3 research outputs found
Resolving the Role of Lipoxygenases in the Initiation and Execution of Ferroptosis
Lipoxygenases (LOXs)
have been implicated as central players in
ferroptosis, a recently characterized cell death modality associated
with the accumulation of lipid hydroperoxides: the products of LOX
catalysis. To provide insight on their role, human embryonic kidney
cells were transfected to overexpress each of the human isoforms associated
with disease, 5-LOX, p12-LOX, and 15-LOX-1, which yielded stable cell
lines that were demonstrably sensitized to ferroptosis. Interestingly,
the cells could be rescued by less than half of a diverse collection
of known LOX inhibitors. Furthermore, the cytoprotective compounds
were similarly potent in each of the cell lines even though some were
clearly isoform-selective LOX inhibitors. The cytoprotective compounds
were subsequently demonstrated to be effective radical-trapping antioxidants,
which protect lipids from autoxidation, the autocatalytic radical
chain reaction that produces lipid hydroperoxides. From these data
(and others reported herein), a picture emerges wherein LOX activity <i>may</i> contribute to the cellular pool of lipid hydroperoxides
that initiate ferroptosis, but lipid autoxidation drives the cell
death process
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Lipidomics Reveals Dramatic Physiological Kinetic Isotope Effects during the Enzymatic Oxygenation of Polyunsaturated Fatty Acids Ex Vivo
Arachidonic acid
(AA, 20:4) is an omega-6 polyunsaturated fatty
acid (PUFA) and the main precursor to the class of lipid mediators
known as eicosanoids. The enzymes that catalyze the oxygenation of
AA begin by abstracting hydrogen from one of three bis-allylic carbons
within 1,4-<i>cis</i>,<i>cis</i>-diene units.
Substitution of deuterium for hydrogen has been shown to lead to massive
kinetic isotope effects (KIE) for soybean lipoxygenase (sLOX) oxygenation
of linoleic acid (LA, 18:2). Yet, experimental determination of the
KIE during oxygenation of AA and LA by mammalian enzymes including
cyclooxygenase (COX) and lipoxygenase (LOX) has revealed far lower
values. All prior studies investigating the KIE of PUFA oxygenation
have relied on <i>in vitro</i> systems using purified enzymes
and were limited by availability of deuterated substrates. Here we
demonstrate the use of macrophages as an <i>ex vivo</i> model
system to study the physiological KIE (PKIE) during enzymatic AA oxygenation
by living cells using a newly synthesized library of deuterated AA
isotopologues. By extending lipidomic UPLC-MS/MS approaches to simultaneously
quantify native and deuterated lipid products, we were able to demonstrate
that the magnitude of the PKIE measured in macrophages for COX and
LOX oxygenation of AA is similar to KIEs determined in previous reports
using the AA isotopologue deuterated at carbon 13 (C13). However,
for the first time we show that increasing the number of deuterated
bis-allylic carbons to include both C10 and C13 leads to a massive
increase in the PKIE for COX oxygenation of AA. We provide evidence
that hydrogen(s) present at C10 of AA play a critical role in the
catalysis of prostaglandin and thromboxane synthesis. Furthermore,
we discovered that deuteration of C10 promotes the formation of the
resolving lipid mediator lipoxin B4, likely by interfering with AA
cyclization and shunting AA to the LOX pathway under physiological
conditions
Unusual Kinetic Isotope Effects of Deuterium Reinforced Polyunsaturated Fatty Acids in Tocopherol-Mediated Free Radical Chain Oxidations
Substitution
of −CD<sub>2</sub>– at the reactive
centers of linoleic and linolenic acids reduces the rate of abstraction
of D by a tocopheryl radical by as much as 36-fold, compared to the
abstraction of H from a corresponding −CH<sub>2</sub>–
center. This H atom transfer reaction is the rate-determining step
in the <i>tocopherol-mediated peroxidation</i> of lipids
in human low-density lipoproteins, a process that has been linked
to coronary artery disease. The unanticipated large kinetic isotope
effects reported here for the tocopherol-mediated oxidation of linoleic
and linolenic acids and esters suggests that tunneling makes this
process favorable