Cholesterol Biosynthesis: Site, Pathway, Steps, Regulation, Significance

• Cholesterol is the most important sterol in the human body.
• Almost half of the cholesterol in the body derives from de novo biosynthesis.
• The liver and intestines are major contributors to endogenous production.
• The liver is the primary site of de novo cholesterol synthesis, accounting for approximately 80% of total cholesterol synthesis in mammals.
•  HMG-CoA reductase (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is the key regulatory and rate-limiting enzyme.

• The synthesis starts from acetyl-CoA, which is derived from protein, carbohydrate, and fat metabolism.
• The process is energy-intensive and requires ATP and NADPH.
• To produce one mole of cholesterol, 18 moles of acetyl-CoA, 36 moles of ATP, and 16 moles of NADPH are required.

Site and Significance of Cholesterol Biosynthesis

• Biosynthesis of cholesterol primarily occurs in the liver’s cytoplasm and endoplasmic reticulum.
• This synthesis provides cholesterol, which is essential for cellular homeostasis, contributing to membrane rigidity and fluidity.
• It is the necessary precursor to the steroid hormones such as glucocorticoids, mineralocorticoids, and sex hormones produced in the endocrine tissues.
• Synthesis of bile acids out of cholesterol facilitates the excretion of cholesterol and the digestion of lipids in the diet.
• Vitamin D is synthesized from a cholesterol backbone and regulates calcium homeostasis and bone mineralization.

The Pathway Breakdown: From Acetyl-CoA to Cholesterol

The synthesis of cholesterol may be learnt in 5 stages

1. Synthesis of HMG CoA
2. Formation of mevalonate (6C)
3. Production of isoprenoid units (5C)
4. Synthesis of squalene (30C)
5. Conversion of squalene to cholesterol (27C).

1. Formation of HMG-CoA: The Initial Condensation Steps

• The process starts with the two molecules of acetyl-CoA, which combine to start the biosynthesis of cholesterol.
• These two acetyl-CoA molecules are condensed to acetoacetyl-CoA, which is catalyzed by the enzyme thiolase in the cytosol, also known as acetyl-CoA acetyl transferase 2 (ACAT2).
• Acetoacetyl-CoA then reacts with a 3rd molecule of acetyl-CoA to produce HMG-CoA.
• This reaction is catalyzed by the cytosolic HMG-CoA synthase (encoded by HMGCS1).
• The HMG-CoA thus formed acts as a precursor of mevalonate synthesis, which is the subsequent stage of cholesterol biosynthesis.

2. The Rate-Limiting Step: Synthesis of Mevalonate by HMG-CoA Reductase

• HMG-CoA is transformed into mevalonate by HMG-CoA reductase (HMGR), which is an endoplasmic reticulum-bound enzyme.
• Two molecules of NADPH are required for this reduction, making it NADPH-dependent.
• This is the rate-limiting and most important regulatory step in cholesterol biosynthesis.

3. From Mevalonate to Squalene: Formation of Isoprenoid Units

• Mevalonate is phosphorylated by mevalonate kinase and phosphomevalonate kinase to form mevalonate-5-pyrophosphate, requiring ATP.
• Mevalonate-5-pyrophosphate is decarboxylated by the ATP-dependent decarboxylase enzyme, mevalonate-5-pyrophosphate decarboxylase, to give isopentenyl pyrophosphate (IPP) and CO 2.
• IPP is isomerized to dimethylallyl pyrophosphate (DMAPP) by isopentenyl pyrophosphate isomerase.
• IPP and DMAPP condense to form geranyl pyrophosphate (GPP, 10-carbon molecule), catalyzed by geranyl pyrophosphate synthase.
• GPP combines with another IPP to produce farnesyl pyrophosphate (FPP, 15-carbon molecule) with the help of the farnesyl pyrophosphate synthase (FDPS).
• Two molecules of FPP are condensed by squalene synthase (FDFT1) to give squalene (30-carbon molecules).

4. Cyclization of Squalene: Creating the Steroid Nucleus (Lanosterol)

• Squalene is cyclized in two steps to produce lanosterol.
• In the first step, squalene epoxidase (SQLE) introduces an epoxide at the 2,3 position, forming 2,3-oxidosqualene; this reaction requires NADPH and O₂.
• The second step involves the lanosterol synthase (LSS), which is a catalyst that helps in catalyzing the cyclization of 2, 3-oxidosqualene to create lanosterol, the initial steroid nucleus.
• This step establishes the four-ring steroid structure, which is essential for all downstream cholesterol and steroid derivatives.

5. Final Processing: Conversion of Lanosterol to Cholesterol

• The conversion of Lanosterol to cholesterol occurs through 19 enzymatic reactions that include demethylation, desaturation, isomerization and reduction.
• The primary human pathway is the Kandutsch-Russell pathway, producing 7-dehydrocholesterol as the immediate precursor.
• Key reactions include reduction of carbon atoms from 30 to 27, removal of two methyl groups at C4 and one at C14, shift of a double bond from C8 to C5, and reduction of the double bond between C24 and C25.
• Important intermediates include desmethyl lanosterol, zymosterol, cholestadienol, and desmosterol.
• The last step is the conversion of 7-dehydrocholesterol into cholesterol with the help of DHCR7.

Regulation of Biosynthesis: Hormonal Control and the SREBP Pathway

Hormonal control 

• HMG-CoA reductase is the rate-limiting enzyme in cholesterol biosynthesis, and it regulates the biosynthesis of cholesterol.
• There are two forms of HMG-CoA reductase, the dephosphorylated (active) and the phosphorylated (inactive) forms.
• Insulin and thyroxine stimulate cholesterol production by increasing the production of the active dephosphorylated enzyme.
• The synthesis of cholesterol is inhibited by glucagon and glucocorticoids, which stabilize the phosphorylated form.

 SREBP Pathway (Transcriptional Regulation)

• SREBPs (Sterol Regulatory Element-Binding Proteins) are transcription factors that are produced in the ER membrane as inactivated precursors. 
• At low cellular cholesterol, SREBPs are bound by SCAP (SREBP cleavage-activating protein) and delivered to the Golgi.
•  Site-1 and site-2 proteases cleave SREBP in the Golgi, releasing the active domain to the nucleus. 
• Nuclear SREBP stimulates genes of HMG-CoA reductase, HMG-CoA synthase, and other enzymes of the mevalonate pathway that produce cholesterol. 
• ER cholesterol elevation inhibits the cleavage of SREBP through INSIG proteins, reducing the transcription of cholesterol biosynthetic genes.
•  SREBP is also inhibited by oxysterols (e.g., 25-hydroxycholesterol) as another feedback inhibitor.

Clinical Pharmacology: How Statins Inhibit Cholesterol Synthesis

• The statins are FDA-approved medications used to treat hyperlipidemia and hypercholesterolemia.
• They are reversible competitive inhibitors of the HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis.
• Structurally, statins are analogs of HMG-CoA, which means that they can compete with the natural substrate to bind the enzyme.
• HMG-CoA reductase inhibition prevents the conversion of HMG-CoA to mevalonate, thereby inhibiting the de novo synthesis of cholesterol in the liver.
• The low intracellular cholesterol triggers the transcriptional regulation via SREBP and increases the expression of LDL receptors.
• Higher LDL receptors promote the cellular absorption and clearance of blood LDL cholesterol.
• The net pharmacologic action has been lowering plasma LDL cholesterol and cardiovascular risk levels.
• Atorvastatin, simvastatin, and lovastatin are common statins, and they are fungal-derived HMG-CoA reductase inhibitors.

The Fate of Cholesterol: Bile Acid Synthesis and Esterification

Bile Acid Synthesis

• The only place where cholesterol is converted to primary bile acid (cholic acid, CA; chenodeoxycholic acid, CDCA) is in the liver.
• There are two pathways, the classic (neutral) pathway and the alternative (acidic) pathway.
• Cholesterol 7α-hydroxylase (CYP7A1) catalyzes the rate-limiting first step in the classic pathway, converting cholesterol to 7α-hydroxycholesterol.
• Sterol 12α-hydroxylase (CYP8B1) oxidizes C-12 to cholic acid; the lack of which gives CDCA.
• CYP27A1 catalyzes side-chain oxidation, which results in C24 bile acids.
• The alternative pathway begins with CYP27A1 converting cholesterol to 27-hydroxycholesterol, then hydroxylated by CYP7B1 to CDCA.
• Primary bile acids are conjugated with glycine or taurine in the liver and secreted into bile.
• Bacterial enzymes present in the intestine change the primary bile acids into secondary bile acids (deoxycholic acid, lithocholic acid).
• Bile acids play a crucial role in the process of lipid digestion, the elimination of cholesterol, and reverse cholesterol transport between the peripheral tissues and the liver.

Esterification

• Cholesterol is esterified to promote hydrophobicity in order to be safely stored or transported.
• The addition of the fatty acid to the hydroxyl group of cholesterol in the liver is catalyzed by ACAT (acyl-CoA cholesterol acyltransferase). Forming cholesteryl esters (CEs).
• LCAT (lecithin cholesterol acyltransferase) is the one that carries out the same esterification in plasma with HDL particles.
• Cholesteryl esters are more hydrophobic than free cholesterol, allowing storage in lipid droplets or incorporation into VLDL for transport.
• VLDL transports cholesteryl esters, triglycerides, and phospholipids to tissues to synthesize membranes, produce steroid hormones, and vitamin D.
• Esterification retards the toxic accumulation of free cholesterol in cells and facilitates the regulated cholesterol dispersion.
• This is necessary to maintain cholesterol homeostasis and lipoprotein-mediated transportation.

Conclusion 

• Cholesterol is a key sterol, and almost half of cholesterol is synthesized de novo, primarily in the liver and the intestine.
• The production of cholesterol takes place in the cytoplasm and endoplasmic reticulum and involves the use of acetyl-CoA, ATP, and NADPH.
• The pathway goes through five steps: formation of HMG-CoA, mevalonate synthesis, isoprenoid units’ formation, squalene formation, and cholesterol formation.
• The rate-limiting and most important regulatory enzyme of the pathway is HMG-CoA reductase.
• Cholesterol plays a critical role in maintaining membrane integrity, synthesizing steroid hormones, making bile acids and synthesizing vitamin D.
• It is regulated through the hormonal mechanisms in which insulin stimulates and glucagon suppresses the production of cholesterol.
• The SREBP pathway gives transcriptional control in response to intracellular cholesterol.
• Statins reduce cholesterol by blocking HMG-CoA reductase competitively and stimulating the expression of the LDL receptor.
• The liver converts cholesterol to bile acids that help in the digestion of lipids and the excretion of cholesterol.
• ACAT and LCAT facilitation of esterification of cholesterol allows safe storage and transportation of cholesteryl esters.

Reference 

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   https://themedicalbiochemistrypage.org/cholesterol-synthesis-metabolism-and-regulation/