Cholesterol biosynthesis and its regulation, role of HMG-CoA reductase and SREBP protein

cholesterol

Cholesterol synthesis in essentially all tissues, but the liver, intestines, adrenal cortex and reproductive organs (ovaries, seeds and placenta) contribute the most to the cholesterol pool. Similar to fatty acid synthesis, all carbon atoms in cholesterol are provided by acetate and NADPH provides reducing equivalents.

This endergonic metabolic pathway is driven by hydrolysis of the thioester bond in acetyl-CoA and hydrolysis of ATP.

Cholesterol synthesis requires the enzymatic equipment of the cytosol and the smooth ER membrane.

This metabolic pathway is responsible for changes in cholesterol concentration and therefore there are regulatory mechanisms that regulate the rate of cholesterol synthesis in the body based on the rate of cholesterol excretion. An imbalance in this regulation can lead to an increase in the concentration of cholesterol in the plasma and thus the chance of developing cardiovascular diseases.

Synthesis

This metabolic pathway takes place in the endoplasmic reticulum and involves several enzymatic reactions. The first two reactions of cholesterol synthesis are similar to the production of ketone bodies, and thus their final product is 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA).

First, acetoacetyl-CoA is formed from two molecules of acetyl-CoA by the action of thiolase.

Subsequently, another molecule of acetyl-CoA is added using HMG-CoA synthase, resulting in HMG-CoA, a six-carbon compound.

• Liver cells contain two isozymes of HMG-CoA synthase. The cytosolic enzyme is involved in the synthesis of cholesterol and the mitochondrial form is important in the production of ketone bodies.

The next step of the pathway, the reduction of HMG-CoA to mevalonate, is catalyzed by HMG-CoA reductase and is the main regulatory step of the entire synthetic pathway. It takes place in the cytosol, uses two molecules of NADPH as a reducing agent, and secretes CoA, making it an irreversible step.

• HMG-CoA reductase is an ER membrane protein that has a catalytic domain directed into the cytosol.

Next steps of the metabolic pathway:

(1) Mevalonate is converted to 5-pyrophosphomevalonate in two steps, both of which involve the transfer of a phosphate group from ATP.

(2) The five-carbon isoprene unit, isopentenyl pyrophosphate (IPP), is formed by decarboxylation of 5-pyrophosphomevalonate. This reaction requires ATP.  IPP is isomerized to 3,3-dimethylallyl pyrophosphate (DPP).

(3) IPP and DPP condensation to form ten-carbon geranyl pyrophosphate (GPP).

(4) Another IPP molecule subsequently condenses with GPP to form 15-carbon farnesyl pyrophosphate (FPP).

(5) Two FPP molecules react together to release pyrophosphate.  Subsequently, the 30-carbon compound squalene is formed by reduction. Squalene is formed from 6 isoprenoid units. Since 3 ATP are required to convert mevalonate to IPP, a total of 18 ATP are required to form squalene.

(6) Squalene is converted to the sterol lanosterol by a series of reactions catalyzed by enzymes of the endoplasmic reticulum using molecular oxygen and NADPH. Hydroxylation of squalene stimulates its cyclization to lanosterol.

(7) The conversion of lanosterol into cholesterol is a multi-step process, the goal of which is to shorten the carbon chain from 30 to 27 carbons, remove two methyl groups on carbon no. 4, move the double bond from carbon no. 8 to carbon no. 5 and reduce the double bond  between carbons 24 and 25.

Smith-Lemli-Opitz syndrome (SLOS) is a relatively common autosomal recessive disorder of cholesterol synthesis that is caused by a partial deficiency of 7-dehydrocholesterol-7-reductase, an enzyme involved in double bond displacement. SLOS is one of several multisystem syndromes causing malformation of the embryo and are associated with insufficient cholesterol synthesis.

Regulation

HMG-CoA reductase, the key enzyme of the pathway, is the main regulatory point of cholesterol biosynthesis and is subject to various regulatory mechanisms.

cholesterol metabolism

Expression of the HMG-CoA reductase gene is controlled by the transcription factor SREBP-2 (sterol regulatory element-binding protein-2), which binds to DNA at the cis-acting sterol regulatory element (SRE) site of the reductase gene.

SREBP is an integral protein of the ER membrane that associates with the second ER membrane protein – SCAP (SREBP cleavage-activating protein)

When the level of sterols in the cell decreases, the SREBP-SCAP complex leaves the ER and moves to the Golgi apparatus. In the Golgi apparatus, SREBP is gradually subjected to the action of two proteases, resulting in a soluble fragment that enters the nucleus, binds to the SRE and is used as a transcription factor. This results in increased synthesis of HMG-CoA reductase and subsequently increased synthesis of cholesterol.

If the level of sterols in the cell is increased, the sterols bind to the sterol-sensitive domain of SCAP and induce binding of SCAP to other ER membrane proteins. This has the effect of suspending the formation of the SCAP-SREBP complex in the ER, thereby inactivating SREBP and thus inhibiting cholesterol synthesis.

The reductase itself is sensitive to the level of sterols. If the level of sterols in the cell rises, then the reductase binds to other proteins of the ER membrane. The binding leads to ubiquitination and proteasome degradation of the reductase.

HMG-CoA reductase activity is controlled covalently by the activity of AMP-activated protein kinase (AMPK) and phosphoprotein phosphatase. The phosphorylated form of reductase is inactive and its dephosphorylated form is active. AMPK is activated by AMP and thus cholesterol synthesis, like fatty acid synthesis, is dependent on the level of ATP (decreases with reduced ATP level).

The amount of HMG-CoA reductase is hormonally controlled. Increased amounts of insulin and thyroxine increase reductase gene expression. Glucagon and glucocorticoids will reduce it.

Statin drugs (e.g. atorvastatin, fluvastein, lovastatin, pravastatin, etc.) are structurally analogous to HMG-CoA and are therefore competitive inhibitors of HMG-CoA reductase. They are used to lower cholesterol levels in patients with hypercholesterolemia.

Sources

MATOUŠ, Bohuslav, et al. Základy lékařské chemie a biochemie. 1. vydání. Praha : Galén, 2010. 540 s.