Catabolism of 4-Hydroxyacids and 4-Hydroxynonenal via 4-Hydroxy-4-phosphoacyl-CoAs

4-Hydroxyacids are products of ubiquitously occurring lipid peroxidation (C₉, C₆) or drugs of abuse (C₄, C₅). We investigated the catabolism of these compounds using a combination of metabolomics and mass isotopomer analysis. Livers were perfused with various concentrations of unlabeled and labeled saturated 4-hydroxyacids (C₄ to C₁₁) or 4-hydroxynonenal. All the compounds tested form a new class of acyl-CoA esters, 4-hydroxy-4-phosphoacyl-CoAs, characterized by liquid chromatography-tandem mass spectrometry, accurate mass spectrometry, and ³¹P-NMR. All 4-hydroxyacids with five or more carbons are metabolized by two new pathways. The first and major pathway, which involves 4-hydroxy-4-phosphoacyl-CoAs, leads in six steps to the isomerization of 4-hydroxyacyl-CoA to 3-hydroxyacyl-CoAs. The latter are intermediates of physiological β-oxidation. The second and minor pathway involves a sequence of β-oxidation, α-oxidation, and β-oxidation steps. In mice deficient in succinic semialdehyde dehydrogenase, high plasma concentrations of 4-hydroxybutyrate result in high concentrations of 4-hydroxy-4-phospho-butyryl-CoA in brain and liver. The high concentration of 4-hydroxy-4-phospho-butyryl-CoA may be related to the cerebral dysfunction of subjects ingesting 4-hydroxybutyrate and to the mental retardation of patients with 4-hydroxybutyric aciduria. Our data illustrate the potential of the combination of metabolomics and mass isotopomer analysis for pathway discovery.     

Direct interaction of Rab4 with syntaxin 4

In the present study, we examined the possible interaction between Rab4 and syntaxin 4, both having been implicated in insulin-induced GLUT4 translocation. Rab4 and syntaxin 4 were coimmunoprecipitated from the lysates of electrically permeabilized rat adipocytes. The interaction between the two proteins was reduced by insulin treatment and increased by the addition of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). An in vitro binding assay revealed that the bacterially expressed Rab4 was bound to a glutathione S-transferase fusion protein containing the cytoplasmic domain of syntaxin 4 (GST-syntaxin 4-(1-273)) but not to syntaxin 1A or vesicle-associated membrane protein-2. The interaction between Rab4 and syntaxin 4 seemed to be regulated by the guanine nucleotide status of Rab4, because 1) GTPgammaS treatment of the cells significantly increased, but guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS) treatment decreased the amount of Rab4 pulled down with GST-syntaxin 4-(1-273) from the cell lysates; 2) GTPgammaS loading on Rab4 caused a marked increase in the affinity of Rab4 to syntaxin 4 whereas GDPbetaS loading had little effect; and 3) a GTPase-deficient mutant of Rab4 (Rab4(Q67L)), but not a GTP-binding-defective mutant (Rab4(S22N)), was bound to GST-syntaxin 4-(1-273). Although insulin stimulated [gamma-(32)P]GTP binding to Rab4 in a time-dependent fashion, its effect on the Rab4 interaction with syntaxin 4 was apparently biphasic; an initial increase in Rab4 associated with syntaxin 4 was followed by a gradual dissociation of the GTPase from syntaxin 4. Finally, the binding of Rab4(Q67L) to GST-syntaxin 4-(1-273) was inhibited by munc-18c in a dose-dependent manner, indicating that GTP-loaded Rab4 binds to syntaxin 4 in the open conformation. These results suggest that 1) Rab4 interacts with syntaxin 4 in a direct and specific manner, and 2) the interaction is regulated by the guanine nucleotide status of Rab4 as well as by the conformational status of syntaxin 4.     

A short path to synthesis of 4-deoxy-4-fluoroglucosaminides: methylumbelliferyl N-acetyl-4-deoxy-4-fluoro-beta-D-glucosaminide

A convenient method of synthesis of 1,6-anhydro-4-deoxy-2-O-tosyl-4-fluoro-beta-D-glucopyranose by fusion of 1,6;3,4-dianhydro-2-O-tosyl-beta-D-galactopyranose with 2,4,6-trimethylpyridinium fluoride was found. By successive action of ammonia, methyl trifluoroacetate, and acetic anhydride, the resulting compound was transformed into 1,6-anhydro-3-O-acetyl-2,4-dideoxy-2-trifluoroacetamido-4-fluoro-beta-D-glucopyranose, which was converted into 3,6-di-O-acetyl-2,4-dideoxy-2-trifluoroacetamido-4-fluoro-alpha-D-glucopyranosyl fluoride by the reaction with HF/Py. The resulting fluoride was further used as a glycosyl donor in the synthesis of methylumbelliferyl N-acetyl-4-deoxy-4-fluoro-beta-D-glucosaminide.     

Synergistic activation of eIF4A by eIF4B and eIF4G

eIF4A is a key component in eukaryotic translation initiation; however, it has not been clear how auxiliary factors like eIF4B and eIF4G stimulate eIF4A and how this contributes to the initiation process. Based on results from isothermal titration calorimetry, we propose a two-site model for eIF4A binding to an 83.5 kDa eIF4G fragment (eIF4G-MC), with a high- and a low-affinity site, having binding constants K_D of ∼50 and ∼1000 nM, respectively. Small angle X-ray scattering analysis shows that the eIF4G-MC fragment adopts an elongated, well-defined structure with a maximum dimension of 220 Å, able to span the width of the 40S ribosomal subunit. We establish a stable eIF4A–eIF4B complex requiring RNA, nucleotide and the eIF4G-MC fragment, using an in vitro RNA pull-down assay. The eIF4G-MC fragment does not stably associate with the eIF4A–eIF4B–RNA-nucleotide complex but acts catalytically in its formation. Furthermore, we demonstrate that eIF4B and eIF4G-MC act synergistically in stimulating the ATPase activity of eIF4A.     

Hydration pattern of A4T4 and T4A4 DNA: a molecular dynamics study

Hydration pattern and energetics of 'A-tract' containing duplexes have been studied using molecular dynamics on 12-mer self-complementary sequences 5'-d(GCA4T4GC)-3' and 5'-d(CGT4A4CG)-3'. The structural features for the simulated duplexes showed correlation with the corresponding experimental structures. Analysis of the hydration pattern confirmed that water network around the simulated duplexes is more conformation specific rather than sequence specific. The calculated heat capacity change upon duplex formation showed that the process is entropically driven for both the sequences. Furthermore, the theoretical free energy estimates calculated using MMPBSA approach showed a higher net electrostatic contribution for A4T4 duplex formation than for T4A4, however, energetically both the duplexes are observed to be equally stable.     

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs

Eukaryotic initiation factor (eIF) 4E, the mRNA 5'-cap-binding protein, mediates the association of eIF4F with the mRNA 5'-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (approximately 30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras-expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization.     

Modulation of the helicase activity of eIF4A by eIF4B, eIF4H, and eIF4F.

Eukaryotic initiation factor (eIF) 4A is a DEAD box RNA helicase that works in conjunction with eIF4B, eIF4H, or as a subunit of eIF4F to unwind secondary structure in the 5'-untranslated region of mRNA, which facilitates binding of the mRNA to the 40 S ribosomal subunit. This study demonstrates how the helicase activity of eIF4A is modulated by eIF4B, eIF4H, or as a subunit of eIF4F. Results indicate that a linear relationship exists between the initial rate or amplitude of unwinding and duplex stability for all factor combinations tested. eIF4F, like eIF4A, behaves as a non-processive helicase. Either eIF4B or eIF4H stimulated the initial rate and amplitude of eIF4A-dependent duplex unwinding, and the magnitude of stimulation is dependent on duplex stability. Furthermore, eIF4A (or eIF4F) becomes a slightly processive helicase in the presence of eIF4B or eIF4H. All combinations of factors tested indicate that the rate of duplex unwinding is equivalent in the 5' --> 3' and 3' --> 5' directions. However, the optimal rate of unwinding was dependent on the length of the single-stranded region of the substrate when different combinations of factors were used. The combinations of eIF4A, eIF4A + eIF4B, eIF4A + eIF4H, and eIF4F showed differences in their ability to unwind chemically modified duplexes. A simple model of how eIF4B or eIF4H affects the duplex unwinding mechanism of eIF4A is proposed.     

Catabolism of leukotriene A4 into B4, C4, and D4 by rat liver subcellular fractions

[3H]Leukotriene A4 was incubated with various subcellular fractions of rat liver homogenates. After solvent extraction and purification on C18 Sep-Pak cartridges, tritiated products migrating on reversed-phase HPLC with authentic unlabelled leukotriene C4, D4 and B4 were observed. The identity of leukotriene C4 was confirmed through enzymatic conversion into D4 by gamma-glutamyl transpeptidase as well as by bioassay on the rat stomach fundus after HPLC purification. The contractile response to the extracted material was blocked by the SRS antagonist, FPL 55712. Leukotriene B4 synthesis was located in the 100 000 X g supernatant, while C4 synthesis was present in the corresponding pellet. Leukotriene C4 formation was enhanced when reduced glutathione was supplemented in the incubation medium. These results demonstrate the presence in rat liver of various enzymatic steps in leukotriene A4 catabolism.     

Metabolism of LTC4 to LTD4 and LTE4 in isolated guinea pig lung lobes

Leukotrienes are known to be easily metabolized to other substances. But the metabolic fates of LTC4 and LTD4 have not been established in the intact lung. In this investigation we perfused isolated guinea pig lung lobes and injected synthesized LTC4 and LTD4. The effluent was assayed by HPLC. LTD4 and LTE4 were detected following perfusion of LTC4, and LTE4 was detected following perfusion of LTD4. These results suggest that perfused guinea pig lung lobes may metabolize LTC4 to LTD4 and LTE4, and LTD4 to LTE4.     

Characterizing the interaction of the mammalian eIF4E-related protein 4EHP with 4E-BP1

Eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) represses translation initiation by binding to eukaryotic initiation factor 4E (eIF4E). 4E-BP1 also binds to the eIF4E homologous protein (4EHP). We show that eIF4E-binding mutants of 4E-BP1 (Y54A and L59A) fail to form heterodimeric complexes with wild-type 4EHP. In addition, the W95A mutant of 4EHP, similar to a homologous mutation in eIF4E, inhibits its binding to wild-type 4E-BP1. Interestingly, 4EHP over-expression instigates a negative feedback loop that inhibits upstream signaling to 4E-BP1 and ribosomal protein S6 kinase 1 (S6K1) whereas the 4E-BP1-binding-deficient mutant of 4EHP(W95A) was unable to trigger this feedback loop. Thus, the interaction of 4EHP with 4E-BP1 is necessary for this observed impaired signaling to 4E-BP1 and S6K1.     

Formation of 4-ethoxy-4'-nitrosodiphenylamine in the reaction of the phenacetin metabolite 4-nitrosophenetol with glutathione

Phenacetin, a constituent of several analgesic and antipyretic formulations has been made responsible for a variety of toxic and carcinogenic actions. 4-Nitrosophenetol, the N-oxydation product of intermediate 4-phenetidine, forms methemoglobin and binds covalently to sulfhydryl groups of proteins and glutathione. In the reaction of 4-nitrosophenetol with glutathione and other thiols an intermediate so-called "semimercaptal" is formed from which N-(thiol-S-yl)-4-phenetidine S-oxide, N-(thiol-S-yl)-4-phenetidine and 4-phenetidine derive. Besides thiol adducts, a yellow compound is formed which was isolated as a pure crystalline product (elemental analysis) and identified by FAB-MS, EI-MS, 13C-, 1H-NMR, and UV-VIS spectroscopy as 4-ethoxy-4'-nitrosodiphenylamine. This nitrosoarene is formed by an unknown mechanism from 4-nitrosophenetol and 4-phenetidine under liberation of ethanol. In human erythrocytes this compound is easily reduced to 4-amino-4'-ethoxydiphenylamine (FAB-MS, EI-MS, 13C-NMR). During the reaction of 4-nitrosophenetol with red cells only traces of 4-ethoxy-4'-nitrosodiphenylamine were formed, whereas up to 10% appeared as the reduction product 4-amino-4'-ethoxydiphenylamine. This latter compound is unstable in red cells and is metabolized further to unidentified products.     

Non-cross-reactivity of antibodies to murine LDH-C4 with LDH-A4 and LDH-B4

The induction of infertility by immunization with the sperm-specific lactate dehydrogenase, LDH-C4, suggests its use in a contraceptive vaccine. Development of an immunological contraceptive for human use, however, requires that there be no cross-reactions with somatic tissues. We have demonstrated, using enzyme-linked immunoabsorbence, solid-phase radioimmunoassay, and competitive inhibition radioimmunoassay, that antisera to LDH-C4 is specific and does not cross-react with the somatic isozymes, LDH-A4 and LDH-B4.     

Biotransformation of 4-halophenols to 4-halocatechols using Escherichia coli expressing 4-hydroxyphenylacetate 3-hydroxylase

Escherichia coli cells, expressing 4-hydroxyphenylacetate 3-hydroxylase, fully transformed 4-halogenated phenols to their equivalent catechols as single products in shaken flasks. 4-Fluorophenol was transformed at a rate 1.6, 1.8, and 3.4-fold higher than the biotransformation of 4-chloro-, 4-bromo-, and 4-iodo-phenol, respectively. A scale-up from shaken flask to a 5 L stirred tank bioreactor was undertaken to develop a bioprocess for the production of 4-substituted halocatechols at higher concentrations and scale. In a stirred tank reactor, the optimized conditions for induction of 4-HPA hydroxylase expression were at 37 °C for 3 h. The rate of biotransformation of 4-fluorophenol to 4-fluorocatechol by stirred tank bioreactor grown cells was the same at 1 and 4.8 mM (5.13 μmol/min/g CDW) once the ratio of biocatalyst (E. coli CDW) to substrate concentration (mM) was maintained at 2:1. At 10.8 mM 4-fluorophenol, the rate of 4-fluorocatechol formation decreased by 4.7-fold. However, the complete transformation of 1.3 g of 4-fluorophenol (10.8 mM) to 4-fluorocatechol was achieved within 7 h in a 1 L reaction volume. Similar to 4-fluorophenol, other 4-substituted halophenols were completely transformed to 4-halocatechols at 2 mM within a 1–2 h period. An increase in 4-halophenol concentration to 4.8 mM resulted in a 2.5–20-fold decrease in biotransformation efficiency depending on the substrate tested. Organic solvent extraction of the 4-halocatechol products followed by column chromatography resulted in the production of purified products with a final yield of between 33% and 38%.     

Determinants of 4 alpha-phorbol sensitivity in transmembrane domains 3 and 4 of the cation channel TRPV4

TRPV4, a Ca(2+)-permeable member of the vanilloid subgroup of the transient receptor potential (TRP) channels, is activated by cell swelling and moderate heat (>27 degrees C) as well as by diverse chemical compounds including synthetic 4 alpha-phorbol esters, the plant extract bisandrographolide A, and endogenous epoxyeicosatrienoic acids (EETs; 5,6-EET and 8,9-EET). Previous work identified a tyrosine residue located in the first half of putative transmembrane segment 3 (TM3) as a crucial determinant for the activation of TRPV4 by its most specific agonist 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), suggesting that 4 alpha-PDD interacts with the channel through its transmembrane segments. To obtain insight in the 4 alpha-PDD-binding site and in the mechanism of ligand-dependent TRPV4 activation, we investigated the consequences of specific point mutations in TM4 on the sensitivity of the channel to different chemical and physical stimuli. Mutations of two hydrophobic residues in the central part of TM4 (Leu(584) and Trp(586)) caused a severe reduction of the sensitivity of the channel to 4 alpha-PDD, bisandrographolide A, and heat, whereas responses to cell swelling, arachidonic acid, and 5,6-EET remained unaffected. In contrast, mutations of two residues in the C-terminal part of TM4 (Tyr(591) and Arg(594)) affected channel activation of TRPV4 by all stimuli, suggesting an involvement in channel gating rather than in interaction with agonists. Based on a comparison of the responses of WT and mutant TRPV4 to 4 alpha-PDD and different 4 alpha-phorbol esters, we conclude that the length of the fatty acid moiety determines the ligand binding affinity and propose a model for the interaction between 4 alpha-phorbol esters and the TM3/4 region of TRPV4.     

Characterization of leukotriene A4 and B4 biosynthesis

We have studied LTA4 and LTB4 synthesis in a cell-free system from RBL-1 cells. All the enzymes leading to the formation of LTB4 from arachidonic acid are localized in the soluble fraction (100,000 x g supernatant) of these cells. The formation of LTA4 and LTB4 is complete by 10 min. When we varied the arachidonic acid concentration from 1 to 300 microM, the synthesis of LTB4 leveled off at 30 microM and of LTA4 at 100 microM while 5-HETE had not reached a plateau at 300 microM. This enzyme system has the capacity to generate relatively large amounts of 5-HETE and LTA4 and only a relatively small amount of LTB4. Therefore, the rate limiting step is not the 5-lipoxygenase, the first step in the pathway, but the conversion of LTA4 to LTB4. This is in contrast to cyclooxygenase pathway where the first step is rate limiting. A second addition of arachidonic acid at submaximal concentration for LTA4 synthesis did not produce any additional LTA4 or LTB4. Further study of this phenomenon showed that the 5-lipoxygenase and LTA-synthase were inactivated with time by preincubation with arachidonic acid and that peroxy fatty acids seem to be the inactivating species.     

Contractions of D4Z4 on 4qB subtelomeres do not cause facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is associated with contractions of the D4Z4 repeat in the subtelomere of chromosome 4q. Two allelic variants of chromosome 4q (4qA and 4qB) exist in the region distal to D4Z4. Although both variants are almost equally frequent in the population, FSHD is associated exclusively with the 4qA allele. We identified three families with FSHD in which each proband carries two FSHD-sized alleles and is heterozygous for the 4qA/4qB polymorphism. Segregation analysis demonstrated that FSHD-sized 4qB alleles are not associated with disease, since these were present in unaffected family members. Thus, in addition to a contraction of D4Z4, additional cis-acting elements on 4qA may be required for the development of FSHD. Alternatively, 4qB subtelomeres may contain elements that prevent FSHD pathogenesis.     

Bal31 sensitivity of micronuclear sequences homologous to C4A4/G4T4 repeats in Oxytricha nova

The sequence similarity and functional equivalence of telomeres from macronuclear linear DNA molecules in Oxytricha and telomeric sequences of true mitotic/meiotic chromosomes suggest that the (C4A4)n/(G4T4)n sequences found at macronuclear telomeres may also function as micronuclear telomeres in Oxytricha. In this study, radioactively labeled (C4A4)n have been hybridized to micronuclear DNA samples that have been treated with the enzyme Bal31, which has double-stranded exonuclease activity. A time course of digestion shows that approximately 50% of the micronuclear sequences that hybridize to a C4A4 probe disappear with mild digestion by Bal31, suggesting that these sequences are telomeric. The remainder of the hybridizing sequences are not degraded any more rapidly than the total genomic DNA. All of the C4A4/G4T4 sequences that can be detected by hybridization of C4A4 probes to Southern-blotted restriction enzyme digests of micronuclear DNA occur in regions of the genome that are highly resistant to restriction enzyme digestion and show a clustering of sites reminiscent of telomeres in other organisms. We propose that the micronuclear C4A4 hybridizable sequences that are Bal31 resistant may be located near the telomere and within telomere-associated repetitive sequences that are immediately internal to telomeric (Bal31 sensitive) C4A4 hybridizeable sequences.     

CD4 ligation promotes the IL-4-independent development of IL-4-producing clones from naive CD4(+) T cells.

The signals that trigger IL-4-independent IL-4 synthesis by conventional CD4(+) T cells are not yet defined. In this study, we show that coactivation with anti-CD4 mAb can stimulate single naive CD4(+) T cells to form IL-4-producing clones in the absence of APC and exogenous IL-4, independently of effects on proliferation. When single CD4(+) lymph node cells from C57BL/6 mice were cultured with immobilized anti-CD3epsilon mAb and IL-2, 65-85% formed clones over 12-14 days. Coimmobilization of mAb to CD4, CD11a, and/or CD28 increased the size of these clones but each exerted different effects on their cytokine profiles. Most clones produced IFN-gamma and/or IL-3 regardless of the coactivating mAb. However, whereas 0-6% of clones obtained with mAb to CD11a or CD28 produced IL-4, 10-40% of those coactivated with anti-CD4 mAb were IL-4 producers. A similar response was observed among CD4(+) cells from BALB/c mice. Most IL-4-producing clones were derived from CD4(+) cells of naive (CD44(low) or CD62L(high)) phenotype and the great majority coproduced IFN-gamma and IL-3. The effect of anti-CD4 mAb on IL-4 synthesis could be dissociated from effects on clone size since anti-CD4 and anti-CD11a mAb stimulated formation of clones of similar size which differed markedly in IL-4 production. Engagement of CD3 and CD4 in the presence of IL-2 is therefore sufficient to induce a substantial proportion of naive CD4(+) T cells to form IL-4-producing clones in the absence of other exogenous signals, including IL-4 itself.