Download Isoprenoid metabolism: cholesterol and the others

Isoprenoid metabolism: cholesterol and the others
Jan Trnka, Charles University
Winter Semester 2009
A. What are isoprenoids good for?
Isoprenoids are an extremely diverse group of organic compounds
composed of a varying number of isoprene (2-methyl-1,3-butadiene)
units. Without isoprenoids a rose would not smell sweet by any
name (geraniol), carrots would be white (β-carotene) and hemp
would literally be just a weed (∆9 -tetrahydrocannabinol).
These plant-derived chemicals play important roles in our lives.
Carotenoids are the source of retinal and retinoic acid (vitamin A
derivatives), we use natural latex (polyisoprene) to make condoms,
gloves and chewing gum, and complex mixtures of volatile isoprenoids in flower extracts and spices are the daily bread of perfume
manufacturers and top chefs.
While plants are veritable chemical factories capable of synthesising thousands of isoprenoid derivatives, animals such as us can
make some too. Probably the most abundant isoprenoid in our
body is cholesterol.
isoprene
OH
geraniol
OH
H
H
O
∆9-THC
β-carotene
H3C
CH3
CH3
CH3
H
H
H3C
H
HO
Cholesterol is a polycyclic isoprenoid and its ring structure is
common to all steroids. The rings are labelled from the left as A, B,
C and D.
Cholesterol contains 27 carbon atoms and, as we will see later, it
is formed from six five-carbon isoprene units (three carbon atoms
are removed later in the process). As a polycyclic hydrocarbon with
only one hydroxyl group it is fairly hydrophobic and practically insoluble in water. Cholesterol is an important component of cellular
membranes in animals, where it regulates membrane fluidity by
disrupting the relatively uniform pattern set by phospholipids.
Cholesterol is also the raw material for the synthesis of bile acids
necessary for a proper digestion of fats in the intestines, steroid
hormones of the adrenal glands (corticosteroids), sex hormones
(œstrogens, progestagens and androgens) and finally a hormone
called for historical reasons vitamin D3 .
Despite the bad press cholesterol has been receiving in the last
few decades it is an essential component of our bodies without
which we could not survive for one second. The major portion of
cholesterol is synthesised in the liver with the remaining amount
D
C
A
B
HO
Despite the way its structure is often
depicted (above) cholesterol is not
a perfectly planar molecule. The
saturated rings generally assume the
so called “chair” conformation and the
steroid nucleus is somewhat creased.
H3C
CH3
H
CH3
HO
H
H
H
H3C
CH3
Note also that the hydrogens and
methyl groups sticking from the ring
joints are facing opposite ways. This
means that the rings are fused in
trans configuration. The four rings
in cholesterol and steroid hormones
are joined trans, trans, trans. Bile acids
have cis, trans, trans configuration
while cardiac glycosides (digitonin)
are cis, trans, cis. The upper face of
the steroid nucleus is referred to as β
and the lower face as α. These labels
are used to describe the configuration
of substituents—the two methyl
groups (C18, C19) and the hydroxyl in
cholesterol are all β.
isoprenoid metabolism: cholesterol and the others
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coming from the diet and extrahepatic tissues. In the following
paragraphs we will look at the synthesis, transport and further
metabolism (including excretion) of cholesterol in humans and we
will briefly mention other isoprenoid products.
B. Cholesterol synthesis: 18 × acetyl-CoA
Making “activated isoprene”
The entire molecule of cholesterol is made of acetate units. AcetylCoA formed in mitochondria by the β-oxidation of fatty acids or
decarboxylation of pyruvate is transported into the cytoplasm (as
citrate) and used to make acetoacetyl-CoA (two acetyl-CoA) and then
hydroxymethylglutaryl-CoA (hmg-CoA, three acetyl-CoA). These
two steps appear identical to the synthesis of ketone bodies but
the crucial difference is that ketone bodies are made inside the
mitochondrial matrix while steroid synthesis takes place in the
cytosol.
The next step is both important and somewhat unusual. It is the
rate limiting step of the whole pathway and as such it determines
how much cholesterol (and other isoprenoids) we can make per
unit time. hmg-CoA is reduced by the enzyme hmg-CoA reductase
to mevalonate. The unusual thing about this step is that a carboxyl
group (or, more specifically, a thioester group) is reduced all the
way to hydroxyl—this is rarely seen in mammalian biochemistry.
The reaction requires two molecules of nadph, which is produced
e.g. in the pentose phosphate pathway.
The activity of hmg-coa reductase is regulated by phosphorylation, which has an inhibitory effect. The enzyme responsible for this regulation is amp-activated kinase (ampk). The complete signaling cascade is fairly complex but the main signal determining the rate of cholesterol synthesis is the availability of atp.
When atp levels decline, the amount of amp increases, which activates ampk and ampk in turn inhibits hmg-CoA reductase. This
means that starved cells will not embark on a hugely expensive
synthesis such as that of cholesterol and, on the other hand, in
times of plenty cholesterol will be produced in abundance.
The phosphorylation of hmg-CoA is also regulated by two important metabolic hormones: insulin and glucagon. Both act indirectly through changing levels of another nucleotide, cyclic amp . An
increase in camp stimulates the phosphorylation of hmg-CoA reductase (by inhibiting protein phosphatases) and therefore inhibits
its activity. Insulin decreases and glucagon increases the intracellular concentration of camp, which means that insulin stimulates the
synthesis of cholesterol while glucagon inhibits it. This once again
fits into the overall story: when we are well fed and have plenty of
energy to spare, insulin is secreted, which stimulates the synthesis
of cholesterol. Glucagon secreted during fasting does the opposite.
O
O
O
CoA
CoA
S
S
O
O
CoA
S
CoA
S
CoA-SH
CoA-SH
OH
OH
S
O
Enz
Enz
S
O
O
H2O
S
S
CoA
CoA
O
O
O
OH
O
CoA
CoA
S
HO
S
HMG-CoA
acetoacetyl-CoA
2 NADPH 2 NADP+
O
OH
O
O
OH
CoA
HO
S
HO
OH
mevalonate
hmg-CoA reductase as the rate limiting step is the obvious pharmacological target to decrease the synthesis
of cholesterol. A major group of
cholesterol-lowering drugs—statins—
inhibit this enzyme. These fungal
metabolites (found also in common
edible mushrooms—oyster mushroom)
contain a hmg-like domain and competitively inhibit the binding of the
substrate.
HO
O
OH
H3C
O
HO
S
O
CoA
HMG-CoA
O
O
O
H
lovastatin
ATP
AMP
AMP
ATP
INSULIN
GLUCAGON
GLUCAGON
INSULIN
isoprenoid metabolism: cholesterol and the others
Cholesterol metabolism is also regulated at the level of gene expression. Genes for enzymes necessary for its synthesis, transport
and degradation respond to amounts of cholesterol and its derivatives in the body. Sterol response element binding protein (srebp) is
the responsible entity. It is normally inserted in the membrane of
the endoplasmic reticulum and when there is plenty of cholesterol
around it binds several “protective” proteins, which keep it where it
is. When the concentration of steroids drops, the “protective” proteins disassociate from srebp, which stimulates its translocation to
the Golgi apparatus. Once there, srebp is cleaved by proteases and
its dna-binding domain translocates into the nucleus to increase
the transcription of genes responsible for cholesterol synthesis,
including hmg-CoA reductase.
For a detailed review see for example
Bengoechea-Alonso MT, Ericsson J.
srebp in signal transduction: cholesterol metabolism and beyond. Current
Opinion in Cell Biology (2007) 19:215-222
O
Mevalonate formed by the reduction of hmg-CoA is then
diphosphorylated on the newly formed hydroxyl group. Two phosphate groups are sequentially transferred from two molecules of
atp resulting in mevalonate-5-diphosphate and 2 adp. In the following step the other hydroxyl group (on carbon 3) is also phosphorylated, which destabilises the carboxyl group and leads to a
decarboxylation accompanied by the formation of a double bond.
The result is a molecule often alluded to as “activated isoprene,”
chemically isopentenyl diphosphate (ester of 3-methylbut-3-ene-1-ol or
“isopentenol” and diphosphoric acid). This is the direct isoprenoid
precursor.
OH
HO
OH
2 ATP
2 ADP
O
O
OH
O
P
O-
O
O-
O-
ATP
ADP
OO
O
O-
P
O
O
O
P
HO
O
P
O-
O
O-
O-
CO2 + Pi
O
O
P
O-
P
O
O-
O-
isopentenyl diphosphate
Linking “isoprenes” together
Before we discuss the remaining steps leading to cholesterol,
let’s stop for a moment and talk about other isoprenoids made in
our cells and their functions. Farnesyl diphosphate and geranylgeranyl diphosphate (formed by dimerisation of geranyl-PP) are
used for the “prenylation” of some proteins. Several signalling pro-
O
P
HO
O
In the first part of the metabolic pathway we formed a five-carbon
activated isoprene unit. The remaining steps leading to all isoprenoids are effectively little more than joining these units together
and rearranging the resulting molecules. Cholesterol is essentially
made in three stages: a) adding three five-carbon units together;
b) attaching two of these 15-carbon compounds into one chain and
c) making this 30-carbon chain into a cycle while removing three
carbon atoms.
In order to do this, some of isopentenyl diphosphate is isomerised to dimethylallyl diphosphate (dmapp)—the double bond
is simply moved to the right. These two isomers are then joined
together and form the first proper isoprenoid, geranyl diphosphate
(containing 10 carbon atoms). Another molecule of isopentenyl-PP
is attached to geranyl diphosphate and a fifteen-carbon isoprenoid
called farnesyl diphosphate is formed.
3
+
5
c.
b.
a.
5
+
15
5
15
+
27
30
15
O
O
P
O
O-
P
O
O
O-
O-
O
P
O
O-
P
O
O-
dimethylallyl diphosphate
30
O-
isoprenoid metabolism: cholesterol and the others
4
teins (e.g the small G-protein Ras) are attached to the inner leaf
of the cytoplasmic membrane by means of these long isoprenoid
chains. Since Ras plays an important role in certain types of cancer,
the inhibition of the attachment of a farnesyl chain and resulting
disruption of its function is an interesting pharmacological target.
Farnesyl diphosphate is also the starting material for the synthesis of the isoprenoid side chain of coenzyme Q or ubiquinone,
which is an essential electron carrier in the mitochondrial electron
transport chain. It is important to bear in mind that the inhibition
of the isoprenoid pathway does not only reduce the synthesis of
cholesterol but also of these other important metabolites.
Let’s come back to cholesterol. Two molecules of farnesyl diphosphate synthesised in the previous step are linked tail-to-tail to form
a 30-carbon acyclic isoprenoid called squalene, which is then cyclised and trimmed in 19 steps into cholesterol. In the process three
carbon atoms are removed as CO2 and a hydroxyl group is added.
Cholesterol synthesis occurs predominantly in the liver but all
tissues need it for their survival and function. This is why there is a
transport system for cholesterol and other lipids from the liver (and
the intestine—absorbed from the diet) to the rest of the body.
Lipoproteins
Cholesterol, triacylglycerols (tag) and phospholipids cannot be
easily transported dissolved in the blood especially in the quantities
required. They are too hydrophobic and too abundant to bind
to plasma albumin as for example free fatty acids do. They are
therefore packaged together into small droplets of fat (≈20-1000 nm
in diameter) surrounded by protein molecules. These droplets are
called lipoproteins.
The core of lipoproteins is composed of a variable mixture of
esterified cholesterol (cholesterol with a fatty acid bound to the hydroxyl) and tag. The interface between the hydrophobic core and
the surrounding aqueous environment is provided by a layer of
phospholipids, free cholesterol and special proteins called apolipoproteins.
Lipoproteins are classified based on their density. Since fat is
less dense than proteins the overall density of a lipoprotein particle
depends on the ratio of the two components. The more fat, the
lower density.
The largest lipoproteins with the lowest density are chylomicrons. They are made in the small intestine (enterocytes) from lipids
absorbed from digested food. To start with they contain apolipoprotein B-48 and are secreted into lymph, which bypasses the liver
and is drained into systemic circulation via the thoracic duct. There
chylomicrons acquire other apolipoproteins by contact with hdl
(see below) and these apoproteins allow tissues (e.g adipose tissue)
to extract free fatty acids from these particles. Once a chylomicron
When a blood sample is taken from
an individual shortly after a fatty
meal and blood cells are removed by
centrifugation the remaining plasma
is visibly milky due to suspended
lipoproteins.
apolipoproteins
hydrophobic
core
small lipoprotein
Apo-protein is a general name for
a protein missing its non-protein
component, e.g. an enzyme with its
coenzyme removed is called an apoenzyme. When all lipids are removed
from a lipoprotein all that is left are
apolipoproteins.
Some apolipoproteins are responsible
for keeping the particle together and
some of them bind to specific receptors
on target cells or activate enzymes
responsible for the removal of tag or
cholesterol from lipoproteins.
isoprenoid metabolism: cholesterol and the others
has travelled around the body and got rid of some of its contraband
it is endocytosed in the liver and broken down.
The liver also produces its own lipoproteins. The largest of them
and with the second lowest density are very low density lipoproteins
or vldl. These are packed with tag and cholesterol synthesised in
the liver and carry apolipoprotein B-100.
The life of vldl is very similar to that of chylomicrons. As they
float in the blood stream they acquire further apolipoproteins from
hdl, and peripheral tissues can remove fat from the particles thus
increasing the particles’ protein/fat ratio and their density. This
process results in intermediate density lipoproteins or idl and finally
low density lipoproteins or ldl.
ldl contain mostly cholesterol and their job is to deliver it to
extrahepatic tissues which cannot synthesise their own—and we
know how important cholesterol is. These target tissues absorb
whole ldl particles by receptor-mediated endocytosis.
The final lipoprotein particle with the highest density of them all
has already been mentioned a couple of times—high density lipoproteins or hdl. The function of hdl is exactly opposite to all the other
members of the family. Apart from donating some of its apolipoproteins to its cousins, its main job is to transport excess cholesterol
from peripheral tissues back to the liver. When they are born hdl are
equipped with some phospholipids and a lot of proteins (hence
their high density). Some of these proteins stimulate peripheral
cells to give up their cholesterol and others activate an enzyme,
which esterifies cholesterol with fatty acids to make it more hydrophobic. The resulting cholesteryl esters are then packed into the
lipoprotein core and carried back to the liver for repackaging or
further metabolism.
clinical note: Several large epidemiological studies have shown
that in addition to total plasma cholesterol the ldl/hdl ratio is a
good predictor of cardiovascular risk. Given the opposite functions
of the two lipoprotein particles and the fact that many severe cardiovascular diseases are caused by the deposition of cholesterol in
arteries this should not be a surprise for anyone with some knowledge of lipoprotein metabolism.
C. The fate of cholesterol
Cholesterol is an expensive molecule for any organism and since
natural selection did not anticipate fast food restaurants we have
evolved to conserve it. There is therefore no mechanism for the
excretion of cholesterol. Cholesterol is, however, metabolised into
various products, some of which find their way out of the organism.
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ApoB-100 is the full version of the protein of which ApoB-48 is a truncated
form. Liver makes the former and
intestines the latter.
tag from lipoproteins are hydrolysed
by lipoprotein lipase into fatty acids
and monoacylglycerol, which are then
taken up by the target tissue. Lipoprotein lipase is located on capillary
endothelium.
The enzyme is lecithin:cholesterol
acyltransferase or lcat. Lecithin, a
phospholipid (phosphatidylcholine),
donates fatty acids required for the
reaction.
isoprenoid metabolism: cholesterol and the others
6
Bile acids
Bile is secreted by the liver into the duodenum to enable the digestion of fats from the diet. Its role is to emulsify droplets of fat,
i.e. break them down into much smaller sizes so that digestive enzymes (mainly pancreatic lipase) can effectively hydrolyse them.
Bile therefore acts in a similar way as washing up liquid or a detergent.
In addition to essentially inert components such as water, inorganic ions and bile pigments (mostly bilirubin, a degradation
product of hæm) bile contains the following detergents: fatty acids,
cholesterol, phospholipids and bile acids.
Bile acids are carboxylic acids derived from cholesterol. The
steroid polycyclic nucleus can be made more hydrophilic by adding
polar groups such as hydroxyls or carboxyls. Furthermore, recall
that cholesterol has two faces, α and β. We can therefore make a
Janus-like molecule with a hydrophobic and a hydrophilic face
(like cholic acid pictured on the right). The non-polar face would
stick to a fat globule while its opposite number would be more
than happy to face water. This amphiphilic behaviour is what makes
detergents work: hydrophobic molecules such as fats do not want
to face aqueous environment (for entropic reasons) and therefore
clump together in an attempt to minimise the surface area exposed
to water. Detergents prevent that and keep hydrophobic droplets
very small and dispersed.
Bile acids are synthesised in hepatocytes in a series of steps involving hydroxylation of the steroid nucleus and oxidative removal
of last three carbon atoms from the side chain leaving a carboxyl
group. This carboxyl can be conjugated via a peptide bond with an
amino acid such as glycine (e.g. glycocholic acid) or taurine (e.g.
taurocholic acid). These acids are then secreted with the bile into
the intestine where they do their important job.
Keeping with the fact that cholesterol is an important and expensive molecule these bile acids are not meant to be lost—indeed,
they are recycled. Bile acids are re-absorbed by enterocytes and
transported in the blood stream back to the liver, less than 10 % is
lost in fæces. Some bile acids are modified by intestinal bacteria
forming secondary bile acids, which are also recycled (e.g. deoxycholic acid).
Steroid hormones
Cholesterol is also the starting material for the synthesis of several signaling molecules. Steroid hormones can be divided based
on their effects and chemical structure into progestagens (a.k.a.
gestagens), mineralo- and glucocorticoids, androgens, œstrogens
and vitamin D3 . These hydrophobic hormones easily cross cellular
membranes and exert their primary effects by binding to intracellular receptors, which then attach to nuclear dna and regulate the
transcription of specific genes.
hydrophobic H3C
CH3
H
CH3
H
COOHO
H
H
OH
hydrophilic
OH
From Greek ἀμφί- meaning both and
φιλεῖν to love
This recycling of bile acids can be
blocked for therapeutic purposes.
Since bile acids are made from cholesterol by increasing their excretion
we can decrease the amount of cholesterol in the body. Bile acid recycling
can be prevented using a positively
charged insoluble resin which, when
ingested, will bind negatively charged
bile acids and block their re-uptake.
The resin with bound bile acids is then
excreted. One such resin approved for
lowering plasma cholesterol is called
cholestyramine.
Vitamin D3 is technically not a
steroid—it does not contain the full
steroid nucleus—but is synthesised
from cholesterol and will be dealt with
at the end of this chapter.
isoprenoid metabolism: cholesterol and the others
The first step in steroid hormone synthesis is the removal of
the side chain by the side chain cleavage or scc enzyme in the inner
mitochondrial membrane. Only two carbon atoms are left sticking from ring D and one of them carries a keto group. This first
committed precursor of all steroid hormones therefore contains 21
carbon atoms and is called pregnenolone. This molecule has long
been thought of as merely a pro-hormone but it appears to play an
important role in the central nervous system (neurosteroids).
Pregnenolone is only one step away from the first real hormone,
progesterone. All that is needed for this synthesis is the isomerisation
of the double bond and oxidation of the hydroxyl. The resulting
configuration of ring A is a recurring feature in steroid hormones.
Progesterone, as the name suggests, is a progestagen produced
in the corpus luteum and later in the placenta and its role is to
prepare the body for pregnancy (gestation) and then sustain it.
Progesterone can be converted in a few steps into the most
abundant glucocorticoid, cortisol. Three new hydroxyl groups are
introduced into the molecule. Glucocorticoids are produced in the
adrenal cortex (hence ‘corticoids’) and function as major regulators
of metabolic and immune functions. The adrenal cortex also produces mineralocorticoids such as aldosterone, which regulates the
handling of inorganic ions in kidneys.
A complete removal of the side chain (already severely truncated
by scc) from progesterone gives rise to a major androgen, testosterone, which therefore contains only 19 carbon atoms. As a male sex
hormone produced primarily in the Leydig cells of the testes it is
responsible for many aspects of male anatomy, physiology and
possibly behaviour such as post-pubertal hair growth pattern (and
hair loss pattern later in life), increased muscle and bone growth or
enhanced protein synthesis (‘anabolic’ effects).
Testosterone, perhaps surprisingly, can be converted in one step
to its physiological opposite, an œstrogen called œstradiol. The
enzyme aromatase removes the methyl group between rings A and
B and by creating a third double bond it forms an aromatic system.
Œstrogens therefore contain only 18 carbon atoms. They are female
sex hormones and as such are primarily synthesised in the ovaries.
However, aromatase is present in many other tissues, most notably
adipose tissue, which explains higher levels of œstrogens in obese
individuals both female and male. Œstrogens promote female
characteristics such as typical fat deposition (breasts, hips) or body
hair pattern but also increase plasma hdl and decrease ldl leading
to a significantly lower cardiovascular risk in women compared to
men.
Vitamin D was discovered in the 1920s in cod liver oil as a
substance capable of preventing rickets in dogs. Rickets, a disorder
of bone formation, became widespread in Europe with the advent
of the industrial revolution as more and more poor people came to
live in polluted cities without access to proper nutrition.
O
O
HO
pregnenolone
O
O
progesterone
OH
O
HO
OH
O
cortisol
OH
O
testosterone
OH
HO
oestradiol
7
isoprenoid metabolism: cholesterol and the others
However, the fact that vitamin D can be acquired in the diet
proved misleading. As was discovered later it is not really a vitamin; we can and do make it in our own skin. We will see shortly
that it would be much more correct to call it pro-hormone D.
The starting material for the synthesis of vitamin D is 7-dehydrocholesterol (easily made from cholesterol) and the magic ingredient
is ultraviolet light. An energetic photon absorbed by the parent molecule causes ring B to open, which is then followed by a rotation of
ring A resulting in cholecalciferol or vitamin D3 . The thing the proletariat were missing in the dark slums of late 18th century London
and Manchester was not (only) proper food but sunlight!
Cholecalciferol is then converted into the final hormone by
double hydroxylation: one in the liver and the other in kidneys.
The resulting 1,25-dihydroxycholecalciferol or calcitriol regulates the
absorption of calcium and phosphate in the intestine and kidneys.
1
7
HO
6
UV
1
7
HO
6
25
7
6
HO
1
cholecalciferol
© Jan Trnka 2010. This document was created with TEX/LATEX using
the tufte-handout document class typeset in Palatino. Please send
your comments or corrections to jan.trnka[at]lf3.cuni.cz
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