Transcript Lecture 4 - Edward Dennis - University of California San Diego

BIOM 209/CHEM 210/PHARM 209
Glycerolipid and Phospholipid
Metabolism, Signaling and Lipidomics
Professor Edward A. Dennis
Department of Chemistry and Biochemistry
Department of Pharmacology, School of Medicine
University of California, San Diego
Copyright/attribution notice: You are free to copy, distribute, adapt and transmit this tutorial or
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Attribution: Edward A. Dennis (2010) “LIPID MAPS Lipid Metabolomics Tutorial” www.lipidmaps.org
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Types of Biological Lipids
• Lipids derived from ketoacyl units
– Fatty acids (inc. Prostaglandins and wax esters)
– Glycerolipids
– Glycerophospholipids
– Sphingolipids
– Saccharolipids
– Polyketides
• Lipids derived from isoprene units
– Sterols
– Prenols (inc. terpenes and Fat Soluble Vitamins)
• Mixtures
– Lipoproteins
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Definitions
Glycerophospholipid = an amphipathic
lipid in which two fatty acyl groups are
attached to a glycerol-3-phosphate whose
phosphate group is linked to a polar group
X=head group
Usually a saturated FA
Phosphatidic acid = the simplest
glycerophospholipid -the precursor to
other phospholipids and to triacylglycerols.
Also called diacylglycerol-3-phosphate.
Usually an unsaturated FA
Triacylglycerol = a lipid in which three
fatty acids are esterified by a glycerol
backbone.
It is the major form of energy storage in
humans. Also called a triglyceride.
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Glycerol-3-Phosphate Synthesis
• sn-glycerol-3-phosphate
is the backbone of
triglycerides and
phospholipids
• sn-glycerol-3-phosphate
has the R configuration
at C2
Backbone of
phospholipids
and
triglycerides
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Biosynthesis of Phosphatidic Acid
• Precursors
– Fatty acids
– sn-glycerol-3-phosphate
• sn-glycerol-3-phosphate is
produced from the
– Reduction of DHAP by
glycerol phosphate
dehydrogenase OR
– Phosphorylation of glycerol
by glycerol kinase and ATP
• Acyl transferases perform two
successive esterifications with
fatty acyl Co A to generate
phosphatidic acid
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Biosynthesis of Triacylglycerol
• Phosphatidic acid
phosphatase removes the
phosphate producing 1,2Diacylglycerol
• An acyl transferase
transfers an acyl CoA to
position 3.
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Biosynthesis of Glycerophospholipids
glycerol
phosphodiester
• Glycerophospholipids (or phospholipids) can be made from
– Phosphatidic acid OR
– Diacylglycerol
• There are many different head groups which can be linked
to the C3 of glycerol by a phosphodiester bond
• Cytidine triphosphate (CTP) provides the synthetic energy
in the synthesis of all PLs
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Strategies for Phospholipid Synthesis
Strategy 1: Headgroup activated with CDP
Strategy 1: The polar head group
is activated before being attached
to the lipid
– Used during the synthesis of PE
and PC
Strategy 2: The hydrophobic tail
of diacylglycerol is activated
rather than the polar head group
– Used during the synthesis of PI
and PG
Strategy 2: Diacylglycerol activated with CDP
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De Novo Synthesis of Phosphatidylcholine (PC)
• PC is the most abundant
phospholipid in eukaryotic cells
• PC is also known as lecithin
De Novo Synthesis
• Choline is phosphorylated
• Cytidyltransferase makes CDPcholine
• C3 OH groups of DAG attacks
the phosphoryl groups of the
activated CDP-choline
displacing CMP and yielding the
glycerophospholipid
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De Novo Synthesis of PE
• PE is the second most
abundant phospholipid in
eukaryotic cells
De Novo Synthesis
• Ethanolamine is
phosphorylated
• Cytidyltransferase makes
CDP-ethanolamine
• C3 OH groups of DAG attacks
the phosphoryl groups of the
activated CDP-ethanolamine
or displacing CMP and yielding
the glycerophospholipid
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Synthesis of PS
Phosphatidylserine (PS) is
synthesized from PE by a
head group exchange
Phosphatidylserine
synthase 2
Bacteria can make PS de
novo because they have a
PS synthase which adds
serine to diacylglycerol(Strategy 1 mechanism)
Mammals do not make PS
de novo because they lack
this type of PS synthase
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Interconversion of PS, PE and PC
• PS decarboxylase in the
mitochondria can convert PS to
PE
– Bacteria can do this too!
• A calcium-activated transferase
can exchange ethanolamine for
the serine of PS
– This reaction occurs in the ER
and Golgi
• In mammals, PE can undergo 3
successive methylations to yield
PC
– This reaction occurs in the ER
of liver
– S-adenosylmethionine is the
methyl donor
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De Novo Synthesis of PI
Synthesis of PI
Phosphatidic acid attacks the
phosphoryl group of CTP to form
activated CDP-diacylglycerol
• Inositol attacks CDPdiacylglycerol
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Phosphatidylinositol Phosphorylation
These OH groups
can also be esterified
with PO32Phosphatidylinositol (PI)
• PI can be phosphorylated to different degrees
• PIP2 = phosphatidylinositol 4,5-bisphosphate is very important in
signal transduction
– When a receptor G protein is activated it can mediate the cleavage of
PIP2 to DG and IP3
– DG activates protein kinase C which adds phosphates to certain
proteins
– IP3 mobilizes intracellular Ca and activates certain cell processes
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De Novo Synthesis of PG
Synthesis of PG
• The C1 OH group of glycerol3-phosphate attacks CDPdiacylglycerol
• The phosphoryl group is
hydrolyzed to form PG
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Cardiolipin
• First isolated from heart tissue
• Many autoimmune diseases, such as lupus, are associated
with anti-cardiolipin antibodies for unknown reasons
• Formed by the condensation of two molecules of PG with
the elimination of one molecule of glycerol
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Plasmalogens
• About 20% of eukaryotic glycerophospholipids
are plasmalogens. They are found in varying
amounts in different tissues.
• Plasmalogens contain a hydrocarbon chain
linked to glycerol C1 by a vinyl ether linkage.
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Major Components of Membranes
• Proteins
–
–
–
–
Channels & Pumps
Structural proteins
Receptors
Reaction enzymes
• Lipids
– Sterols (Cholesterol)
– Glycerophospholipids
– Glycerolipids (neutral
lipids)
– “Surfactant”
– Sphingolipids
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Lipid Bilayers
• In aqueous solution, amphiphilic
molecules form micelles to
eliminate the contact of the
hydrophobic tails with water, but
allow the polar heads to be in
contact with it.
• The diameter of the micelle
depends upon the length of the
tail.
• A suspension of PLs can form
liposomes, closed, self-sealing
solvent-filled vesicles, that have
only a single bilayer.
• Liposomes serve as models of
biological membranes.
Figures: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Life at
the Molecular Level, 2nd ed. Reprinted with permission of John Wiley & Sons, Inc.
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Movement in Bilayers
Key Principle:
Things on one side tend to
stay on that side...
…unless specifically moved by a
carrier protein called a “flippase.”
Some Implications:
• Lipid populations on each side
can be different
• Receptors aim out
• Embedded/anchored enzymes
localize reactions to only one
side
• Ion pumps move the same ions
the same direction
• Etc.
Figure: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Life at
the Molecular Level, 2nd ed. Reprinted with permission of John Wiley & Sons, Inc.
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DPPC = Surfactant
• Dipalmitoyl phosphatidylcholine (DPPC)
• Main function: reduces surface tension at
the alveoli-air interface (increases
compliance)
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Diagnostics: the L/S Ratio
• Phosphatidylcholine (PC) is produced
by type II alveolar epithelial cells in the
fetal lung. During gestation it is found
in the amniotic fluid.
PC
22
18
Amniotic Fluid14
Concentration
(mg/dl)
10
• Sphingomyelin remains at a low
level throughout gestation. It
can be used as a baseline for
comparison
• The L/S ratio is close to 1:1 until
the 30-35th week of gestation.
At this time, lecithin content
increases dramatically attaining
a ratio of greater than 2:1. This
indicates pulmonary maturity.
Sphingomyelin
6
2
18
22
26 30 34 38
Term
Gestation (weeks)
• This test is very accurate in
determining the absence of RDS;
but patients with a ratio <2:1 may
not have RDS.
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Digestion of Fats
Figure: Lehninger AL, Nelson DL, Cox MM (1993),
Principles of Biochemistry, 2nd ed. Worth Publishers, Inc.
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Bile Acids/Salts
R2 = OH
R2 = NH – CH2 – COOH
R2 = NH – CH2 – CH2 – SO3H
R1 = OH
Cholic acid
Glycocholic acid
Taurocholic acid
R1 = H
Chenodeoxycholic acid
Glycochenodeoxycholic acid
Taurochenodeoxycholic acid
• Bile salts act as detergents in the digestive tract to
emulsify triglycerides and phospholipids into micelles.
Bile salts are a highly oxidized form of cholesterol.
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Micelles
• The hydrophobic surface of the bile salt
associates with TAGs, and a number of these
aggregate to form a micelle.
• This allows the association of pancreatic lipase
which liberates free fatty acids in a smaller micelle
which can be absorbed through the mucosa.
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Definitions
• Lipases or Acyl Hydrolases
– Triacylglycerol Lipase – the general term.
– Lingual Lipase- found in saliva for pre-digestion.
– Pancreatic Lipase- produced by the pancreas for
digestion.
– Lipoprotein lipase- found on capillary endothelial
cells. It hydrolyzes TAG in chylomicrons and VLDLs.
– Hormone Sensitive Lipase- generates FAs when
energy is needed. Found in adipose tissue.
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Definitions
• Pancreatic Lipase
– Only removes FAs from the 1 and 3 positions
– Single polypeptide chain of 48,000 MW
– Requires colipase, a 10,000 MW cofactor, which helps the
association of pancreatic lipase with the lipid/water interface
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Lineweaver-Burk Plot of Lipase Activity
• When TAG is mixed
with water, it forms two
layers.
• When the water and
TAG are shaken, they
mix together forming
microemulsions.
• The finer the emulsion,
the greater the activity
of the enzyme due to
increased surface area
• 1/v versus surface area
is the same for both
Lipase Action
Coarse emulsion
Fine emulsion
1/v
1/[S]
Fine
Coarse
1/v
1/[Surface Area]
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Phospholipase Sites of Action
1-palmitoyl, 2-oleoyl-phosphatidylinositol- 4’,5’ bisphosphate (PIP2)
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More Definitions
• Pancreatic Phospholipase A2
– Removes FAs from the 2 position
– 124 aa single polypeptide chain of 14,000 MW
– Produced as a zymogen. Trypsin hydrolyzes a
peptide bond at position 7 of the zymogen to make the
active enzyme.
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Lipases and Phospholipases
Lipases are unique because their substrates are lipids, not small molecules.
• At low concentrations, DiC7PC forms monomers
• At higher concentrations, it forms micelles.
• The concentration at which micelles form is called the critical micelle
concentration (CMC).
DiC7PC
PLA2 on DiC7PC
Mixed Micelles with detergent
Micelles
V
Figure: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Life at
the Molecular Level, 2nd ed. Reprinted with permission of John Wiley & Sons, Inc.
Monomers
[S] CMC
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New Thematic Review
Series on
Phospholipases in Lipid
Signaling and
Disease