BiochemistrySixth Edition

Chapter 26The Biosynthesis of Membrane

Lipids and Steroids

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

Cholesterol is made from Acetyl CoA

27 C atoms of cholesterol are derived from AcetylCoA in a 3 stage synthetic process.

Stage 1: Synthesis of isopentenyl pyroP, an activated isoprene unit that is the key building block of cholesterol

Stage 2: Condensation of 6 molecules of isopentenyl pyroP to form squalene

Stage 3: Squalene cyclizes and the tetracyclic product is subsequently converted into cholesterol

The synthesis of mevalonate, which is activated as isopentenyl pyrophosphate, initiates the synthesis of cholesterol

Stage 1: Making isopentenyl pyrophosphate from acetylCoA

The synthesis of mevalonate is the committed step in cholesterol formation

• Enzyme: HMG-CoA reductase• Mevalonate is converted into 3-isopentenyl pyroP in 3

consecutive reactions requiring ATP.

Squalene (C30) is synthesized from 6 molecules of isopentenyl pyroP (C5)

Stage 2: Squalene is synthesized from isopentenyl pyrophosphate

Reaction series: C5 C10 C15 C30

1. Isopentenyl pyrophosphate isomerization2. Dimethylallyl pyroP and isopentenyl pyroP condenses to form geranyl

pyroP.3. The same kind of attack takes place again

– Geranyl pyroP is converted into an allylic carbonium ion and attacked by isopentenyl pyroP, resulting in C15 farsenyl pyroP

– Geranyl transferase catalyzes each of these reactions. The last step in the synthesis of squalene is a reductive tail-to-

tail condensation of two molecules of farnesyl pyroP catalyzed by the ER enzyme squalene synthase

Squalene cyclizes to form cholesterol

Stage 3: The final stage of cholesterol biosynthesis starts with the cyclization of squalene

• Squalene is activated by conversion into squalene epoxide.• Squalene epoxide is then cyclized to lanosterol by

oxidosqualene cyclase.• Lanosterol is converted to cholesterol in a multistep process by

– removal of the 3 methyl groups– the reduction of 1 double bond by NADPH– the migration of the other double bond.

Cholesterol Synthesis Broken down into Five Steps

1. Synthesis of mevalonic acid from Acetyl-CoA • Conversion of HMG-CoA into mevalonate by HMG-CoA

reductase is the rate limiting step!

2. Formation of isoprenoid units from mevalonic acid.3. Six activated isoprene units undergo condensation

to form Squalene.4. Squalene is converted into Lanosterol (in animals).5. Cholesterol is formed from lanosterol after several

further steps that includes the loss of three methyl groups.

Regulation of cholesterol biosynthesis

In mammals, it is regulated by intracellular cholesterol concentration and by the hormones glucagon and insulin.• The rate limiting step is the conversion of HMG-CoA into mevalonate;

the enzyme is HMG-CoA reductase.– It is allosterically inhibited by cholesterol and mevalonate.

• Insulin favors cholesterol synthesis.• Glucagon inhibits cholesterol synthesis.

High intracellular cholesterol – activates ACAT (acyl CoA-cholesterol acyl transferase), increasing

esterification of cholesterol for storage.– causes reduced production of the LDL receptor, slowing the uptake of

cholesterol from the blood.

HMG-CoA reductase

Regulation of cholesterol biosynthesis

Translational control of cholesterol

Feedback regulation is primarily mediated by the amount of HMG-CoA reductase activity. This enzyme is controlled in multiple ways.

1. The rate of synthesis of reductase mRNA is controlled by the sterol regulatory element binding protein (SREBP). • SREBP is a transcription factor and binds to a specific site on DNA called

SRE (sterol regulatory element). • SREBP is attached to ER or nuclear membrane when cholesterol level is

normal.• When cholesterol is low, SRBEP is released by proteolytic cleavage and

moves to DNA and binds to SRE to start making HMG-CoA reductase.• When cholesterol is high, the proteolytic release of the SREBP is blocked,

and the SREBP in the nucleus is rapidly degraded.

Regulation continue

2. Nonsterol metabolites derived from mevalonate inhibit translation of reductase mRNA

3. Degradation of reductase carefully controlled.

4. Phosphorylation decreases the activity of the reductase. This enzyme, like Acetyl CoA carboxylase, is turned off by an AMP-activated protein kinase. Therefore, cholesterol synthesis stops when the ATP level is low.

Lipoproteins

Cholesterol is carried in the blood plasma by plasma lipoproteins.

They are molecular aggregates of specific carrier proteins called “apolipoproteins”

Different combinations of lipids and proteins produce particles of different densities, ranging from VLDL to HDL which may be separated by ultracentrifugation and visualized by EM.

At least 9 different apoproteins are found.

The protein component of lipoprotein has a specific function determined by its point of synthesis, lipid composition, and apolipoprotein content.

Composition of serum lipoproteins

CM85% TAG2% protein8% phospholipid5% Cholesterol-esters

VLDL60% TAG10% protein15% phospholipid15% Cholesterol

LDL8% TAG22% protein20% phospholipid50% Cholesterol

HDL3% TAG50% protein30% phospholipid17% Cholesterol

The blood levels of certain lipoproteins can serve diagnostic purposes

Bad cholesterol (LDL) Good cholesterol (HDL)

– HDL 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.

– HDL protects us from heart attacks. Why??

For a healthy person LDL/HDL ratio is 3.5

Serum lipoproteins

They are complexes of lipids and specific proteins called “apoproteins”

Classified according to increasing density– CM (chylomicron)– VLDL (very low density)– LDL (low density), IDL (intermediary density)– HDL (high density)

They function to keep lipids soluble as they transport them in the serum.

LDL plays a central role in cholesterol metabolism

Cells outside the liver and intestine obtain cholesterol from the plasma

Specifically, their primary source of cholesterol is the LDL

The process of LDL uptake is called receptor mediated endocytosis.

Uptake of cholesterol by receptor mediated endocytosis

LDL receptor

Domain 1 – Amino-terminal region• Cys rich sequence of about 40 residues that is

repeated , with some variation, 7times• Site of LDL binding• Ca also binds here.

Domain 4• Very rich in Ser and Thr• Contains O-linked sugars that may function as

struts to keep the receptor extended from the membrane so that the LDL-binding domain is accessible to LCL

Domain 2• Homologous to EGF• Repeated 3 times , and in between the second

and third repeat is the third domain

Domain 5• 22 hydrophobic residues• Spans the plasma membrane

Domain 3• Similar to the blades of the transducin β

subunit• Exposure to the low-pH environment of the

lysosomes causes the propeller-like structures to interact with the LDL-binding domain. This interaction displaces the LDL, which is then digested by the lysosome.

Domain 6• Has 50 residues• Emerges on the cytoplasmic side of the

membrane• Controls the interaction of the receptor with

coated pits and participates in endocytosis

115 kd protein 6 domains

LDL receptor

The absence of the LDH receptor leads to hypercholesteremia

In familial hypercholesterolemia:– The total concentration of cholesterol and LDL in the blood plasma is

markedly elevated in this genetic disorder.– The result of a mutation at a single autosomal locus.

The desirable cholesterol level is <200 mg/dL. But the levels for those with the genetic disorder are:– 680 mg/dL in homozygotes– 300 mg/dL in heterozygotes

Cholesterol is deposited in various tissues because of the increased concentration of LDL cholesterol in plasma.– Xanthomas, nodules of cholesterol, are prominent in the skin and tendons.– LDL can be oxidized to form oxLDL that can be taken up by immune system

cells, called macrophages. The engorged macrophages form foam cells that are trapped in the walls of the blood vessels and contribute to the formation of atherosclerotic plaques.

atherosclerosis

Foam cells are trapped in the blood vessel wall making it thick, contributing to the formation of atherosclerosis plaques.

Most homozygotes die of CAD in childhood.

Why HDL is good cholesterol?– Possibly, the HDL-associated protein destroys the oxLDL.

Molecular defect: NO LDL RECEPTOR...

Atherosclerotic plaque

The clinical management of cholesterol levels can be understood at a biochemical level

Homozygous familial hypercholesterolemia can be treated only by a liver transplant!

With heterozygous familial hypercholesterolemia, the goal is to increase the gene expression so that more LDL receptors are made.– If the cells are deprived of cholesterol, mRNA production for the

LDL receptor would increase.– How do we deprive cells of cholesterol?

1. By inhibiting intestinal reabsorption of bile salts. Bile salts are cholesterol derivatives that increase the absorption of dietary cholesterol and dietary fats (achieved by positively charged polymers).

2. Blocking de novo synthesis of cholesterol with statins, like lovastatin. These drugs are competitive inhibitors of HMG-CoA reductase.

Lovastatin

Important derivatives of cholesterol

Bile salts• Highly effective detergents• Made in the liver• Stored in the gallbladder

Building block for 5 major classes of steroid hormones• Progesterone• Androgen• Estrogen• Glucocorticoids• Mineralocorticoids

Vit D

Nomenclature of steroid hormones

The rings in steroids are denoted by the letters A, B, C and D

Cholesterol has 2 angular methyl groups– The C-19 methyl group is attached to C-10.– The C-18 methyl group is attached to C-13.

C18 and C19 methyl groups lie above the plane containing the 4 rings– A substituent above the plane is termed b oriented.– A substituent below the plane is termed a oriented.

How do we make steroid hormones? Cholesterol has 27 carbon whereas steroid hormones contain 21 or

fewer carbon. Need to remove 6C unit from cholesterol to form pregnenolone

– Cholesterol side chain is hydroxylated at C-20 and C-22 – The bond between these carbon atoms is subsequently cleaved by

desmolase

– Rate limiting step

Pregnenolone is next oxidized and then isomerized to progesterone.

Progesterone is further modified by a series of hydroxylation reactions to other steroid hormones. These enzymes are mixed-function oxidases requiring NADPH and oxygen.

desmolasecholesterol pregnenolone

Steroid hormones All steroid hormones are derived from cholesterol.

– Mineralocorticoids• Control mineral absorption • Example: aldosterone

– Glucocorticoids• Regulate gluconeogenesis and decrease inflammatory response• Example: cortizon

– Androgens• Sex hormones

– These hormones are effective at very low concentrations and are, therefore, synthesized in relatively small quantities.

Adrenal cortex has three histological zones that are exclusive steroid producers. – Together, the zones can produce all classes of steroid hormones. – Each zone has cells that make different steroid hormones.

Steroid hormone synthesis

Cholesterol (C27)

pregnenolone

desmolase

3-b-OHsteroiddehydrogenase

DeficiencyNo gluco, mineralo or sex hEarly deathprogesteron

17-a-OHprogesteron

testosteron

estradiol

11-deoxycortisol

cortisol

21-a-OHlase

1-deoxycorticosterone

Aldosteron

11-b-OHlase

17-a-OHlaseCommon form of CAHAldosteron and cortisol sex hormones

Cortisol, aldosteron sex hormones

Congenital AdrenalHyperplasia (CAH)

IMPORTANT A defect in the activity or amount of an enzyme in

steroid hormone synthesis pathway can lead to BOTH– a deficiency in the synthesis of hormones beyond the

affected step AND– an excess in the hormones or metabolites before that

step. Therefore severe metabolic imbalances may

occur.

Secretion of adrenal steroid hormones

Adrenal cortical hormone secretion is controlled by the hypothalamus, to which the pituitary gland is attached.

When the body is stressed, released factors travel to the anterior lobe that produce and secrete ACTH (adrenocorticotropic hormone).

ACTH is often called “stress hormone”• Stimulates adrenal cortex to make mineralocorticoids and

glucocorticoids (collectively called corticosteroids)

Corticosteroids bind to their specific receptors and do their action

Actions of corticosteroids Aldosteron

• Stimulates reabsorption of Na and excretion of K Cortisol

• Increased gluconeogenesis• Anti-inflammatory action

Estrogens• Controls menstrual cycle• Promotes female secondary sex characteristics

Progesteron• Maturation of fertilized ovum

Testosteron• Stimulates spermatogenesis• Promotes male secondary sex characteristics

Vit D is derived from cholesterol by the ring-splitting activity of light

Cholesterol is also Vit D precursor. Vit D plays an important role in Ca and phosphorous

metabolism 7-dehydrocholesterol (provitD3) is photolyzed by the UV

light of sunlight to preVit D3. This spontaneously isomerized to D3, D3 is then converted to Calcitriol, the active hormone in the liver and kidney.

Vit D deficiency in children causes rickets Recommended daily intake of Vit D is 400 iu. In adults Vit D deficiency causes osteomalacia (softening

of bones)

BiochemistrySeventh Edition

CHAPTER 26The Biosynthesis of Membrane

Lipids and Steroids

Copyright © 2012 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

Q.

Mice were divided into four groups, two of which were fed a normal diet and two of which were fed a cholesterol-rich diet. HMG-CoA reductase mRNA and protein from liver were then isolatedAnd quantified. Graph A shows the results of the mRNA isolation.a. What is the effect of cholesterol feeding on the amount of HMG-CoA reductase mRNA?b. What is the purpose of also isolating the mRNA for the protein actin?

HMG-CoA reductase protein was isolated by precipitation with a monoclonal antibody to HMG-CoA reductase. The amount of HMG-CoA protein in each group is shown in graph B.

c. What is the effect of the cholesterol diet on the amount of HMG-CoA reductase protein?d. Why is this result surprising in light of the results in graph A?e. Suggest possible explanations for the results in graph B.