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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 26.3The Complex Regulation of Cholesterol Biosynthesis Takes Place at Several Levels

Cholesterol can be obtained from the diet or it can be synthesized de novo. An adult on a low-cholesterol diet typically synthesizes about 800 mg of cholesterol per day. The liver is the major site of cholesterol synthesis in mammals, although the intestine also forms significant amounts. The rate of cholesterol formation by these organs is highly responsive to the cellular level of cholesterol. This feedback regulation is mediated primarily by changes in the amount and activity of 3-hydroxy-3-methylglutaryl CoA reductase (Figure 26.14). As discussed in Section 26.2.1, this enzyme catalyzes the formation of mevalonate, the committed step in cholesterol biosynthesis. HMG CoA reductase is controlled in multiple ways:

Figure 26.14. HMG-CoA Reductase.

Figure 26.14

HMG-CoA Reductase. Image mouse.jpg The structure of a portion of the tetrameric enzyme is shown.

1.

The rate of synthesis of reductase mRNA is controlled by the sterol regulatory element binding protein (SREBP). This transcription factor binds to a short DNA sequence called the sterol regulatory element (SRE) on the 5′ side of the reductase gene. In its inactive state, the SREBP is anchored to the endoplasmic reticulum or nuclear membrane. When cholesterol levels fall, the amino-terminal domain is released from its association with the membrane by two specific proteolytic cleavages. The released protein migrates to the nucleus and binds the SRE of the HMG-CoA reductase gene, as well as several other genes in the cholesterol biosynthetic pathway, to enhance transcription. When cholesterol levels rise, the proteolytic release of the SREBP is blocked, and the SREBP in the nucleus is rapidly degraded. These two events halt the transcription of the genes of the cholesterol biosynthetic pathways.

2.

The rate of translation of reductase mRNA is inhibited by nonsterol metabolites derived from mevalonate as well as by dietary cholesterol.

3.

The degradation of the reductase is stringently controlled. The enzyme is bipartite: its cytosolic domain carries out catalysis and its membrane domain senses signals that lead to its degradation. The membrane domain may undergo a change in its oligomerization state in response to increasing concentrations of sterols such as cholesterol, making the enzyme more susceptible to proteolysis. Homologous sterol-sensing regions are present in the protease that activates SREBP. The reductase may be further degraded by ubiquitination and targeting to the 26S proteasome under some conditions. A combination of these three regulatory devices can regulate the amount of enzyme over a 200-fold range.

4.

Phosphorylation decreases the activity of the reductase. This enzyme, like acetyl CoA carboxylase (which catalyzes the committed step in fatty acid synthesis, Section 22.5), is switched off by an AMP-activated protein kinase. Thus, cholesterol synthesis ceases when the ATP level is low.

As we will see shortly, all four regulatory mechanisms are modulated by receptors that sense the presence of cholesterol in the blood.

26.3.1 Lipoproteins Transport Cholesterol and Triacylglycerols Throughout the Organism

Cholesterol and triacylglycerols are transported in body fluids in the form of lipoprotein particles. Each particle consists of a core of hydrophobic lipids surrounded by a shell of more polar lipids and apoproteins. The protein components of these macromolecular aggregates have two roles: they solubilize hydrophobic lipids and contain cell-targeting signals. Lipoprotein particles are classified according to increasing density (Table 26.1): chylomicrons, chylomicron remnants, very low density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Ten principal apoproteins have been isolated and characterized. They are synthesized and secreted by the liver and the intestine.

Table 26.1. Properties of plasma lipoproteins.

Table 26.1

Properties of plasma lipoproteins.

Triacylglycerols, cholesterol, and other lipids obtained from the diet are carried away from the intestine in the form of large chylomicrons (180– 500 nm in diameter; Section 22.1.2). These particles have a very low density (d<0.94 g cm-3) because triacylglycerols constitute ~99% of their content. Apolipoprotein B-48 (apo B-48), a large protein (240 kd), forms an amphipathic spherical shell around the fat globule; the external face of this shell is hydrophilic. The triacylglycerols in chylomicrons are released through hydrolysis by lipoprotein lipases. These enzymes are located on the lining of blood vessels in muscle and other tissues that use fatty acids as fuels and in the synthesis of fat. The liver then takes up the cholesterol-rich residues, known as chylomicron remnants.

The liver is a major site of triacylglycerol and cholesterol synthesis (Figure 26.15). Triacylglycerols and cholesterol in excess of the liver's own needs are exported into the blood in the form of very low density lipoproteins (d<1.006 g cm-3). These particles are stabilized by two lipoproteins—apo B-100 and apo E (34 kd). Apo B-100, one of the largest proteins known (513 kd), is a longer version of apo B-48. Both apo B proteins are encoded by the same gene and produced from the same initial RNA transcript. In the intestine, RNA editing (Section 28.3.2) modifies the transcript to generate the mRNA for apo B-48, the truncated form. Triacylglycerols in very low density lipoproteins, as in chylomicrons, are hydrolyzed by lipases on capillary surfaces. The resulting remnants, which are rich in cholesteryl esters, are called intermediate-density lipoproteins(1.006 < d < 1.019 g cm-3). These particles have two fates. Half of them are taken up by the liver for processing, and half are converted into low-density lipoprotein (1.019 < d < 1.063 g cm-3) by the removal of more triacylglycerol.

Figure 26.15. Site of Cholesterol Synthesis.

Figure 26.15

Site of Cholesterol Synthesis. Electron micrograph of a part of a liver cell actively engaged in the synthesis and secretion of very low density lipoprotein (VLDL). The arrow points to a vesicle that is releasing its content of VLDL particles. [Courtesy (more...)

Low-density lipoprotein is the major carrier of cholesterol in blood. This lipoprotein particle has a diameter of 22 nm and a mass of about 3 million daltons (Figure 26.16). It contains a core of some 1500 esterified cholesterol molecules; the most common fatty acyl chain in these esters is linoleate, a polyunsaturated fatty acid. A shell of phospholipids and unesterified cholesterols surrounds this highly hydrophobic core. The shell also contains a single copy of apo B-100, which is recognized by target cells. The role of LDL is to transport cholesterol to peripheral tissues and regulate de novo cholesterol synthesis at these sites, as described in Section 26.3.3. A different purpose is served by high-density lipoprotein (1.063 < d < 1.21 g cm-3), which picks up cholesterol released into the plasma from dying cells and from membranes undergoing turnover. An acyltransferase in HDL esterifies these cholesterols, which are then either rapidly shuttled to VLDL or LDL by a specific transfer protein or returned by HDL to the liver.

Figure 26.16. Schematic Model of Low-Density Lipoprotein.

Figure 26.16

Schematic Model of Low-Density Lipoprotein. The LDL particle is approximately 22 nm (220 Å) in diameter.

26.3.2 The Blood Levels of Certain Lipoproteins Can Serve Diagnostic Purposes

Image caduceus.jpg High serum levels of cholesterol cause disease and death by contributing to the formation of atherosclerotic plaques in arteries throughout the body. This excess cholesterol is present in the form of the low density lipoprotein particle, so-called “bad cholesterol.”The ratio of cholesterol in the form of high density lipoprotein, sometimes referred to as “good cholesterol,” to that in the form of LDL can be used to evaluate susceptibility to the development of heart disease. For a healthy person, the LDL/HDL ratio is 3.5.

High-density lipoprotein functions as a shuttle that moves cholesterol throughout the body. HDL binds and esterifies cholesterol released from the peripheral tissues and then transfers cholesteryl esters to the liver or to tissues that use cholesterol to synthesize steroid hormones. A specific receptor mediates the docking of the HDL to these tissues. The exact nature of the protective effect of HDL levels is not known; however, a possible mechanism is discussed in Section 26.3.5.

26.3.3 Low-Density Lipoproteins Play a Central Role in Cholesterol Metabolism

Cholesterol metabolism must be precisely regulated to prevent atherosclerosis. The mode of control in the liver, the primary site of cholesterol synthesis, has already been discussed: dietary cholesterol reduces the activity and amount of 3-hydroxy-3-methylglutaryl CoA reductase, the enzyme catalyzing the committed step. The results of studies by Michael Brown and Joseph Goldstein are sources of insight into the control of cholesterol metabolism in nonhepatic cells. In general, cells outside the liver and intestine obtain cholesterol from the plasma rather than synthesizing it de novo. Specifically, their primary source of cholesterol is the low-density lipoprotein. The process of LDL uptake, called receptor-mediated endocytosis, serves as a paradigm for the uptake of many molecules.

The steps in the receptor-mediated endocytosis of LDL are as follows (see Figure 12.40).

1.

Apolipoprotein B-100 on the surface of an LDL particle binds to a specific receptor protein on the plasma membrane of nonhepatic cells. The receptors for LDL are localized in specialized regions called coated pits, which contain a specialized protein called clathrin.

2.

The receptor-LDL complex is internalized by endocytosis, that is, the plasma membrane in the vicinity of the complex invaginates and then fuses to form an endocytic vesicle (Figure 26.17).

3.

These vesicles, containing LDL, subsequently fuse with lysosomes, acidic vesicles that carry a wide array of degradative enzymes. The protein component of the LDL is hydrolyzed to free amino acids. The cholesteryl esters in the LDL are hydrolyzed by a lysosomal acid lipase. The LDL receptor itself usually returns unscathed to the plasma membrane. The round-trip time for a receptor is about 10 minutes; in its lifetime of about a day, it may bring many LDL particles into the cell.

4.

The released unesterified cholesterol can then be used for membrane biosynthesis. Alternatively, it can be reesterified for storage inside the cell. In fact, free cholesterol activates acyl CoA:cholesterol acyltransferase (ACAT), the enzyme catalyzing this reaction. Reesterified cholesterol contains mainly oleate and palmitoleate, which are monounsaturated fatty acids, in contrast with the cholesterol esters in LDL, which are rich in linoleate, a polyunsaturated fatty acid (see Table 24.1). It is imperative that the cholesterol be reesterified. High concentrations of unesterified cholesterol disrupt the integrity of cell membranes.

Figure 26.17. Endocytosis of LDL Bound to Its Receptor.

Figure 26.17

Endocytosis of LDL Bound to Its Receptor. (A) Electron micrograph showing LDL (conjugated to ferritin for visualization, dark spots) bound to a coated-pit region on the surface of a cultured human fibroblast cell. (B) Micrograph showing this region invaginating (more...)

The synthesis of LDL receptor is itself subject to feedback regulation. The results of studies of cultured fibroblasts show that, when cholesterol is abundant inside the cell, new LDL receptors are not synthesized, and so the uptake of additional cholesterol from plasma LDL is blocked. The gene for the LDL receptor, like that for the reductase, is regulated by SREBP, which binds to a sterol regulatory element that controls the rate of mRNA synthesis.

26.3.4 The LDL Receptor Is a Transmembrane Protein Having Five Different Functional Regions

The amino acid sequence of the human LDL receptor reveals the mosaic structure of this 115-kd protein, which is composed of six different types of domain (Figure 26.18). The amino-terminal region of the mature receptor consists of a cysteine-rich sequence of about 40 residues that is repeated, with some variation, seven times to form the LDL-binding domain (Figure 26.19). A set of conserved acidic side chains in this domain bind calcium ion; this metal ion lies at the center of each domain and, along with disulfide bonds formed from the conserved cysteine residues, stabilizes the three-dimensional structure. Protonation of these glutamate and aspartate side chains of the receptor in lysosomes leads to the release of calcium and hence to structural disruption and the release of LDL from its receptor. A second region of the LDL receptor includes two types of recognizable domains, three domains homologous to epidermal growth factor and six repeats that are similar to the blades of the transducin β subunit (Section 15.2.2). The six repeats form a propeller-like structure that packs against one of the EGF-like domains (Figure 26.20). An aspartate residue forms hydrogen bonds that hold each blade to the rest of the structure. These interactions, too, would most likely be disrupted at the low pH in the lysosome.

Figure 26.18. LDL Receptor Domains.

Figure 26.18

LDL Receptor Domains. A schematic representation of the amino acid sequence of the LDL receptor showing six types of domain.

Figure 26.19. Structure of Cysteine-Rich Domain.

Figure 26.19

Structure of Cysteine-Rich Domain. Image mouse.jpg This calcium-binding cysteine-rich domain is repeated seven times at the amino terminus of the LDL receptor.

Figure 26.20. Structure of Propeller Domain.

Figure 26.20

Structure of Propeller Domain. Image mouse.jpg The six-bladed propeller domain and an adjacent EGF-like domain of the LDL receptor.

The third region contains a single domain that is very rich in serine and threonine residues and contains O-linked sugars. These oligosaccharides may function as struts to keep the receptor extended from the membrane so that the LDL-binding domain is accessible to LDL. The fourth region contains the fifth type of domain, which consists of 22 hydrophobic residues that span the plasma membrane. The final region contains the sixth type of domain; it consists of 50 residues and emerges on the cytosolic side of the membrane, where it controls the interaction of the receptor with coated pits and participates in endocytosis. The gene for the LDL receptor consists of 18 exons, which correspond closely to the structural units of the protein. The LDL receptor is a striking example of a mosaic protein encoded by a gene that was assembled by exon shuffling.

26.3.5 The Absence of the LDL Receptor Leads to Hypercholesteremia and Atherosclerosis

Image caduceus.jpg The results of Brown and Goldstein's pioneering studies of familial hypercholesterolemia revealed the physiologic importance of the LDL receptor. The total concentration of cholesterol and LDL in the plasma is markedly elevated in this genetic disorder, which results from a mutation at a single autosomal locus. The cholesterol level in the plasma of homozygotes is typically 680 mg dl-1, compared with 300 mg dl-1 in heterozygotes (clinical assay results are often expressed in milligrams per deciliter, which is equal to milligrams per 100 milliliters). A value of < 200 mg dl-1 is regarded as desirable, but many people have higher levels. In familial hypercholesterolemia, cholesterol is deposited in various tissues because of the high concentration of LDL cholesterol in the plasma. Nodules of cholesterol called xanthomas are prominent in skin and tendons. Of particular concern is the oxidation of the excess blood LDL to form oxidized LDL (oxLDL). The oxLDL is taken up by immune-system cells called macrophages, which become engorged to form foam cells. These foam cells become trapped in the walls of the blood vessels and contribute to the formation of atherosclerotic plaques that cause arterial narrowing and lead to heart attacks (Figure 26.21). In fact, most homozygotes die of coronary artery disease in childhood. The disease in heterozygotes (1 in 500 people) has a milder and more variable clinical course. A serum esterase that degrades oxidized lipids is found in association with HDL. Possibly, the HDL-associated protein destroys the oxLDL, accounting for HDL's ability to protect against coronary disease.

Figure 26.21. An Atherosclerotic Plaque.

Figure 26.21

An Atherosclerotic Plaque. A plaque (marked by an arrow) blocks most of the lumen of this blood vessel. The plaque is rich in cholesterol. [Courtesy of Dr. Jeffrey Sklar.]

The molecular defect in most cases of familial hypercholesterolemia is an absence or deficiency of functional receptors for LDL. Receptor mutations that disrupt each of the stages in the endocytotic pathway have been identified. Homozygotes have almost no functional receptors for LDL, whereas heterozygotes have about half the normal number. Consequently, the entry of LDL into liver and other cells is impaired, leading to an increased plasma level of LDL. Furthermore, less IDL enters liver cells because IDL entry, too, is mediated by the LDL receptor. Consequently, IDL stays in the blood longer in familial hypercholesterolemia, and more of it is converted into LDL than in normal people. All deleterious consequences of an absence or deficiency of the LDL receptor can be attributed to the ensuing elevated level of LDL cholesterol in the blood.

26.3.6 The Clinical Management of Cholesterol Levels Can Be Understood at a Biochemical Level

Image caduceus.jpg Homozygous familial hypercholesterolemia can be treated only by a liver transplant. A more generally applicable therapy is available for heterozygotes and others with high levels of cholesterol. The goal is to reduce the amount of cholesterol in the blood by stimulating the single normal gene to produce more than the customary number of LDL receptors. We have already observed that the production of LDL receptors is controlled by the cell's need for cholesterol. Therefore, in essence, the strategy is to deprive the cell of ready sources of cholesterol. When cholesterol is required, the amount of mRNA for the LDL receptor rises and more receptor is found on the cell surface. This state can be induced by a two-pronged approach. First, the intestinal reabsorption of bile salts is inhibited. Bile salts are cholesterol derivatives that promote the absorption of dietary cholesterol and dietary fats (Section 22.1.1). Second, de novo synthesis of cholesterol is blocked.

The reabsorption of bile is impeded by oral administration of positively charged polymers, such as cholestyramine, that bind negatively charged bile salts and are not themselves absorbed. Cholesterol synthesis can be effectively blocked by a class of compounds called statins (e.g., lovastatin, which is also called mevacor; Figure 26.22). These compounds are potent competitive inhibitors (Ki < 1 nM) of HMG-CoA reductase, the essential control point in the biosynthetic pathway. Plasma cholesterol levels decrease by 50% in many patients given both lovastatin and inhibitors of bile-salt reabsorption. Lovastatin and other inhibitors of HMG-CoA reductase are widely used to lower the plasma cholesterol level in people who have atherosclerosis, which is the leading cause of death in industrialized societies.

Figure 26.22. Lovastatin, a Competitive Inhibitor of HMG-CoA Reductase.

Figure 26.22

Lovastatin, a Competitive Inhibitor of HMG-CoA Reductase. The part of the structure that resembles the 3-hydroxy-3-methylglutaryl moiety is shown in red.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22336