19 research outputs found
The Adductomics of Isolevuglandins: Oxidation of IsoLG Pyrrole Intermediates Generates Pyrrole–Pyrrole Crosslinks and Lactams
Isoprostane endoperoxides generated by free radical-induced oxidation of arachidonates, and prostaglandin endoperoxides generated through enzymatic cyclooxygenation of arachidonate, rearrange nonenzymatically to isoprostanes and a family of stereo and structurally isomeric γ-ketoaldehyde seco-isoprostanes, collectively known as isolevuglandins (isoLGs). IsoLGs are stealthy toxins, and free isoLGs are not detected in vivo. Rather, covalent adducts are found to incorporate lysyl ε-amino residues of proteins or ethanolamino residues of phospholipids. In vitro studies have revealed that adduction occurs within seconds and is uniquely prone to cause protein–protein crosslinks. IsoLGs accelerate the formation of the type of amyloid beta oligomers that have been associated with neurotoxicity. Under air, isoLG-derived pyrroles generated initially are readily oxidized to lactams and undergo rapid oxidative coupling to pyrrole–pyrrole crosslinked dimers, and to more highly oxygenated derivatives of those dimers. We have now found that pure isoLG-derived pyrroles, which can be generated under anoxic conditions, do not readily undergo oxidative coupling. Rather, dimer formation only occurs after an induction period by an autocatalytic oxidative coupling. The stable free-radical TEMPO abolishes the induction period, catalyzing rapid oxidative coupling. The amine N-oxide TMAO is similarly effective in catalyzing the oxidative coupling of isoLG pyrroles. N-acetylcysteine abolishes the generation of pyrrole–pyrrole crosslinks. Instead pyrrole-cysteine adducts are produced. Two unified single-electron transfer mechanisms are proposed for crosslink and pyrrole-cysteine adduct formation from isoLG-pyrroles, as well as for their oxidation to lactams and hydroxylactams
Acetylation of αA-crystallin in the human lens: Effects on structure and chaperone function
Abstractα-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a Nε-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein
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
Cysteinyl leukotriene-like metabolites are generated in retinal pigment epithelial cells through glutathionylation/reduction of an oxidatively truncated fragment of arachidonate
γ-Hydroxyalkenals, 4-hydroxynonenal (HNE) and phospholipid esters of 4-hydroxy-8-oxooctenoic acid (HOOA-PL), are produced from the alkyl and carboxyl termini of arachidonyl phospholipids by radical-induced oxidative cleavage. Metabolism of HNE by Michael addition of glutathione (GSH) followed by reduction of the aldehyde carbonyl produces a GSH derivative of 1,4-dihydroxynonane (DHN)-GSH. Analogous biochemistry was anticipated to produce a GSH derivative of 5,8-dihydroxyoctanoic acid (DHOA-GSH) that has structural and functional similarity to the cysteinyl leukotriene (LT)C4. We now report that exposure of human retinal pigment epithelial cells to CoCl2, an in vitro model of hypoxia-induced oxidative stress, generates DHOA-GSH and two products of its peptidolysis, DHOA-CysGly and DHOA-Cys that resemble LTD4 and LTE4. Identification of these metabolites was confirmed by unambiguous chemical syntheses that also provided a heavy isotope labeled quantitative standard 13C215N-DHOA-GSH. The availability of pure samples of these arachidonate metabolites will enable assessment of their biological activities, and testing the hypothesis that øLTs promote pathological inflammation by serving as LT receptor agonists. Because LT biosynthetic enzymes, e.g., 5-lipoxygenase, are not involved in the generation of øLTs in vivo, inhibitors of LT biosynthesis, e.g., Zileuton, are not expected to prevent the generation of øLTs. On the other hand, if øLTs are leukotriene receptor agonists, then the therapeutic effects of leukotriene receptor antagonist drugs, e.g., Montelukast, may include inhibition not only of LT-induced but also øLT-induced LT receptor activation and signaling
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
4‑Hydroxy-7-oxo-5-heptenoic Acid (HOHA) Lactone is a Biologically Active Precursor for the Generation of 2‑(ω-Carboxyethyl)pyrrole (CEP) Derivatives of Proteins and Ethanolamine Phospholipids
2-(ω-Carboxyethyl)Âpyrrole
(CEP) derivatives of proteins were
previously shown to have significant pathological and physiological
relevance to age-related macular degeneration, cancer and wound healing.
Previously, we showed that CEPs are generated in the reaction of ε-amino
groups of protein lysyl residues with 1-palmityl-2-(4-hydroxy-7-oxo-5-heptenoyl)-<i>sn</i>-glycero-3-phosphatidylcholine (HOHA-PC), a lipid oxidation
product uniquely generated by oxidative truncation of docosahexanenate-containing
phosphatidylcholine. More recently, we found that HOHA-PC rapidly
releases HOHA-lactone and 2-lyso-PC (<i>t</i><sub>1/2</sub> = 30 min at 37 °C) by nonenzymatic transesterification/deacylation.
Now we report that HOHA-lactone reacts with Ac-Gly-Lys-OMe or human
serum albumin to form CEP derivatives in vitro. Incubation of human
red blood cell ghosts with HOHA-lactone generates CEP derivatives
of membrane proteins and ethanolamine phospholipids. Quantitative
analysis of the products generated in the reaction HOHA-PC with Ac-Gly-Lys-OMe
showed that HOHA-PC mainly forms CEP-dipeptide that is not esterified
to 2-lysophosphatidycholine. Thus, the HOHA-lactone pathway predominates
over the direct reaction of HOHA-PC to produce the CEP-PC-dipeptide
derivative. Myleoperoxidase/H<sub>2</sub>O<sub>2</sub>/NO<sub>2</sub><sup>–</sup> promoted in vitro oxidation of either 1-palmityl-2-docosahexaneoyl-<i>sn</i>-glycero-3-phosphatidylcholine (DHA-PC) or docosahexaenoic
acid (DHA) generates HOHA-lactone in yields of 0.45% and 0.78%, respectively.
Lipid oxidation in human red blood cell ghosts also releases HOHA-lactone.
Oxidative injury of ARPE-19 human retinal pigmented epithelial cells
by exposure to H<sub>2</sub>O<sub>2</sub> generated CEP derivatives.
Treatment of ARPE-19 cells with HOHA-lactone generated CEP-modified
proteins. Low (submicromolar), but not high, concentrations of HOHA-lactone
promote increased vascular endothelial growth factor (VEGF) secretion
by ARPE-19 cells. Therefore, HOHA-lactone not only serves as an intermediate
for the generation of CEPs but also is a biologically active oxidative
truncation product from docosahexaenoate lipids
Detection and Biological Activities of Carboxyethylpyrrole Ethanolamine Phospholipids (CEP-EPs)
Oxidation of docosahexaenoate phospholipids
produces 4-hydroxy-7-oxo-hept-5-eonyl
phospholipids (HOHA-PLs) that react with protein lysyl ε-amino
residues to generate 2-ω-carboxyethylpyrrole (CEP) derivatives,
endogenous factors that induce angiogenesis in the retina and tumors.
It seemed likely, but remained unproven, that HOHA-PLs react with
ethanolamine phospholipids (EPs) <i>in vivo</i> to generate
CEP-EPs. We now show that CEP-EPs are present in human blood at 4.6-fold
higher levels in age-related macular degeneration plasma than in normal
plasma. We also show that CEP-EPs are pro-angiogenic, inducing tube
formation by human umbilical vein endothelial cells by activating
Toll-like receptor 2. CEP-EP levels may be a useful biomarker for
clinical assessment of AMD risk and CEP-associated tumor progression
and a tool for monitoring the efficacy of therapeutic interventions