Avanti Polar Lipids Research Articles

Identification of a Domain That Mediates Association of Platelet-activating Factor Acetylhydrolase with High Density Lipoprotein

The plasma form of platelet-activating factor (PAF) acetylhydrolase (PAF-AH), also known as lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) inactivates potent lipid messengers such as PAF and modified phospholipids generated in settings of oxidant stress. In humans, PAF-AH circulates in blood in fully active form and associates with high and low density lipoproteins (HDL and LDL). Several studies suggest that the location of PAF-AH affects both the catalytic efficiency and the function of the enzyme in vivo. The distribution of PAF-AH among lipoproteins varies widely among mammals. Here, we report that mouse and human PAF-AHs associate with human HDL particles of different density. We made use of this observation in the development of a binding assay to identify domains required for association of human PAF-AH with human HDL. Sequence comparisons among species combined with domain-swapping and site-directed mutagenesis studies led us to the identification of C-terminal residues necessary for the association of human PAF-AH with human HDL. Interestingly, the region identified is not conserved among PAF-AHs, suggesting that PAF-AH interacts with HDL particles in a manner that is unique to each species. These findings contribute to our understanding of the mechanisms responsible for association of human PAF-AH with HDL and may facilitate future studies aimed at precisely determining the function of PAF-AH in each lipoprotein particle.

Yeast Pgc1p (YPL206c) Controls the Amount of Phosphatidylglycerol via a Phospholipase C-type Degradation Mechanism

The product of the open reading frame YPL206c, Pgc1p, of the yeast Saccharomyces cerevisiae displays homology to bacterial and mammalian glycerophosphodiester phosphodiesterases. Deletion of PGC1 causes an accumulation of the anionic phospholipid, phosphatidylglycerol (PG), especially under conditions of inositol limitation. This PG accumulation was not caused by increased production of phosphatidyl-glycerol phosphate or by decreased consumption of PG in the formation of cardiolipin, the end product of the pathway. PG accumulation in the pgc1Delta strain was caused rather by inactivation of the PG degradation pathway. Our data demonstrate an existence of a novel regulatory mechanism in the cardiolipin biosynthetic pathway in which Pgc1p is required for the removal of excess PG via a phospholipase C-type degradation mechanism.

Detailed Mechanisms of the Inactivation of Factor VIIIa by Activated Protein C in the Presence of Its Cofactors, Protein S and Factor V

Factor VIIIa is inactivated by a combination of two mechanisms. Activation of factor VIII by thrombin results in a heterotrimeric factor VIIIa that spontaneously inactivates due to dissociation of the A2 subunit. Additionally, factor VIIIa is cleaved by the anticoagulant serine protease, activated protein C, at two cleavage sites, Arg(336) in the A1 subunit and Arg(562) in the A2 subunit. We previously characterized an engineered variant of factor VIII which contains a disulfide bond between the A2 and the A3 subunits that prevents the spontaneous dissociation of the A2 subunit following thrombin activation. Thus, in the absence of activated protein C, this variant has stable activity following activation by thrombin. To isolate the effects of the individual activated protein C cleavage sites on factor VIIIa, we engineered mutations of the activated protein C cleavage sites into the disulfide bond-cross-linked factor VIII variant. Arg(336) cleavage is 6-fold faster than Arg(562) cleavage, and the Arg(336) cleavage does not fully inactivate factor VIIIa when A2 subunit dissociation is blocked. Protein S enhances both cleavage rates but enhances Arg(562) cleavage more than Arg(336) cleavage. Factor V also enhances both cleavage rates when protein S is present. Factor V enhances Arg(562) cleavage more than Arg(336) cleavage as well. As a result, in the presence of both activated protein C cofactors, Arg(336) cleavage is only twice as fast as Arg(562) cleavage. Therefore, both cleavages contribute significantly to factor VIIIa inactivation.

An analysis of the role of a retroendocytosis pathway in ABCA1-mediated cholesterol efflux from macrophages

The ATP binding cassette transporter A-1 (ABCA1) is critical for apolipoprotein-mediated cholesterol efflux, an important mechanism employed by macrophages to avoid becoming lipid-laden foam cells, the hallmark of early atherosclerotic lesions. It has been proposed that lipid-free apolipoprotein A-I (apoA-I) enters the cell and is resecreted as a lipidated particle via a retroendocytosis pathway during ABCA1-mediated cholesterol efflux from macrophages. To determine the functional importance of such a pathway, confocal microscopy was used to characterize the internalization of a fully functional apoA-I cysteine mutant containing a thiol-reactive fluorescent probe in cultured macrophages. ApoA-I was also endogenously labeled with (35)S-methionine to quantify cellular uptake and to determine the metabolic fate of the internalized protein. It was found that apoA-I was specifically taken inside macrophages and that a small amount of intact apoA-I was resecreted from the cells. However, a majority of the label that reappeared in the media was degraded. We estimate that the mass of apoA-I retroendocytosed is not sufficient to account for the HDL produced by the cholesterol efflux reaction. Furthermore, we have demonstrated that lipid-free apoA-I-mediated cholesterol efflux from macrophages can be pharmacologically uncoupled from apoA-I internalization into cells. On the basis these findings, we present a model in which the ABCA1-mediated lipid transfer process occurs primarily at the membrane surface in macrophages, but still accounts for the observed specific internalization of apoA-I.

Unexpected Complexity in the Mechanisms That Target Assembly of the Spectrin Cytoskeleton

The spectrin cytoskeleton assembles within discrete regions of the plasma membrane in a wide range of animal cell types. Although recent studies carried out in vertebrate systems indicate that spectrin assembly occurs indirectly through the adapter protein ankyrin, recent studies in Drosophila have established that spectrin can also assemble through a direct ankyrin-independent mechanism. Here we tested specific regions of the spectrin molecule for a role in polarized assembly and function. First, we tested mutant beta-spectrins lacking ankyrin binding activity and/or the COOH-terminal pleckstrin homology (PH) domain for their assembly competence in midgut, salivary gland, and larval brain. Remarkably, three different assembly mechanisms operate in these three cell types: 1) neither site was required for assembly in salivary gland; 2) only the PH domain was required in midgut copper cells; and 3) either one of the two sites was sufficient for spectrin assembly in larval brain. Further characterization of the PH domain revealed that it binds strongly to lipid mixtures containing phosphatidylinositol 4,5-bisphosphate (PIP(2)) but not phosphatidylinositol 3,4,5-trisphosphate. A K8Q mutation in the lipid binding region of the PH domain eliminated the PIP(2) interaction in vitro, yet the mutant protein retained full biological function in vivo. Reporter gene studies revealed that PIP(2) and the spectrin PH domain codistribute with one another in cells but not with authentic wild type alphabeta-spectrin. Thus, it appears that the PH domain imparts membrane targeting activity through a second mechanism that takes precedence over its PIP(2) binding activity.

The relationship between the metabolism of sphingomyelin species and the hemolysis of sheep erythrocytes induced by Clostridium perfringens α-toxin

Clostridium perfringens alpha-toxin induces the hemolysis of sheep erythrocytes by activating the metabolism of sphingomyelin (SM) via a GTP binding protein in membranes. alpha-Toxin stimulated the formation of 15-N-nervonoyl sphingosine (C24:1-ceramide), which was identified by positive ion fast atom bombardment-MS and 1H-NMR spectroscopy. C24:1-ceramide stimulated the toxin-induced hemolysis of saponin-pretreated sheep erythrocytes and increased the production of sphingosine 1-phosphate (S1P) in the cells, but N-lignoceroyl sphingosine did not. These events elicited by the toxin in the presence of C24:1-ceramide were significantly attenuated by treatment with dihydrosphingosine, a sphingosine kinase inhibitor. TLC showed that the level of C24:1-ceramide was highest among the ceramides with an unsaturated bond in the fatty acyl chain in the detergent-resistant membranes (DRMs). The toxin specifically bound to DRMs rich in cholesterol, resulting in the hydrolysis of N-nervonoic sphingomyelin (C24:1-SM) in DRMs. Treatment of the cells with pertussis toxin (PT) inhibited the alpha-toxin-induced formation of C24:1-ceramide from C24:1-SM in DRMs and hemolysis, indicating that endogenous sphingomyelinase, which hydrolyzes C24:1-SM to C24:1-ceramide, is controlled by PT-sensitive GTP binding protein in membranes. These results show that the toxin-induced metabolism of C24:1-SM to S1P in DRMs plays an important role in the toxin-induced hemolysis of sheep erythrocytes.

Probing Structure-Function Relationships and Gating Mechanisms in the CorA Mg2+ Transport System

Recent crystal structures of the CorA Mg(2+) transport protein from Thermotoga maritima (TmCorA) revealed an unusually long ion pore putatively gated by hydrophobic residues near the intracellular end and by universally conserved asparagine residues at the periplasmic entrance. A conformational change observed in an isolated funnel domain structure also led to a proposal for the structural basis of gating. Because understanding the molecular mechanisms underlying ion channel and transporter gating remains an important challenge, we have undertaken a structure-guided engineering approach to probe structure-function relationships in TmCorA. The intracellular funnel domain is shown to constitute an allosteric regulatory module that can be engineered to promote an activated or closed state. A periplasmic gate centered about a proline-induced kink of the pore-lining helix is described where "helix-straightening" mutations produce a dramatic gain-of-function. Mutation to the narrowest constriction along the pore demonstrates that a hydrophobic gate is operational within this Mg(2+)-selective transport protein and likely forms an energetic barrier to ion flux. We also provide evidence that highly conserved acidic residues found in the short periplasmic loop are not essential for TmCorA function or Mg(2+) selectivity but may be required for proper protein folding and stability. This work extends our gating model for the CorA-Alr1-Mrs2 superfamily and reveals features that are characteristic of an ion channel. Aspects of these results that have broader implications for a range of channel and transporter families are highlighted.

BIM and tBID Are Not Mechanistically Equivalent When Assisting BAX to Permeabilize Bilayer Membranes

BIM and tBID are two BCL-2 homology 3 (BH3)-only proteins with a particularly strong capacity to trigger BAX-driven mitochondrial outer membrane permeabilization, a crucial event in mammalian apoptosis. However, the means whereby BIM and tBID fulfill this task is controversial. Here, we used a reconstituted liposomal system bearing physiological relevance to explore systematically how the BAX-permeabilizing function is influenced by interactions of BIM/BID-derived proteins and BH3 motifs with multidomain BCL-2 family members and with membrane lipids. We found that nanomolar dosing of BIM proteins sufficed to reverse completely the inhibition of BAX permeabilizing activity exerted by all antiapoptotic proteins tested (BCL-2, BCL-X(L), BCL-W, MCL-1, and A1). This effect was reproducible by a peptide representing the BH3 motif of BIM, whereas an equivalent BID BH3 peptide was less potent and more selective, reversing antiapoptotic inhibition. On the other hand, in the absence of BCL-2-type proteins, BIM proteins and the BIM BH3 peptide were inefficient, directly triggering the BAX-permeabilizing function. In contrast, tBID alone potently assisted BAX to permeabilize membranes at least in part by producing a structural distortion in the lipid bilayer via BH3-independent interaction of tBID with cardiolipin. Together, these results support the notion that BIM and tBID follow different strategies to trigger BAX-driven mitochondrial outer membrane permeabilization with strong potency.

Kinetic Analysis of the Interaction of the C1 Domain of Protein Kinase C with Lipid Membranes by Stopped-flow Spectroscopy

The diacylglycerol (DG)/phorbol ester-dependent translocation of conventional protein kinase C (PKC) isozymes is mediated by the C1 domain, a membrane-targeting module that also selectively binds phosphatidylserine (PS). Using stopped-flow spectroscopy, we dissect the contribution of DG/phorbol esters (C1 ligand) and PS in driving the association and dissociation of the C1 domain from membranes. Specifically, we examine the binding to membranes of the C1B domain of PKCbeta with a substituted Trp (Y123W) whose fluorescence is quenched upon binding to membranes. Binding of this construct (C1Bbeta-Y123W) to phospholipid vesicles is cooperative with respect to PS content and dependent on C1 ligand, as previously characterized. Stopped-flow analysis reveals that the apparent association rate (k(on)(app)), but not the apparent dissociation rate (k(off)(app)), is highly sensitive to PS content: the 60-fold increase in membrane affinity for vesicles containing no PS compared with 40 mol % PS results primarily from a robust (30-fold) increase in k(on)(app) with little effect (2-fold) on k(off)(app). Membrane affinity is also controlled by the content and structure of the C1 ligand. In contrast to PS, these ligands markedly alter k(off)(app) with smaller effects on k(on)(app). We also show that the affinity for phorbol ester-containing membranes is 2 orders of magnitude higher than that for DG-containing membranes primarily resulting from differences in k(off)(app). Our data are consistent with a model in which the C1 domain is recruited to the membrane via an initial weak electrostatic interaction with PS, followed by a rapid two-dimensional search for ligand, the binding of which retains the domain at the membrane. Thus, PS drives the initial encounter, and DG/phorbol esters retain the domain on membranes. The decreased effectiveness of DG compared with phorbol esters in retaining the C1 domain on membranes contributes to the molecular dichotomy of the rapid, transient nature of DG-dependent PKC signaling versus the chronic hyperactivity of phorbol ester-activated PKC.

Different Mechanisms Regulate Lysophosphatidic Acid (LPA)-dependent Versus Phorbol Ester-dependent Internalization of the LPA1 Receptor

Lysophosphatidic acid (LPA) stimulates cells by activation of five G-protein-coupled receptors, termed LPA 1-5. The LPA 1 receptor is the most widely expressed and is a major regulator of cell migration. In this study, we show that phorbol ester (PMA)-induced internalization of the LPA(1) receptor requires clathrin AP-2 complexes, protein kinase C, and a distal dileucine motif (amino acids 352 and 353) in the cytoplasmic tail but not beta-arrestin. Agonist-dependent internalization of LPA 1, however, requires a cluster of serine residues (amino acids 341-347) located proximal to the dileucine motif, beta-arrestin, and to a lesser extent clathrin AP-2. The serine cluster of LPA 1 is required for beta-arrestin2-GFP translocation to the plasma membrane and signal desensitization. In contrast, the dileucine motif (IL) is required for both basal and PMA-induced internalization. Evidence for the beta-arrestin independence of PMA-induced internalization of LPA 1 comes from the observations that beta-arrestin2-GFP is not recruited to the plasma membrane upon PMA treatment and that LPA 1 is readily internalized in beta-arrestin1/2 knock-out mouse embryonic fibroblasts. These results indicate that distinct molecular mechanisms regulate agonist-dependent and PMA-dependent internalization of the LPA 1 receptor.

Enhancement of the enzymatic activity of activated coagulation factor IX by anti-factor IX antibodies

Enhancement of the enzymatic activity of activated coagulation factor IX by anti-factor IX antibodies

The Key Residue for Substrate Transport (Glu14) in the EmrE Dimer Is Asymmetric

Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli multidrug transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms a functional dimer to transport a diverse array of aromatic, positively charged substrates in a proton/drug antiport fashion. Here, we report (13)C chemical shifts of the essential residue Glu(14) within the binding pocket. To ensure a native environment, EmrE was reconstituted into E. coli lipids. Experiments were carried out using one- and two-dimensional double quantum filtered (13)C solid state NMR. For an unambiguous assignment of Glu(14), an E25A mutation was introduced to create a single glutamate mutant. Glu(14) was (13)C-labeled using cell-free expression. Purity, labeling, homogeneity, and functionality were probed by mass spectrometry, NMR spectroscopy, freeze fracture electron microscopy, and transport assays. For Glu(14), two distinct sets of chemical shifts were observed that indicates structural asymmetry in the binding pocket of homodimeric EmrE. Upon addition of ethidium bromide, chemical shift changes and altered line shapes were observed, demonstrating substrate coordination by both Glu(14) in the dimer.

Dimeric Structure of Human Na+/H+ Exchanger Isoform 1 Overproduced in Saccharomyces cerevisiae

The Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H(+) in exchange for one extracellular Na(+). The human NHE1 isoform is involved in heart disease and cell growth and proliferation. Although details of NHE1 regulation and transport are being revealed, there is little information available on the structure of the intact protein. In this report, we demonstrate overexpression, purification, and characterization of the human NHE1 (hNHE1) protein in Saccharomyces cerevisiae. Overproduction of the His-tagged protein followed by purification via nickel-nitrilotriacetic acid-agarose chromatography yielded 0.2 mg of pure protein/liter of cell culture. Reconstitution of hNHE1 in proteoliposomes demonstrated that the protein was active and responsive to an NHE1-specific inhibitor. Circular dichroism spectroscopy of purified hNHE1 revealed that the protein contains 41% alpha-helix, 23% beta-sheet, and 36% random coil. Size exclusion chromatography indicated that the protein-detergent micelle was in excess of 200 kDa, consistent with an hNHE1 dimer. Electron microscopy and single particle reconstruction of negatively stained hNHE1 confirmed that the protein was a dimer, with a compact globular domain assigned to the transmembrane region and an apical ridge assigned to the cytoplasmic domain. The transmembrane domain of the hNHE1 reconstruction was clearly dimeric, where each monomer had a size and shape consistent with the predicted 12 membrane-spanning segments for hNHE1.

The Atg12-Atg5 Conjugate Has a Novel E3-like Activity for Protein Lipidation in Autophagy

Autophagy is a bulk degradation process in eukaryotic cells; autophagosomes enclose cytoplasmic components for degradation in the lysosome/vacuole. Autophagosome formation requires two ubiquitin-like conjugation systems, the Atg12 and Atg8 systems, which are tightly associated with expansion of autophagosomal membrane. Previous studies have suggested that there is a hierarchy between these systems; the Atg12 system is located upstream of the Atg8 system in the context of Atg protein organization. However, the concrete molecular relationship is unclear. Here, we show using an in vitro Atg8 conjugation system that the Atg12-Atg5 conjugate, but not unconjugated Atg12 or Atg5, strongly enhances the formation of the other conjugate, Atg8-PE. The Atg12-Atg5 conjugate promotes the transfer of Atg8 from Atg3 to the substrate, phosphatidylethanolamine (PE), by stimulating the activity of Atg3. We also show that the Atg12-Atg5 conjugate interacts with both Atg3 and PE-containing liposomes. These results indicate that the Atg12-Atg5 conjugate is a ubiquitin-protein ligase (E3)-like enzyme for Atg8-PE conjugation reaction, distinctively promoting protein-lipid conjugation.

Phosphatidylinositol 4-Phosphate 5-Kinase Reduces Cell Surface Expression of the Epithelial Sodium Channel (ENaC) in Cultured Collecting Duct Cells

Ubiquitination of ENaC subunits has been shown to negatively regulate the cell surface expression of ENaC channels. We have previously demonstrated that epsin links ubiquitinated ENaC to clathrin adaptors for clathrin-mediated endocytosis. Epsin is thought to directly modify the curvature of membranes upon binding to phosphatidylinositol 4,5-bisphosphate (PIP2) where it recruits clathrin and stimulates lattice assembly. Murine phosphatidylinositol 4-phosphate 5-kinase alpha (PI5KIalpha) has been shown to enhance endocytosis in a PIP2-dependent manner. We tested the hypothesis that PI5KIalpha-mediated PIP2 production would negatively regulate ENaC current by enhancing epsin-mediated endocytosis of the channel. Expression of PI5KIalpha decreased ENaC currents in Xenopus oocytes by 80%, entirely because of a decrease in cell surface ENaC levels. Catalytically inactive mutants of PI5Kalpha had no effect on ENaC activity. Expression of the PIP2 binding region of epsin increased ENaC current in oocytes, an effect completely reversed by co-expression of PI5KIalpha. Overexpression of epsin reduced amiloride-sensitive current in CCD cells. Overexpression of PI5KIalpha enhanced membrane PIP2 levels and reduced apical surface expression of ENaC in CCD cells, down-regulating amiloride-sensitive current. Knockdown of PI5KIalpha with isoform-specific siRNA resulted in a 4-fold enhancement of ENaC activity. PI5KIalpha localized exclusively to the apical plasma membrane domain when overexpressed in mouse CCD cells, consistent for a role in regulating PIP2 production at the apical plasma membrane. We conclude that membrane turnover events regulating ENaC surface expression and activity in oocytes and CCD cells can be regulated by PI5KIalpha.

Anionic lipids activate group IVA cytosolic phospholipase A2 via distinct and separate mechanisms

Previously, ceramide-1-phosphate (C1P) and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] were demonstrated to be potent and specific activators of group IVA cytosolic phospholipase A2 (cPLA2alpha). In this study, we hypothesized that these anionic lipids functionally activated the enzyme by distinctly different mechanisms. Indeed, surface plasmon resonance and surface dilution kinetics demonstrated that C1P was a more potent effector than PI(4,5)P2 in decreasing the dissociation constant of the cPLA2alpha-phosphatidylcholine (PC) interaction and increasing the residence time of the enzyme on the vesicles/micelles. PI(4,5)P2, in contrast to C1P, decreased the Michaelis-Menten constant, increasing the catalytic efficiency of the enzyme. Furthermore, PI(4,5)P2 activated cPLA2alpha with a stoichiometry of 1:1 versus C1P at 2.4:1. Lastly, PI(4,5)P2, but not C1P, increased the penetration ability of cPLA2alpha into PC-rich membranes. Therefore, this study demonstrates two distinct mechanisms for the activation of cPLA2alpha by anionic lipids. First, C1P activates cPLA2alpha by increasing the residence time of the enzyme on membranes. Second, PI(4,5)P2 activates the enzyme by increasing catalytic efficiency through increased membrane penetration.

Protein Kinase Cϵ Regulates γ-Aminobutyrate Type A Receptor Sensitivity to Ethanol and Benzodiazepines through Phosphorylation of γ2 Subunits

Ethanol enhances gamma-aminobutyrate (GABA) signaling in the brain, but its actions are inconsistent at GABA(A) receptors, especially at low concentrations achieved during social drinking. We postulated that the epsilon isoform of protein kinase C (PKCepsilon) regulates the ethanol sensitivity of GABA(A) receptors, as mice lacking PKCepsilon show an increased behavioral response to ethanol. Here we developed an ATP analog-sensitive PKCepsilon mutant to selectively inhibit the catalytic activity of PKCepsilon. We used this mutant and PKCepsilon(-/-) mice to determine that PKCepsilon phosphorylates gamma2 subunits at serine 327 and that reduced phosphorylation of this site enhances the actions of ethanol and benzodiazepines at alpha1beta2gamma2 receptors, which is the most abundant GABA(A) receptor subtype in the brain. Our findings indicate that PKCepsilon phosphorylation of gamma2 regulates the response of GABA(A) receptors to specific allosteric modulators, and, in particular, PKCepsilon inhibition renders these receptors sensitive to low intoxicating concentrations of ethanol.

Genetic Ablation of Calcium-independent Phospholipase A2γ Leads to Alterations in Mitochondrial Lipid Metabolism and Function Resulting in a Deficient Mitochondrial Bioenergetic Phenotype

Previously, we identified a novel calcium-independent phospholipase, designated calcium-independent phospholipase A(2) gamma (iPLA(2)gamma), which possesses dual mitochondrial and peroxisomal subcellular localization signals. To identify the roles of iPLA(2)gamma in cellular bioenergetics, we generated mice null for the iPLA(2)gamma gene by eliminating the active site of the enzyme through homologous recombination. Mice null for iPLA(2)gamma display multiple bioenergetic dysfunctional phenotypes, including 1) growth retardation, 2) cold intolerance, 3) reduced exercise endurance, 4) greatly increased mortality from cardiac stress after transverse aortic constriction, 5) abnormal mitochondrial function with a 65% decrease in ascorbate-induced Complex IV-mediated oxygen consumption, and 6) a reduction in myocardial cardiolipin content accompanied by an altered cardiolipin molecular species composition. We conclude that iPLA(2)gamma is essential for maintaining efficient bioenergetic mitochondrial function through tailoring mitochondrial membrane lipid metabolism and composition.

LPT1 Encodes a Membrane-bound O-Acyltransferase Involved in the Acylation of Lysophospholipids in the Yeast Saccharomyces cerevisiae

Phospholipids are major components of cellular membranes that participate in a range of cellular processes. Phosphatidic acid (PA) is a key molecule in the phospholipid biosynthetic pathway. In Saccharomyces cerevisiae, SLC1 has been identified as the gene encoding lysophosphatidic acid acyltransferase, which catalyzes PA synthesis. However, despite the importance of PA, disruption of SLC1 does not affect cell viability (Nagiec, M. M., Wells, G. B., Lester, R. L., and Dickson, R. C. (1993) J. Biol. Chem. 268, 22156-22163). We originally aimed to identify the acetyl-CoA:lyso platelet-activating factor acetyltransferase (lysoPAF AT) gene in yeast. Screening of a complete set of yeast deletion clones (4741 homozygous diploid clones) revealed a single mutant strain, YOR175c, with a defect in lysoPAF AT activity. YOR175c has been predicted to be a member of the membrane-bound O-acyltransferase superfamily, and we designated the gene LPT1. An Lpt1-green fluorescent protein fusion protein localized at the endoplasmic reticulum. Other than lysoPAF AT activity, Lpt1 catalyzed acyltransferase activity with a wide variety of lysophospholipids as acceptors, including lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, and lysophosphatidylserine. A liquid chromatography-mass spectrometry analysis indicated that lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in the Deltalpt1 mutant strain. Although the Deltalpt1 mutant strain did not show other detectable defects, the Deltalpt1 Deltaslc1 double mutant strain had a synthetic lethal phenotype. These results indicate that, in concert with Slc1, Lpt1 plays a central role in PA biosynthesis, which is essential for cell viability.

ApoA-I cleaved by transthyretin has reduced ability to promote cholesterol efflux and increased amyloidogenicity

A fraction of plasma transthyretin (TTR) circulates in HDL through binding to apolipoprotein A-I (apoA-I). Moreover, TTR is able to cleave the C terminus of lipid-free apoA-I. In this study, we addressed the relevance of apoA-I cleavage by TTR in lipoprotein metabolism and in the formation of apoA-I amyloid fibrils. We determined that TTR may also cleave lipidated apoA-I, with cleavage being more effective in the lipid-poor prebeta-HDL subpopulation. Upon TTR cleavage, discoidal HDL particles displayed a reduced capacity to promote cholesterol efflux from cholesterol-loaded THP-1 macrophages. In similar assays, TTR-containing HDL from mice expressing human TTR in a TTR knockout background had a decreased ability to perform reverse cholesterol transport compared with similar particles from TTR knockout mice, reinforcing the notion that cleavage by TTR reduces the ability of apoA-I to promote cholesterol efflux. As amyloid deposits composed of N-terminal apoA-I fragments are common in the atherosclerotic intima, we assessed the impact of TTR cleavage on apoA-I aggregation and fibrillar growth. We determined that TTR-cleaved apoA-I has a high propensity to form aggregated particles and that it formed fibrils faster than full-length apoA-I, as assessed by electron microscopy. Our results show that apoA-I cleavage by TTR may affect HDL biology and the development of atherosclerosis by reducing cholesterol efflux and increasing the apoA-I amyloidogenic potential.