1 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.)

2 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.

3 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

4 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.

5

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.

6

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.