27 research outputs found
Total Synthesis Confirms the Molecular Structure Proposed for Oxidized Levuglandin D<sub>2</sub>
Levuglandins (LG)ÂD<sub>2</sub> and
LGE<sub>2</sub> are γ-ketoaldehyde
levulinaldehyde derivatives with prostanoid side chains produced by
spontaneous rearrangement of the endoperoxide intermediate PGH<sub>2</sub> in the biosynthesis of prostaglandins. Covalent adduction
of LGs with the amyloid peptide Aβ<sub>1–42</sub> promotes
formation of the type of oligomers that have been associated with
neurotoxicity and are a pathologic hallmark of Alzheimer’s
disease. Within 1 min of their generation during the production of
PGH<sub>2</sub> by cyclooxygenation of arachidonic acid, LGs are sequestered
by covalent adduction to proteins. In view of this high proclivity
for covalent adduction, it is understandable that free LGs have never
been detected <i>in vivo</i>. Recently a catabolite, believed
to be an oxidized derivative of LGD<sub>2</sub> (ox-LGD<sub>2</sub>), a levulinic acid hydroxylactone with prostanoid side chains, was
isolated from the red alga <i>Gracilaria edulis</i> and
detected in mouse tissues and in the lysate of phorbol-12-myristate-13-acetate-treated
THP-1 cells incubated with arachidonic acid. Such oxidative catabolism
of LGD<sub>2</sub> is remarkable because it must be outstandingly
efficient to prevail over adduction with proteins and because it requires
a unique dehydrogenation. We now report a concise total synthesis
that confirms the molecular structure proposed for ox-LGD<sub>2</sub>. The synthesis also produces ox-LGE<sub>2</sub>, which readily undergoes
allylic rearrangement to Δ<sup>6</sup>-ox-LGE<sub>2</sub>
Bioactive 4‑Oxoheptanedioic Monoamide Derivatives of Proteins and Ethanolaminephospholipids: Products of Docosahexaenoate Oxidation
Oxidative
stress causes lipid-derived oxidative modification of
biomolecules that has been implicated in many pathological states.
Phospholipids containing polyunsaturated fatty acids are major targets
of free radical-initiated oxidation. Phospholipids that incorporate
docosahexaenoate (DHA) are highly enriched in important neural structures
including the brain and retina, where DHA comprises 40% and 60% of
total fatty acids, respectively. Oxidative fragmentation of 2-docosahexaenoyl-1-palmityl-<i>sn</i>-glycerophosphocholine generates esters of 4-hydroxy-7-oxohept-5-enoic
acid (HOHA) and 4-keto-7-oxohept-5-enoic acid (KOHA) with 2-lysophosphatidylcholine,
HOHA-PC, and KOHA-PC. Covalent HOHA adducts that incorporate the primary
amino groups of proteins and ethanolamine phospholipids in carboxyethylpyrrole
(CEP) derivatives were detected immunologically with anti-CEP antibodies
in human tumors, retina, and blood. Now, we generated an anti-OHdiA
antibody to test the hypothesis that KOHA adducts, which incorporate
the primary amino groups of proteins or ethanolamine phospholipids
in 4-oxo-heptanedioic (OHdiA) monoamide derivatives, are present in
vivo. However, whereas the anti-CEP antibody is highly specific and
does not cross-react with the OHdiA monoamide epitope, the anti-OHdiA
monoamide antibody cross-reacted with CEP epitopes making it of little
value as an analytical tool for OHdiA monoamides but suggesting the
possibility that OHdiA monoamides would exhibit receptor-mediated
biological activity similar to that of CEP. An LC-MS/MS method was
developed that allows quantification of OHdiA derivatives in biological
samples. We now find that KOHA-PC forms OHdiA monoamide adducts of
proteins and ethanolamine phospholipids and that OHdiA-protein levels
are significantly higher than OHdiA-ethanloamine phospholipid levels
in blood from healthy human subjects, 0.45 μM and 0.18 μM,
respectively (<i>n</i> = 3, and <i>p</i> = 0.027).
OHdiA monoamide epitopes are angiogenic, causing TLR2-dependent adhesion
and tube formation by human umbilical vein endothelial cells. OHdiA
monoamide epitopes are only slightly less potent than CEP epitopes
that contribute to the pathological angiogenesis of age-related macular
degeneration and tumor growth
Low-Density Lipoprotein Has an Enormous Capacity To Bind (<i>E</i>)-4-Hydroxynon-2-enal (HNE): Detection and Characterization of Lysyl and Histidyl Adducts Containing Multiple Molecules of HNE
(<i>E</i>)-4-Hydroxynon-2-enal (HNE), an electrophilic bifunctional cytotoxic lipid peroxidation product, forms covalent adducts with nucleophilic side chains of amino acid residues. HNE-derived adducts have been implicated in many pathophysiological processes including atherosclerosis, diabetes, and Alzheimer’s disease. Tritium- and deuterium-labeled HNE (<i>d</i><sub>4</sub>-HNE) were used orthogonally to study adduction with proteins and individual nucleophilic groups of histidyl, lysyl, and cysteine residues. Using tritium-labeled HNE, we detected the binding of 486 molecules of HNE per low-density lipoprotein (LDL) particle, significantly more than the total number of all reactive nucleophiles in the LDL particle. This suggests the formation of adducts that incorporate multiple molecules of HNE with some nucleophilic amino acid side chains. We also found that the reaction of a 1:1 mixture of <i>d</i><sub>4</sub>-HNE and <i>d</i><sub>0</sub>-HNE with <i>N</i>-acetylhistidine, <i>N</i>-acetyl-Gly-Lys-OMe, or <i>N</i>-acetyl cysteine generates 1:1, 2:1, and 3:1 adducts, which exhibit unique mass spectral signatures that aid in structural characterization. A domino-like reaction of initial 1:1 HNE Michael adducts of histidyl or lysyl nucleophiles with multiple additional HNE molecules forms 2:1 and 3:1 adducts that were structurally characterized by tandem mass spectrometry
Molecular Structures of Isolevuglandin-Protein Cross-Links
Isolevuglandins (isoLGs) are stereo
and structurally isomeric γ-ketoaldehydes
produced through free radical-induced oxidation of arachidonates.
Some isoLG isomers are also generated through enzymatic cyclooxygenation.
Post-translational modification of proteins by isoLGs is associated
with loss-of-function, cross-linking and aggregation. We now report
that a low level of modification by one or two molecules of isoLG
has a profound effect on the activity of a multi subunit protease,
calpain-1. Modification of one or two key lysyl residues apparently
suffices to abolish catalytic activity. Covalent modification of calpain-1
led to intersubunit cross-linking. Hetero- and homo-oligomers of the
catalytic and regulatory subunits of calpain-1 were detected by SDS–PAGE
with Western blotting. <i>N</i>-Acetyl-glycyl-lysine methyl
ester and β-amyloid(11–17) peptide EVHHQKL were used
as models for characterizing the cross-linking of protein lysyl residues
resulting from adduction of iso[4]ÂLGE<sub>2</sub>. Aminal, bispyrrole,
and trispyrrole cross-links of these two peptides were identified
and fully characterized by mass spectrometry. Aminal and bispyrrole
dimers were both detected. Furthermore, a complex mixture of derivatives
of the bispyrrole cross-link containing one or more additional atoms
of oxygen was found. Interesting differences are evident in the predominant
cross-link type generated in the reaction of iso[4]ÂLGE<sub>2</sub> with these peptides. More aminal cross-links versus bispyrrole are
formed during the reaction of the dipeptide with iso[4]ÂLGE<sub>2</sub>. In contrast, more bispyrrole versus aminal cross-links are formed
during the reaction of EVHHQKL with iso[4]ÂLGE<sub>2</sub>. It is tempting
to speculate that the EVHHQKL peptide–pyrrole modification
forms noncovalent aggregates that favor the production of covalent
bispyrrole cross-links because β-amyloid(11–17) tends
to spontaneously oligomerize
The Oxidative Stress Product Carboxyethylpyrrole Potentiates TLR2/TLR1 Inflammatory Signaling in Macrophages
<div><p>Oxidative stress is key in the pathogenesis of several diseases including age-related macular degeneration (AMD), atherosclerosis, diabetes, and Alzheimer's disease. It has previously been established that a lipid peroxidation product, carboxyethylpyrrole (CEP), accumulates in the retinas of AMD patients. Retinal infiltrating macrophages also accumulate in the retinas of both AMD patients and in a murine model of AMD. We therefore investigated the ability of CEP-adducts to activate innate immune signaling in murine bone-marrow derived macrophages (BMDMs). We found that CEP specifically synergizes with low-dose TLR2-agonists (but not agonists for other TLRs) to induce production of inflammatory cytokines. Moreover, CEP selectively augments TLR2/TLR1-signaling instead of TLR2/TLR6-signaling. These studies uncover a novel synergistic inflammatory relationship between an endogenously produced oxidation molecule and a pathogen-derived product, which may have implications in the AMD disease process and other oxidative stress-driven pathologies.</p></div
4‑Hydroxy-7-oxo-5-heptenoic Acid Lactone Induces Angiogenesis through Several Different Molecular Pathways
Oxidative stress
and angiogenesis have been implicated not only
in normal phenomena such as tissue healing and remodeling but also
in many pathological processes. However, the relationships between
oxidative stress and angiogenesis still remain unclear, although oxidative
stress has been convincingly demonstrated to influence the progression
of angiogenesis under physiological and pathological conditions. The
retina is particularly susceptible to oxidative stress because of
its intensive oxygenation and high abundance of polyunsaturated fatty
acyls. In particular, it has high levels of docosahexanoates, whose
oxidative fragmentation produces 4-hydroxy-7-oxo-5-heptenoic acid
lactone (HOHA-lactone). Previously, we found that HOHA-lactone is
a major precursor of 2-(ω-carboxyethyl)Âpyrrole (CEP) derivatives,
which are tightly linked to age-related macular degeneration (AMD).
CEPs promote the pathological angiogenesis of late-stage AMD. We now
report additional mechanisms by which HOHA-lactone promotes angiogenesis.
Using cultured ARPE-19 cells, we observed that HOHA-lactone induces
secretion of vascular endothelial growth factor (VEGF), which is correlated
to increases in reactive oxygen species and decreases in intracellular
glutathione (GSH). Wound healing and tube formation assays provided,
for the first time, in vitro evidence that HOHA-lactone induces the
release of VEGF from ARPE-19 cells, which promotes angiogenesis by
human umbilical vein endothelial cells (HUVEC) in culture. Thus, HOHA-lactone
can stimulate vascular growth through a VEGF-dependent pathway. In
addition, results from MTT and wound healing assays as well as tube
formation experiments showed that GSH-conjugated metabolites of HOHA-lactone
stimulate HUVEC proliferation and promote angiogenesis in vitro. Previous
studies demonstrated that HOHA-lactone, through its CEP derivatives,
promotes angiogenesis in a novel Toll-like receptor 2-dependent manner
that is independent of the VEGF receptor or VEGF expression. The new
studies show that HOHA-lactone also participates in other angiogenic
signaling pathways that include promoting the secretion of VEGF from
retinal pigmented epithelial cells
CEP Biomarkers as Potential Tools for Monitoring Therapeutics
<div><p>Background</p><p>Carboxyethylpyrrole (CEP) adducts are oxidative modifications derived from docosahexaenoate-containing lipids that are elevated in ocular tissues and plasma in age-related macular degeneration (AMD) and in rodents exposed to intense light. The goal of this study was to determine whether light-induced CEP adducts and autoantibodies are modulated by pretreatment with AL-8309A under conditions that prevent photo-oxidative damage of rat retina. AL-8309A is a serotonin 5-HT<sub>1A</sub> receptor agonist.</p> <p>Methods</p><p>Albino rats were dark adapted prior to blue light exposure. Control rats were maintained in normal cyclic light. Rats were injected subcutaneously 3x with 10 mg/kg AL-8309A (2 days, 1 day and 0 hours) before light exposure for 6 h (3.1 mW/cm<sup>2</sup>, λ=450 nm). Animals were sacrificed immediately following light exposure and eyes, retinas and plasma were collected. CEP adducts and autoantibodies were quantified by Western analysis or ELISA.</p> <p>Results</p><p>ANOVA supported significant differences in mean amounts of CEP adducts and autoantibodies among the light + vehicle, light + drug and dark control groups from both retina and plasma. Light-induced CEP adducts in retina were reduced ~20% following pretreatment with AL-8309A (n = 62 rats, <i>p</i> = 0.006) and retinal CEP immunoreactivity was less intense by immunohistochemistry. Plasma levels of light-induced CEP adducts were reduced at least 30% (n = 15 rats, <i>p</i> = 0.004) by drug pretreatment. Following drug treatment, average CEP autoantibody titer in light exposed rats (n = 22) was unchanged from dark control levels, and ~20% (<i>p</i> = 0.046) lower than in vehicle-treated rats.</p> <p>Conclusions</p><p>Light-induced CEP adducts in rat retina and plasma were significantly decreased by pretreatment with AL-8309A. These results are consistent with and extend previous studies showing AL-8309A reduces light-induced retinal lesions in rats and support CEP biomarkers as possible tools for monitoring the efficacy of select therapeutics.</p> </div
CEP selectively enhances Pam3CSK4-signaling in a TLR4-independent manner.
<p>(<b>A, B</b>) BMDMs from either wild-type (WT) or <i>Tlr4−/−</i> mice were co-stimulated as indicated with Pam3CSK4 (1.0 ng/mL) and sham-BSA (10 µg/mL) or CEP-BSA (10 µg/mL) for 9 hours. <i>Tnf</i> or <i>Il12a</i> expression was assessed by qPCR (n = 3). (<b>C, D</b>) Immuno-depletion assays were performed as described in the Methods & Materials section. sham-BSA (1 µg/mL) or CEP-BSA (1 µg/mL) were immuno-depleted with either IgG1-isotype control or anti-CEP antibody. Supernatants were either combined or not with Pam3CSK4 (1.0 ng/mL) for stimulation of BMDMs. <i>Tnf</i> or <i>Il12a</i> expression was assessed by qPCR (n = 3). (<b>E, F</b>) BMDMs were stimulated with the indicated combinations of Pam3CSK4 (1 ng/mL) and either sham-adducted (10 µg/mL) or CEP-adducted (10 µg/mL) BSA, HSA, or transferrin (Tran) for 9 hours followed by qPCR expression analysis of <i>Tnf</i> and <i>Il12</i> (n = 3) *P<0.05. Results are mean +/− SEM.</p
Pretreatment with AL-8309A reduces light-induced CEP immunoreactivity in rat plasma.
<p>A summary is shown of average CEP adduct levels in rat plasma following <i>in </i><i>vivo</i> blue light exposure with or without pretreatment with AL-8309A (from Western analyses in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076325#pone.0076325.s004" target="_blank">Figure S4</a>). Log<sub>10</sub> transformed CEP optical density measurements were normalized to the mean dark control per analysis and average ratios transformed to linear scale. Δ represents the difference in average ratios between animals groups, the p-values are from the 2-sided t-test and error bars reflect standard deviation. The number of animals assayed in each group is indicated (n).</p
CEP immunoreactivity in rat retina.
<p>A summary is shown of CEP adduct levels in rat retina following blue light exposure with or without pretreatment with AL-8309A (from Western analyses in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076325#pone.0076325.s002" target="_blank">Figure S2</a>). Log10 transformed CEP optical density measurements were normalized to the mean dark control per analysis, outliers were eliminated and average ratios were transformed to linear scale. Δ represents the difference in average ratios between animals groups, the p-values are from the 2-sided t-test and error bars reflect standard deviation. The number of animals assayed in each group is indicated (n).</p