Differential selectivity of protein modification by the cyclopentenone prostaglandins PGA1 and 15-deoxy-Delta12,14-PGJ2: role of glutathione

Cyclopentenone prostaglandins (cyPG) with antiinflammatory and antiproliferative properties have been envisaged as leads for the development of therapeutic agents. Because cyPG effects are mediated in part by the formation of covalent adducts with critical signaling proteins, it is important to assess the specificity of this interaction. By using biotinylated derivatives of 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)-B) and PGA(1) (PGA(1)-B) we herein provide novel evidence for the differential selectivity of protein modification by distinct cyPG. The marked quantitative and qualitative differences in the binding of 15d-PGJ(2)-B and PGA(1)-B to cellular proteins were related to a differential reactivity in the presence of glutathione (GSH), both in vitro and in intact cells. Therefore GSH levels may influence not only the intensity but also the specificity of cyPG action.     

Induction of heat shock protein 70 by herbimycin A and cyclopentenone prostaglandins in smooth muscle cells

This study characterizes Hsp70 induction in human smooth muscle cells (SMC) by herbimycin A and cyclopentenone prostaglandins. The magnitude of Hsp70 induction by cyclopentenone prostaglandins was 8- to 10-fold higher than induction by herbimycin A. Hsp70 induction by delta12PGJ2 was first observed at 10 microM, rose to 4000-5000 ng/mL within one log unit and a maximum response was not observed; concentrations of delta12PGJ2 higher than 30 microM were toxic to the cells. A maximum response with herbimycin A (500 ng/mL) was reached at 0.05 microM and maintained to 1 microM without toxicity. Both, delta12PGJ2 and herbimycin A, were inhibited by dithiothreitol (DTT, 100 microM) at lower concentrations and became less sensitive to inhibition at higher concentrations. Hsp70 induction after incubation of SMC with delta12PGJ2 followed by addition of herbimycin A was significantly higher than Hsp70 induction after incubation with herbimycin A followed by addition of delta12PGJ2. When cells were incubated with [3H]-PGJ2, followed by protein denaturation, substantial radioactivity remained protein-bound suggesting that the prostaglandin must be covalently bound. Covalent binding was largely insensitive to DTT. Maximal Hsp70 induction was observed after 5 minutes of exposure of the cells to herbimycin A followed by a 20 hour recovery period in agent-free medium. Cells required 3-4 hours of exposure to delta12PGJ2 followed by a 20 hour recovery period in order to see high Hsp70 induction. Binding of the heat shock factor (HSF) to the heat shock element (HSE) in the presence of herbimycin A or delta12PGJ2, and the effects of DTT, mirrored the results of Hsp70 induction. The results suggest that probable differences between the 2 agents are at the level of the signal transduction prior to HSF activation.     

Inhibition of growth in oral squamous carcinoma cells by cyclopentenone prostaglandins: comparison with chemotherapeutic agents

Four cyclopentenone prostaglandins (CPPGs) and PGE₂ caused significant dose-dependent inhibition in growth of human oral squamous carcinoma cells (SCC-15). The rank order of their potency was PGJ₂>PGA₁>16, 16-dimethyl PGA₁>PGA₂>PGE₂. In a follow-up experiment it was found that the mean per cent inhibition in cell growth by PGJ₂ and Δ¹²-PGJ₂ at 10⁻⁵ M was 61.22 and 63.81, while that of 5-fluorouracil and methotrexate was 36.67 and 38.86, respectively. Δ¹²-PGJ₂ and PGJ₂ induced significant dose-dependent inhibition in nuclear DNA synthesis (i.e. cell proliferation). Combining vitamin E succinate with lower concentrations of CPPGs enhanced significantly their inhibitory effect on nuclear DNA synthesis of cancer cells.     

Further studies on the site-specific protein modification by microbial transglutaminase

A guinea pig liver transglutaminase (G-TGase)-mediated procedure for the site-specific modification of chimeric proteins was recently reported. Here, an alternative method with advantages over the recent approach is described. This protocol utilizes a microbial transglutaminase (M-TGase) instead of the G-TGase as the catalyst. M-TGase, which has rather broad structural requirements as compared to the G-TGase, tends to catalyze an acyl transfer reaction between the gamma-carboxamide group of a intact protein-bound glutamine residue and various primary amines. To demonstrate the applicability of the M-TGase-catalyzed protein modification in a drug delivery system, we have utilized recombinant human interleukin 2 (rhIL-2) as the target protein and two synthetic alkylamine derivatives of poly(ethyleneglycol) (PEG12; MW 12 kDa) and galactose-terminated triantennary glycosides ((Gal)(3))) as the modifiers. For the M-TGase-catalyzed reaction with PEG12 and (Gal)(3), 1 mol of alkylamine was incorporated per mole of rhIL-2, respectively. Peptide mapping of (Gal)(3)-modified rhIL-2 ((Gal)(3)-rhIL-2) by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) suggested that the Gln74 residue in rhIL-2 was site specifically modified with (Gal)(3). The PEG12-rhIL-2 and (Gal)(3)-rhIL-2 conjugates retained full bioactivity relative to the unmodified rhIL-2. In pharmacokinetic studies, PEG12-rhIL-2 was eliminated more slowly from the circulation than rhIL-2, whereas (Gal)(3)-rhIL-2 accumulated in the liver via hepatic asialoglycoprotein receptor binding. The results of this study expand the applicability of the TGase-catalyzed methodology for the preparation of protein conjugates for clinical use.     

Role of liquid membrane phenomenon in the biological actions of prostaglandins: studies on prostaglandin E1 and prostaglandin F2 alpha

Modifications in the transport of relevant permeants to their respective sites of action due to the liquid membranes formed by prostaglandins in association with lecithin and cholesterol have been discussed in the light of biological actions of prostaglandins. It has been shown that the phenomenon of liquid membrane formation is likely to make a significant contribution to the biological actions.     

Further studies on prostaglandins. III. Effect of prostaglandins on thyroid activity as assessed by 125I.

The effect of crude prostaglandins extraced from prostate glands of camels as well as that PGE1, on the thyroid activity of male immature Boskat rabbits, was investigated. The iodinated amino acid fractions (T1, T2, T3 and T4) in the serum of the injected groups were determined in control and treated animals. Cytological work on sections of the thyroid glands of the treated and control rabbits were performed to study the effect on subcellular level. The results indicate that both crude and pure prostaglandins have a stimulating effect on thyroid activity. This was indicated by the increased 125I uptake of the thyroids of rabbits and evidenced histologically. PGE1, was found to be more effective as compared with the crude prostaglandins. The indinated amino acid and hormone fractions in the serum of the treated groups showed that T2 was increased as a result of injection of the crude prostaglandins, and T4 was increased after PGE1 injection. Tables and figures explained diversed effect.     

Taking it step by step: mechanistic insights from structural studies of ubiquitin/ubiquitin-like protein modification pathways

Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a diverse array of cellular pathways through post-translational attachment to protein substrates. Ub/Ubl-mediated signaling is initiated through E1, E2, and E3-mediated conjugation, transduced by proteins that recognize Ub/Ubl-modified substrates, and terminated by proteases which remove the Ub/Ubl from the substrate. Recent structural studies have elucidated mechanisms pertinent to Ub/Ubl conjugation, recognition, and deconjugation, highlighting essential steps during Ub/Ubl modification that illustrate common and divergent mechanistic themes within this important process.     

Nmr studies on linear homo-oligopeptides: A perspective view

The use of high-resolution nmr to determine the structure of linear homo-oligopeptides in solution is described. Complete assignments of NH and α-CH resonances were elucidated based upon a guest–host series of compounds and selective α-deuteration of residues in the chain. The conformations for oligomethionines and oligoglutamates in dimethylsulfoxide, chloroform, and trifluoroethanol are discussed, and molecular models are described. Recent studies of L -glutamate peptides attached to polyoxyethylene are also included. These peptide–polyoxyethylene conjugates are soluble in a broad range of solvents, such as dimethyl-sulfoxide, chloroform, trifluoroethanol, and water. Preliminary conclusions are drawn concerning the effects of amine terminal end groups and the nature of the solvent on the conformations of these peptide derivatives.     

How our view of animal phylogeny was reshaped by molecular approaches: lessons learned

In the late 1980s, researchers began applying molecular sequencing tools to questions of deep animal phylogeny. These advances in sequencing were accompanied with improvements in computation and phylogenetic methods, and served to significantly reshape our understanding of metazoan evolution. Prior to this time, researchers asserted phylogenetic hypotheses based on their experience with taxa and to some degree, their authority. Molecular phylogenetic tools provided discrete methods and objective characters for reconstructing phylogeny. Nonetheless, major changes to widely accepted views, such as animal phylogeny, take time to be accepted. Development and acceptance of our current understanding of animal evolution occurred in three main phases: initial hypotheses based on 18S data, confirmation with additional molecular markers, and continued refinement with phylogenomics. With the advent of ideas such as Lophotrochozoa and Ecdysozoa, flaws in the traditional view became apparent. We now understand that complex morphological and embryological features (e.g., segmentation, coelom formation, development of body cavities) are much more evolutionarily plastic than previously recognized. Here, I explore how the transition from the traditional to the modern phylogenetic understanding of animal phylogeny occurred and examine some implications of this change in understanding. As the field moves forward, the utility of morphological and embryological characters for reconstruction of deep animal phylogeny should be discouraged. Instead, these characters should be interpreted in the light of independent phylogeny.     

Molecular basis of enzyme inactivation by an endogenous electrophile 4-hydroxy-2-nonenal: identification of modification sites in glyceraldehyde-3-phosphate dehydrogenase

4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation-derived reactive aldehyde, is a potent inhibitor of sulfhydryl enzymes, such as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It has been suggested that HNE exerts an inhibitory effect on the enzyme due to the modification of the cysteine residue (Cys-149) at the catalytic site generating the HNE-cysteine Michael addition-type adduct [Uchida, K., and Stadtman, E. R. (1993) J. Biol. Chem. 268, 6388-6393]. In the study presented here, to elucidate the mechanism for the inactivation of GAPDH by HNE, we attempted to identify the modification sites of the enzyme by monitoring the formation of the HNE Michael adducts by mass spectrometric methods. Incubation of GAPDH (1 mg/mL) with 1 mM HNE in 50 mM sodium phosphate buffer (pH 7.4) at 37 degrees C resulted in a time-dependent loss of enzyme activity, which was associated with the covalent binding of HNE to the enzyme. To identify the site of modification of GAPDH by HNE, both the HNE-pretreated and untreated GAPDH were digested with trypsin and V8 protease, and the resulting peptides were subjected to electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS). This technique identified five peptides, which contained the HNE adducts at His-164, Cys-244, Cys-281, His-327, and Lys-331 and revealed that both His-164 and Cys-281 were very rapidly modified at 5 min, followed by Cys-244 at 15 min and His-327 and Lys-331 at 30 min. These observations and the observation that the HNE modification of the catalytic center, Cys-149, was not observed suggest that the HNE inactivation of GAPDH is not due to the modification of the catalytic center but to the selective modification of amino acids primarily located in the surface of the GAPDH molecule.     

Proteomic analysis of protein expression and oxidative modification in r6/2 transgenic mice: a model of Huntington disease

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by motor, psychiatric, and cognitive symptoms. The genetic defect responsible for the onset of the disease, expansion of CAG repeats in exon 1 of the gene that codes for huntingtin on chromosome 4, has been unambiguously identified. On the other hand, the mechanisms by which the mutation causes the disease are not completely understood yet. However, defects in energy metabolism of affected cells may cause oxidative damage, which has been proposed as one of the underlying molecular mechanisms that participate in the etiology of the disease. In our effort to investigate the extent of oxidative damage occurring at the protein level, we used a parallel proteomic approach to identify proteins potentially involved in processes upstream or downstream of the disease-causing huntingtin in a well established HD mouse model (R6/2 transgenic mice). We have demonstrated that the expression levels of dihydrolipoamide S-succinyltransferase and aspartate aminotransferase increase consistently over the course of disease (10-week-old mice). In contrast, pyruvate dehydrogenase expression levels were found to be decreased in 10-week-old HD transgenic mice compared with young (4-week-old) mice. Our experimental approach also led to the identification of oxidatively modified proteins. Six proteins were found to be significantly oxidized in old R6/2 transgenic mice compared with either young transgenic mice or non-transgenic mice. These proteins are alpha-enolase, gamma-enolase (neuron-specific enolase), aconitase, the voltage-dependent anion channel 1, heat shock protein 90, and creatine kinase. Because oxidative damage has proved to play an important role in the pathogenesis and the progression of Huntington disease, our results for the first time identify specific oxidatively modified proteins that potentially contribute to the pathogenesis of Huntington disease.     

Breadth by depth: expanding our understanding of the repair of transposon-induced DNA double strand breaks via deep-sequencing

The transposases of DNA transposable elements catalyze the excision of the element from the host genome, but are not involved in the repair of the resulting double-strand break. To elucidate the role of various host DNA repair and damage response proteins in the repair of the hairpin-ended double strand breaks (DSBs) generated during excision of the maize Ac element in Arabidopsis thaliana, we deep-sequenced hundreds of thousands of somatic excision products from a variety of repair- or response-defective mutants. We find that each of these repair/response defects negatively affects the preservation of the ends, resulting in an enhanced frequency of deletions, insertions, and inversions at the excision site. The spectra of the resulting repair products demonstrate, not unexpectedly, that the canonical nonhomologous end joining (NHEJ) proteins DNA ligase IV and KU70 play an important role in the repair of the lesion generated by Ac excision. Our data also indicate that auxiliary NHEJ repair proteins such as DNA ligase VI and DNA polymerase lambda are routinely involved in the repair of these lesions. Roles for the damage response kinases ATM and ATR in the repair of transposition-induced DSBs are also discussed.     

Heteronuclear NMR studies of the specificity of the post-translational modification of biotinyl domains by biotinyl protein ligase

The lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes and the biotinyl domains of biotin-dependent enzymes have homologous structures, but the target lysine residue in each domain is correctly selected for posttranslational modification by lipoyl protein ligase and biotinyl protein ligase, respectively. We have applied two-dimensional heteronuclear NMR spectroscopy to investigate the interaction between the apo form of the biotinyl domain of the biotin carboxyl carrier protein of acetyl-CoA carboxylase and the biotinyl protein ligase (BPL) from Escherichia coli. Heteronuclear multiple quantum coherence NMR spectra of the 15N-labelled biotinyl domain were recorded in the presence and absence of the ligase and backbone amide 1H and 15N chemical shifts were evaluated. Small, but significant, changes in chemical shift were found in two regions, including the tight beta-turn that houses the lysine residue targetted for biotinylation, and the beta-strand 2 and the loop that precedes it in the domain. When compared with the three-dimensional structure, sequence alignments of other biotinyl and lipoyl domains, and mutagenesis data, these results give a clear indication of how the biotinyl domain is both recognised by BPL and distinguished from the structurally related lipoyl domain to ensure correct posttranslational modification.     

Central activation of thermogenesis by prostaglandins: dependence on CRF

Central (intracerebroventricular) injections of PGE2, PGF2 alpha caused dose dependent increases in oxygen consumption (VO2) and colonic temperature in conscious rats. The effects of combined injection of maximal doses of CRF and PGE2 were additive, whereas PGF2 alpha and CRF were not. A CRF receptor antagonist, (alpha helical CRF 9-41, 25 micrograms icv) markedly inhibited the effects of PGF2 alpha on VO2 and temperature, but did not affect the actions of PGE2. These data indicate that PGF2 alpha and PGE2 stimulate thermogenesis by two different mechanisms, the former depending on CRF release. PGF2 alpha may be involved in the thermogenic actions of interleukin-1 beta which is also a potent thermogenic agent acting via CRF.     

Model studies on iTRAQ modification of peptides: sequence-dependent reaction specificity

A multiplexed peptide quantification strategy using the iTRAQ reagent has been described for relative measurements of peptides in digested protein mixtures. To validate the chemical specificity of the iTRAQ reaction, we have performed a detailed study of iTRAQ reactivity with two sets of synthetic peptides. The first set of peptides had sequences of Tyr-Xaa-Ser-Glu-Gly-Leu-Ser-Lys and Tyr-Xaa-Ser-Glu-Tyr-Leu-Ser-Lys where Xaa = Ala, Pro, Trp, Tyr, or Glu and was designed to study the extent of O-acylation by iTRAQ, especially hydroxyl-containing residues in different positions. The second set of peptides included Ala-Ser-Glu-His-Ala-Xaa-Tyr-Gly where Xaa = Ser, Thr, or Tyr and was selected to investigate the effect of histidyl residues separated by one amino acid residue from seryl, tyrosyl, or threonyl residues. Our findings indicated that, in addition to variable levels of O-acylation of nonsequence-specific hydroxyl-containing residues, significant sequence-specific O-acylation of seryl, threonyl, and tyrosyl hydroxyls occurred when separated one residue removed from a histidyl residue, that is, (Tyr/Ser)-Xaa-His or His-Xaa-(Tyr/Ser/Thr). This behavior was verified by a separate spiking experiment of one of the first set of peptides into Escherichia coli protein extracts, followed by retention time targeted LC-MS/MS to demonstrate the occurrence of modifications in a complex mixture. These sequence-dependent O-acylation modifications can be confounding factors to accurate MS quantification. Reversal of peptide O-acylation by the iTRAQ reagent can be accomplished by reaction with hydroxylamine with virtually no cleavage of N-acylation and is a recommended modification of the iTRAQ protocol for many applications.     

New silk protein: modification of silk protein by gene engineering for production of biomaterials.

The interest in silk fibroin morphology and structure have increased due to its attractiveness for bio-related applications. Silk fibers have been used as sutures for a long time in the surgical field, due to the biocompatibility of silk fibroin fibers with human living tissue. In addition, it has been demonstrated that silk can be used as a substrate for enzyme immobilization in biosensors. A more complete understanding of silk structure would provide the possibility to further exploit silk fibroin for a wide range of new uses, such as the production of oxygen-permeable membranes and biocompatible materials. Silk fibroin-based membranes could be utilized as soft tissue compatible polymers. Baculovirus-mediated transgenesis of the silkworm allows specific alterations in a target sequence. Homologous recombination of a foreign gene downstream from a powerful promoter, such as the fibroin promoter, would allow the constitutive production of a useful protein in the silkworm and the modification of the character of silk protein. A chimeric protein consisted of fibroin and green fluorescent protein was expressed under the control of fibroin in the posterior silk gland and the gene product was spun into the cocoon layer. This technique, gene targeting, will lead to the modification and enhancement of physicochemical properties of silk protein.