Cholesterol Biosynthesis and Regulation

Overview and Biological Significance of Cholesterol

Cholesterol is an essential molecule in many animal species, including humans, serving as a vital structural component of cellular membranes and acting as a necessary metabolic precursor for the synthesis of steroid hormones and bile acids. Despite its importance, cholesterol is not required in the mammalian diet because all cells have the enzymatic machinery to synthesize it from simple precursors. However, there is a strong clinical implication linking high levels of cholesterol to various cardiovascular diseases.

The molecular structure of cholesterol consists of $27$ carbon atoms. Notably, every single one of these carbon atoms is provided by a single metabolic precursor: acetate. In the metabolic pathway transitioning from acetate to the final cholesterol molecule, isoprene units serve as the essential intermediates.

The Four Stages of Cholesterol Biosynthesis

The biosynthesis of cholesterol is derived entirely from acetyl-CoA. The scientific elucidation of this complex biosynthetic pathway was the work of several prominent researchers in the late $1950s$, including Konrad Bloch, Feodor Lynen, John Cornforth, and George Popják.

The process occurs in four distinct stages. The first stage involves the condensation of three acetate units to form the six-carbon intermediate known as mevalonate. The second stage consists of the conversion of mevalonate into activated isoprene units. In the third stage, six of these $5$-carbon isoprene units undergo polymerization to form squalene, a linear $30$-carbon molecule. The final stage involves the cyclization of squalene to form the characteristic four rings of the steroid nucleus. Following these stages, a series of further changes, including various oxidations and the removal or migration of methyl groups, occur to produce the final cholesterol molecule.

Stage One: Synthesis of Mevalonate from Acetate

The initial stage of biosynthesis focuses on the formation of mevalonate. This occurs through three primary reactions. In the first reaction, two molecules of acetyl-CoA undergo condensation to form acetoacetyl-CoA. During the second reaction, this acetoacetyl-CoA condenses with a third molecule of acetyl-CoA to produce common six-carbon compound $\beta$-hydroxy-$\beta$-methylglutaryl-CoA, abbreviated as HMG-CoA.

The first two reactions are catalyzed by the enzymes acetyl-CoA acetyltransferase and HMG-CoA synthase, respectively. The third reaction constitutes the committed step of the entire pathway: the reduction of HMG-CoA to mevalonate. This step requires two molecules of NADPH, each of which donates two electrons. The enzyme responsible for this step is HMG-CoA reductase, an integral membrane protein located in the smooth endoplasmic reticulum (ER). HMG-CoA reductase serves as the major point of metabolic regulation for the pathway.

Stage Two: Conversion of Mevalonate to Activated Isoprenes

During the second stage, mevalonate is converted into two forms of activated isoprenes through a process requiring the transfer of three phosphate groups from three ATP molecules. Initially, a phosphate attaches to the $C-5$ hydroxyl group of mevalonate. Subsequent phosphorylations result in the formation of the intermediate $3$-phospho-$5$-pyrophosphomevalonate. The phosphate group located at the $C-3$ position serves as an effective leaving group.

This loss of phosphate and a decarboxylation step produces a double bond in the resulting five-carbon product, $\triangle^3$-isopentenyl pyrophosphate. This molecule is the first of two activated isoprenes central to cholesterol formation. The second activated isoprene, dimethylallyl pyrophosphate, is produced through the isomerization of $\triangle^3$-isopentenyl pyrophosphate.

Stage Three: Condensation of Isoprene Units to Form Squalene

In the third stage, isopentenyl pyrophosphate and dimethylallyl pyrophosphate undergo a head-to-tail condensation. During this reaction, one pyrophosphate group is displaced, resulting in the formation of a $10$-carbon chain called geranyl pyrophosphate. This molecule then undergoes another head-to-tail condensation with a second molecule of isopentenyl pyrophosphate to yield a $15$-carbon intermediate known as farnesyl pyrophosphate.

Finally, two molecules of farnesyl pyrophosphate join together in a head-to-head fashion. This specific condensation eliminates both pyrophosphate groups to form squalene. Squalene is a $30$-carbon molecule, featuring $24$ carbons in its main chain and $6$ carbons arranged as methyl group branches.

Stage Four: Cyclization of Squalene to the Steroid Nucleus

All sterols are characterized by four fused rings that constitute the steroid nucleus. Sterols are chemically classified as alcohols because they possess a hydroxyl group at the $C-3$ position. To initiate cyclization, the enzyme squalene monooxygenase adds a single oxygen atom from $O_2$ to the end of the linear squalene chain, forming an epoxide intermediate called squalene $2,3$-epoxide.

The double bonds within the squalene $2,3$-epoxide are positioned such that a concerted reaction can convert the linear structure into a cyclic one. In animal cells, this cyclization produces lanosterol, which contains the four characteristic rings. Lanosterol is then converted to cholesterol through a series of approximately $20$ additional reactions involving the migration and removal of various methyl groups. While animals produce cholesterol, other organisms produce different sterols via similar pathways: plants produce stigmasterol and fungi produce ergosterol.

Regulation of Cholesterol Synthesis and Transport

Mammals possess complex systems to regulate cholesterol production based on intracellular concentrations, as excess cholesterol cannot be catabolized for fuel and must be excreted. Regulation is influenced by the cellular supply of ATP and by the hormones glucagon and insulin. The major site of regulation is the conversion of HMG-CoA to mevalonate via the enzyme HMG-CoA reductase.

Short-term regulation involves reversible covalent modification. Specifically, HMG-CoA reductase is phosphorylated by AMP-dependent protein kinase (AMPK). This phosphorylation allows the enzyme to react to high concentrations of AMP, which signals low ATP levels. When ATP levels are low, cholesterol synthesis slows down, while catabolic pathways are stimulated to generate more ATP. Hormonally, glucagon stimulates the phosphorylation (inactivation) of HMG-CoA reductase, whereas insulin promotes its dephosphorylation, which activates the enzyme and favors cholesterol synthesis.

Long-Term Transcriptional Regulation via SREBPs

Long-term regulation is achieved by adjusting the number of HMG-CoA reductase molecules through transcriptional control. This system is governed by sterol regulatory element-binding proteins (SREBPs). When these proteins are newly synthesized, they are embedded in the ER membrane. Only the soluble regulatory domain fragment of an SREBP can function as a transcription activator.

At high levels of cholesterol or oxysterol, SREBPs are held in the ER as part of a complex with SREBP cleavage-activating protein (SCAP) and a third protein called Insig (insulin-induced gene protein). SCAP and Insig serve as sterol sensors. When sterol levels are sufficiently high, the Insig-SCAP-SREBP complex remains anchored in the ER. If sterol levels decline, Insig is Targeted for degradation by ubiquitin, and the SCAP-SREBP complex is escorted by secretory proteins to the Golgi complex.

In the Golgi, two proteolytic cleavages release the SREBP regulatory fragment. This fragment then enters the nucleus and activates the transcription of target genes, including the gene for HMG-CoA reductase and the LDL receptor protein. Once sterol levels rise again, the proteolytic release of SREBP is blocked, and any existing active domains are degraded by proteasomes. Additionally, Insig can sense high cholesterol and trigger the attachment of ubiquitin molecules directly to HMG-CoA reductase, leading to its degradation.

Pathophysiology of Cardiovascular Disease and Atherosclerosis

Because animal cells cannot catabolize cholesterol, any excess must be removed through excretion or conversion into bile salts. When levels exceed the body's requirements, pathological accumulations known as plaques can obstruct blood vessels, causing atherosclerosis. This condition is linked to high levels of LDL-cholesterol (often called "bad cholesterol"). Conversely, there is a negative correlation between levels of HDL ("good cholesterol") and arterial disease.

Plaque formation begins when LDL molecules containing partially oxidized fatty acyl groups accumulate in the extracellular matrix of the epithelial cells lining the arteries. This attracts monocytes, which differentiate into macrophages. These macrophages take up the oxidized LDL and its cholesterol contents. Unlike other cells, macrophages cannot limit their sterol uptake; as they accumulate cholesteryl esters and free cholesterol, they transform into "foam cells."

As foam cells accumulate excess cholesterol, they eventually undergo apoptosis. Over time, the remnants of these cells, along with scar tissue and extracellular matrix material, form large plaques that occlude the artery. Occasionally, a plaque may break loose and travel to a narrower region of the artery in the heart or brain, resulting in a heart attack or stroke.

Familial Hypercholesterolemia and Therapeutic Treatments

Familial hypercholesterolemia is a condition where blood cholesterol levels are extremely high, often leading to severe atherosclerosis in childhood. This is caused by a defective LDL receptor, which prevents the receptor-mediated uptake of cholesterol from the blood. Consequently, because extracellular cholesterol cannot enter the cells to regulate intracellular production, endogenous synthesis continues unabated despite high serum levels.

Treatments for elevated serum cholesterol include a class of drugs known as statins, which inhibit HMG-CoA reductase. Another drug, Ezetimibe, is used to control cholesterol levels. Additionally, research is undergoing to develop new drugs that target intestinal liver X receptors (LXRs), which activate SREBP1C. To counter plaque formation, HDL participates in reverse cholesterol transport, picking up stored cholesterol from tissues and foam cells and carrying it back to the liver. This process is facilitated by ATP-binding cassette (ABC) transporters; the human genome encodes $48$ such transporters, approximately half of which are involved in lipid transport.