BIOC 303: Cholesterol

Cholesterol Synthesis: Acetate

• cholesterol is a 27 C compound, in which all C atoms are provided by acetate

Cholesterol Synthesis: Isoprene

• isoprene units are essential intermediates in the pathway

Cholesterol Synthesis Steps

• condensation of 3 acetate units to form a six-carbon intermediate, mevalonate

• conversion of mevalonate to activated isoprene units

• polymerization of six 5-carbon isoprene units to form the 30 carbon liner squalene

• cyclization os squalene to form the 4 rings of the steroid nucleus, with a further series of changes (oxidations, removal or migration of methyl groups) to produce cholesterol

Cholesterol Synthesis Figure

Stage 1: Mevalonate Synthesis from Acetate

• two molecules of acetyl-CoA condense to form acetoacetyl-CoA, catalyzed by acetyl-CoA acetyl transferase

• HMG-CoA synthase catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form β-hydroy-β-methylglutaryl-CoA (HMG CoA)

  • both are Claisen condensations and the standard equilibrium in both favors degradation to acetyl-CoA

    • however the HMG-CoA is rapidly used after synthesis in subsequent reactions

• reduction of HMG-CoA to mevalonate by HMG-CoA reductase

  • the committed step; major point of regulation on the pathway to cholesterol

  • requires 2 NADPH

Stage 1: Mevalonate Synthesis from Acetate FIGURE

Stage 2: Conversion of Mevalonate to 2 Activates Isoprenes

• 3 phosphate groups are transferred from 3 ATP molecules to mevalonate to produce the intermediate, 3-phospho-5-pyrophosphomevalonate

  • 3 ATP for each activated isoprene → total of 18 ATP 

• one of the added phosphates (on the C3 of ⬆ intermediate) makes a good leaving group

  • it leaves and at the same time, a nearby carboxyl group also leaves, forming a double bond in the molecule, yielding ∆³-isopentyl pyrophosphate (IPP)

    • first of 2 activates isoprenes

• isomerization of IPP yields the second activated isoprene, dimethylallyl pyrophosphate (some are isomerized but not all to create 2 pools)

Stage 2: Conversion of Mevalonate to 2 Activates Isoprenes FIGURE

Stage 3: Condensation of 6 Activated Isoprene Units to form Squalene

• Isopentyl PP and dimethylallyl PP undergo head-to-tail condensation to form geranyl PP (10 C chain)

  • one PP group is displaced

• geranyl PP undergoes head-to-tail condensation with isopentyl PP to yield farnesyl PP (15 C intermediates)

• 2 molecules of farnesyl PP join head to head, eliminating both PP groups, to form squalene (30 C’s; 24 in main chain, 6 in methyl group branches)

Stage 3: Condensation of 6 Activated Isoprene Units to form Squalene FIGURE

Stage 4: Conversion of Squalene to the Steroid Nucleus

• squalene monooxygenase adds one O atom from O₂ to the end of the squalene chain, forming squalene 2,3-epoxide

  • requires NADPH

  • a mixed-function oxidase: the other O atom is reduced by NADPH to H₂O

• the double bonds of squalene 2,3-epoxide are positioned in a way that can convert the linear squalene epoxide to a cyclic structure

  • this cyclization results in lanosterol, which contains the 4 ring steroid nucleus

• lanosterol is converted to cholesterol in a series of 20 rxns

Stage 4: Conversion of Squalene to the Steroid Nucleus FIGURE

Several Fates of Cholesterol

• cholesterol is primarily synthesized in the liver

• a small fraction of cholesterol made there is incorporated into the membranes of hepatocytes

• most of it is exported as: bile acids, biliary cholesterol or cholesteryl esters

  • cholesteryl esters are formed in the liver by acyl-CoA cholesterol acyltransferase (ACAT)

• in other tissues, cholesterol is converted into steroid hormones (in adrenal cortex and gonads) or into vitamin D hormone (liver and kidney)

  • these hormones are biological signals acting thru nuclear receptor proteins

Bile Acids

• fluid stored in the gallbladder and excreted into the small intestine to aid in the digestion of fatty meals

• relatively hydrophobic cholesterol derivatives that serve as emulsifiers in the intestine

• bile contains small amounts of cholesterol (biliary cholesterol)

• helps remove excess cholesterol from the intestine and facilitates excretion

Dietary Fibers on Bile

• can enhance the excess cholesterol effect by binding to bile and interfering with bile reabsorption in the intestines, leading to increased bile excretion in the feces

• more cholesterol is then used to make bile

• available in oats and barley

Acyl-CoA Cholesterol Acyltransferase (ACAT)

• catalyzes the synthesis of cholesteryl esters in the liver

• catalyzes the transfer of a fatty acid from coenzyme A to the hydroxyl group of cholesterol

  • this converts cholesterol into a more hydrophobic form that isn’t sufficiently amphipathic anymore to function appropriately in membranes

• cholesteryl esters are transported in secreted lipoprotein particles to other tissues that use cholesterol, or are stored in the liver in lipid droplets

Reverse Cholesterol Pathway

• HDL’s contain lecithin-cholesterol acyltransferase (LCAT) which catalyzes the formation of cholesteryl esters from lecithin (phosphatidylcholine) and cholesterol

• HDL picks up cholesterol and cholesteryl-esters from cholesterol-rich extrahepatic cells and returns it to the liver for unloading

• much of this cholesterol is converted to bile salts → stored in gallbladder

Why is Reverse Cholesterol Transport Useful?

• HDL can get cholesterol from LDL and macrophages that”

  • don’t have access to liver/ aren’t brought back to liver

• circulating LDL isn’t good, as it gets eaten up by macrophages, which break down cholesteryl-esters into cholesterol

  • cholesterol crystallizes and becomes plaque

• when HDL’s are empty again, they can go thru the system again and get more cholesterol

Reverse Cholesterol Transport: Step 1

HDLs are synthesized, containing LCAT

• LCAT on surfaces of nascent (newly forming) HDL particles converts the cholesterol and phosphatidylcholine of chylomicron and VLDL remnants to cholesteryl esters

  • this begins to form a core, transforming the disk-shaped nascent HDL to a mature, spherical HDL particle

Reverse Cholesterol Transport: Step 2

Nascent HDL can also pick up cholesterol from cholesterol-rich extrahepatic cells

• including macrophages, and foam cells (formed from macrophages)

Reverse Cholesterol Transport: Step 3

Mature HDL then returns to the liver, where the cholesterol is unloaded via the scavenger receptor SR-BI

Reverse Cholesterol Transport: Step 4

Some of the cholesteryl esters in HDL can be transferred to LDL by the cholesteryl ester transfer protein

Receptor Mediated Endocytosis: Details

• each LDL particle in the bloodstream contains apoB-100, which is recognized by LDL receptors present in the plasma membranes of cells that need to take up cholesterol

• LDL receptors: receptors in the hepatocyte plasma membrane that take up LDL not taken up by peripheral tissues and cells

  • synthesized in the ER and modified by Golgi complex, then transported to the plasma membrane

• ApoB-100 is also present in VLDL, but its receptor binding domain is not available for binding to the LDL receptor

  • conversion of VLDL to LDL exposes the receptor-binding domain of apoB-100

Receptor Mediated Endocytosis: Steps

• LDL receptors are synthesized and transported to plasma membrane, where they are available to bind apoB-100

• binding of LDL to an LDL receptor initiates endocytosis

• this conveys LDL and its receptor into the cell within an endosome

• the receptor containing portions of the endosome membrane bud off and are returned to the cell surface, to function again in LDL uptake

• the endosome fuses with a lysosome which contains enzymes that hydrolyze the cholesteryl esters

  • this releases cholesterol and fatty acids into the cytosol

• the apoB-100 is also degraded to amino acids that are released to the cytosol

Cholesterol Synthesis/Transport Regulation

• cholesterol synthesis is a complex and energy expensive process

• excess cholesterol cannot be catabolized for use as fuel and must be excreted

  • thus advantageous to regulate the biosynthesis of cholesterol to complement dietary intake

• in mammals, cholesterol production is regulated by intracellular cholesterol concentration, supply of ATP and by glucagon and insulin

• the committed step in the pathway to cholesterol is HMG-CoA → mevalonate

Cholesterol Synthesis/Transport Regulation: Short-Term

Minute to minute

• short term regulation of existing HMG-CoA reductase activity is accomplished by reversible covalent alteration

• phosphorylation by the AMP-dependent protein kinase (AMPK), which senses high AMP conc. (low ATP conc.)

• thus when ATP levels drop, the synthesis of cholesterol slows and catabolic pathways for the generation of ATP are stimulated

• hormones also act on HMG-CoA: glucagon stimulates its phosphorylation (inactivation), insulin promotes dephosphorylation (activation, favoring cholesterol synthesis)

• high intracellular concs. of cholesterol reduce transcription of the genes encoding HMG-CoA reductase and promote its proteolytic degradation.

Dysregulation of Cholesterol Metabolism 

• can lead to cardiovascular disease

• excess cholesterol can be removed only by excretion or by conversion to bile salts

• atherosclerosis: the obstruction of blood vessels from the pathological accumulation of cholesterol (plaques)

  • linked to high levels of cholesterol and LDL

• macrophages can’t limit their uptake of sterols and with increasing accumulation of cholesteryl esters and free cholesterol, they become foam cells

  • overtime, the cells undergo apoptosis (cell death)

Plaque from Cholesterol

• initiated when LDL containing partially oxidized fatty acyl groups adheres to and accumulates in the extracelluar matrix of epithelial cells lining arteries

• over time, arteries become progressively occluded as plaques consisting of extracellular matrix material, scar tissue and foam cell remnants gradually grow larger

• within the cholesterol-rich plaques, cholesterol can crystallize

• occasionally, a plaque breaks loose from the site of its formation and is carried thru the blood to a narrowed region of an artery in the brain or heart, causing a stroke or heart attack

  • or cause vascular injury

Statins

• drug class used to treat patients with elevated serum cholesterol

• resembled mevalonate

• competitive inhibitors of HMG-CoA reductase

  • prevents things downstream of mevalonate

• HMG-CoA Reductase step is a good step to target for restricting cholesterol synthesis because it’s the commitment step

Familial Hypercholesterolemia

• characterized by extremely high blood levels of cholesterol due to a defective LDL receptor

• this prevents the normal uptake of LDL by liver and peripheral tissues, resulting in high blood levels of LDL and of the cholesterol it carries

• cholesterol accumulates in foam cells and contributes to the formation of atherosclerotic plaques

  • increased probability of developing atherosclerosis

Reverse Cholesterol Transport

• process by which HDL removes cholesterol from peripheral tissues and carries it to the liver, protecting against atherosclerosis (reducing the potential damage from foam cell buildup)

• cholesterol movement out of cells requires transporters.

  • ApoA-I interacts with ABCA1 transporter in a cholesterol rich cell

  • ABCA1 transports a load of cholesterol from inside the cell to the outer surface of the plasma membrane, where lipid-free/-poor apoA-I picks it up, then transporting it to the liver

  • another transporter, ABCG1 interacts with mature HDL, facilitating the movement of cholesterol out of the cell and into the HDL

  • As HDL matures, it gains more cholesterol (with ABCG1 also helping)

  • the resulting HDL particle then transports it to the liver

Reverse Cholesterol Transport FIGURE

Familial HDL Deficiency and Tangier Disease

Familial HDL Deficiency: HDL levels are very low

Tangier Disease: HDL levels are almost undetectable

• both genetic disorders are the result of mutations in the ABCA1 protein

• ApoA-I in cholesterol-depleted HDL cannot take up cholesterol from cells that lack ABCA1 protein, and apoA-I and cholesterol-poor HDL are rapidly removed from the blood and destroyed 

Lipid Nanoparticles (LNP)

• allow for the transport of negatively charged nucleic acid polymers thru the body to the target tissue (easiest ones currently are blood cells and liver, but this is rapidly advancing)

• different structural lipids have distinct properties and propensities for forming lipid structures, these can be leveraged to carry different types of cargo

• pH dependent ionization of structural lipid head groups enables the uptake of the nucleic acid, as well as release during endocytosis

• we are using these particles in lots of interesting ways to deliver drugs and therapeutics

• flexible and can escape endosomes

Formation and Structure of a Lipoplex

• involves the electrostatic attraction and binding of anionic nucleic acids (like DNA) to cationic lipid vesicles

  • process driven by the + charge of the lipid headgroups and - charged DNA backbone

• the strong attraction causes lipid bilayers to cluster together (aggregate) and sometimes disrupt (rupture) because the nucleic acid inserts b/w them

• the result is a layered (multiamellar) structure, where sheets of lipid alternate with layers of condensed nucleic acid, like a sandwich 

If you want:

• aqueous interior → bilayer

• hydrophobic interior → monolayer

Formation of Lipid Nano Particle

• lipids are dissolved in an organic solvent (eg. ethanol)

• mixed rapidly with an aqueous solution containing nucleic acids (eg. mRNA)

• rapid mixing triggers self-assembly into LNPs

Mechanism of LNP mRNA Deliver

• LNPs carrying mRNA are introduced (eg. by injection)

• cells take up LNPs by endocytosis

• LNPs disrupt the endosome membrane via pH ionization, releasing mRNA into the cytoplasm

• released mRNA is translated into protein by ribosomes

siRNA to target PCSK9

• siRNA is a drug therapy, treating high cholesterol

• reduces LDL cholesterol by inhibiting PCSK9 protein production in the liver

• siRNA enters liver cells often via LNPs

• in the cytoplasm, siRNA incorporates into the RISC complex

• RISC-siRNA complex binds PCSK9 mRNA, causing mRNA degradation

• reduced PCSK9 protein increases LDL receptor availability on hepatocytes, resulting in more LDL cholesterol cleared from the blood

Normal PCSK9 Activity

• PCSK9 directs LDL receptors to lysosomes, where they are degraded and can’t return to the cell surface

• this leads to less cholesterol being cleared form the blood