Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.     

A NOVEL APPROACH TO SOLID PHASE CHEMICAL SYNTHESIS OF OLIGONUCLEOTIDE mRNA CAP ANALOGS

A novel approach for the synthesis of 5′-capped 2′-O-methyloligoribonucleotides on a disulfide-tethered solid support is described. The key step of the synthesis is ZnCl₂ promoted coupling of m⁷GDP imidazolide to a fully deprotected oligonucleotide 5′-phosphate on-support. By this methodology m⁷G⁵′pppm²′Apm²′Upm^2′Ap has been prepared.     

Lack of an Efficient Endoplasmic Reticulum-localized Recycling System Protects Peroxiredoxin IV from Hyperoxidation

Typical 2-Cys peroxiredoxins are required to remove hydrogen peroxide from several different cellular compartments. Their activity can be regulated by hyperoxidation and consequent inactivation of the active-site peroxidatic cysteine. Here we developed a simple assay to quantify the hyperoxidation of peroxiredoxins. Hyperoxidation of peroxiredoxins can only occur efficiently in the presence of a recycling system, usually involving thioredoxin and thioredoxin reductase. We demonstrate that there is a marked difference in the sensitivity of the endoplasmic reticulum-localized peroxiredoxin to hyperoxidation compared with either the cytosolic or mitochondrial enzymes. Each enzyme is equally sensitive to hyperoxidation in the presence of a robust recycling system. Our results demonstrate that peroxiredoxin IV recycling in the endoplasmic reticulum is much less efficient than in the cytosol or mitochondria, leading to the protection of peroxiredoxin IV from hyperoxidation.     

Identification of thiol groups and a disulfide crosslink site in bovine myelin proteolipid protein

The existence of disulfide crosslinks limits the number of possible folded structures a protein can assume. Thus localization of disulfide and thiol groups is a key to understanding the conformation and orientation of myelin proteolipid protein (PLP) in the myelin membrane. [¹⁴C]Carboxamidomethylated PLP was fragmented with chymotrypsin, and the resulting mixture was partially separated by reversed-phase HPLC. Purified ¹⁴C-labeled peptides and a disulfide containing peptide were characterized by amino acid analysis. These experiments showed that Cys-32 and Cys-34 are free thiols, and are presumably on the interior of the cell or within the membrane bilayer, and that Cys-200 and Cys-219 are joined by a disulfide bond, and are probably located on the extracellular face of the membrane. Sequence analysis experiments indicate that Cys-5, Cys-6 and Cys-9 are linked by disulfides, probably to other parts of the protein on the extracellular face of the membrane.     

Characterization of messenger RNA by direct translation from agarose gels

A method for characterizing nanogram quantities of poly(A)-containing messenger RNAs that have been fractionated according to size by electrophoresis through agarose gels has been developed. The mRNAs from Friend leukemia cells were identified by the protein products they encode, as determined by slicing the agarose gel and directly translating the enclosed mRNA with an extract from rabbit reticulocytes that had been treated with micrococcal nuclease. A number of parameters which affect the efficiency of translation in this system have been examined. These include the sensitivity of the in vitro translational system to RNA, the agarose concentration, the incubation temperature, and the addition of either exogeneous tRNA or RNasin. The procedure is rapid, simple, reproducible, and applicable for the fractionation and characterization of mRNAs from any source.     

Stereoisomeric flavour compounds XXXV: Chiral aroma compounds of sherry I. Optically pure stereoisomers of solerone and solerole

The enantioseparation of the sherry aroma components 5-oxo-4-hydroxyhexanoic acid γ-lactone (solerone) and 4,5-dihydroxyhexanoic acid γ-lactone (solerole) is achieved, using Chiraspher (Merck) as the chiral HPLC phase and the optical purity ascertained directly by HRGC with heptakis(3-O-acetyl-2,6-di-O-pentyl)-β-cyclodextrin (Lipodex D) as the chiral stationary phase. The absolute configurations of 4,5-dihydroxyhexanoic acid γ-lactones are assigned by ¹H-NMR spectral data of diastereomeric α-methoxy-α-trifluoromethylphenylacetic acid (MTPA) esters, according to Mosher's model. Sensory qualities of the isomers are given.     

Chiral stationary phase high-performance liquid chromatographic resolution and absolute configuration of enantiomeric benzo[a]pyrene diol-epoxides and tetrols

Enantiomers of diastereomeric benzo[a]pyrene (BP) diol-epoxides, r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-BP (BP 7,8-diol-anti-9,10-epoxide), r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-BP (BP 7,8-diol-syn-9,10-epoxide), r-9,t-10-dihydroxy-t-7,8-epoxy-7,8,9,10-tetrahydro-BP (BP 9,10-diol-anti-7,8-epoxide), and several 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes (BP tetrols) were resolved by high-performance liquid chromatography (HPLC) using columns packed with either (R)-N-(3,5-dinitrobenzoyl)phenylglycine[(R)-DNBPG] or (S)-N-(3,5-dinitrobenzoyl)leucine [(S)-DNBL], which is either ionically or covalently bonded to gamma-aminopropylsilanized silica. Resolution of enantiomers was confirmed by ultraviolet-visible absorption and circular dichroism spectral analyses. Resolved enantiomers of BP diol-epoxides were each hydrolyzed in acidic solution to a pair of diastereomeric tetrols which were separated by reversed-phase HPLC. Absolute stereochemistries of enantiomeric diol-epoxides were deduced by the absolute configuration of their hydrolysis products.     

Location in Muscarinic Acetylcholine Receptors of Sites for [3H]Propylbenzilcholine Mustard Binding and for Phosphorylation with Protein Kinase C

Muscarinic acetylcholine receptors purified from porcine cerebra or atria were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), and then the labeled receptors were subjected to limited hydrolysis with trypsin, V8 protease, and lysyl endopeptidase, followed by analysis involving sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, autoradiography, or immunostaining. The labeled peptides were located on the basis of their reactivity with antibodies raised against three synthetic peptides with partial sequences of the m1 or m2 receptor, and of their sensitivity to endoglycosidase F, which was taken as evidence that they contain glycosylation sites near the N terminus. The [3H]PrBCM-binding site in both cerebral and atrial receptors was found to be located between the N terminus and the second intracellular loop, because the size of the smallest deglycosylated peptide that contained both the [3H]PrBCM-binding and glycosylation sites was approximately 16 kDa. Cerebral receptors were 32P-phosphorylated with protein kinase C, and the major phosphorylation sites in cerebral muscarinic receptors were found to be located in a C-terminal segment including a part of the third intracellular loop, because a 32P-labeled peptide of 12-14 kDa reacted with anti-(m1 C-terminal peptide) antiserum. The presence of an intramolecular disulfide bond, probably between Cys 98 and Cys 178 in the first and second extracellular loops, respectively, was suggested by the finding that a peptide of approximately 17 kDa containing the [3H]PrBCM-binding site, but not the glycosylation sites, was partly converted to a peptide of approximately 12 kDa on treatment with beta-mercaptoethanol.     

Haptoglobin in Carnivora: a unique molecular structure in bear, cat and dog haptoglobins

Haptoglobin (Hp), a hemoglobin-binding protein in plasma, consists of α and β subunits and has a tetra-chain arrangement (β-α-α-β) connected by disulfide bridges in most mammals so far examined. Dog Hp has been reported to be unique compared with other Hps in respect that (1) the two αβ units are joined by a non-covalent interaction rather than a disulfide bridge and (2) the α chain has an oligosaccharide-binding sequence (Asn-X-Ser/Thr) and is glycosylated. To determine whether the unique structures of dog Hp are common in the Carnivora, we purified Hps from sera of bear and cat, and analyzed their subunit structure and partial amino acid sequences. The analyses by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under both reducing and non-reducing conditions, revealed that bear and cat Hps have similar subunit arrangements to dog Hp, suggesting the absence of a disulfide bridge between two α chains. This was confirmed by amino acid sequence analysis of the α chains: that is, Cys₁₅ participating in the inter-α chain disulfide bridge was replaced by Val in bear or Leu in cat and dog. Thus, the unique subunit arrangement of Hp reported in dog may be common in the Carnivora. In contrast to dog Hp, however, α chains of bear and cat Hps were found not to have the typical oligosaccharide binding sequence on their α chains and were not glycosylated.     

Effect of disulfide bridge formation on the NMR spectrum of a protein: Studies on oxidized and reduced Escherichia coli thioredoxin

Summary As a prelude to complete structure calculations of both the oxidized and reduced forms of Escherchia coli thioredoxin (M_r 11 700), we have analyzed the NMR data obtained for the two proteins under identical conditions. The complete aliphatic ¹³C assignments for both oxidized and reduced thioredoxin are reported. Correlations previously noted between ¹³C chemical shifts and secondary structure are confirmed in this work, and significant differences are observed in the C and C shifts between cis- and trans-proline, consistent with previous work that identifies this as a simple and unambiguous method of identifying cis-proline residues in proteins. Reduction of the disulfide bond in the active-site Cys³²-Gly-Pro-Cys³⁵ sequence causes changes in the ¹H, ¹⁵N and ¹³C chemical shifts of residues close to the active site, some of them quite far distant in the amino acid sequence. Coupling constants, both backbone and side chain, show some differences between the two proteins, and the NOE connectivities and chemical shifts are consistent with small changes in the positions of several side chains, including the two tryptophan rings (Trp²⁸ and Trp³¹). These results show that, consistent with the biochemical behavior of thioredoxin, there are minimal differences in backbone configuration between the oxidized and reduced forms of the protein.