scholarly journals Accumulation of 15-deoxy-Δ12,14-prostaglandin J2 adduct formation with Keap1 over time: effects on potency for intracellular antioxidant defence induction

2008 ◽  
Vol 411 (2) ◽  
pp. 297-306 ◽  
Author(s):  
Joo Yeun Oh ◽  
Niroshini Giles ◽  
Aimee Landar ◽  
Victor Darley-Usmar
Keyword(s):  
Cell Signalling ◽  
Adduct Formation ◽  
Covalent Modification ◽  
Erythroid Cell ◽  
Covalent Adduct ◽  
Electrophilic Lipids ◽  
Covalent Adducts ◽  
Subsequent Activation ◽  
Prostaglandin J2

The COX (cyclo-oxygenase) pathway generates the reactive lipid electrophile 15d-PGJ2 (15-deoxy-Δ12,14-prostaglandin J2), which forms covalent protein adducts that modulate cell signalling pathways. It has been shown that this regulates important biological responses, including protection against oxidative stress, and supports the proposal that 15d-PGJ2 has pharmacological potential. Protective pathways activated by 15d-PGJ2 include those controlling the synthesis of the intracellular antioxidants GSH and the enzyme HO-1 (haem oxygenase-1). The induction of the synthesis of these intracellular antioxidants is, in large part, regulated by covalent modification of Keap1 (Kelch-like erythroid cell-derived protein with cap‘n’collar homology-associated protein 1) by the lipid and the subsequent activation of the EpRE (electrophile-response element). For the first time, we show that the potency of 15d-PGJ2 as a signalling molecule in endothelial cells is significantly enhanced by the accumulation of the covalent adduct with 15d-PGJ2 and endogenous Keap1 over the time of exposure to the prostaglandin. The consequence of this finding is that signalling initiated by electrophilic lipids differs from agonists that do not form covalent adducts with proteins because the constant generation of very low concentrations of 15d-PGJ2 can lead to induction of GSH or HO-1. In the course of these studies we also found that a substantial amount (97–99%) of exogenously added 15d-PGJ2 is inactivated in the medium and does not enter the cells to initiate cell signalling. In summary, we propose that the accumulation of covalent adduct formation with signalling proteins provides a mechanism through which endogenous intracellular formation of electrophilic lipids from COX can exert an anti-inflammatory effect in vivo.

scholarly journals Destabilizing missense mutations in the tumour suppressor protein p53 enhance its ubiquitination in vitro and in vivo

2006 ◽  
Vol 397 (2) ◽  
pp. 355-367 ◽  
Author(s):  
Harumi Shimizu ◽  
David Saliba ◽  
Maura Wallace ◽  
Lee Finlan ◽  
Patrick R. R. Langridge-Smith ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Covalent Modification ◽  
High Molecular Mass ◽  
Mutant Form ◽  
Dependent Manner ◽  
Ubiquitination System ◽  
Covalent Adducts ◽  
In Cells

p53 ubiquitination catalysed by MDM2 (murine double minute clone 2 oncoprotein) provides a biochemical assay to dissect stages in E3-ubiquitin-ligase-catalysed ubiquitination of a conformationally flexible protein. A mutant form of p53 (p53F270A) containing a mutation in the second MDM2-docking site in the DNA-binding domain of p53 (F270A) is susceptible to modification of long-lived and high-molecular-mass covalent adducts in vivo. Mutant F270A is hyperubiquitinated in cells as defined by immunoprecipitation and immunoblotting with an anti-ubiquitin antibody. Transfection of His-tagged ubiquitin along with p53R175H or p53F270A also results in selective hyperubiquitination in cells under conditions where wild-type p53 is refractory to covalent modification. The extent of mutant p53R175H or p53F270A unfolding in cells as defined by exposure of the DO-12 epitope correlates with the extent of hyperubiquitination, suggesting a link between substrate conformation and E3 ligase function. The p53F270A:6KR chimaeric mutant (where 6KR refers to the simultaneous mutation of lysine residues at positions 370, 372, 373, 381, 382 and 386 to arginine) maintains the high-molecular-mass covalent adducts and is modified in an MDM2-dependent manner. Using an in vitro ubiquitination system, mutant p53F270A and the p53F270A:6KR chimaeric mutant is also subject to hyperubiquitination outwith the C-terminal domain, indicating direct recognition of the mutant p53 conformation by (a) factor(s) in the cell-free ubiquitination system. These data identify an in vitro and in vivo assay with which to dissect how oligomeric protein conformational alterations are linked to substrate ubiquitination in cells. This has implications for understanding the recognition of misfolded proteins during aging and in human diseases such as cancer.

scholarly journals Cellular mechanisms of redox cell signalling: role of cysteine modification in controlling antioxidant defences in response to electrophilic lipid oxidation products

2004 ◽  
Vol 378 (2) ◽  
pp. 373-382 ◽  
Author(s):  
Anna-Liisa LEVONEN ◽  
Aimee LANDAR ◽  
Anup RAMACHANDRAN ◽  
Erin K. CEASER ◽  
Dale A. DICKINSON ◽  
...  
Keyword(s):  
Lipid Oxidation ◽  
Molecular Mechanisms ◽  
Intact Cell ◽  
Cell Signalling ◽  
Oxidation Products ◽  
Oxidized Lipids ◽  
Related Factor ◽  
Lipid Oxidation Products ◽  
Electrophilic Lipids ◽  
Prostaglandin J2

The molecular mechanisms through which oxidized lipids and their electrophilic decomposition products mediate redox cell signalling is not well understood and may involve direct modification of signal-transduction proteins or the secondary production of reactive oxygen or nitrogen species in the cell. Critical in the adaptation of cells to oxidative stress, including exposure to subtoxic concentrations of oxidized lipids, is the transcriptional regulation of antioxidant enzymes, many of which are controlled by antioxidant-responsive elements (AREs), also known as electrophile-responsive elements. The central regulator of the ARE response is the transcription factor Nrf2 (NF-E2-related factor 2), which on stimulation dissociates from its cytoplasmic inhibitor Keap1, translocates to the nucleus and transactivates ARE-dependent genes. We hypothesized that electrophilic lipids are capable of activating ARE through thiol modification of Keap1 and we have tested this concept in an intact cell system using induction of glutathione synthesis by the cyclopentenone prostaglandin, 15-deoxy-Δ12,14-prostaglandin J2. On exposure to 15-deoxy-Δ12,14-prostaglandin J2, the dissociation of Nrf2 from Keap1 occurred and this was dependent on the modification of thiols in Keap1. This mechanism appears to encompass other electrophilic lipids, since 15-A2t-isoprostane and the lipid aldehyde 4-hydroxynonenal were also shown to modify Keap1 and activate ARE. We propose that activation of ARE through this mechanism will have a major impact on inflammatory situations such as atherosclerosis, in which both enzymic as well as non-enzymic formation of electrophilic lipid oxidation products are increased.

Oxidative biotransformation of oxazepam to reactive and nonreactive products in rat, mouse and human microsomes

1995 ◽  
Vol 14 (10) ◽  
pp. 779-786 ◽  
Author(s):  
RJ Griffin ◽  
LT Burka ◽  
KB Demby
Keyword(s):  
Carboxylic Acid ◽  
Oxidative Metabolism ◽  
Adduct Formation ◽  
Protein Adducts ◽  
Major Metabolite ◽  
Cyclohexene Oxide ◽  
F344 Rat ◽  
Covalent Adduct

1 The oxidative metabolism of oxazepam by human, B6C3F1 mouse and F344 rat microsomes was examined. The major metabolite in all three species was 6-chloro-4- phenyl-2(1H)-quinazolinecarboxylic acid (CPQ-carboxylic acid). In addition, rat microsomes produced 4'-hydroxyox azepam and oxazepam-dihydrodiol in NADPH-containing incubations. 2 Covalent protein adducts were increased by the addi tion of NADPH to rat and mouse microsomes but not human microsomes. The magnitude of adduct formation was rats > mice > humans. 3 Formation of oxazepam-dihydrodiol was reduced by the addition of cyclohexene oxide and GSH to the incuba tions. Two additional metabolites were produced under these conditions. One of these was tentatively identified as a GSH conjugate. Covalent adduct formation was unaffect ed by GSH or cyclohexene oxide. 4 These results suggest that adduct formation occurred via an unknown reactive product rather than via oxazepam-epoxide, and that the relative rates of oxidative metabolism in vitro parallel that in vivo for the three species examined.

ATP potently modulates anion channel-mediated excitatory amino acid release from cultured astrocytes

2002 ◽  
Vol 283 (2) ◽  
pp. C569-C578 ◽  
Author(s):  
Alexander A. Mongin ◽  
Harold K. Kimelberg
Keyword(s):  
Amino Acid ◽  
Excitatory Amino Acid ◽  
Atp Release ◽  
Anion Channel ◽  
P2y Receptors ◽  
Cell Swelling ◽  
Channel Blockers ◽  
Medium Osmolarity ◽  
Subsequent Activation

Volume-dependent ATP release and subsequent activation of purinergic P2Y receptors have been implicated as an autocrine mechanism triggering activation of volume-regulated anion channels (VRACs) in hepatoma cells. In the brain ATP is released by both neurons and astrocytes and participates in intercellular communication. We explored whether ATP triggers or modulates the release of excitatory amino acid (EAAs) via VRACs in astrocytes in primary culture. Under basal conditions exogenous ATP (10 μM) activated a small EAA release in 70–80% of the cultures tested. In both moderately (5% reduction of medium osmolarity) and substantially (35% reduction of medium osmolarity) swollen astrocytes, exogenous ATP greatly potentiated EAA release. The effects of ATP were mimicked by P2Y agonists and eliminated by P2Y antagonists or the ATP scavenger apyrase. In contrast, the same pharmacological maneuvers did not inhibit volume-dependent EAA release in the absence of exogenous ATP, ruling out a requirement of autocrine ATP release for VRAC activation. The ATP effect in nonswollen and moderately swollen cells was eliminated by a 5–10% increase in medium osmolarity or by anion channel blockers but was insensitive to tetanus toxin pretreatment, further supporting VRAC involvement. Our data suggest that in astrocytes ATP does not trigger EAA release itself but acts synergistically with cell swelling. Moderate cell swelling and ATP may serve as two cooperative signals in bidirectional neuron-astrocyte communication in vivo.

scholarly journals Influence of Dopamine on Fluorescent Advanced Glycation End Products Formation Using Drosophila melanogaster

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 453
Author(s):  
Ana Filošević Vujnović ◽  
Katarina Jović ◽  
Emanuel Pištan ◽  
Rozi Andretić Waldowski
Keyword(s):  
Drosophila Melanogaster ◽  
Advanced Glycation End Products ◽  
Model Organism ◽  
Covalent Modification ◽  
Advanced Glycation ◽  
Biomarkers Of Aging ◽  
Glycation End Products ◽  
End Products

Non-enzymatic glycation and covalent modification of proteins leads to Advanced Glycation End products (AGEs). AGEs are biomarkers of aging and neurodegenerative disease, and can be induced by impaired neuronal signaling. The objective of this study was to investigate if manipulation of dopamine (DA) in vitro using the model protein, bovine serum albumin (BSA), and in vivo using the model organism Drosophila melanogaster, influences fluorescent AGEs (fAGEs) formation as an indicator of dopamine-induced oxidation events. DA inhibited fAGEs-BSA synthesis in vitro, suggesting an anti-oxidative effect, which was not observed when flies were fed DA. Feeding flies cocaine and methamphetamine led to increased fAGEs formation. Mutants lacking the dopaminergic transporter or the D1-type showed further elevation of fAGEs accumulation, indicating that the long-term perturbation in DA function leads to higher production of fAGEs. To confirm that DA has oxidative properties in vivo, we fed flies antioxidant quercetin (QUE) together with methamphetamine. QUE significantly decreased methamphetamine-induced fAGEs formation suggesting that the perturbation of DA function in vivo leads to increased oxidation. These findings present arguments for the use of fAGEs as a biomarker of DA-associated neurodegenerative changes and for assessment of antioxidant interventions such as QUE treatment.

scholarly journals Frequency-preference response in covalent modification cycles under substrate sequestration conditions

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Juliana Reves Szemere ◽  
Horacio G. Rotstein ◽  
Alejandra C. Ventura
Keyword(s):  
Frequency Response ◽  
Covalent Modification ◽  
Protein Abundance ◽  
Dynamic Features ◽  
Signaling Systems ◽  
Low Pass ◽  
Low Pass Filters ◽  
Periodic Inputs ◽  
Preferred Frequency Response

AbstractCovalent modification cycles (CMCs) are basic units of signaling systems and their properties are well understood. However, their behavior has been mostly characterized in situations where the substrate is in excess over the modifying enzymes. Experimental data on protein abundance suggest that the enzymes and their target proteins are present in comparable concentrations, leading to substrate sequestration by the enzymes. In this enzyme-in-excess regime, CMCs have been shown to exhibit signal termination, the ability of the product to return to a stationary value lower than its peak in response to constant stimulation, while this stimulation is still active, with possible implications for the ability of systems to adapt to environmental inputs. We characterize the conditions leading to signal termination in CMCs in the enzyme-in-excess regime. We also demonstrate that this behavior leads to a preferred frequency response (band-pass filters) when the cycle is subjected to periodic stimulation, whereas the literature reports that CMCs investigated so far behave as low-pass filters. We characterize the relationship between signal termination and the preferred frequency response to periodic inputs and we explore the dynamic mechanism underlying these phenomena. Finally, we describe how the behavior of CMCs is reflected in similar types of responses in the cascades of which they are part. Evidence of protein abundance in vivo shows that enzymes and substrates are present in comparable concentrations, thus suggesting that signal termination and frequency-preference response to periodic inputs are also important dynamic features of cell signaling systems, which have been overlooked.

Enantiospecificity of Covalent Adduct Formation by Benzo[a]pyrene anti-Diol Epoxide with Human Serum Albumin

1994 ◽  
Vol 7 (6) ◽  
pp. 829-835 ◽  
Author(s):  
Billy W. Day ◽  
Paul L. Skipper ◽  
Joseph Zaia ◽  
Kuldip Singh ◽  
Steven R. Tannenbaum
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Adduct Formation ◽  
Diol Epoxide ◽  
Covalent Adduct

scholarly journals Cell signalling by reactive lipid species: new concepts and molecular mechanisms

2012 ◽  
Vol 442 (3) ◽  
pp. 453-464 ◽  
Author(s):  
Ashlee Higdon ◽  
Anne R. Diers ◽  
Joo Yeun Oh ◽  
Aimee Landar ◽  
Victor M. Darley-Usmar
Keyword(s):  
Cell Death ◽  
Redox Regulation ◽  
Molecular Mechanisms ◽  
Chemical Reactivity ◽  
Cell Signalling ◽  
Significant Proportion ◽  
Covalent Modification ◽  
Lipid Species ◽  
Electrophilic Stress ◽  
Cellular Antioxidants

The process of lipid peroxidation is widespread in biology and is mediated through both enzymatic and non-enzymatic pathways. A significant proportion of the oxidized lipid products are electrophilic in nature, the RLS (reactive lipid species), and react with cellular nucleophiles such as the amino acids cysteine, lysine and histidine. Cell signalling by electrophiles appears to be limited to the modification of cysteine residues in proteins, whereas non-specific toxic effects involve modification of other nucleophiles. RLS have been found to participate in several physiological pathways including resolution of inflammation, cell death and induction of cellular antioxidants through the modification of specific signalling proteins. The covalent modification of proteins endows some unique features to this signalling mechanism which we have termed the ‘covalent advantage’. For example, covalent modification of signalling proteins allows for the accumulation of a signal over time. The activation of cell signalling pathways by electrophiles is hierarchical and depends on a complex interaction of factors such as the intrinsic chemical reactivity of the electrophile, the intracellular domain to which it is exposed and steric factors. This introduces the concept of electrophilic signalling domains in which the production of the lipid electrophile is in close proximity to the thiol-containing signalling protein. In addition, we propose that the role of glutathione and associated enzymes is to insulate the signalling domain from uncontrolled electrophilic stress. The persistence of the signal is in turn regulated by the proteasomal pathway which may itself be subject to redox regulation by RLS. Cell death mediated by RLS is associated with bioenergetic dysfunction, and the damaged proteins are probably removed by the lysosome-autophagy pathway.

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