"The anonymity that is the fate of nearly every scientist as the
work of one generation blends almost without a trace into that of
the next is a small price to pay for its unending progress, the
great long march of human reason. To feel that one has contributed
to this splendid enterprise, on however small a scale, is reward
enough for labor at the end of the day"
--Eugene P. Kennedy, Harvard Medical School
Numerous signal
transduction processes involve lipids as signaling molecules. Many
of these molecules are generated by phospholipases such as
phospholipase A2,
which releases fatty acids like arachidonic acid, and lysophospholipids. Each of these products is implicated in signal
transduction processes, but also serves as a precursor for platelet
activating factor or the eicosanoids. The eicosanoids
are a large family of bioactive mediators that derive from the enzymatic
oxygenation of arachidonic acid. Prostaglandins, leukotrienes, thromboxane, lipoxins, are all members of the eicosanoid
family. Circulating monocytes and tissue macrophages are major
sources of these compounds. The eicosanoids are biomedically important because they
mediate all four signs of inflammation, namely heat, redness,
swelling and pain. Controlling the formation of eicosanoids has been
found to be of great benefit for the treatment of acute and chronic
inflammatory diseases.
Lipid signaling
is also key to the development of cardiovascular disease, one of the
most prevalent inflammatory disorders. Atherosclerosis is the
primary cause for cardiovascular disease, and diabetes increases the
risk several-fold by enhancing the formation and/or progression of
atherosclerotic lesions, a process in which abnormally-activated
monocytes and macrophages appear to play a major role. In diabetes,
these cells appear to be in a proinflammatory state, releasing
elevated amounts of cytokines and eicosanoids that perpetuate the
inflammatory condition. Monocytes/macrophages from diabetic patients
have been found to exhibit enhanced expression of Toll-like
receptors 2 and 4. These receptors sense bacterial pathogens
but also endogenous danger molecules such as saturated free fatty
acids, typically present at elevated amounts in obese individuals.
Our
current research focuses primarily on the lipid signaling enzymes
phospholipase A2
and phosphatidate phosphohydrolase (phosphatidic acid-specific
phospholipase C; lipin). The latter is a key enzyme in the de novo
pathway for glycerolipid biosynthesis, providing an excellent
example that enzymes involved in this pathway may also act to
initiate intracellular signaling. General events that we are interested in
include (i) the spatiotemporal regulation of these phospholipases in
a cellular context, which we study utilizing advanced microscopy
techniques, (ii) pharmacological manipulation of enzymatic activity
both in intact cells and in vitro, (iii) analysis of lipid
metabolite production by state-of-the-art mass spectrometry (lipidomics
& metabolipidomics), and (iv) the physiological functioning of
phospholipases in animal models.
Ongoing studies in our labs focus on the localization and stimulus-driven
translocation of different members of the phospholipase A2
and lipin families. Phospholipase A2s cleave the fatty acid at the sn-2 position of
phospholipids and thus constitute the earliest regulatory point of
the eicosanoid biosynthetic cascade. Lipins dephosphorylate
phosphatidic acid to form diacylglycerol, which can be used for the
biosynthesis of glycerophospholipids and triacylglycerol, and may
function as intracellular signalers as well. Current studies are being
carried out by transfecting chimeric constructs of green fluorescent
protein (GFP) (or any of its colored varieties) with the appropriate phospholipase. GFP
is placed at either the N- or C-termini. of the
enzymes. These constructs provide a very useful tool to visualize
the intracellular movements of the enzymes
in response to the different stimuli. Mutagenesis studies are also
being conducted to pinpoint the specific amino acids of the phospholipase A2s
and lipins that are implicated in the movement among intracellular
compartments.
Another of our goals is to apply a lipidomics approach to the
study of the mechanisms governing the availability and oxidative
metabolism of free arachidonic acid during activation of
macrophages by stimuli of the innate immune response. Availability
of free arachidonate is a limiting step for the synthesis of eicosanoids.
While the pathways of fatty acid uptake, incorporation and remodeling in
glycerolipids are well documented, the individual lipid species in
which arachidonate is stored and released from have not been identified.
This is so because of the impossibility of traditional methods for
lipid separation (i.e. thin-layer chromatography, liquid
chromatography) to differentiate among individual lipids within
various classes and subclasses. This is now possible with the advent
of electrospray mass spectrometry (ESI-MS). Application of this
technology to the field of lipid biochemistry has been a major
breakthrough in profiling the lipidomes of cells and tissues in
physiological and pathophysiological conditions. We are conducting lipidomic analyses of all the lipid molecular species involved in
arachidonic acid homeostasis, from those that act as acceptors of the fatty acid
to those from which the fatty acid is liberated for subsequent eicosanoid synthesis, and including as well a full survey of arachidonate-derived
oxygenated metabolites.
In the context of these studies,
we
have described a number of novel arachidonate-containing lipids, the
levels of which increase during cell activation. We are currently
investigating the metabolic pathways involved in their biosynthesis
as well possible biological processes mediated by these species.
Finally, we have begun studies aimed at
defining the regulation of lipid droplet formation in cells involved
in inflammation. Lipid droplets are
cytosolic inclusions present in most eukaryotic cells that contain a
core rich in neutral lipids such as triacylglycerol and cholesteryl
esters and are surrounded by a phospholipid monolayer decorated with
a variety of proteins. Initially regarded as inert neutral
lipid-storage compartments, the interest for lipid droplets has
increased recently because of their association with
diabetes and atherosclerosis. Our results have defined group IVA
phospholipase
A2
as a key regulator of lipid droplet formation. Also, subcellular
localization studies have shown that lipin-1 localizes permanently
on the surface of these organelles, thus suggesting a metabolic or
regulatory role for this enzyme. Lipidomic analyses of the
composition of lipid droplets formed under various conditions, have
uncovered the presence of unusual
fatty acids in these organelles. Some of these fatty acids might play a role in regulating specific
cellular responses.
All of our lines of
research rely heavily on biochemical and analytical methods to
identify specific reactions and the mechanisms through which the
products of said reactions are formed. With this information, we
expect to delineate pathways responsible for disease. In summary, in
our laboratory we combine a range of chemical, biochemical,
pharmacological, and molecular cell biology techniques to study
pathophysiologically-relevant problems involving alterations in
lipid metabolism and signaling.
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Support for
our studies currently comes from the Spanish Ministry of Science,
Innovation and Universities (Grants PID2022-140764OB-I00, and CIBERDEM-ISCIII
CB07/08/0004). |
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Research Support History |
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