Biochemistry: Phospholipid and cholesterol metabolism

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2011.09.13.. TÁMOP – 4.1.2-08/2/A/KMR-2009-0006 1 Development of Complex Curricula for Molecular Bionics and Infobionics Programs within a consortial* framework**

Consortium leader

PETER PAZMANY CATHOLIC UNIVERSITY

Consortium members

SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER

The Project has been realised with the support of the European Union and has been co-financed by the European Social Fund ***

**Molekuláris bionika és Infobionika Szakok tananyagának komplex fejlesztése konzorciumi keretben

***A projekt az Európai Unió támogatásával, az Európai Szociális Alap társfinanszírozásával valósul meg.

PETER PAZMANY CATHOLIC UNIVERSITY SEMMELWEIS

UNIVERSITY

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Semmelweis University

ORGANIC AND BIOCHEMISTRY

Lipids forming structural elements of cells

http://semmelweis-egyetem.hu/

(Szerves és biokémia)

(Szerkezetalkotó lipidek)

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Biochemistry: Phospholipid and cholesterol metabolism

Lecture objectives

At the end of the presentation the participant will be able:

1) To define the role of phospholipids and cholesterol in the maintenance of cellular compartments

2) To discuss the metabolism of phospholipids

3) To interpret the regulation of phospholipid synthesis 4) To understand the major sources of cholesterol

5) To discuss the principles of cholesterol transport in the body 6) To interpret the mechanisms for disposal of excess cholesterol

7) To understand the biological role of the regulatory mechanisms of cholesterol metabolism

8) To define alternative routes for cellular uptake of cholesterol

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Biochemistry: Phospholipid and cholesterol metabolism Biosynthesis of phospholipids

common steps of triacylglycerol and phospholipid synthesis 1

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Biochemistry: Phospholipid and cholesterol metabolism Biosynthesis of phospholipids

common steps of triacylglycerol and phospholipid synthesis 2

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Biochemistry: Phospholipid and cholesterol metabolism Biosynthesis of phospholipids

branching point of triacylglycerol and phospholipid synthesis

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Biochemistry: Phospholipid and cholesterol metabolism

Two strategies for the attachment of the phospholipid polar head

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Biochemistry: Phospholipid and cholesterol metabolism

Attachment of the polar group of phosphatidylcholine

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Synthesis of phosphatidylserine by “base exchange”

Biochemistry: Phospholipid and cholesterol metabolism

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Biochemistry: Phospholipid and cholesterol metabolism

Synthesis of phosphatidylethanolamine

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Biochemistry: Phospholipid and cholesterol metabolism

Synthesis of phosphatidylcholine in the liver

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Biochemistry: Phospholipid and cholesterol metabolism

Interconversions of phospholipids

PC: phosphatidylcholine;

PS: phosphatidylserine;

PE: phosphatidylethanolamine

PSS: phosphatidylserine synthase (I and II);

PSD: phosphatidylserine decarboxylase;

ser: serine;

cho: choline;

eth: ethanolamine

p-eth: phosphoethanolamine;

cdp-eth: CDP-ethanolamine

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Biochemistry: Phospholipid and cholesterol metabolism

Remodeling of phospholipids for the formation of lung surfactant

^DEF

The acyltransferase forming the ester bond at C-2 of monoacylglycerol inserts unsaturated fatty acid, which should be exchanged for palmitic acid.

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Biochemistry: Phospholipid and cholesterol metabolism

Regulation of phosphatidylcholine synthesis by membrane composition

Functional domains of CTP-choline cytidylyltransferase (CCCT)

CCCT is active when bound to membrane. Its affinity for Lβ state membranes (PC)

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Biochemistry: Phospholipid and cholesterol metabolism

Regulation of phosphatidylcholine synthesis by hormones

Phosphorylation impairs the membrane binding of CCCT (inactivation), whereas

dephosphorylation favors binding (activation).

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Biochemistry: Phospholipid and cholesterol metabolism

Coordinated regulation of phosphatidylcholine and triacylglycerol synthesis by insulin

In the liver insulin triggers triacylglycerol synthesis through the regulation of

diacylglycerol acyltransferase (DGAT) in a manner similar to the regulation of CCCT. Thus, the major constituents of VLDL are synthesized in parallel.

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Biochemistry: Phospholipid and cholesterol metabolism

Cholesterol

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Biochemistry: Phospholipid and cholesterol metabolism

Overview of cholesterol homeostasis

Cholesterol content of the body (70 kg):

- total: 140 g

- membranes: 120 g - blood plasma: 10 g

Daily loss: 1 g

-0.5 g as bile acids (BA)

-0.5 g as free cholesterol (FC)

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Biochemistry: Phospholipid and cholesterol metabolism Role of cholesterol in the membranes

Cholesterol moderates the fluidity of the phospholipid bilayers.

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Role of cholesterol in the membranes

Biochemistry: Phospholipid and cholesterol metabolism

Cholesterol forms rafts^DEF in the membranes for stable assembly of protein complexes.

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Biochemistry: Phospholipid and cholesterol metabolism

Sources of the C-atoms of cholesterol

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Biochemistry: Phospholipid and cholesterol metabolism

Biosynthesis of cholesterol

18 acetyl-CoA 36 ATP

20 NADPH

21 enzymes

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Biochemistry: Phospholipid and cholesterol metabolism

Scheme of cholesterol biosynthesis

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Biochemistry: Phospholipid and cholesterol metabolism Initial reactions of cholesterol biosynthesis

1. Condensation of three activated acetate subunits

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Biochemistry: Phospholipid and cholesterol metabolism

Rate-limiting step of cholesterol biosynthesis

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Biochemistry: Phospholipid and cholesterol metabolism

2. Formation of activated isoprene subunits

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Biochemistry: Phospholipid and cholesterol metabolism

3. Condensation of activated isoprene subunits

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Biochemistry: Phospholipid and cholesterol metabolism

Elongation of isoprenyl derivatives

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Biochemistry: Phospholipid and cholesterol metabolism

Formation of squalene

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Biochemistry: Phospholipid and cholesterol metabolism

Final stage of cholesterol synthesis

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Biochemistry: Phospholipid and cholesterol metabolism

Formation of the storage form of cholesterol

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Biochemistry: Phospholipid and cholesterol metabolism

Catabolism of cholesterol: synthesis of bile acids

Basic facts about bile acid synthesis

Water solubility increases: 0.3 g/L (cholesterol) Æ 200 g/L (bile acids)

Typical reactions: hydroxylation, oxidation/reduction, β-oxidation-like cleavage of the side chain

Final products: bile acid + propionate

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Biochemistry: Phospholipid and cholesterol metabolism

7a-Hydoxylase:

committed step of bile acid synthesis

- hydroxylation^DEF at C-7 (ER) occurs at various derivatives

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Biochemistry: Phospholipid and cholesterol metabolism

Ring structure modification

- hydroxylation at C-12 (ER) - oxidation/reduction (cytosol)

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Biochemistry: Phospholipid and cholesterol metabolism

Side chain cleavage

- step 11 occurs in mitochondria - steps 12-16

occur in peroxisomes

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Biochemistry: Phospholipid and cholesterol metabolism

Conjugation of bile acids

- step 17 occurs in peroxisomes

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Biochemistry: Phospholipid and cholesterol metabolism

Enterohepatic recycling

^DEF

of bile acids

95 % of the bile acids in the intestine are reabsorbed with the help of the ileal bile

acid transporter (IBAT^DEF, a membrane carrier) and the ileal bile acid binding protein (IBABP, an intracellular carrier).

Part of the primary bile acids are converted to secondary bile acids through

deconjugation and removal of the 7α- hydroxy group.

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Overview of the cholesterol traffic in the body

Biochemistry: Phospholipid and cholesterol metabolism

The daily loss of approximately 1 g cholesterol is compensated by dietary cholesterol (0.5 g) and de novo synthesis (0.5 g).

Although all human cells can produce cholesterol, half of the endogenous synthesis occurs in the liver. The storage and transport form of cholesterol, the cholesteryl esters (CE) is formed by acyl-CoA cholesterol acyl transferase

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Biochemistry: Phospholipid and cholesterol metabolism

LDL and cholesterol transport

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Nobel prize for medicine and physiology 1985

“for their discoveries concerning the regulation of cholesterol metabolism"

Biochemistry: Phospholipid and cholesterol metabolism

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Biochemistry: Phospholipid and cholesterol metabolism

Function of the LDL receptor

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Biochemistry: Phospholipid and cholesterol metabolism Intracellular processing of the LDL-carried cholesterol

NPC1^DEF: Nieman- Pick C1 protein, an intra-

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Biochemistry: Phospholipid and cholesterol metabolism

Control points on the two routes of cellular cholesterol

1. Activity of HMG-CoA reductase

2. Number of LDL receptors

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Biochemistry: Phospholipid and cholesterol metabolism

Common motif in the regulatory effects of cellular cholesterol

The cholesterol sensors are integral membrane proteins with 8 transmembrane

domains. Depending on the cholesterol content of the membrane they adopt different conformations

resulting in changes of enzyme activity or binding affinities.

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Biochemistry: Phospholipid and cholesterol metabolism 1

^st

Level of control: enzyme activity

Increase in the cholesterol content of membranes results in inhibition of HMG-CoA reductase (allosteric effect).

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Biochemistry: Phospholipid and cholesterol metabolism

2

^nd

Level of control: gene expression

SRE: sterol response element SREBP: SRE binding protein SCAP^DEF: SREBP^DEF cleavage activator protein

bHLH: basic helix-loop-helix domain (DNA binding domain) S1P: site 1 protease

S2P: site 2 protease

Binding of bHLH to SRE^DEF activates the transcription of

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Biochemistry: Phospholipid and cholesterol metabolism The sensor of the SREBP pathway: SCAP

High membrane cholesterol increases the binding affinity of SCAP for INSIG^DEF and the ternary INSIG-SCAP-SREBP complex cannot move from the endoplasmic reticulum to the Golgi.

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Biochemistry: Phospholipid and cholesterol metabolism

Summary of the function of the SREBP pathway

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Biochemistry: Phospholipid and cholesterol metabolism

3

^rd

Level of control: life span of the protein

In the presence of lanosterol (a signal of active de novo cholesterol synthesis)

HMG-CoA reductase binds INSIG, which mediates interaction with a ubiquitinating^DEF complex (gp78, Ubc7, VCP). Ubiquitin^DEF is a short peptide tag, which directs proteins to degradation in a multi-protease particle

(proteasome^DEF).

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Biochemistry: Phospholipid and cholesterol metabolism

The fate of excess cellular cholesterol

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Biochemistry: Phospholipid and cholesterol metabolism Structure of the nascent HDL particle

phospholipid

ApoA1

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Biochemistry: Phospholipid and cholesterol metabolism Cholesterol transfer to HDL

Major route

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Biochemistry: Phospholipid and cholesterol metabolism

Structure of the ABCA1

^DEF

transporter

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Biochemistry: Phospholipid and cholesterol metabolism

Spatial model of the ABCA1 transporter

NBD: ATP binding domain TMD: transmembrane domains

P: hydrophobic pore (the arrow in B shows its opening in the hydrophobic region of the cell membrane)

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Biochemistry: Phospholipid and cholesterol metabolism

Function of the ABCA1 transporter in the loading of HDL with cholesterol

binding of HDL through ApoA1

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Biochemistry: Phospholipid and cholesterol metabolism

Cholesteryl ester formation on the surface of HDL

ApoA1 activator

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Biochemistry: Phospholipid and cholesterol metabolism

Reverse cholesterol transport

^DEF

(from peripheral tissues to the liver)

CETP: cholesteryl ester transfer protein (exchanges TG for CE shuttling between TRL and HDL)

TRL: triglyceride (TG) rich lipoprotein (VLDL and chylomicrons)

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Biochemistry: Phospholipid and cholesterol metabolism

Two major routes for cholesterol uptake in the liver

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Biochemistry: Phospholipid and cholesterol metabolism

Overview of the disposal of excess cholesterol

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Biochemistry: Phospholipid and cholesterol metabolism

Control mechanisms in the disposal of excess cholesterol

Metabolite receptors^DEF: transcriptional regulators which form heterodimers when specific ligands (metabolites) bind to them. The heterodimers trigger transcription of target genes.

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Nuclear receptor Ligand Target gene Biological function LXR (liver X

receptor)

oxysterol CYP7A1 ↑ ABCA1 ↑ Idol ↑

Bile acid synthesis↑

Cholesterol efflux ↑ LDL receptor ↓ FXR (farnesoid X

receptor)

bile acids IBAT ↑ IBABP ↑ BSEP ↑ SHP ↑

Bile acid reabsorption ↑ Bile acid reabsorption ↑ Bile acid excretion ↑ gene repression

SHP (small

heterodimer partner)

none CYP7A1 ↓ Bile acid synthesis ↓

Metabolite receptors in the control of cholesterol disposal Biochemistry: Phospholipid and cholesterol metabolism

Idol: induced destruction of LDL-receptor (ubiquitinating protein)

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Coordinated action of SREBP, LXR, FXR in the control of cholesterol homeostasis

Biochemistry: Phospholipid and cholesterol metabolism

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Biochemistry: Phospholipid and cholesterol metabolism

Coordinated regulation of bile acid synthesis and recycling

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Biochemistry: Phospholipid and cholesterol metabolism

Coordinated regulation of peripheral and hepatic disposal

of cholesterol

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Biochemistry: Phospholipid and cholesterol metabolism Functions of scavenger receptors

- endocytosis of various waste products

- high affinity for modified (e.g.

oxidized) LDL

- no regulation by cellular cholesterol

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Biochemistry: Phospholipid and cholesterol metabolism

Structure of scavenger receptors

^DEF

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Messages to take home

1) Phospholipids and cholesterol form the flexible structural boundaries of the cellular compartments

2) Phospholipids are synthesized from phosphatidic acid following two different strategies to attach the polar head

3) Phospholipid synthesis is regulated by the cellular requirements and hormonal effects

4) Cellular cholesterol is derived from two sources (endogenous de novo synthesis and blood-borne cholesterol)

5) LDL carries cholesterol of dietary origin and from the liver

6) Excess cholesterol is loaded into HDL and transported to the liver for disposal in the bile as free cholesterol or bile acids

7) Cholesterol metabolism is under the control of sensor systems that detect the amount of cholesterol in the cellular membranes

8) The points of control of cholesterol metabolism are the uptake, the de novo synthesis and the disposal.

Biochemistry: Phospholipid and cholesterol metabolism

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Comprehension problem 1

Which metabolic pathway uses the reaction catalyzed by phosphatidic acid phosphatase?

1: triglyceride synthesis 2: triglyceride degradation

3: phosphatidyl choline synthesis 4: phosphatidyl choline degradation

Biochemistry: Phospholipid and cholesterol metabolism

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Comprehension problem 2

Which statement is true regarding the phosphocholine citydilyl transferase?

A: It is active in phosphorylated state.

B: Its membrane-bound form is active.

C: Its free cytosolic form is active.

D: Phosphatidyl choline activates it.

E: Its higher activity results in inhibition of phosphatidyl choline synthesis.

Biochemistry: Phospholipid and cholesterol metabolism

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Comprehension problem 3

Which statement is true regarding the biological functions of cholesterol?

A: Most of body cholesterol is found in the cell membranes.

B: Most of body cholesterol serves the synthesis of hormones.

C: Most of body cholesterol is found in the blood.

D: Most of body cholesterol serves the generation of energy.

Biochemistry: Phospholipid and cholesterol metabolism

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Comprehension problem 4

Which metabolic pathway uses the reaction catalyzed by 3- hydroxy 3-methyl glutaryl-CoA reductase?

A: glycolysis

B: citric acid cycle

C: oxidation of fatty acids D: synthesis of fatty acids E: synthesis of cholesterol

Biochemistry: Phospholipid and cholesterol metabolism

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Comprehension problem 5

Where is the 3-hydroxy 3-methyl glutaryl-CoA reductase localized within the cell?

A: in the free cytosol

B: in the membrane of the endoplasmic reticulum C: in the matrix of mitochondria

D: in the inner membrane of mitochondria

Biochemistry: Phospholipid and cholesterol metabolism

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Comprehension problem 6

Which statement is true regarding the LDL receptor?

1: It recognizes apo B100 protein.

2. It binds oxidized LDL with higher affinity than the native LDL.

3. It is found in extrahepatic tissues.

4. It is found in liver.

5. It takes up HDL too.

A: 1,2 B: 1,2,3 C: 1,3,4 D: 2,3 E: 4,5

Biochemistry: Phospholipid and cholesterol metabolism

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Comprehension problem 7

Which factor exerts the most direct effect on the increase of LDL receptors in the plasma membrane of liver cells?

A: Increase in the blood cholesterol level.

B: Increase in bile acid synthesis.

C: Consumption of cholesterol-rich meal.

D: Increase in the blood LDL level.

Biochemistry: Phospholipid and cholesterol metabolism

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Comprehension problem 8

Which statements are true regarding the synthesis of cholesterol in mammalians?

1: Cholesterol is synthesized only in the liver.

2: The rate of cholesterol synthesis is constant.

3: Cholesterol inhibits the activity of 3-hydroxy 3-methyl glutaryl- CoA reductase

4: Cholesterol inhibits the transcription of 3-hydroxy 3-methyl glutaryl-CoA reductase

5: Cholesterol inhibits the degradation of 3-hydroxy 3-methyl glutaryl-CoA reductase

A: 1,2 B: 3,4 C: 4,5 D: 3,5 E: 1,3

Biochemistry: Phospholipid and cholesterol metabolism

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Comprehension problem 9

Which statements are true regarding the biological role of HDL?

A: Its major function is to carry cholesterol from the tissues to the liver.

B: Its major function is to carry cholesterol from the liver to the tissues.

C: Its major function is to carry cholesterol from the intestine to the liver.

D: Its major function is to carry cholesterol from the intestine to the tissues.

Biochemistry: Phospholipid and cholesterol metabolism

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Comprehension problem 10

Which statements are true regarding the lecithin cholesterol acyl transferase?

1: It is one of the enzymes of cholesterol synthesis.

2: It forms cholesteryl esters.

3: It is localized in the membrane of the endoplasmic reticulum.

4: Apo AI is its activator.

5: It is active in phosphorylated state.

A: 1,2 B: 2,3 C: 4,5 D: 2,4 E: 3,5

Biochemistry: Phospholipid and cholesterol metabolism

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Recommended literature

Orvosi Biokémia (Ed. Ádám Veronika): pp.168-175,193-200 Orvosi patobiokémia (Ed. Mandl, Machovich): pp. 441-454

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Biochemistry: Phospholipid and cholesterol metabolism

Answers to comprehension problems:

1. A; 2. B; 3. A; 4. E; 5. B; 6. C; 7. B; 8. B; 9. A; 10. D