells. In mammals, there are two CDS enzymes at the ER, CDS1 and CDS2. In this review, we summarise the de novo synthesis of PI at the ER and the enzymes involved in its subsequent remodelling to acquire its characteristic acyl chains. We discuss how CDS, the rate limiting enzymes in PI synthesis are regulated by different mechanisms. During phospholipase C signalling, the CDS1 enzyme is specifically upregulated by cFos via protein kinase C.     

Lysophosphatidylcholine acyltransferase 3 knockdown-mediated liver lysophosphatidylcholine accumulation promotes very low density lipoprotein production by enhancing microsomal triglyceride transfer protein expression

After de novo biosynthesis phospholipids undergo extensive remodeling by the Lands' cycle. Enzymes involved in phospholipid biosynthesis have been studied extensively but not those involved in reacylation of lysophosphopholipids. One key enzyme in the Lands' cycle is fatty acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), which utilizes lysophosphatidylcholine (LysoPC) and fatty acyl-CoA to produce various phosphatidylcholine (PC) species. Four isoforms of LPCAT have been identified. In this study we found that LPCAT3 is the major hepatic isoform, and its knockdown significantly reduces hepatic LPCAT activity. Moreover, we report that hepatic LPCAT3 knockdown increases certain species of LysoPCs and decreases certain species of PC. A surprising observation was that LPCAT3 knockdown significantly reduces hepatic triglycerides. Despite this, these mice had higher plasma triglyceride and apoB levels. Lipoprotein production studies indicated that reductions in LPCAT3 enhanced assembly and secretion of triglyceride-rich apoB-containing lipoproteins. Furthermore, these mice had higher microsomal triglyceride transfer protein (MTP) mRNA and protein levels. Mechanistic studies in hepatoma cells revealed that LysoPC enhances secretion of apoB but not apoA-I in a concentration-dependent manner. Moreover, LysoPC increased MTP mRNA, protein, and activity. In short, these results indicate that hepatic LPCAT3 modulates VLDL production by regulating LysoPC levels and MTP expression.     

LPS impairs phospholipid synthesis by triggering beta-transducin repeat-containing protein (beta-TrCP)-mediated polyubiquitination and degradation of the surfactant enzyme acyl-CoA:lysophosphatidylcholine acyltransferase I (LPCAT1)

Acyl-CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a relatively newly described and yet indispensable enzyme needed for generation of the bioactive surfactant phospholipid, dipalmitoylphosphatidylcholine (DPPtdCho). Here, we show that lipopolysaccharide (LPS) causes LPCAT1 degradation using the Skp1-Cullin-F-box ubiquitin E3 ligase component, β-transducin repeat-containing protein (β-TrCP), that polyubiquitinates LPCAT1, thereby targeting the enzyme for proteasomal degradation. LPCAT1 was identified as a phosphoenzyme as Ser(178) within a phosphodegron was identified as a putative molecular recognition site for glycogen synthase kinase-3β (GSK-3β) phosphorylation that recruits β-TrCP docking within the enzyme. β-TrCP ubiquitinates LPCAT1 at an acceptor site (Lys(221)), as substitution of Lys(221) with Arg abrogated LPCAT1 polyubiquitination. LPS profoundly reduced immunoreactive LPCAT1 levels and impaired lung surfactant mechanics, effects that were overcome by siRNA to β-TrCP and GSK-3β or LPCAT1 gene transfer, respectively. Thus, LPS appears to destabilize the LPCAT1 protein by GSK-3β-mediated phosphorylation within a canonical phosphodegron for β-TrCP docking and site-specific ubiquitination. LPCAT1 is the first lipogenic substrate for β-TrCP, and the results suggest that modulation of the GSK-3β-SCFβ(TrCP) E3 ligase effector pathway might be a unique strategy to optimize dipalmitoylphosphatidylcholine levels in sepsis.     

Inositol depletion regulates phospholipid metabolism and activates stress signaling in HEK293T cells

Inositol plays a significant role in cellular function and signaling. Studies in yeast have demonstrated an “inositol-less death” phenotype, suggesting that inositol is an essential metabolite. In yeast, inositol synthesis is highly regulated, and inositol levels have been shown to be a major metabolic regulator, with its abundance affecting the expression of hundreds of genes. Abnormalities in inositol metabolism have been associated with several human disorders. Despite its importance, very little is known about the regulation of inositol synthesis and the pathways regulated by inositol in human cells. The current study aimed to address this knowledge gap. Knockout of ISYNA1 (encoding myo-inositol-3-P synthase 1) in HEK293T cells generated a human cell line that is deficient in de novo inositol synthesis. ISYNA1-KO cells exhibited inositol-less death when deprived of inositol. Lipidomic analysis identified inositol deprivation as a global regulator of phospholipid levels in human cells, including downregulation of phosphatidylinositol (PI) and upregulation of the phosphatidylglycerol (PG)/cardiolipin (CL) branch of phospholipid metabolism. RNA-Seq analysis revealed that inositol deprivation induced substantial changes in the expression of genes involved in cell signaling, including extracellular signal-regulated kinase (ERK), and genes controlling amino acid transport and protein processing in the endoplasmic reticulum (ER). This study provides the first in-depth characterization of the effects of inositol deprivation on phospholipid metabolism and gene expression in human cells, establishing an essential role for inositol in maintaining cell viability and regulating cell signaling and metabolism.     

Lysophosphatidylcholine Acyltransferase1 Overexpression Promotes Oral Squamous Cell Carcinoma Progression via Enhanced Biosynthesis of Platelet-Activating Factor

Background

The relevance of lysophosphatidylcholine acyltransferase1 (LPCAT1), a cytosolic enzyme in the remodeling pathway of phosphatidylcholine metabolism, in oral squamous cell carcinoma (OSCC) is unknown. We investigated LPCAT1 expression and its functional mechanism in OSCCs.

Methods

We analyzed LPCAT1 mRNA and protein expression levels in OSCC-derived cell lines. Immunohistochemistry was performed to identify correlations between LPCAT1 expression levels and primary OSCCs clinicopathological status. We established LPCAT1 knockdown models of the OSCC-derived cell lines (SAS, Ca9-22) for functional analysis and examined the association between LPCAT1 expression and the platelet-activating factor (PAF) concentration and PAF-receptor (PAFR) expression.

Results

LPCAT1 mRNA and protein were up-regulated significantly (p<0.05) in OSCC-derived cell lines compared with human normal oral keratinocytes. Immunohistochemistry showed significantly (p<0.05) elevated LPCAT1 expression in primary OSCCs compared with normal counterparts and a strong correlation between LPCAT1-positive OSCCs and tumoral size and regional lymph node metastasis. In LPCAT1 knockdown cells, cellular proliferation and invasiveness decreased significantly (p<0.05); cellular migration was inhibited compared with control cells. Down-regulation of LPCAT1 resulted in a decreased intercellular PAF concentration and PAFR expression.

Conclusion

LPCAT1 was overexpressed in OSCCs and correlated with cellular invasiveness and migration. LPCAT1 may contribute to tumoral growth and metastasis in oral cancer.     

Localized de novo phospholipid synthesis drives autophagosome biogenesis

ABSTRACT

During (macro)autophagy, cells form transient organelles, termed autophagosomes, to target a broad spectrum of substrates for degradation critical to cellular and organismal health. Driven by rapid membrane assembly, an initially small vesicle (phagophore) elongates into a large cup-shaped structure to engulf substrates within a few minutes in a double-membrane autophagosome. In particular, how autophagic membranes expand has been a longstanding question. Here, we summarize our recent work that delineates a pathway that drives phagophore expansion by localized de novo phospholipid synthesis. Specifically, we found that the conserved acyl-CoA synthetase Faa1 localizes to nucleated phagophores to locally activate fatty acids for de novo phospholipid synthesis in the neighboring ER. These newly synthesized phospholipids are then preferentially incorporated into autophagic membranes and drive the expansion of the phagophore into a functional autophagosome. In summary, our work uncovers molecular principles of how cells coordinate phospholipid synthesis and flux with autophagic membrane formation during autophagy.

Abbreviations: ACS: acyl-CoA synthestases; CoA: coenzyme A; ER: endoplasmic reticulum     

Nuclear Enrichment of Folate Cofactors and Methylenetetrahydrofolate Dehydrogenase 1 (MTHFD1) Protect de Novo Thymidylate Biosynthesis during Folate Deficiency

Folate-mediated one-carbon metabolism is a metabolic network of interconnected pathways that is required for the de novo synthesis of three of the four DNA bases and the remethylation of homocysteine to methionine. Previous studies have indicated that the thymidylate synthesis and homocysteine remethylation pathways compete for a limiting pool of methylenetetrahydrofolate cofactors and that thymidylate biosynthesis is preserved in folate deficiency at the expense of homocysteine remethylation, but the mechanisms are unknown. Recently, it was shown that thymidylate synthesis occurs in the nucleus, whereas homocysteine remethylation occurs in the cytosol. In this study we demonstrate that methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), an enzyme that generates methylenetetrahydrofolate from formate, ATP, and NADPH, functions in the nucleus to support de novo thymidylate biosynthesis. MTHFD1 translocates to the nucleus in S-phase MCF-7 and HeLa cells. During folate deficiency mouse liver MTHFD1 levels are enriched in the nucleus >2-fold at the expense of levels in the cytosol. Furthermore, nuclear folate levels are resistant to folate depletion when total cellular folate levels are reduced by >50% in mouse liver. The enrichment of folate cofactors and MTHFD1 protein in the nucleus during folate deficiency in mouse liver and human cell lines accounts for previous metabolic studies that indicated 5,10-methylenetetrahydrofolate is preferentially directed toward de novo thymidylate biosynthesis at the expense of homocysteine remethylation during folate deficiency.     

Phosphatidylthreonine and Lipid-Mediated Control of Parasite Virulence

The major membrane phospholipid classes, described thus far, include phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). Here, we demonstrate the natural occurrence and genetic origin of an exclusive and rather abundant lipid, phosphatidylthreonine (PtdThr), in a common eukaryotic model parasite, Toxoplasma gondii. The parasite expresses a novel enzyme PtdThr synthase (TgPTS) to produce this lipid in its endoplasmic reticulum. Genetic disruption of TgPTS abrogates de novo synthesis of PtdThr and impairs the lytic cycle and virulence of T. gondii. The observed phenotype is caused by a reduced gliding motility, which blights the parasite egress and ensuing host cell invasion. Notably, the PTS mutant can prevent acute as well as yet-incurable chronic toxoplasmosis in a mouse model, which endorses its potential clinical utility as a metabolically attenuated vaccine. Together, the work also illustrates the functional speciation of two evolutionarily related membrane phospholipids, i.e., PtdThr and PtdSer.     

Cellular responses to excess phospholipid

Phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells, and its synthesis is controlled by the activity of CDP:phosphocholine cytidylyltransferase (CCT). Enforced CCT expression accelerated the rate of PtdCho synthesis. However, the amount of cellular PtdCho did not increase as a result of the turnover of both the choline and glycerol components of PtdCho. Metabolic labeling experiments demonstrated that cells compensated for elevated CCT activity by the degradation of PtdCho to glycerophosphocholine (GPC). Phospholipase D-mediated PtdCho hydrolysis and phosphocholine formation were unaffected. Most of the GPC produced in response to excess phospholipid production was secreted into the medium. Cells also degraded the excess membrane PtdCho to GPC when phospholipid formation was increased by exposure to exogenous lysophosphatidylcholine or lysophosphatidylethanolamine. The replacement of the acyl moiety at the 1-position of PtdCho with a non-hydrolyzable alkyl moiety prevented degradation to GPC. Accumulation of alkylacyl-PtdCho was associated with the inhibition of cell proliferation, demonstrating that alternative pathways of degradation will not substitute. GPC formation was blocked by bromoenol lactone, implicating the calcium-independent phospholipase A2 as a key participant in the response to excess phospholipid. Owing to the fact that PtdCho is biosynthetically converted to PtdEtn, excess PtdCho resulted in overproduction and exit of GPE as well as GPC. Thus, general membrane phospholipid homeostasis is achieved by a balance between the opposing activities of CCT and phospholipase A2.     

FKBP38 Protects Bcl-2 from Caspase-dependent Degradation

The cellular processes that regulate Bcl-2 at the posttranslational levels are as important as those that regulate bcl-2 synthesis. Previously we demonstrated that the suppression of FK506-binding protein 38 (FKBP38) contributes to the instability of Bcl-2 or leaves Bcl-2 unprotected from degradation in an unknown mechanism. Here, we studied the underlying molecular mechanism mediating this process. We first showed that Bcl-2 binding-defective mutants of FKBP38 fail to accumulate Bcl-2 protein. We demonstrated that the FKBP38-mediated Bcl-2 stability is specific as the levels of other anti-apoptotic proteins such as Bcl-X^L and Mcl-1 remained unaffected. FKBP38 enhanced the Bcl-2 stability under the blockade of de novo protein synthesis, indicating it is posttranslational. We showed that the overexpression of FKBP38 attenuates reduction rate of Bcl-2, thus resulting in an increment of the intracellular Bcl-2 level, contributing to the resistance of apoptotic cell death induced by the treatment of kinetin riboside, an anticancer drug. Caspase inhibitors markedly induced the accumulation of Bcl-2. In caspase-3-activated cells, the knockdown of endogenous FKBP38 by small interfering RNA resulted in Bcl-2 down-regulation as well, which was significantly recovered by the treatment with caspase inhibitors or overexpression of FKBP38. Finally we presented that the Bcl-2 cleavage by caspase-3 is blocked when Bcl-2 binds to FKBP38 through the flexible loop. Taken together, these results suggest that FKBP38 is a key player in regulating the function of Bcl-2 by antagonizing caspase-dependent degradation through the direct interaction with the flexible loop domain of Bcl-2, which contains the caspase cleavage site.     

Phosphorylation of Lysophosphatidylcholine Acyltransferase 2 at Ser34 Enhances Platelet-activating Factor Production in Endotoxin-stimulated Macrophages

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that elicits various cellular functions under physiological and pathological conditions. We have recently identified two enzymes involved in PAF production: lysophosphatidylcholine acyltransferase-1 (LPCAT1) and LPCAT2. We found that LPCAT2 is highly expressed in inflammatory cells and is activated by lipopolysaccharide (LPS) treatment through Toll-like receptor 4. However, the molecular mechanism for the activation remains elusive. In this study, Phos-tag SDS-PAGE revealed the LPS-induced phosphorylation of LPCAT2. Furthermore, mass spectrometry and mutagenesis analyses identified Ser³⁴ of LPCAT2 as the phosphorylation site to enhance the catalytic activities. The experiments using inhibitors and siRNA against MAPK cascades demonstrated that LPCAT2 phosphorylation through LPS-TLR4 signaling may directly depend on MAPK-activated protein kinase 2 (MAPKAP kinase 2 or MK2). These findings develop a further understanding of both PAF production and phospholipid remodeling triggered by inflammatory stimuli. Specific inhibition of the PAF biosynthetic activity by phosphorylated LPCAT2 will provide a novel target for the regulation of inflammatory disorders.     

The dynamics of cardiolipin synthesis post-mitochondrial fusion

Alteration in mitochondrial fusion may regulate mitochondrial metabolism. Since the phospholipid cardiolipin (CL) is required for function of the mitochondrial respiratory chain, we examined the dynamics of CL synthesis in growing Hela cells immediately after and 12 h post-fusion. Cells were transiently transfected with Mfn-2, to promote fusion, or Mfn-2 expressing an inactive GTPase for 24 h and de novo CL biosynthesis was examined immediately after or 12 h post-fusion. Western blot analysis confirmed elevated Mfn-2 expression and electron microscopic analysis revealed that Hela cell mitochondrial structure was normal immediately after and 12 h post-fusion. Cells expressing Mfn-2 exhibited reduced CL de novo biosynthesis from [1,3-³H]glycerol immediately after fusion and this was due to a decrease in phosphatidylglycerol phosphate synthase (PGPS) activity and its mRNA expression. In contrast, 12 h post-mitochondrial fusion cells expressing Mfn-2 exhibited increased CL de novo biosynthesis from [1,3-³H]glycerol and this was due to an increase in PGPS activity and its mRNA expression. Cells expressing Mfn-2 with an inactive GTPase activity did not exhibit alterations in CL de novo biosynthesis immediately after or 12 h post-fusion. The Mfn-2 mediated alterations in CL de novo biosynthesis were not accompanied by alterations in CL or monolysoCL mass. [1-¹⁴C]Oleate incorporation into CL was elevated at 12 h post-fusion indicating increased CL resynthesis. The reason for the increased CL resynthesis was an increased mRNA expression of tafazzin, a mitochondrial CL resynthesis enzyme. Ceramide-induced expression of PGPS in Hela cells or in CHO cells did not alter expression of Mfn-2 indicating that Mfn-2 expression is independent of altered CL synthesis mediated by elevated PGPS. In addition, Mfn-2 expression was not altered in Hela cells expressing phospholipid scramblase-3 or a disrupted scramblase indicating that proper CL localization within mitochondria is not essential for Mfn-2 expression. The results suggest that immediately post-mitochondrial fusion CL de novo biosynthesis is “slowed down” and then 12 h post-fusion it is “upregulated”. The implications of this are discussed.