Cholesterol
How can the lipoprotein groups be remodelled extracellularly by lipases?
-Esterification of cholesterol by an acyltransferase -Transfer of CEs, TGs and PLs between lipoproteins (specific lipid-transfer proteins) -uptake of some particles of cholesterol and PLs exported by cells -Association and dis-association of apolipoproteins from particles
What is the rate limiting step in the synthesis of bile acids?
enzyme cytochrome P450 cholesterol 7a-hydroxylase or CYP7A1 activity (activated by LXR and LRH-1 together)
What is familial hypercholesterolemia?
have 5x levels of cholesterol in blood, 650-1000mg/ml, homozygous individuals die early of heart attack in 20's, heterozygous individuals have an increased risk of heart attack in 30-40's. These individuals have problems with their uptake and degradation of LDL into their tissues.
How are bile acids made simply?
have extra OH groups produced by microsomal Cyctochrome P450 mixed function oxidases (CYP7A)- insert hydroyl groups that can be esterified to make that area more hydrophillic. Addition of 7-OH is the first and rate limiting step of bile acid sythesis- COOH group on side-chain is often conjugated to amino acids (e.g. glycine or taurine) to give bile salts (glycocholate and taurocholate) . some can be hydroxylated at 12-OH.
What are the effects of bile-acid binding resins e.g. Cholestyramine?
increase loss of bile salts by preventing IBAT taking them up from intestine, decreased bile acid return to the liver by NTCP and decreased binding of Bile acids to FRX. Decreased repression of CYP7A Resulting in increased bile acid production, decreased intracellular cholesterol conc and increased LDL receptor= decreased LDL in blood, decreased atherosclerosis + heart attacks + strokes.
What does low cholesterol do to the SCAP/SREBP complex?
nuclear SREBP released (can be produced very quickly) - transcription can occur of cholesterol as bHLH travels to nucleus with SREPB. Insig-1(2) no longer binds to SCAP.
What does high cholesterol do to the SCAP/SREBP complex?
SCAP binds tightly to membrane bound Insig-1(2)- cannot travel to golgi
What is the role of sterol sensing domain in HMG-CoA reductase?
-The domain in this enzyme senses high levels of cholesterol and steroidal precursors of cholesterol Triggers rapid ubiquitin-dependent proteasomal degradation of the enzyme Reduced cholesterol synthesis Involves cholesterol-dependent binding of Insig-1(2)
How is HMG-CoA reductase degraded?
1. Lanosterol triggers the binding of HMG-CoA reductase to Insig-1or 2. 2. E3 ubiquitin ligase, gp78 (attached to Insig), ubiquitinates the HMG-CoA reductase 3. The ubiquitinised HMG-CoA is removed from the membrane by VCP and delivered to a proteasome for degradation. Step 3 triggered by geranylgeraniol through an undefined mechanism that likely involves a geranylgeranylated protein, such as one of the Rab proteins.
What are the three fates of HDL?
1. Straight to the liver where there are SR-BI 2. Into steriodogenic cells and turned into steroid hormones. 3. Turned into cholesterol esters and then transported with other lipoproteins to the liver. (transfer of cholesterol)
What are the four phenotypes of having a mutation in the LDL receptor (in FH individuals?
1. reductions in amount of LDL receptor made 2. LDL receptor is made, but it fails to migrate to plasma membrane (fails to target cell surface) 3. LDL receptor is in plasma membrane but it fails to bind LDL 4. LDL receptor is in plasma membrane and binds LDL, but it fails to cluster in coated pits (fails to be endocytosed)
What are the general features o the SREBP (Sterol response element binding protein)?
1150 amino acid membrane protein in ER- Nterminal is basic helix-loop-helix transcription factor (bHLH), C-termial contains regulatory domain- both face cytosol. Transcription factor requires two proteolytic cleavage to be released- S1P in luminal loop (site projecting to the lumen of the ER. Cleaves Arg-Ser-Val-Leu-Ser) and S2P (metalloproteasejust within first transmembrane spanning domain) in transmembrane domain.
What is the tangier disease phenotype thought to be caused by ?
A mutation in the ATP binding cassette protein (ABC1) transporter-has 12 membrane spanning domains. It is upregulated by sterol loading and is found on chromosome 9. examples of other ABC proteins= CFTR- cystic fibrosis membrane transport protein that controls Cl- secretion -ABCB4- acts in liver to export phosphatidylcholine into bile -ABCB11- acts in liver to export bile into bile salts
What is the mechaism for cholesterol leaving the cell?
Accumulation of choelsterol in cell= activation of ACAT. Stored cholesterol can be mobilised when cells are incubated with Apo-A1, cholesterol hydrolysed and moved to the cell membrane- collected by apolipoprotein and if in form of HDL Apo-AI is re-esterifies the cholesterol with LCAT and is stored in the lipid core of the HDL molecule.
What is step one of cholesterol biosynthesis?
Acetyl CoA to HMG-CoA and melavonate. Condensation of acetoacetyl CoA with another acetyl CoA molecule to form HMG-CoA (catalysed by Acetyl-CoA synthase. Occurs in mitochondria. This is then converted to melavonate as a CoA-SH is lost and two NADPHs used- catalysed by HMG-CoA reductase- found in the ER- rate limiting step. This enzyme is regulated in relation to cholesterol levels (transcription + degradation).
What are the major protein types in HDL and LDL respectively?
Apolipoprotein-1- activates LCAT. Apolipoprotein-B100- large protein
What is the importance of a tyrosine amino acid residue in the cytoplasmic facing C-terminal region of the LDL receptor?
Asn-Pro-Tyr is a sorting signal that acts as an endocytosis motif that interacts with AP2 protiens (if the tyrosine residue is missing then LDL is not able to cluster in the clathrin coated pits as there is no interaction with AP2)
Why must the scavenger receptor be used by HDL for passing cholesterol to liver of steroidogenic tissues?
Because HDL cannot be internalised, it binds to the SR-BI- lipids + cholesterol are passed to the tissue and then HDL goes off to collect other PL/cholesterol cargo
What does the sterol sensing domain of SCAP do?
Binding of a sterol to the sterol sensing domain must prevent SCAP dependent cellular trafficking to the golgi and S1P processing
How does the liver scerete cholesterol directly into bile?
Cholesterol almost insoluble but very soluble in phosphatidyl choline (PC)- a little bit is secreted at the same time as bile. Cholesterol and Phospholipids can be secreted with bile.
What is CETP?
Cholesterol ester transferase protein- used by HDL to transfer cholesterol esters to other lipoprotiens
How are steroid hormones formed from cholesterol?
Cholesterol esters found in hormone producing tissues in the body. CE hydrolysed to Cholesterol which is taken in to the mitochondria and is converted to pregenolone by cytochrome P450 enzyme. Many hormones stem from pregenolone.
When theres an excess of cholesterol what does SCAP do?
Cholesterol negatively regulates ER-to-Golgi transport by binding to Scap, thereby changing its conformation and triggering the binding of Scap to Insig, an ER anchor protein. Insig prevents the binding of Scap to COPII proteins, thereby halting transport of SREBPs to the Golgi.
How does cholesterol regulate its own synthesis + uptake?
Cholesterol released from lysosome- moves to ER- suppresses action of HMG-CoA reductase here (reduces transcription + degradation increased). activates acyl-CoA acyl transferase (ACAT) which forms cholesterol esters. Can also reduce transcription of LDL receptor so the cell does not accumulate more cholesterol.
What are the effects of statins e.g. Lovostatin?
Inhibit HMG-CoA reductase, decreased cholesterol and Insig1/Scap interaction. Increase Nuclear SREBP and LDLreceptors. Decreases plasma LDL and hence atherosclerosis- heart attacks etc.
How is HDL produced?
by an efflux of cholesterol in tissues.
What is the physiology of cholesterol metabolism in enterocytes?
End of small intestine, majority of bile acids are retrieved by a bile acid transporter. ~95% of bile acids are reabsorbed and returned to the liver. ~5% are secreted. Unrecovererd bile acids are replaced by resynthesis (0.5 g/ day) in the liver.
How is bile acid levels regulated in enterocytes?
FRX promotes IBABP (bile acid binding protein) + promotes exportation of bile acids out ABCC3.
the nuclear receptor FXR is activated by binding of bile acids, what is its function?
FXR binds bile acids and plays a key role in regulating enterohepatic circulation of bile acids FXR is expressed in the liver and intestinal epithelial cells FXR represses synthesis of cholesterol 7α-hydroxylase (CYP7A gene) Stimulates the expression of I-BABP (intracellular bile acid binding protein) and transport proteins (e.g. ABCB11) that mediate cellular export of bile salts Represses NTCP
What is the fourth step of cholesterol synthesis?
Formation of lanosterol from cyclisation squalene (can be a regulated step) and from there 20 reactions to form cholesterol.
What are the four major classes of lipoproteins?
HDL- high density lipoproteins (higher conc of proteins to fats) LDL- low density lipoproteins (two above are mainly cholesterol esters in core) VLDL- very low density lipoproteins (mainly triglyceorls) Chylomicrons- lowest density
Structure of lipoproteins?
Have a hydrophobic core- containing triglycerols then a hydrophillic outside consisting of apolipoproteins (protein layer).
Features of structure of cholesterol?
It is a derivative of a saturated tetracycline hydrocarbon. Contains 3 five membered rings and one 6 membered ring. Aliphatic chain at C-17 CH3 groups at C13 and C10 Double bond in second ring Hydroxyl (OH group at ring one) Cholesterol is flat and compact and is a sterol.
What is SCAP?
It is a sterol sensing domain- 1276 amino acids- 8 membrane spanning domains in ER- 5 alpha helical cholesterol binding domains . WD repeats at carboxy terminal fold like a propeller and mediate protein-protein interactions. SCAP found in tight complex with SREBP and this complex is required for S1P cleavage (regulatory region of SREBP binds to WD repeats of SCAP)
What catalyses the esterification of cholesterol esters?
LCAT - Lecitthin cholesterol acyl transferase (blood) + ACAT- Acyl-CoA cholesterol acyl transferase (tissues). Esterified with a fatty acid at 3'-OH group. Present with long chain fatty acids (cholesterol esters) or free cholesterol.
What is the fate of LDL particles?
LDL binds to the receptor via the B100 Apolipoprotein- taken into cell via coated vesicle- vesicles fuse to make an endosome with a proton pump- inside becomes acidic- LDL detaches from its receptor (which is recylced to membrane)- LDL- apolipoprotein B-100 into free amino acids and cholesterol esters into free cholesterol.
How does high LDL cause atherosclerosis?
LDL is oxidised in plasma and cells, (hydroxylation of cholesterol, peroxidation of unsaturated fatty acids and oxidation of amino aicds in the apoprotein. Oxidised LDL taken up by white blood cells through Scavenger receptor (unregulated) resulting in accumulation forming foam cells. Chemotactic signals for more white blood cells to accumulate with cholesterol- cluster at atherosclerotic plaques and cause thickening/damage to blood vessel.
How is cholesterol provided to the body?
Little over half (800mg) is synthesized in body- rest obtained from diet- digested dietary fats (fats emulsified by bile saltes then broken into triacylglycerols by pancreatic lipase)- carried in blood by lipoproteins to the liver- small micelles absorbed in intestine- chylomicrons taken to liver.
What is step 2 of cholesterol synthesis?
Mevalonate forms isoprenoid units- cytosol- mevalonate is phosphorylated successively 3 times- then theres an ATP dependent decarboxylation- (transelimination)- Isopentenyl pyrophosphate (IPP) isomerizes to give dimethylallyl pyrophosphate (DMP)
How is SR-AI/AII responsible for accumulation of cholesterol by foam cells?
Oxidation of unsaturated fatty acids generates aldehydes which can modify proteins and other components of LDLs The modified LDLs bind to the Scaveger Receptor (SR-AI/II) as the Normal function of SRs is to promote endocytic destruction of damaged molecules, cells etc SRs not normally part of cellular cholesterol metabolism so their levels are not regulated.
What are some essential roles of cholesterol?
Precursor to hormones and vitamin D. Essential component of cell membranes, make up 25% membrane lipids in nerve cells. Needed for digestion of fats. Together with spingomyelin forms 'rafts' of caveolae in the membrane where signalling molecules and membrane proteins are concentrated.
How is LDL taken up in to tissues?
Receptor mediated endocytosis- LDL binds and is taken into a clathrin coated pit. Adaptor protein complex 2 (AP2) is the one required for endocytosis of the plasma membrane components. Dynamin is at the top of the coated pit and contracts like a rubber band to seal off the vesicle.
What is RIP?
Regulated Intramembrane Proteolysis : RIP important for controlling cholesterol metabolism Also important for activation of transcription factors in Notch signalling pathway Generation of toxic amyloid β-peptides in Alzheimer's disease
How do SREBP (ER) and S1P (golgi) meet?
SCAP is formed (glycosylation) in the ER (it is a glycoprotein) and its sugar chains are processed as glycoproteins in the golgi. SCAP binds to SREBP causes its retention in the ER. Cholesterol induces a conformational change to SCAP. Overexpression of SCAP sterol-sensing domain permits SCAP-SREBP traffic to Golgi suggesting a saturable SCAP binding site in ER. When cells have adequate cholesterol SREBP is found in ER complexed with SCAP and Insig-1(2) Insig-1(2) binding prevents ER->Golgi transport of SREBP/SCAP When cholesterol levels in ER membranes drop, Insig-1(2) no longer binds to SCAP and SREBP/SCAP moves to the Golgi S1P and S2P cleavages occur to give nuclear SREBP
When cells are derived of cholesterol what does SCAP do?
Scap escorts SREBPs from the ER to Golgi by binding to Sec24, a component of the Sar1/Sec23/Sec24 complex of the COPII protein coat. Once in the Golgi, the SREBPs are proteolytically processed to generate their nuclear forms that activate genes for cholesterol synthesis and uptake
What is the function of LXR (Liver X receptor)?
Senses oxysterols (e.g. hydroxylated forms of cholesterol) which increase when cholesterol levels are high Stimulates cholesterol 7-hydroxylase (CYP7A) Stimulates expression of ABC transporters that export cholesterol into bile (ABCG5/8) or onto lipoproteins (ABCA1) in blood Promotes lipoprotein production
What is the 3rd step of cholesterol synthesis?
Six isoprenoid units come together to form squalene. Cytosol- three 5C isoprenoid units come together + IPP and DPP to form geranyl pyrophosphate + an extra 5C unit to form Farensyl pyrophosphate. Squalene formation occurs in the ER as two Farensyl pyrophosphate groups (15C) join tail to tail (condensation) to form squalene (C30).
What are some ways to reduce serum cholesterol?
Switch unsaturated fatty acids for poly and mono saturated fatty acids (stimultes excretion of cholesterol into intestine, and oxidation of choelsterol into bile acids.) Exercise can increase HDL content Surgical procedures to interrupt the enterohepatic circulation of bile acids or drugs which block the uptake of cholesterol. Drugs- Fungal inhibitors of HMG-CoA reductase- (lovostatin + metastatin)- block cholesterol synthesis + up regulate LDL recptors for uptake of circulating LDL.
How can cholesterol be taken up by cells from blood?
Taken up as LDL through LDL receptor.
Bile acids are taken up and exit via what channels in liver cells?
Taken up from blood by NTPC/Na+ channel and exit via ABCB11 channel
Where is the only place that Apo-AI can be degraded?
The kidney
How are bile acids related to cholesterol?
They are derived from cholesterol- only way cholesterol can be removed from body- 20-30g made a day- 0.5g bile acids lost. 800mg of cholesterol made a day- half of this used to make bile acids. Most bile acids are recycled from the lower intestine to the liver (enterohepatic circulation)- another way of regulating cholesterol levels.
How is it ensured that cholesterol synthesis is not terminated before the cell creates sufficient cholesterol for the cell?
Two SREBP have to bind to SCAP at same time for it to be inactivated- Insig-1 and Cholesterol
What is the main site of cholesterol synthesis?
Usually liver, somtimes brain and intestines.
Lipoproteins are made in the ER how can they be modified in circulation?
VLDL- formed liver + Chylomicrons- formed in intestines- released into blood- remodelled in capillaries by lipoprotein lipase into smaller chylomicron remnants which are then taken up by liver. VLDL synthesised in liver- released- remodelled in circulation (on cell surfaces) to IDL then to LDL.
How are bile acids taken up from the instestinal lumen by intestinal cells?
Via the IBAT/Na+ channel
What is the source of all carbons in the 27C cholesterol structure?
acetyl CoA
After S1P and S2P cleave transcription factor from SREBP what happens?
amino-terminal domain then travels to the nucleus and binds SREs in the enhancers of genes encoding enzymes of cholesterol biosynthesis, triglyceride biosynthesis and lipid uptake. Example- HMG-CoA reductase, HMG-CoA synthase farnesyl diphosphate synthase and squalene synthase. Other targets include enzymes involved in fatty acid and triglyceride synthesis (acetyl CoA carboxylase, fatty acid synthase). SERBPs also enhance the transcription of the LDL receptor gene. When cholesterol rises in the cell the activity of SERBPs decreases as S1P is regulated by cholesterol levels (S2P is too indirectly as SIP has to be cleaved before S2P can be).
What is the simple mechanism for reverse cholesterol transport?
cholesterol efflux is rapid 0.1% of total cell cholesterol per min. cholesterol is picked up by apolipoprotein A-I containing HDL particles
How is bile acid levels regulated in hepatocytes?
cholesterol gets taken up by LDL receptor - oxysterols are produced suggesting to cell that cholesterol is high- binds to LXR which stimulates CYP7A1 by LRH-1 to promote production of bile acids. Bile acids intermediate + FXR suppress CYP7A1 and NTC respectively- reducing bile acid production.
How does the ABC1 protein work?
it is expressed ubiquotously and exports cholesterol and phospholipids from cells that can then be taken up by HDL. HDL particles pickup cholesterol and group to become nascent HDL particles. They can then become cholesterol rich HDL and there is SR-BI (scavenger receptor) mediated selective uptake of cholesterol esters. Mutation in ABC1 cholesterol and phospholipids build up in cell.
What is the structure of clathrin?
made up of three heavy and three light chains (basci unit = triskelion) - forms a clathrin coat/lattice by polymerisation and creates a selective molecule filter. Adaptor proteins provide a link between the receptor tail and the globular end of the heavy chain of clathrin.
What protective roles does HDL play against atherosclerosis?
trasgenic expression of Apo-A1 suppresses atherosclerosis, HDLs can promote removal of cholesterol from cells by reversal transport- takes to liver and can be converted to bile acids. A serum esterase, Paroxanase, able to destroy oxLDL is associated with HDL. HDL has anti-inflammatory properties.
