session 4 cholesterol - lsu health new orleansbiosynthesis of cholesterol in order to ﬁnd a drug...

cholesterol structure

• most plasma cholesterol is in the esterified form (not found in cells or membranes)

• cholesterol functions in all membranes (drives formation of lipid microdomains)

• cholesterol is the precursor for steroid hormones

• note 4 fused rings, single dbl bond, single hydroxyl, acyl chain at C17

Cholesterol FAQs

• Cholesterol is the second most abundant fraction in blood besides glucose.

• All the carbon atoms in cholesterol come from Acetyl-CoA.

• Energy for synthesis comes from hydrolysis of thioester bonds of acetyl CoA and ATP hydrolysis.

• Synthesis occurs in the cytoplasm, with key enzymes found in the membrane of the ER.

Cholesterol promotes the

“liquid-ordered” phase of

membranes• cholesterol has limited

flexibility and is amphipathic.

• it stiffens the membrane and regulates permeability.

• it can interact with and affect the structure of integral membrane proteins (e.g. lipid rafts)

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Cholesterol is less soluble in (artificial) membranes high in

unsaturated acyl chains

S.R. Wassall, W. Stillwell / Chemistry and Physics of Lipids 153 (2008) 57–63

Journal of Lipid Research, Vol. 44, 655-667, April 2003

compound as cholesterol must be considered a great ac-complishment. It is fortunate that two imaginative youngbiochemists happened to be students in Munich at a timewhen the famous steroids chemists Wieland and Windaus aswell as other outstanding biochemists such as Hans Fischerand Rudolph Willstätter were educators at the University.After 1934 and the rise of the Nazis, it became impossiblefor Bloch and Lynen to work together because of Bloch’sJewish ancestry. However, it was fortunate that their workcould be resumed after the end of the war in 1945. Thefull mechanism of sterol biosynthesis was gradually solvedstep by step by Bloch and his group in USA and by Lynenand his group in West Germany. The main interest of thesetwo groups was to elucidate the rate-limiting step in thebiosynthesis of cholesterol in order to find a drug that wouldprevent hypercholesterolaemia. In his Nobel lecture, Blochproposed that the reduction of hydroxymethylglutaryl CoA

to mevalonic acid may be the homeostatic control mecha-nism in the biosynthesis of cholesterol, a hypothesis, whichis now known to be correct.

References

1. Ružička L. The isoprene rule and the biogenesis of terpeniccompounds. Experientia 1953; 9: 35–7.

2. Robinson R. J Chem Soc Ind Lond 1934; 53: 1062–3.3. Channon HJ. The biological significance of the unsaponifiable

matter of oils: experiments with the unsaturated hydrocarbon,squalene (spinacene). Biochem J 1926; 20: 400–8.

4. Lynen F. Der Weg von der. ‘‘Aktivierten Essigsäuse’’ zu denterpenen und den fettsäuren. Les Prix Nobel. Stockholm:Norstedt & Sons, 1965: 205–45.

5. Bloch K. The biological synthesis of cholesterol. Les Prix Nobel.Stockholm: Norstedt & Son, 1965: 179–203.

6. Rittenberg D, Schoenheimer R. Deuterium as an indicator inthe study of intermediary metabolism. XI. Further studies onthe biological uptake of deuterium into organic substances,with special reference to fat and cholesterol formation. J BiolChem 1937; 121: 235–53.

7. Sonderhoff R, Thomas H. Liebigs Ann Chem 1937; 247: 104.8. Lynen F. Liebigs Ann Chem 1939; 539: 1.9. Lynen F. Liebigs Ann Chem 1942; 552: 270.10. Lynen F, Reichert U, Rueff L. Biological degradation of acetic

acid. VI. Isolation and chemical nature of activated acetic acid.Liebigs Ann Chem 1951; 574: 1–32.

11. Lynen F. The Harvey lectures. New York: Academic Press Inc.,1954; 48: 210.

12. Bloch K, Rittenberg DJ. An estimation of acetic acid formationin the rat. J Biol Chem 1945; 159: 45–58.

13. Ottke RC, Tatum EL, Zabin I, Bloch K. Isotopic acetate andisovalerate in the synthesis of ergosterol byNeurospora. J BiolChem 1951; 189: 429–33.

Figure 6 Biosynthesis of terpenes. From Lynen (4).

Figure 7 Biosynthesis of fatty acids according to a hypothetic structure of amulti-enzyme complex. From Lynen (4).

1226 ª2009 The Author/Journal Compilation ª2009 Foundation Acta Pædiatrica/Acta Pædiatrica 2009 98, pp. 1223–1227

1964 Nobel Prize for discovery of biosynthesis of cholesterol Zetterström

Lynen F. DerWegvonder. ‘‘AktiviertenEssigsa ̈use’’ zuden terpenen und den fettsa ̈ uren. Les Prix Nobel. Stockholm: Norstedt & Sons, 1965: 205–45.

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Cholesterol synthesis initially follows that of ketone bodies

3 cytoplasmic acetyl CoA molecules are sequentially condensed to form HMG CoA (6 carbons)

mitochondrialHMG-CoA Synthase

cytoplasmicHMG-CoA Synthase

Cholesterol biosynthesis includes over 30 enzymatic steps that occur in the cytoplasm and outer membrane of the ER

Mito_HMG-CoA

Synthase

Cyto_HMG-CoA

Synthase

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Biochemistry, Vol. 39, No. 8, 2000

Beginning with squalene, sterol-carrier-protein keeps the remaining

intermediates soluble

Squalene epoxide is converted to lanosterol in concerted fashion

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The committed step of de novo cholesterol biosynthesis is

catalyzed by HMG CoA reductase

• The reduction of HMG CoA by HMG CoA reductase results in the oxidation of two NADPH and results in mevalonate.

• HMG CoA reductase is a membrane protein of the ER: catalytic domain projects into the cytoplasm.

• Target of statin drugs

The committed step of de novo cholesterol biosynthesis is

catalyzed by HMG CoA reductase

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HMG-CoA-reductase forms a homotetramer and utilizes NADPH

and CoA cofactors

Cell Research (2008) 18:609–621

HMG-CoA-r has a cytoplasmic catalytic domain and a membrane

sterol sensing domain

HMG-CoA-reductase utilizes two NADPH for each HMG-CoA

1. NADPH reduces HMG carbon.2. His donates a proton and cleaves CoA from HMG.3. Second NADPH protonates HMG carbonyl to form

Mevalonate.

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• Inhibitory phosphorylation of HMG-CoA-r at SER872 (e.g. by AMP-dependent protein kinase) and regulated by hormones.

Fine control of cholesterol homeostasis

Coarse control of cholesterol homeostasis

1. Regulation of the transcription of HMG-CoA-r (and others) by SCAP/SREBP (sterol-response-element-binding-protein)

Sterol-accelerated degradation of HMG CoA reductase612npg

Cell Research | Vol 18 No 6 | June 2008

For further insight into the pathway for degradation of HMG2p in yeast, readers are referred to several excellent reviews [20, 22, 23].

Insigs, polytopic proteins of the ER that mediate sterol-accelerated degradation of HMG CoA reduc-tase

Crucial insights into the mechanism for sterol-acceler-ated degradation of reductase have emerged from com-parisons made between reductase and Scap (the SREBP cleavage-activating protein). Similar to reductase, Scap contains two distinct domains: a hydrophobic N-terminal domain that spans the membrane eight times and a hydro-philic C-terminal domain that projects into the cytosol [24]. The C-terminal domain of Scap mediates a constitutive association with SREBPs; this interaction is required for Scap-dependent translocation of SREBPs from the ER to Golgi in sterol-deprived cells (Figure 3). Upon arrival in the Golgi, SREBPs encounter a pair of proteases (designated site-1 and site-2 proteases) that act successively to release soluble fragments from the membrane into the cytosol [25-29]. These processed forms of SREBPs then migrate from the cytosol into the nucleus and stimulate target gene expression, which results in increased synthesis and uptake of sterols [6]. The subsequent accumulation of sterols in ER membranes prevents proteolytic activation of SREBPs by blocking exit of Scap-SREBP complexes from the ER; transcription of SREBP target genes declines and choles-terol synthesis and uptake are suppressed. Inhibition of ER to Golgi transport of SREBPs results from sterol-induced binding of Scap to ER retention proteins called Insig-1 and Insig-2 [30, 31]. Insig binding occludes a cytosolic binding site in Scap recognized by COPII proteins, which incorporate cargo molecules into vesicles that deliver ER-derived proteins to the Golgi [32]. Scap-Insig binding is mediated by a segment of Scap’s membrane domain that includes transmembrane helices 2-6 [25, 30]. A similar stretch of transmembrane helices is found in at least four other polytopic membrane proteins (including the Niemann Pick C1 protein, Patched, Dispatched, and reductase), all of which have been postulated to interact with sterols. Thus, the region has become known as the sterol-sensing domain [33]. The importance of the sterol-sensing domain in regu-

within the region disrupt Insig binding, thereby relieving mutant Scap-SREBP complexes from sterol-mediated ER retention [30, 31, 34-36].

The recognition of sequence resemblances between the sterol-sensing domains of Scap and reductase stimulated an appraisal of a role for Insigs in degradation of reduc-

tase. This effort led to the following observations, which considered together divulge the action of at least one of the Insig proteins in sterol-accelerated degradation of reductase. First, when overexpressed by transfection in Chinese hamster ovary (CHO) cells, reductase cannot be degraded when the cells are treated with sterols [37]. Co-expression of Insig-1 restores sterol-accelerated degrada-tion of reductase, suggesting the saturation of endogenous Insigs by the overexpressed reductase. Second, reduction of both Insig-1 and Insig-2 by RNA interference (RNAi) abolishes sterol-accelerated degradation of endogenous reductase [38]. Third, mutant CHO cells lacking both Insigs are impervious to sterol-stimulated degradation of reductase as well as sterol-mediated inhibition of SREBP processing [39].

Degradation of reductase coincides with sterol-induced binding of its membrane domain to Insigs [37], an action that requires a tetrapeptide sequence, YIYF, located in the second transmembrane segment of reductase (see Figure 2B) [38]. A mutant form of reductase in which the YIYF sequence is mutated to alanine residues no longer binds to Insigs and the enzyme is not subject to rapid degrada-tion. The YIYF sequence is also present in the second

Figure 3 Model for sterol-regulated Scap-SREBP pathway. SCAP

cells, Scap facilitates export of SREBPs from the ER to the Golgi apparatus, where two proteases, Site-1 protease (S1P) and Site-2

sterol response element (SRE) in the enhancer/promoter region of

target genes declines.

Lumen

ERReg

SCAP SREBP Nucleus

SRE

Sterols

S1P S2PLumen

Golgi

Coarse control of cholesterol homeostasis

2. INSIG protein senses sterol concentration and regulates ubiquitin-mediated protein degradation of HMG-CoA-Reductase

Sterol-accelerated degradation of HMG CoA reductase618npg

Cell Research | Vol 18 No 6 | June 2008

studies that directly focus on reductase degradation are required in order to determine the contribution of protein stability to overall regulation of reductase in mice in vivo under various physiologic conditions, such as hypoxia.

The significance of Insig-mediated regulation of reductase in maintenance of cholesterol homeostasis is highlighted by the effectiveness of reductase inhibition in lowering plasma LDL-cholesterol in humans [80]. How-ever, the inhibition of reductase disrupts normal feedback inhibition of the enzyme, and animals respond by develop-ing a compensatory increase in reductase levels in the liver [81, 82]. Remarkably, a similar response has been observed in livers of statin-treated humans as well [83]. Knowledge of the mechanisms for this compensatory increase, par-ticularly the contribution of degradation, may facilitate development of novel drugs that improve the effectiveness of statins, or in some cases provide alternative treatments. Such a drug would be modeled after lanosterol and 24,25-dihydrolanosterol, which selectively stimulate reductase degradation without affecting the Scap-SREBP pathway or LDL-receptor activity. In addition, elucidation of the

underlying mechanisms for sterol-accelerated, ERAD of reductase may have implications for degradation of other

transmembrane conductance regulator (CFTR). Thus, further excitement will undoubtedly ensue once questions posed in this review begin to become clear.

Acknowledgments

Work in the DeBose-Boyd laboratory is supported by grants from the National Institutes of Health (HL20948), the Perot Family Foundation, the American Heart Associa-tion (0540128N), and the W.M. Keck Foundation.

References

1 Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature 1990; 343:425-430.

2 Brown MS, Goldstein JL. Multivalent feedback regulation of HMG CoA reductase, a control mechanism coordinating isopren-oid synthesis and cell growth. J Lipid Res 1980; 21:505-517.

3 Endo A, Kuroda M, Tanzawa K. Competitive inhibition of 3-

Figure 5 Pathway for sterol-accelerated degradation of HMG CoA reductase. Accumulation of 25-hydroxycholesterol, lanosterol,

-

CytosolER

Lumen

gp78HMG CoAreductase

Sterols

gp78

Degradation

gp78

Proteasome

Extraction

Geranylgeraniol

gp78

OH

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Summary of cholesterol homeostasis

Transcription HMG-CoA-r Degradation

HMG-CoA-r

P

[sterol] compensatory regulationphosphorylation

INSIG-mediated degradationdephosphorylation

SREB-mediated transcription

SCIENCE VOL 292 11 MAY 2001

Statins are competitive inhibitors of HMG-CoA-reductase

Statin binding blocks HMG-CoA binding site

HMG

CoA

NADP+

monomer-1monomer-2

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Statin binding blocks the Coenzyme-A binding pocket

• Reduces amount of circulating cholesterol

• not compensated by increased de novo synthesis of cholesterol

• Reduces amount of circulating cholesterol (inhibits absorption)

• compensated for by increased de novo synthesis

Plant sterols Ezetimibe

Plant sterol margarines (Benecol, sitosterol) act through a different mechanism than Ezetimibe

• Recent evidence points to enhanced degradation of HMG-CoA-r.

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Cholesterol is modified by cytochrome p450 enzymes to form steroid hormones

• Unlike fatty acids, sterols cannot be used as an energy source

• The sterol ring nucleus is eliminated from the body by conversion to bile acids and bile salts.

Degradation of Cholesterol

Degradation of Cholesterol

• The theme is for cholesterol to be converted to a relatively soluble amphipathic molecule.

• As a bonus, these molecules are used as emulsifying agents during digestion.

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Lipoprotein particles transport lipids.

Each lipoprotein recruits a different set of lipid transfer proteins

LDL delivers cholesterol directly to the interior of cells

HDL are secreted ‘empty’ and scavenge cholesterol via Apo-A associated LCAT (lecithin-

cholesteral acyltransferase) activity

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LCAT helps to sequester cholesterol (amphipathic) by esterifying it to a (hydrophobic) fatty acid

Lecithin-cholesteryl-acyltransferase (LCAT) is recruited to HDL via Apolipoprotein A1(Apo A)

apolipoprotein A1 homodimer forms an α-helical belt to capture lipids

Biophysical Journal, 88:548-556, 2005

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Friday, October 15, 2010

session 4 cholesterol - lsu health new orleansbiosynthesis of cholesterol in order to ﬁnd a drug...

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