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Chapter 18: Metabolism and Movement of Lipids
Outline:1. Phospholipids and sphingolipids:
synthesis and intracellular movement2. Cholesterol: a multifunctional
membrane lipid3. Lipid movement into and out of cells4. Feedback regulation of cellular lipid
metabolism5. The cell biology of atherosclerosis,
heart attacks, and strokes
Cell use lipids for:1. Storing energy2. Building membranes3. Signaling within and between cells4. Sensing the environment5. Covalently modifying proteins6. Forming specialized permeability barriers (e.g., in skin)7. Protecting cells from highly reactive chemicals
Fatty acids – ATP(mitochodria), triglycerides, phospholipids, plasmalogens, sphingolipidsCholesterol – membrane components, precursors for steroid hormones and other biologically active lipids, fat-soluble vitamins (vit A - light detection, vit D - calcium metabolism, vit E - antioxidants, vit K – blood coagulation)Lipid biogenesis, lipid transport, cholesterol metabolism and atherosclerosis
Fig 18-1 Overview of synthesis of major membrane lipids and their movement into and out of cells
Proteolysis of an integral membrane protein precursor
Nuclear sterol regulatory element-bind proteins
18.1 Phospholipids and sphingolipids: synthesis and intracellular movement
Fatty acids are precursors for phospholipids, sphingolipids, and other membrane components:Fatty acid synthesis: – regulation of membrane synthesis
saturated fatty acids, unsaturated fatty acids, C14, C16, C18(stearate, oleate, linoleate, linolenate) C20(arachidonate)
C14, C16 – synthesized in cytosol from acetyl CoA by acetyl-CoA carboxylase & fatty acid synthetase
Palmitoyl CoA – elongated to 18-24 in ER or mitochondriaDe saturase – located in ER, introduce double bonds, kink -
unsaturated fatty acids – more fluidEssential polyunsaturated FAs – linoleic acid, linolenic acids (亞麻油酸)
Cell division need more membraneNew membrane not old membrane extension, but new synthesis
2
Fig 18-2 Chemical structures of FAs and some of their derivatives
16C, saturated FA
20C, polyunsaturated FA
TG - 3 FFA (free fatty acid) + 1 glycerol
Fatty acid are precursor for phospholipids an other membrane components
Arachidonate, platelet activating factor….
TG - 3 FFA (free fatty acid) + 1 glycerol
Fatty acids are precursor for phospholipids and other membrane component
花生四烯酸
Unesterified FAs move within cells bound to small cytosolic proteins
Fig 18-2 Binding of a FA to the hydrophobic pocket of a fatty acid-binding protein (FABP)
FABP ( ): FA glucosein cardiac muscle
Fatty acid-binding proteins: FABP
Fatty acid
OFABP levels are high in active muscles using fatty acids for energyIn adipocytes + fatty acid stored as TG
- fatty acid use by other cellsIn liver 5% of all cytosolic proteins
Interacts non-covalently with hydorphobic amino acid
FABP expression is regulated for release or up-take FA
3
Incorporation of FAs into membrane lipids takes place on organelle membranes
Fig 18-4 Phospholipid synthesis
Fatty acid didn’t directly pass membraneAcetyl CoA -------- saturated fatty acid
acetyl-CoA carboxylasefatty acid synthase
Annexin V ; binds to anionic phospholipidslong exposure of exoplasmic face of plasma memb.signal for scavenger cells to remove dying cells
Flippases move phospholipids from one membrane leaflet to the opposite leaflet
asymmetric distribution of phospholipids
senescence or apoptosis – disturb the asymmetric distribution
Phosphatidylserine (PS) and phosphatidylethanolamine: cytosolic leaflet
exposure of these anionic phospholipids on the exoplasmic face – signal
for scavenger cells to remove and destroy
Annexin V – a protein that specifically binds to PS phospholipids
fluorescently labeled annexin V– to detect apoptotic cells
flippase: ABC superfamily of small molecule pumps
Fig 18-5 In vitro fluorescence quenching assay can detect phospholipid flippase activity of ABCB4
Yeast sec mutant – at nonpermissive temp: secretory vesicle cannot fuse with plasma membrane – purify the secretory vesicles
(dithionite)
Phospholipid flippase activity of ABCB4
4
18.2 Cholesterol: a multifunctional membrane lipid
50-90% cholesterol is present in plasma membrane and related endocyticvesicle membrane
Storage
release
massive
LCAT: lecithin:cholesterolacyl transferase; convert cholesterol to esterifiedcholesterol
Cholesterol is synthesized by enzymes in the cytosol and ER
(ER integral membrane protein:sterol-sensing domain -TMcatalytic activity-cytosol)
(Cytosol)
(Cytosol)
Rate limiting step
HMG-CoA: β-hydroxy-β-methylglutaryl CoA
Drugs used to inhibit cholesterol synthesis include competitive inhibitors of HMG-CoA Reductase.
Examples include various statin drugs such as lovastatin (Mevacor) and derivatives (e.g., Zocor), Lipitor, etc.
A portion of each statin is analogous in structure to mevalonate or to the postulated mevaldehydeintermediate.
Extensive clinical trials have shown that the statin drugs decrease blood cholesterol and diminish risk of cardiovascular disease.
Massive cholesterol → damage
Excess cholesterol → cholesteryl ester formed by acyl
cholesterol acyl transferase (ACAT) located in ER
membrane → stored →as cytosolic lipid droplets –
interfaced with coat protein (perilipins or perilipin-
related proteins) in mammal cell
Lipid droplets contain: fatty acid, cholesteryl ester
In plant, the related protein coat the surface of lipid
droplets called oil bodies.
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many bioactive molecules are made from cholesterol and its biosynthesic precursors
Cholesterol and phospholipids are transported between organelle by Golgi-independent mechanisms: three hypothesis
Classic secretory pathway disruption (chemical inhibitor, mutation) – do not prevent cholesterol or phodpholipidtransport between membranes although do disrupt transport of proteins and sphingolipids
lipid in the ER – can not move to mitochodrial membrane by classic secretory vesicle transport
Fig 18-8 Proposed mechanism of Golgi-independent transport of cholesterol and phospholipids between membranes
Vesicles transfer lipids, did not across Golgi
Cholesterol and phospholipids are transported between organelles by golgi-independent (non-classic secreted transport systems)-three pathway
membrane
Pathway I
Fig 18-8 Proposed mechanism of Golgi-independent transport of cholesterol and phospholipids between membranes
Membrane-embedded proteins Small lipid-transfer proteins
ER membrane extension for organelle
Pathway II Pathway III
StAR (steroidogenic acute regulatory) protein (key step of cholesterol transport for steroid hormone synthesis)Encoded in nuclear DNA, transfer of cholesterol from the rich outer mitochondiral m embrane to the poor inner membrane; First steps in steroid hormones synthesis-N-terminal targeting sequence to mitochondrial outer membrane-C-terminal START (StAR-related transfer) domain-> Has hydrophobic cholesterol binding pocket
-Key role in moving cholesterol into mitochondrion for steroid hormone synthesisrate-limiting step in steroid hormone synthesis
Ex) mutation in StAR geneCongenital adrenal hyperplasia
-a lethal disease-Drastic reduction in the systhesis of steroid hormone
-Phosphatidylcholine-transfer protein contains START protein
Intracellular cholesterol-transport system (StAR, NPC)
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Current working model of StAR mechanism of action via association of C´ with outer mitochondrial membrane
Cells with nonfunctional Niemann-Pick C1 (NPC1) proteinAccumulate cholesterol in late endosomal/lysosomal vesicles
NPC -integral memb. Protein-Rapidly moving endosome/lysosomal compartment-Contain mutiple membrane spanning segment
Sterol sensing domain
Mutation in NPC1-defect in intracellular cholesterol transport-accumulate excess cholesterol in the late endosomal/lysosomalcompartment
Defect in the regulation of cellular cholesterol metabolism
Rescued by Rab9 (small GTPase)-late endosomal vesicular transport
Intracellular cholesterol-transport system
18.3 Lipid movement into and out of cells
Fatty acid transport proteins (FATPs; cell surface protein CD36), concentration dependentAlbumin – lipid-binding groove, bilirubin (膽紅素) , others
Fatty acid binding protein
Two major pathways for importing and exporting cellular lipids
Poor water solubility, need binding protein
Lipids can be exported or imported in large well-defined lipoprotein complexes
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Cell-surface transports aid in moving fatty acids across the plasma membrane
Hydrophobic fatty acid → pass membrane , but rate limitingFatty acid transport protein (CD36) → help FA → pass membraneFA is hydrophobic did exit in water-environment → protein carrier → formed complex albumin
Cholesterol →Bile acids → bile salt → incorportatecholesterol
formed from cholesterol in the liverstored in the gall bladder in bile as bile salts (sodium and
potassium)utilized during digestion of fats and other lipid substances
(act as detergents)
Bile Salts• Breakdown products of cholesterol• Amphipathic molecules• Function to transport cholesterol in the digestive system
Bile salts are synthesized from cholesterol– Rate limiting Enzyme: 7-α-Hydroxylase– After that conjugated with glycin and taurin and secreted into the bile.
Mixed micelle formed by bile salts, triacylglycerols and pancreaticlipase
detergent character of bile salts is due to the hydrophobic-hydrophilic nature of the molecules
the presence of hydroxyl (or sulfate) and the terminal carboxyl group on the tail gives the molecule its hydrophilic face
the steroid ring with its puckered plane provides the hydrophobic face
Bile acids emulsify (solubilize) fats sopancreatic lipase enzymes can split fattyacids from triglycerides.
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ABC proteins mediate cellular export of phospholipids and cholesterol
Fig 18-11 Major transport proteins in the liver and intestine taking part in the enterohepatic circulation of biliary lipids
Binding protein
Na-linked symporter (NTCP)
IBAT: ileal bile acid transporter (IBAT)I-BABP: intenstinal bile acid-binding protein
95%
β-sitosterolemia :ABCG5 or ABCG8 mutation →cholesterol release X → cholesterol store
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Lipids can be exported or imported in large well defined lipoprotein complexes
Lipoprotein: packages from hundreds to thousands of lipid molecules into water-soluble, macromolecular carriers
Chylomicrons (乳糜微粒): contains highest proportion of lipoidVLDL: very low density lipoproteinLDL: low density lipoproteinHDL: high density lipoprotein
Only VLDL and Chyomicrons are fully formed by ER, via microsomal transfer protein. VLDL released from liver; Chyomicrons released from intestine
Lipid or cholesterol → formed lipoprotein → efficient transport
Lipid transport in the circulation
Lipids are insoluble in plasma. In order to be transported they are combined with specific proteins to form lipoproteins
Non polar lipids in core
(TAG and cholesterol esters)
Proteins (apoproteins)Cholesterol
HOHO
O
R
HOHO
O
R
HOHO
脂蛋白依照組成大約可分為四類:1. 乳糜微粒(chylomicron):負責運送小腸細胞吸收的油脂,血中的濃度以飯後時最高,飯後的血漿呈混濁狀態就是乳糜微粒存在之故,其中的脂肪快速被組織利用與儲存,故濃度很快降低。組成份含三酸甘油酯82%,膽固醇9%,磷脂質7%與蛋白質2%。因為含脂肪量最多,所以密度最低。2. 極低密度脂蛋白(VLDL):主要由肝臟合成,運送肝臟合成的脂肪供其他組織利用。組成份含三酸甘油酯52%,膽固醇22%,磷脂質18%與蛋白質8%。3. 低密度脂蛋白(LDL):乳糜微粒與極低密度脂蛋白之代謝產物,經肝臟轉換而成,含膽固醇濃度最高,負責運送膽固醇供周邊組織利用,也是造成高血膽固醇的主要成分。組成份含三酸甘油酯9%,膽固醇47%,磷脂質23%與蛋白質21%。4. 高密度脂蛋白(HDL):由肝臟與小腸所製造,在血液中可以回收血管壁堆積的膽固醇,死亡細胞釋出的膽固醇,並將膽固醇送回肝臟代謝,有助於保護心臟與血管。組成份含三酸甘油酯3%,膽固醇19%,磷脂質28%與蛋白質50%。
Lipoproteins differ in the ratio of protein to lipids, & in the particular apoproteins & lipids that they contain.
They are classified based on their density:
Chylomicron (largest; lowest in density due to high lipid/protein ratio; highest % weight triacylglycerols)
VLDL (very low density lipoprotein; 2nd highest in triacylglycerols as % of weight)
IDL (intermediate density lipoprotein)LDL (low density lipoprotein, highest in cholesteryl esters as % of weight)
HDL (high density lipoprotein; highest in density due to high protein/lipid ratio)
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Chylomicron &VLDL – TG; LDL &HDL – cholesteryl esters
Pre β-lipoprotein
β-lipoprotein α-lipoproteinLipoprotein Structure
LDL
What are lipoproteins?
• Lipoproteins are protein-lipid complexes.Hydrophobic
lipids (TG, CE,ester) in core;
Hydrophilic lipids (UC,PL) on surface 1. hydrolysis of TG and PL by lipases and esterification of cholesterol by an acyl transferase
2. Transfer of cholesteryl esters, TG, PL between lipoproteins by specific lipid-transfer proteins
3. Uptake by some particles of cholesterol and PL exported from cells4. Association and dissociation of some apolipoproteins from the surface
of the particles
Lipoproteins are made in the ER (with microsomal transfer protein activity), exported by the secretory pathway, and remodeled in the circulation
Chylomicron – intestinal epithelial cells; VLDL – liver cells
LDL, IDL, HDL – remodeled (modifications) for chlymicorn & VLDL
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Apolipoproteins
Apo AI (liver, small intestine)– Structural; activator of lecithin:cholesterol acyltransferase
(LCAT)
Apo AII (liver)– Structural; inhibitor of hepatic lipase; component of ligand for
HDL binding
Apo A-IV (small intestine)– Activator of LCAT; modulator of lipoprotein lipase (LPL)
Apo A-V (liver)– Direct functional role is unknown; regulates TG levels.
Apo B-100 (liver)– Structural; synthesis of VLDL; ligand for LDL-
receptorApo B-48 (small intestine)
– Structural; synthesis of chylomicrons; derived from apo B-100 mRNA following specific mRNA editing
Apo E (liver, macrophages, brain)– Ligand for apoE receptor; mobilization of cellular
cholesterol
Apo C-I (liver)– Activator of LCAT, inhibitor of hepatic TGRL
uptakeApo C-II (liver)
– Activator of LPL, inhibitor of hepatic TGRL uptakeApo C-III (liver)
– Inhibitor of LPL, inhibitor of hepatic TGRL uptake
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High density lipoprotiens HDL
HDL carries “used” cholesterol (as CE) back to the liver. Also donate some CE to circulating VLDL for redistribution to tissues.
HDL taken up by liver and degraded. The cholesterol is excreted as bile salts or repackaged in VLDL for distribution to tissues.
Cholesterol synthesis in the liver is regulated by the cholesterol arriving through HDL (and dietary cholesterol returned by chylomicrons remnants).
Cholesterol (CE) in HDL is referred to as “good cholesterol”
HDL may transfer some cholesterol esters to other lipoproteins. Some remain associated with HDL, which may be taken up by liver &
degraded. HDL thus transports cholesterol from tissues & other lipoproteins to
the liver, which can excrete excess cholesterol as bile acids.
Lipoproteins are made in the ER, exported by the secretorypathway, and remodeled in the circulation
Exogenous/chylomicron pathway (dietary fat)
Endogenous pathway (lipids synthesized by the liver)
HDL metabolism (apolipoprotein transfer, cholesteryl ester transfer, reverse cholesterol transport
Intermediate Density Lipoproteins (IDL)
Liver → VLDL →TG hydrolysis by lipoprotein lipase (extracellularenzyme) → loss some TG and lipoprotein →IDL→ LDL→ bind receptor →absorbed
(a) After having been secreted from the liver
apolipoprotein
Lipid uptake by Intestine →chyomicron→secreted → smaller chylomicron → taken up by hepatocytesreceptor-mediated endocytosis
(b) Dietary lipids are sbsorbed
殘餘物
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HDLs are generated extracellularlyLCAT: lecithin:cholesterol acyl transferase; convert cholesterol to esterified cholesterolCETP: cholesteryl ester-transfer protein
receptor
類固醇產生細胞
HDL is though to be formed after secretion of apolipoprotein A from cells
Contain apoA and very little lipid
, a acceptor for phospholipid and cholesterol from ABCA1
LCAT: in
plasma
lecithin:cholesterol acyl transferase
Cells use several protein-mediated mechanisms to import
lipoprotein lipids:
1. Local, partial extracellular hydrolysis of core TG followed
by transport protein-mediated uptake of the released fatty
acids: – lipoprotein lipase linked by glycosaminoglycan
(GAG) on endothelial cells, FATPs (Fatty acid transport
proteins; muscle cells, adipocytes)
2. Regulated expression of cell-surface lipoprotein receptors
that mediate the direct uptake of lipoprotein lipids:
receptor-mediated endocytosis & selective lipid uptake (
by SB-RI: scavenger receptor, class B, type I)
Analysis of familial hypercholesterolemia revealed the pathway for receptor mediated endocytosis of LDL particles
LDL + LDL-receptor → endocytosisFH: familial hypercholesterolemia, high LDL, LDL-receptor mutantFH: easy death
125I-LDL + apo-B-100 → FH cell → analysis
LDL + LDL-receptor → internalized → to lysosome → proteases →hydorlyze surface apolipoproteins and cholesteryl esterases hydrolyze their core cholesteryl ester → unesterified cholesterol for metab
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LDL receptor-mediated endocytosis
FS: HYPERCHOLESTEROLEMIA
Still bindingDid not metab
Pulse-chase experiment demonstrates precursor-product relations in cellular uptake of LDL
Binding → internalization → degradation
Cholesteryl esters in lipoproteins can be selectively taken up by the receptor SR-BI (scavenger receptor, class B type I)
SR-BI: 1. cluster on microvilli and in the cell surface lipid raft, not in coated pits
as does the LDL receptor 2. Mediate the transfer of lipid across the membrane, not endocytosis of
entire LDL particles
Can bind HDL, LDL, VLDL
Still unclear
Binds HDL, LDL, VLDL
18.4 Feedback regulation of cellular lipid metabolism
Cholesterol level rises:1. HMG-CoA reductase – suppressed2. LDL receptor – suppressed3. Acyl:cholesterol acyl transferase (ACAT) – increased
Cholesterol depepdent transcriptional regulation:• Sterol regulatory element (SRE)• SRE-binding proteins (SREBPs) in ER• SCAP (SREBP cleavage-activating protein), insig-1(2)
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SREBP: serol regulatory elements binding protein
1. DNA binding motif (N-terminal)2. Central membrane-anchoring domain3. C-terminal cytosolic regulatory SCAP
binding domain
SCAP:1. 8 transmembrane helices – sterol sensing
domain2. C-terminal cytosolic SREBP-binding
domain
• Sterol binding domain of SCAP binds tightly to insig-1(2), but only at high cellular cholesterol levels:- block the binding of SCAP to COPII vesicle coat proteins:-prevents incorporation of SCAP/SREBP complex into ER-to-Golgi transport vesicles
Target gene-HMG-CoA reductase-LDL receptor
Membrane bound proteases
C
N
ER-lumen
• In cholesterol loaded cells– Unprocessed SREBP resides in Endoplasmatic Reticulum (ER)– SREBP is tightly complexed with sterol sensor SREBP-cleavage
activating protein (SCAP)– SCAP is attached via its sterol-sensing domain (SSD) to Insulin-
induced gene (INSIG) retention protein– (INSIG-1 and -2)
N
C
SCAP SREBPINSIG
ER-lumen
N
C
• In cholesterol depleted cells– Conformation of SCAP is altered due alteration in
conformation of SSD (sterol sensitive domaine)
C
N
SCAP SREBPINSIG
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ER-lumen
N
C
C
N
SCAP
• In cholesterol depleted cells– Conformation of SCAP is altered due alteration in
conformation of SSD (sterol sensitive domaine)
SREBPINSIG
ER-lumen
N
C
C
N
SCAP SREBPINSIG
• In cholesterol depleted cells– Conformation of SCAP is altered due alteration in
conformation of SSD (sterol sensitive domaine)– Which enables the dissociation of INSIG
Golgi-lumen
N
C
C
N
SCAP SREBP
• In cholesterol depleted cells– Conformation of SCAP is altered due alteration in
conformation of SSD (sterol sensitive domaine)– Which enables the dissociation of INSIG– SREBP is escorted to the Golgi apparatus
SREBP
Golgi-lumen
N
C
C
N
SCAP
• SREBP is cleaved in the Golgi apparatus– In Golgi apparatus SREBP is sequentially cleaved by
two proteases– Site-1 protease (S1P) and Site-2 protease (S2P),
respectively– S1P is cholesterol sensitive (inhibited by cholesterol and
SREBP is retained in Golgi if the content of cholesterol is high)
S1P
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SREBP
Golgi-lumen
C
C
N
SCAPS1P
N
S2P
• SREBP is cleaved in the Golgi apparatus– In Golgi apparatus SREBP is sequentially cleaved by
two proteases– Site-1 protease (S1P) and Site-2 protease (S2P),
respectively– S1P is cholesterol sensitive - low cholesterol content -->
cleavage by S1P, followed by S2P cleavage
SREBP
Golgi-lumen
C
C
N
SCAPS1P
S2P
N
• SREBP is cleaved in the Golgi apparatus– In Golgi apparatus SREBP is sequentially
cleaved by two membrane-bound proteases Site-1 protease (S1P) and Site-2 protease (S2P), respectively
– S1P is a serine protease– S2P is a zinc metalloproteinase
Target gene-HMG-CoA reductase-LDL receptor
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