Placental dimethyl acetal fatty acid derivatives are elevated in preeclampsia.

Preeclampsia (PE) was shown to affect the placental content and the transfer of polyunsaturated fatty acids (PUFA) to the fetus. Plasmalogens, a type of phospholipids with a vinyl-ether link at the sn-1 position, play an antioxidant role and are specifically enriched in PUFA at the sn-2 position. In this study, we characterized plasmalogen-derived dimethyl acetal (DMA) fatty acid derivatives, 16:0 DMA, 18:0 DMA, 9c-/11c-18:1 DMA and PUFA in the placenta of normotensive (n = 20) and PE (n = 20) pregnancies, according to the sampling site: peri-insertion or periphery. Phospholipid fatty acids from the placenta and maternal erythrocytes were identified by gas chromatography mass spectrometry and quantified by flame ionization detection. We found elevated total DMA in the PE placenta by 18% when compared to normotensive controls (p = 0.026). Moreover, the 16:0 DMA account for more than 55% of DMA fatty acids measured in the placenta, and its level is significantly higher in PE than controls (p = 0.018). Also, we found elevated placental PUFA, 20:5(n-3), 22:5(n-3) and a low level of 20:4(n-3) in PE compared to controls. Placental DMA was highly correlated with n-6 and n-3 PUFA in both, normotensive and PE pregnancies. In sum, elevated DMA fatty acids in the PE placenta could be an indirect defensive mechanism against oxidative stress and poor placental fatty acid transfer in PE.

Existence of compensatory defense mechanisms against oxidative stress and hypertension in preeclampsia.

OBJECTIVE: Preeclampsia is a complex obstetrical syndrome characterized by hypertension and proteinuria. This syndrome is associated with oxidative stress, antioxidant imbalance and impaired production of vasoactive eicosanoids such as thromboxane A(2) (TXA(2)), a potent vasoconstrictor, and prostacyclin (PGI(2)), a well-known vasodilator. We hypothesized that there was a relationship between antioxidant vitamins, such as vitamin E and coenzyme Q(10) (CoQ(10)), and the production of vasoactive eicosanoids- PGI(2) and TXA(2)-potentially regulated by pro-oxidants and antioxidants in preeclampsia. METHODS: Therefore, the plasma levels of vitamin E, CoQ(10), TXA(2) and PGI(2) in normotensive (n = 30) and preeclamptic (n = 29) pregnancies were evaluated. Reduced and oxidized forms of vitamin E and CoQ(10) in blood were measured using a HPLC coupled to electrochemical detection. The levels of TXB(2) and 6-keto-PGF(1alpha), stable metabolites of TXA(2) and PGI(2) respectively, were measured by ELISA. RESULTS: The CoQ(10) oxidized/reduced ratio was significantly higher in preeclamptic compared to normotensive pregnancies (p = 0.04). A strong correlation between plasma levels of reduced vitamin E and CoQ(10), corrected for apolipoprotein B, was observed only in preeclampsia (r = 0.69, p < 0.0001). The 6-keto-PGF(1alpha)/TXB(2) ratio was higher in preeclampsia than in controls (p = 0.02), and this ratio was correlated to the oxidized/reduced ratio of both, vitamin E and CoQ(10) in all pregnancies (p <0.023). CONCLUSION: The data indicated that CoQ(10) is a sensitive marker of oxidative stress in preeclampsia. The correlation between vitamin E and CoQ(10) suggested a coordinated defense mechanism against oxidation. Furthermore, the higher 6-keto-PGF(1alpha)/TXB(2) ratio that strongly correlated with oxidative stress markers, suggests a mechanism developed by the maternal cardiovascular system to counteract hypertension during preeclampsia.

High dietary consumption of trans fatty acids decreases brain docosahexaenoic acid but does not alter amyloid-beta and tau pathologies in the 3xTg-AD model of Alzheimer's disease.

Dietary consumption of trans fatty acids (TFA) has increased during the 20th century and is a suspected risk factor for cardiovascular diseases. More recently, high TFA intake has been associated with a higher risk of developing Alzheimer's disease (AD). To investigate the impact of TFA on an animal model genetically programmed to express amyloid-beta (Abeta) and tau pathological markers of AD, we have fed 3xTg-AD mice with either control (0% TFA/total fatty acid), high TFA (16% TFA) or very high TFA (43% TFA) isocaloric diets from 2 to 16 months of age. Effects of TFA on plasma hepatic enzymes, glucose and lipid profile were minimal but very high TFA intake decreased visceral fat of non-transgenic mice. Importantly, dietary TFA increased brain TFA concentrations in a dose-related manner. Very high TFA consumption substantially modified the brain fatty acid profile by increasing mono-unsaturated fatty acids and decreasing polyunsaturated fatty acids (PUFA). Very high TFA intake induced a shift from docosahexaenoic acid (DHA, 22:6n-3) toward n-6 docosapentaenoic acid (DPA, 22:5n-6) without altering the n-3:n-6 PUFA ratio in the cortex of both control and 3xTg-AD mice. Changes in levels of Abeta(40), Abeta(42), tau protein, phosphorylated tau protein and synaptic markers were not statistically significant in the three groups of 3xTg-AD mice, despite a trend toward decreased insoluble tau in very high TFA-fed 3xTg-AD animals. In summary, TFA intake modulated brain fatty acid profiles but had no significant effect on major brain neuropathological hallmarks of AD in an animal model.

N-terminal protein acylation confers localization to cholesterol, sphingolipid-enriched membranes but not to lipid rafts/caveolae.

When variably fatty acylated N-terminal amino acid sequences were appended to a green fluorescent reporter protein (GFP), chimeric GFPs were localized to different membranes in a fatty acylation-dependent manner. To explore the mechanism of localization, the properties of acceptor membranes and their interaction with acylated chimeric GFPs were analyzed in COS-7 cells. Myristoylated GFPs containing a palmitoylated or polybasic region colocalized with cholesterol and ganglioside GM(1), but not with caveolin, at the plasma membrane and endosomes. A dipalmitoylated GFP chimera colocalized with cholesterol and GM(1) at the plasma membrane and with caveolin in the Golgi region. Acylated GFP chimeras did not cofractionate with low-density caveolin-rich lipid rafts prepared with Triton X-100 or detergent-free methods. All GFP chimeras, but not full-length p62(c-yes) and caveolin, were readily solubilized from membranes with various detergents. These data suggest that, although N-terminal acylation can bring GFP to cholesterol and sphingolipid-enriched membranes, protein-protein interactions are required to localize a given protein to detergent-resistant membranes or caveolin-rich membranes. In addition to restricting acceptor membrane localization, N-terminal fatty acylation could represent an efficient means to enrich the concentration of signaling proteins in the vicinity of detergent-resistant membranes and facilitate protein-protein interactions mediating transfer to a detergent-resistant lipid raft core.

Tumor necrosis factor-alpha induces stress fiber formation through ceramide production: role of sphingosine kinase.

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that activates several signaling cascades. We determined the extent to which ceramide is a second messenger for TNF-alpha-induced signaling leading to cytoskeletal rearrangement in Rat2 fibroblasts. TNF-alpha, sphingomyelinase, or C(2)-ceramide induced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, and stress fiber formation. Ly 294002, a phosphatidylinositol 3-kinase (PI 3-K) inhibitor, or expression of dominant/negative Ras (N17) completely blocked C(2)-ceramide- and sphingomyelinase-induced tyrosine phosphorylation of FAK and paxillin and severely decreased stress fiber formation. The TNF-alpha effects were only partially inhibited. Dimethylsphingosine, a sphingosine kinase (SK) inhibitor, blocked stress fiber formation by TNF-alpha and C(2)-ceramide. TNF-alpha, sphingomyelinase, and C(2)-ceramide translocated Cdc42, Rac, and RhoA to membranes, and stimulated p21-activated protein kinase downstream of Ras-GTP, PI 3-K, and SK. Transfection with inactive RhoA inhibited the TNF-alpha- and C(2)-ceramide-induced stress fiber formation. Our results demonstrate that stimulation by TNF-alpha, which increases sphingomyelinase activity and ceramide formation, activates sphingosine kinase, Rho family GTPases, focal adhesion kinase, and paxillin. This novel pathway of ceramide signaling can account for approximately 70% of TNF-alpha-induced stress fiber formation and cytoskeletal reorganization.

Regulation of mitochondrial carbamoyl-phosphate synthetase 1 activity by active site fatty acylation.

In addition to its role in reversible membrane localization of signal-transducing proteins, protein fatty acylation could play a role in the regulation of mitochondrial metabolism. Previous studies have shown that several acylated proteins exist in mitochondria isolated from COS-7 cells and rat liver. Here, a prominent fatty-acylated 165-kDa protein from rat liver mitochondria was identified as carbamoyl-phosphate synthetase 1 (CPS 1). Covalently attached palmitate was linked to CPS 1 via a thioester bond resulting in an inhibition of CPS 1 activity at physiological concentrations of palmitoyl-CoA. This inhibition corresponds to irreversible inactivation of CPS 1 and occurred in a time- and concentration-dependent manner. Fatty acylation of CPS 1 was prevented by preincubation with N-ethylmaleimide and 5'-p-fluorosulfonylbenzoyladenosine, an ATP analog that reacts with CPS 1 active site cysteine residues. Our results suggest that fatty acylation of CPS 1 is specific for long-chain fatty acyl-CoA and very likely occurs on at least one of the essential cysteine residues inhibiting the catalytic activity of CPS 1. Inhibition of CPS 1 by long-chain fatty acyl-CoAs could reduce amino acid degradation and urea secretion, thereby contributing to nitrogen sparing during starvation.

Characterization of rat liver malonyl-CoA decarboxylase and the study of its role in regulating fatty acid metabolism.

In the liver, malonyl-CoA is central to many cellular processes, including both fatty acid biosynthesis and oxidation. Malonyl-CoA decarboxylase (MCD) is involved in the control of cellular malonyl-CoA levels, and functions to decarboxylate malonyl-CoA to acetyl-CoA. MCD may play an essential role in regulating energy utilization in the liver by regulating malonyl-CoA levels in response to various nutritional or pathological states. The purpose of the present study was to investigate the role of liver MCD in the regulation of fatty acid oxidation in situations where lipid metabolism is altered. A single MCD enzyme of molecular mass 50.7 kDa was purified from rat liver using a sequential column chromatography procedure and the cDNA was subsequently cloned and sequenced. The liver MCD cDNA was identical to rat pancreatic beta-cell MCD cDNA, and contained two potential translational start sites, producing proteins of 50.7 kDa and 54.7 kDa. Western blot analysis using polyclonal antibodies generated against rat liver MCD showed that the 50.7 kDa isoform of MCD is most abundant in heart and liver, and of relatively low abundance in skeletal muscle (despite elevated MCD transcript levels in skeletal muscle). Tissue distribution experiments demonstrated that the pancreas is the only rat tissue so far identified that contains both the 50.7 kDa and 54. 7 kDa isoforms of MCD. In addition, transfection of the full-length rat liver MCD cDNA into COS cells produced two isoforms of MCD. This indicated either that both initiating methionines are functionally active, generating two proteins, or that the 54.7 kDa isoform is the only MCD protein translated and removal of the putative mitochondrial targeting pre-sequence generates a protein of approx. 50.7 kDa in size. To address this, we transiently transfected a mutated MCD expression plasmid (second ATG to GCG) into COS-7 cells and performed Western blot analysis using our anti-MCD antibody. Western blot analysis revealed that two isoforms of MCD were still present, demonstrating that the second ATG may not be responsible for translation of the 50.7 kDa isoform of MCD. These data also suggest that the smaller isoform of MCD may originate from intracellular processing. To ascertain the functional role of the 50. 7 kDa isoform of rat liver MCD, we measured liver MCD activity and expression in rats subjected to conditions which are known to alter fatty acid metabolism. The activity of MCD was significantly elevated under conditions in which hepatic fatty acid oxidation is known to increase, such as streptozotocin-induced diabetes or following a 48 h fast. A 2-fold increase in expression was observed in the streptozotocin-diabetic rats compared with control rats. In addition, MCD activity was shown to be enhanced by alkaline phosphatase treatment, suggesting phosphorylation-related control of the enzyme. Taken together, our data demonstrate that rat liver expresses a 50.7 kDa form of MCD which does not originate from the second methionine of the cDNA sequence. This MCD is regulated by at least two mechanisms (only one of which is phosphorylation), and its activity and expression are increased under conditions where fatty acid oxidation increases.

Lipid phosphate phosphatase-1 and Ca2+ control lysophosphatidate signaling through EDG-2 receptors.

The serum-derived phospholipid growth factor, lysophosphatidate (LPA), activates cells through the EDG family of G protein-coupled receptors. The present study investigated mechanisms by which dephosphorylation of exogenous LPA by lipid phosphate phosphatase-1 (LPP-1) controls cell signaling. Overexpressing LPP-1 decreased the net specific cell association of LPA with Rat2 fibroblasts by approximately 50% at 37 degrees C when less than 10% of LPA was dephosphorylated. This attenuated cell activation as indicated by diminished responses, including cAMP, Ca(2+), activation of phospholipase D and ERK, DNA synthesis, and cell division. Conversely, decreasing LPP-1 expression increased net LPA association, ERK stimulation, and DNA synthesis. Whereas changing LPP-1 expression did not alter the apparent K(d) and B(max) for LPA binding at 4 degrees C, increasing Ca(2+) from 0 to 50 micrometer increased the K(d) from 40 to 900 nm. Decreasing extracellular Ca(2+) from 1.8 mm to 10 micrometer increased LPA binding by 20-fold, shifting the threshold for ERK activation to the nanomolar range. Hence the Ca(2+) dependence of the apparent K(d) values explains the long-standing discrepancy of why micromolar LPA is often needed to activate cells at physiological Ca(2+) levels. In addition, the work demonstrates that LPP-1 can regulate specific LPA association with cells without significantly depleting bulk LPA concentrations in the extracellular medium. This identifies a novel mechanism for controlling EDG-2 receptor activation.

Lipid phosphate phosphatase-1 in the regulation of lysophosphatidate signaling.

Mammalian lipid phosphate phosphatases (LPPs, or Type 2 phosphatidate phosphohydrolases) constitute a family of enzymes that belongs to a phosphatase superfamily. The LPPs dephosphorylate a variety of bioactive lipid phosphates including phosphatidate, lysophosphatidate, sphingosine 1-phosphate, and ceramide 1-phosphate. Mouse LPP-1 was stably expressed in rat2 fibroblasts to determine its structural and functional properties. Transduced cells showed increased dephosphorylation of exogenous lysophosphatidate. This result is compatible with mutational studies that show the active site of LPP-1 to be located on the external surface of the plasma membrane. Elevated LPP-1 activity attenuated the ability of lysophosphatidate to stimulate mitogen-activated protein kinase (ERK1 and 2) activities and DNA synthesis. It is concluded that one function of LPP-1 is to dephosphorylate exogenous lysophosphatidate, thereby attenuating cell signaling through endothelial cell differentiation gene (EDG) receptors.

Palmitoylation of apolipoprotein B is required for proper intracellular sorting and transport of cholesteroyl esters and triglycerides.

Apolipoprotein B (apoB) is an essential component of chylomicrons, very low density lipoproteins, and low density lipoproteins. ApoB is a palmitoylated protein. To investigate the role of palmitoylation in lipoprotein function, a palmitoylation site was mapped to Cys-1085 and removed by mutagenesis. Secreted lipoprotein particles formed by nonpalmitoylated apoB were smaller and denser and failed to assemble a proper hydrophobic core. Indeed, the relative concentrations of nonpolar lipids were three to four times lower in lipoprotein particles containing mutant apoB compared with those containing wild-type apoB, whereas levels of polar lipids isolated from wild-type or mutant apoB lipoprotein particles appeared identical. Palmitoylation localized apoB to large vesicular structures corresponding to a subcompartment of the endoplasmic reticulum, where addition of neutral lipids was postulated to occur. In contrast, nonpalmitoylated apoB was concentrated in a dense perinuclear area corresponding to the Golgi compartment. The involvement of palmitoylation as a structural requirement for proper assembly of the hydrophobic core of the lipoprotein particle and its intracellular sorting represent novel roles for this posttranslational modification.

Identification of structurally important domains of lipid phosphate phosphatase-1: implications for its sites of action.

Lipid phosphate phosphatase-1 (LPP-1) dephosphorylates exogenous lysophosphatidate and thereby regulates the activation of lysophosphatidate receptors and cell division. Mutation of seven amino acids in three conserved domains of mouse LPP-1 abolished its activity. A glycosylation site was demonstrated between conserved Domains 1 and 2. LPP-1 is expressed in the plasma membrane, and the present results demonstrate the active site to be located on the outer surface.

Functional roles for fatty acylated amino-terminal domains in subcellular localization.

Several membrane-associating signals, including covalently linked fatty acids, are found in various combinations at the N termini of signaling proteins. The function of these combinations was investigated by appending fatty acylated N-terminal sequences to green fluorescent protein (GFP). Myristoylated plus mono/dipalmitoylated GFP chimeras and a GFP chimera containing a myristoylated plus a polybasic domain were localized similarly to the plasma membrane and endosomal vesicles, but not to the nucleus. Myristoylated, nonpalmitoylated mutant chimeric GFPs were localized to intracellular membranes, including endosomes and the endoplasmic reticulum, and were absent from the plasma membrane, the Golgi, and the nucleus. Dually palmitoylated GFP was localized to the plasma membrane and the Golgi region, but it was not detected in endosomes. Nonacylated GFP chimeras, as well as GFP, showed cytosolic and nuclear distribution. Our results demonstrate that myristoylation is sufficient to exclude GFP from the nucleus and associate with intracellular membranes, but plasma membrane localization requires a second signal, namely palmitoylation or a polybasic domain. The similarity in localization conferred by the various myristoylated and palmitoylated/polybasic sequences suggests that biophysical properties of acylated sequences and biological membranes are key determinants in proper membrane selection. However, dual palmitoylation in the absence of myristoylation conferred significant differences in localization, suggesting that multiple palmitoylation sites and/or enzymes may exist.

Lipid phosphate phosphohydrolase-1 degrades exogenous glycerolipid and sphingolipid phosphate esters.

Lipid phosphate phosphohydrolase (LPP)-1 cDNA was cloned from a rat liver cDNA library. It codes for a 32-kDa protein that shares 87 and 82% amino acid sequence identities with putative products of murine and human LPP-1 cDNAs, respectively. Membrane fractions of rat2 fibroblasts that stably expressed mouse or rat LPP-1 exhibited 3.1-3. 6-fold higher specific activities for phosphatidate dephosphorylation compared with vector controls. Increases in the dephosphorylation of lysophosphatidate, ceramide 1-phosphate, sphingosine 1-phosphate and diacylglycerol pyrophosphate were similar to those for phosphatidate. Rat2 fibroblasts expressing mouse LPP-1 cDNA showed 1.6-2.3-fold increases in the hydrolysis of exogenous lysophosphatidate, phosphatidate and ceramide 1-phosphate compared with vector control cells. Recombinant LPP-1 was located partially in plasma membranes with its C-terminus on the cytosolic surface. Lysophosphatidate dephosphorylation was inhibited by extracellular Ca2+ and this inhibition was diminished by extracellular Mg2+. Changing intracellular Ca2+ concentrations did not alter exogenous lysophosphatidate dephosphorylation significantly. Permeabilized fibroblasts showed relatively little latency for the dephosphorylation of exogenous lysophosphatidate. LPP-1 expression decreased the activation of mitogen-activated protein kinase and DNA synthesis by exogenous lysophosphatidate. The product of LPP-1 cDNA is concluded to act partly to degrade exogenous lysophosphatidate and thereby regulate its effects on cell signalling.

Strain variability and localization of important epitopes on the major structural protein (VP2) of infectious pancreatic necrosis virus.

Infectious pancreatic necrosis virus (IPNV), a birnavirus, is an important pathogen in fish farms. Analyses of viral proteins showed that VP2 is the major structural and immunogenic polypeptide of the virus. All neutralizing monoclonal antibodies (mAbs) against IPNV are specific to VP2 and bind to continuous or discontinuous epitopes. In order to determine which parts of the protein are involved in antigenic variations, five IPNV strains were sequenced over the VP2 coding region. Comparison of the sequences obtained with three previously published strains revealed a central variable domain (positions 183 to 335) which encompasses two hydrophilic hypervariable segments. Viral mutants which escaped neutralization were then selected with anti-VP2 mAbs directed against discontinuous epitopes. Sequencing of three mutants revealed a single amino acid mismatch in each of them. All of these substitutions occurred in the hypervariable segments, suggesting that these regions are involved in the formation of a discontinuous epitope. Finally, expression of different truncated VP2s in Escherichia coli allowed localization of the binding site for neutralizing mAbs which recognize continuous epitopes. One of these mAbs bound to the region adjacent to the C-terminus of the variable domain of VP2, while two others reacted with the central and C-terminal parts of the variable domain. No antibody reacted with the N-terminus of VP2. These results suggest that the variable domain of VP2 and the 20 adjacent amino acids of the conserved C-terminal part are the most important in inducing an immune response for the protection of animals.

Biochemical characterization of a palmitoyl acyltransferase activity that palmitoylates myristoylated proteins.

Dynamic regulation of signal transduction by reversible palmitoylation-depalmitoylation cycles has been recently described. However, further understanding of fatty acylation reactions has been hampered by our lack of knowledge about the specific transferases and thioesterases involved. Here, we describe an assay for the palmitoyl acyltransferase (PAT) that palmitoylates "myrGlyCys" containing members of the Src family of protein tyrosine kinases (PTKs). Since N-myristoylation of Fyn PTK, a member of the Src family, has been shown to be a prerequisite for palmitolylation, a new single plasmid vector that allows overexpression of myristoylated Fyn substrate in Escherichia coli was developed. Purified myristoylated protein substrates were incubated with [125I]iodopalmitoyl CoA, a palmitoyl CoA analog, in the presence of bovine brain lysates. Transfer of radiolabel to the Fyn substrate was detected by SDS-polyacrylamide gel electrophoresis and autoradiography. This assay was used to partially purify and characterize PAT activity from bovine brain. Here, we demonstrate that PAT is a membrane-bound enzyme, which palmitoylates myristoylated Fyn substrates containing a cysteine residue in position three. The PAT activity attached palmitate to Fyn proteins via a thioester linkage and exhibited a fatty acyl CoA preference for long chain fatty acids. It is likely that palmitoylation of Fyn and other Src family members by PAT regulates PTK localization and signaling functions.

Characterization of the small open reading frame on genome segment A of infectious pancreatic necrosis virus.

The genome of infectious pancreatic necrosis virus (IPNV) is composed of two segments of dsRNA. The larger segment contains a small ORF partly overlapping the 5' end of the polyprotein reading frame. Yet very little is known about this possible new gene, which presumably codes for a 17 kDa polypeptide (VP5). The region of the viral genome which encompasses the small ORF was reverse-transcribed and amplified by PCR before cloning and sequencing. Analysis of the sequences obtained from five different virus strains revealed that the small ORF is not found on one of them, and that it is truncated on two others. Moreover, the deduced amino acid sequences did not appear to be well conserved. Despite the large variations between IPNV strains at the genomic level, all predicted VP5 are arginine-rich basic polypeptides. To verify whether the small ORF is translated into protein in fish cells, the 17 kDa polypeptide of the VR-299 strain was expressed as fusion protein in a prokaryotic expression vector and used to produce a specific antiserum. This antiserum reacted with concentrated virus in an immunodot assay indicating that VP5 is synthesized in infected cells, but probably only in small quantities. When tested with 12 other IPNV strains, results were less conclusive than those obtained with strain VR-299. Nevertheless, three of the 12 viruses gave a clearly negative signal in the immunodot assay, suggesting that possibly more than one viral strain lacks the small ORF.

Evidence of a major neutralizable conformational epitope region on VP2 of infectious pancreatic necrosis virus.

A collection of neutralizing monoclonal antibodies (MAbs) produced against the LWVRT 60.1, Jasper and N1 strains of infectious pancreatic necrosis virus (IPNV) were selected for the analysis of VP2 epitopes. Previous characterization of the LW and JA MAbs allowed the identification of continuous and discontinuous epitopes but the topological localization of these sites remained obscure. The ability of these MAbs to differentiate individual epitopes was evaluated by additivity and competition assays using antigen-coated plates and by surface plasmon resonance (SPR), an automated biosensor system that is able to retain the conformation integrity of proteins. IPNV-neutralizing MAbs defined a major, conformational-dependent and immunodominant area where continuous epitopes represent portions of a larger discontinuous epitope. Moreover, weakly neutralizing MAbs could interact with internal sites, located within the foldings of the polypeptide chain. Anti-VP2 MAbs prepared against the European serotype N1 recognized and competed for epitopes present on the North American strain LWVRT 60.1 and Jasper. Attempts to establish the proximity of VP2 and VP3 epitopes have been made by SPR. Results indicate that these major structural proteins do not overlap in the virion.

Dual myristylation and palmitylation of Src family member p59fyn affects subcellular localization.

The Src family consists of nine related tyrosine protein kinases with a common domain structure, including a myristylated N-terminal glycine residue. In this report, we identify cysteine residues within the N-terminal region of the Src family member Fyn which serve as sites for palmitylation. To facilitate detection of protein fatty acylation, p59fyn was overexpressed in COS cells and incubated with radioiodinated fatty acid analogs of myristate (IC13) or palmitate (IC16). Incorporation of both fatty acids into p59fyn was readily observed. Acylation with the palmitate analog was prevented when Gly-2 was mutated to alanine, implying that N-myristylation is required for palmitylation, and when either Cys-3 or Cys-6 was mutated to serine. Palmitylation was shown to alter the distribution of p59fyn between membrane-bound and soluble fractions. In contrast, no incorporation of the palmitate analog into pp60v-src, which lacks N-terminal cysteine residues, was observed. Mutation of Ser-3 of Src to cysteine, but not Ser-6, resulted in incorporation of the palmitate analog. These results serve to delineate sequence elements important for dual acylation of proteins, and further illustrate the utility of radioiodinated fatty acid analogs for studies of protein fatty acid acylation.

Regulation of enzymatic activity by active site fatty acylation. A new role for long chain fatty acid acylation of proteins.

Methylmalonate semialdehyde dehydrogenase (MMSDH) is a mitochondrial enzyme which can be acylated by myristoyl-CoA analogs (Deichaite, I., Berthiaume, L., Peseckis, S. M., Patton, W. F., and Resh, M. D. (1993) J. Biol. Chem. 268, 13788-13747). Here we describe the mechanisms which mediate regulation of the enzymatic activity of bovine MMSDH by long chain fatty acylation. The substrate specificity of the acylation reaction was measured in vitro using purified MMSDH and the coenzyme A derivative of an 125I-labeled long chain fatty acid (13-iodotridecanoate), an analog of myristoyl-CoA. Long chain fatty acyl CoAs (> 8 carbons) were able to inhibit radiolabeling of MMSDH. In order to study the physiological role of the acylation process in vivo, a system using highly purified mitochondria from COS-1 cells overexpressing MMSDH was exploited. MMSDH was shown to be processed properly, targeted to the mitochondrial fraction, and enzymatically active. The extent of fatty acylation of MMSDH as well as of other mitochondrial proteins was correlated with the mitochondrial energy level. Biochemical evidence as well as site-specific mutagenesis of cysteine 319 revealed that this highly conserved active site cysteine of MMSDH was the target of the fatty acylation. Another member of the aldehyde dehydrogenase family, yeast aldehyde dehydrogenase was also covalently modified by [125I]13-iodotridecanoyl-CoA and thereby inactivated. Furthermore, we demonstrate that glutamate dehydrogenase, an enzyme that has been previously shown to be strongly inhibited by palmitoyl-CoA, is fatty acylated by the 125I-labeled myristoyl-CoA analog. Our data suggest that attachment of long chain fatty acids to proteins is a new and potentially widespread type of enzyme regulation mechanism that we denote active site fatty acylation.