Download 1 Cholesterol synthesis, uptake, and regulation I. Structure and

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Cholesterol synthesis, uptake, and regulation
I. Structure and Function Cholesterol is essential to the survival of animal cells,
although not to bacteria. In animal cells most of the cholesterol is in the plasma
membrane, where it forms part of the membrane structure and is present in about a
one to one ratio with phospholipids.
A. Structure. While phospholipids have a small hydrophilic head and two long
fatty acid tails, cholesterol is almost completely hydrophobic except for a single
hydrophilic OH group. Attached to the OH group is a large bulky flat structure
composed of several hydrophobic rings. On the other side of the rings is a single
short hydrophobic tail. (For structure see MBOC p. 481 Fig 10-8.)
phospholipid
2nm
1nm
HO
cholesterol
1.05
nm
0.65
nm
In the case of a membrane composed primarily of unsaturated phospholipids, which
are already loosely packed due to the kinks in their fatty acid tails, cholesterol helps
to rigidify the membrane because its large head is just the right size and shape to
complement the kinks.
On the other hand, in the case of saturated phospholipids, in which the fatty acid
chains are tightly packed, the short, single chains of cholesterol give the
phospholipid chains more room to move around near the tail ends than if they
were next to other phospholipids. This extra freedom of movement increases the
fluidity of the membrane. Since most membranes have a mixture of saturated and
unsaturated lipids, this feature modulates the fluidity of membranes over a range
of temperatures. (See MBOC p. 482 Fig 10-9.)
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B. Mystery of high human LDL cholesterol
The level of cholesterol in human blood is about 175 mg/100 mL. Cholesterol is
normally bound by a protein complex in the blood, called LDL, or low-density
lipoprotein. The actual LD level is about 120 mg/100 mL. However, the optimal
level of LDL is 30 mg/100 mL for any animal cell. That is to say, this level is
sufficient to provide for the needs of the cells. All other animals have this level, as
do newborn humans, but in humans the level starts to rise not long after birth and
reaches an average concentration of 140 mg/100 mL in adults. No one knows why
this seems to happen exclusively in humans.
II. Cholesterol synthesis If you are asked on the final exam who figured out the pathway for cholesterol
synthesis, you should know that Konrad Bloch, an emeritus Harvard professor,
discovered that cholesterol is synthesized from acetate. (See MBOC p. 83 Fig. 2-37 for
overview and V&V p. 692-698 for more details.) The process starts with the
combination of 2 acetylCoA molecules to form acetoacetylCoA. The reaction
involves the formation of a carbanion and is catalyzed by the enzyme thiolase. In the
next step a third molecule of acetylCoA adds to acetoacetylCoA to form 3-hydroxy-3methyl-glutarylCoA (HMG-CoA) in a reaction catalyzed by HMG-CoA synthetase and
similar to that catalyzed by citrate synthetase. HMG-CoA is then reduced in two steps
using 2 NADPH to form first an aldehyde and then an alcohol called mevalonate (a
6-C branched compound). This reduction is catalyzed by HMG-CoA reductase, the
most tightly controlled enzyme in biology and the primary regulatory point in
cholesterol biosynthesis; its concentration varies by as much as 200-fold. In the next
two steps, which are catalyzed by a phosphotransferase and a kinase, two phosphates
from 2 ATP are added to mevalonate to form a pyrophosphate group; the new
compound is called 5-pyrophosphate-mevalonate. In the subsequent decarboxylation
step, catalyzed by a decarboxylase, CO2 leaves and the remaining electron pair moves
into the molecule and displaces the OH group to form the 5-carbon compound
isopentenyl pyrophosphate. An isomerase converts this to dimethylallylpyrophsophate. Several of the 5-carbon units can be added together to form
various compounds.
On the way to forming cholesterol via this mechanism the 10-carbon compound
geranyl-pyrophosphate and the 15-carbon farnesyl-pyrophosphate are formed.
Farnesyl-pyrophosphate serves as the precursor to dolichol, heme A, ubiquinone,
and farnesylated proteins that participate in signal transduction. The next
compound formed is squalene, which in the presence of oxygen is converted by
squalene epoxidase to 2,3-oxidosqualene. (Thus, cholesterol is absent in organisms
which predate the availability of O2). In a series of electron transfer reactions,
oxidocyclase catalyzes the closure of the ring structures to form the 30-carbon
compound lanosterol, from which the 27-carbon compound cholesterol is derived.
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III. Regulation of synthesis by cholesterol uptake
A. Free internal cholesterol reduces HMG-CoA reductase activity
Since cholesterol is required for survival, cells must have a way to acquire it as well
as to make sure they have the right amount; either too much or too little would be
fatal. Animal cells can synthesize cholesterol de novo, but organisms need more
cholesterol than the synthetic pathway can provide in order to make bile acids and
hormones. Therefore cells also have an uptake system which we will cover next.
Through their studies in fibroblasts, Brown and Goldstein found that cholesterol
synthesis is regulated by the levels of cholesterol itself. Since HMG-CoA reductase is
the major site of regulation in the synthetic pathway, they measured its activity in
the presence and absence of lipoprotein, or LDL (which contains cholesterol). When
cells were incubated in the presence of LDL and then the LDL was removed, the
HMG-CoA reductase activity showed a major increase, from 2 to 100 pmol/min/mg.
When LDL was added back to the cells, the activity dropped back to its original levels.
From this result, they concluded that the cell must regulate HMG-CoA reductase by
measuring the amount of cholesterol outside the cell.
B. Structure of LDL Since cholesterol is a hydrophobic molecule, it is not soluble by
itself in LDL but must be packaged into particles. The most common particles are
known as LDL (low density lipoprotein). Of the approximately 200 mg/mL of
cholesterol in human blood, 70% is packaged in LDL, a complex with a MW of 2 x
106. (Some of the remaining 30% is packaged in HDL, high density lipoprotein,
which is considered the good cholesterol.) Of this weight, 75% is lipid and 25% is
protein. The protein part, called apolipoprotein B, is a large (MW = 500,000) single
polypeptide (with a large exon that constitutes about half the protein).
Apolipoprotein B surrounds the complex, while the inner core contains cholesterol
ester, which is cholesterol esterified by fatty acid. Between the cholesterol core and
the protein layer are phospholipids, primarily phosphatidylcholine and
sphingomyelin. The structure is shown below (and also in V&V p. 318 Fig. 11-50 and
MBOC p. 621 Fig 13-29).
phospholipid
cholesterol ester
Apolipoprotein B
C. Interaction of cholesterol with cell-surface receptor
These LDL particles cannot pass through the membrane by themselves. So how was
the cell measuring the external cholesterol level, as amount of LDL or free
cholesterol? Cholesterol can be oxidized to a form that is sufficiently soluble in
serum to be able to directly diffuse into the plasma membrane. When they looked at
HMG-CoA reductase activity in the presence of this modified cholesterol, it caused
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the enzyme activity to drop faster as well, indicating that the key to the regulation of
HMG-CoA reductase activity is the amount of free cholesterol inside the cell.
Knowing that the cell measured external cholesterol levels according to their affect
on internal levels, Brown and Goldstein looked for a cell-surface receptor that might
mediate cholesterol uptake. Since cholesterol in LDL is surrounded by a protein, they
made radioactive LDL and looked to see if it bound to the cell surface; it did, with a
saturation curve. They then asked what the LDL was binding to.
They found a receptor, but to understand how it binds cholesterol it is first necessary
to understand the structure of the cholesterol-LDL complex. The receptor itself is a
type I membrane protein (that is, it has only one transmembrane strand) and is
composed of about 820 amino acids. The C-terminus is in the cytoplasm, but most of
the protein is outside the cell. Near the membrane on the extracellular side is a
region containing several O-linked carbohydrates that seem to act as bristles that keep
that domain rigid. Next is a region with homology to EGF (epidermal growth factor)
precursor, followed by a series of eight repeats that acts as the binding site for LDL.
The repeats are each 42 amino acids, are cross-linked by Cys residues, and contain
several negatively-charged amino acids. This last feature is key to its ability to bind
LDL, because apolipoprotein B has two regions containing positively-charged amino
acids at about the same frequency. The negative charges on the receptor and the
positive charges on LDL thus stick together via electrostatic interactions.
D. Cholesterol uptake by receptor-mediated endocytosis
Once the receptor binds LDL, it transmits the message to HMG-CoA reductase by
internalizing the cholesterol rather than by triggering a receptor-mediated signaling
cascade. The C-terminus of the receptor contains a signal that is recognized by
adaptin in clathrin-coated pits, so when the receptor reaches the plasma membrane it
usually ends up in clathrin-coated pits (indentations in the membrane). (See MBOC
p. 622 Fig 13-30 and V&V p. 32, Fig. 11-56.) The pits bud off into the cytoplasm to
form clathrin-coated vesicles, which then lose their clathrin coats and fuse with
endosomes. (See MBOC p. 624 Fig 13-33 and V&V p. 322 Fig. 11-57.) The low pH in
endosomes, maintained by proton pumps, breaks the electrostatic interactions
between LDL and its receptor, and they then separate to opposite regions of the
endosome that bud off to form separate vesicles. The vesicle with the receptor is
recycled back to the plasma membrane; each receptor is recycled through this
pathway every 10-20 minutes whether or not it binds to LDL. The vesicle with the
LDL fuses with a lysosome, where proteases digest its apolipoprotein B and lipases
break down the cholesterol ester to cholesterol. The cell then has cholesterol trapped
in the lysosome.
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plasma membrane
clathrin-coated
pit
lysosome
- proteases
- lipases
endosome
pH 5
LDL
LDL receptor
clathrin-coated
vesicle
D. Transcriptional regulation by cholesterol
An increase in cholesterol concentration causes a decrease in the activities of HMGCoA reductase, HMG-CoA synthetase and in the number of LDL receptors in the
plasma membrane. An experiment examining the effect of cholesterol on the
mRNA for each of these proteins revealed that part of this regulation occurs at the
level of transcription.
The promoters for these genes, but not for other genes, each contain a region called
the SRE, or steroid response element. A transcription factor is required to bind to the
SRE to turn on the gene, and two such factors were found, SREBP-1 and -2, or SRE
binding protein. Oddly, while the protein that was purified by binding to the SRE
sequence on a DNA binding column had a size of about 60 kD, the gene codes for a
130 kD protein. Examination of the DNA sequence of the SREBP gene revealed that
the encoded protein has two transmembrane regions.
The discrepancy in sizes was explained by the observation that in the presence of
cholesterol the 130 kD protein resides in the ER membrane, as shown below, but
when the cell is depleted of sterols, the SREBP is proteolyzed. The proteolysis occurs
in two steps. The first step, which is regulated by sterols, is a cleavage at an Arg
residue in the ER lumen; this is one example of proteolysis that does not take place
in a proteasome. The second proteolysis takes place in the membrane by a cysteine
protease and depends only on the completion of the first step rather than on the
presence of sterols. After cleavage, the N-terminal part of the protein floats away
from the membrane so that it can enter the nucleus, bind to the SRE sequence and
activate transcritption. The N-terminus contains a helix-loop-helix motif (HLH) that
binds to the SRE DNA.
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C
N
HLH
to nucleus
cytoplasm
second cleavage
(nonregulated)
R
ER lumen
first cleavage
(sterol-regulated)
How does the protease sense the level of sterols? A mutant that didn’t allow the
absence of sterols to signal proteolysis led Brown and Goldstein to discover a 1200
amino acid protein with 8 transmembrane domains called SCAP. The protein is
located in the ER and its multiple transmembrane domain acts as a sterol sensor to
detect changes in membrane fluidity, which reflect changes in cholesterol levels.
When the cholesterol level is low, the sterol sensor signals the protease to cleave
SREBP, and ultimately leads to an increase in cellular cholesterol. This process is
facilitated by the fact the WD repeats in the C-terminus of SCAP interacts with the Cterminus of SREBP, thus keeping the two proteins in proximity.
E. HMG-CoA reductase function and regulation
While HMG-CoA reductase mRNA levels vary by a factor of 10, levels of the enzyme
itself vary by a factor of 200, and it can be destroyed very rapidly. The enzyme, which
is present in the ER, has an N-terminal half with eight transmembrane domains as
well as a C-terminal half that contains the enzyme activity. If the C-terminus is
separated from the N-terminus by experimental manipulation, its activity becomes
independent of cholesterol, suggesting that the N-terminus - the membrane-bound
half - is the cholesterol, or sterol sensor. In the presence of high cholesterol, a
conformational change in the HMG-CoA reductase must tell a protease to destroy the
HMG-CoA reductase.
F. Transfer of cholesterol out of the lysosome
Another protein that uses a sterol sensor is a lysosomal membrane protein.
Cholesterol must get out of the lysosome in order to be used by the cell and to
regulate HMG-CoA reductase. This process requires a machine, as revealed by the
fact that in people with Neeman-Pick’s type C disease, cholesterol gets stuck in their
lysosomes. Since the disease is caused by a genetic mutation, the machine for
exporting cholesterol from lysosomes must involve a protein. This protein contains
multiple transmembrane domains, collectively known as the sterol sensing domain,
or SSD. (Interestingly, SSD is also present in PATCH, which is the receptor for the
developmentally important factor sonic hedgehog, which recently was determined to
have covalently bound cholesterol.)Once the cholesterol gets into the cytoplasm, it
interacts with the ER and is packaged and transported to the plasma membrane,
where it forms part of the membrane structure.