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What molecule is at the center of lipid metabolism?
Acetyl-CoA
Acetyl-CoA —> _______ —> _______ —> ________ —> Cholesterol
HMG-CoA (second arrow with NADPH), Mevalonate (third arrow with ATP), Isoprenoids (fourth arrow with NADPH)
How many carbons are in cholesterol and where do they derive from? Is it polar or non-polar? What family of lipids do they belong?
27 carbon molecule with all carbon atoms deriving from acetyl-CoA. A hydroxyl head group is the only polar group, making cholesterol amphipathic. Cholesterol is a steroid, a subfamily of sterols.
What is the first stage in cholesterol biosynthesis? How many reactions are there in the first step?
Synthesis of mevalonate requires two reactions.
What is the first reaction in the synthesis of mevalonate (step 1 of stage 1 of cholesterol biosynthesis)?
Acetoacetyl CoA and Acetyl CoA are condensed, and one CoA is removed (by water), which is catalyzed by HMG-CoA synthase. This results in HMG-CoA.
What is the second reaction in the synthesis of mevalonate (step 1 of stage 1 of cholesterol biosynthesis)? Where does this reaction occur?
Two options:
HMG-CoA is reduced by 2 NADPH and 2 H+ to produce mevalonate in the CYTOPLASM by HMG-CoA reductase.
HMG-CoA is separated by HMG-CoA lyase into Acetyl CoA and Acetoacetate in the MITOCHONDRIA.
What organ is responsible for the transport of cholesterol?
Liver
What is the rate-limiting step and point of feedback regulation in biosynthesis of cholesterol?
HMG-CoA reductase reaction in Stage 1 reaction 2.
Why might HMG-CoA be reduced in the cytoplasm versus separated in the mitochondria?
When we have the energy to synthesize cholesterol, HMG-CoA will be reduced to mevalonate in the cytoplasm to go on and create cholesterol. If there is not enough energy and the body needs more energy, HMG-CoA will be broken down in the mitochondria and Acetoacetate will go toward ketone body synthesis for cells to get energy.
What are some characteristics of rate-limiting reactions?
Non-reversible, high energy input, highly regulated, and directs the synthesis pathway.
What are the competitive inhibitors of HMG-CoA reductase?
Statins (both natural and synthetic)
What are the two mechanisms of statins?
Inhibits HMG-CoA reductase (binds using hydrophobic group)
Up-regulates the expression of LDL receptors on cell membranes (so dietary LDLs are used now that intracellular cholesterol is low).
About 1/3 of Americans over 40 take statins. What is the method behind prescribing statins?
80% of the cholesterol in the body is synthesized while 20% is dietary. Prescribers will instruct patients to reduce their dietary intake of fats before prescribing statins to lower the body-synthesized cholesterol.
What are Type 1 statins?
Natural fungal products
What are type II statins?
Fully synthetic
The measure of the effectiveness of a substance in inhibiting a specific biological or biochemical function. How do we use this measure?
Half Maximal Inhibitory Concentration (IC50). We want less of the drug for the maximum amount of efficiency to reduce side effects. This is done with a low IC50.
What is the second step of Stage 1 of cholesterol synthesis? What are the three reactions that occur in the second step of stage 1?
Mevalonate is converted to high-energy isoprene intermediates.
Pi transfer from ATP to yield 5-phosphomevalonate.
Pi transfer from ATP to yield 5-pyrophosphomevalonate
ATP-dependent decarboxylation yields 3-isopentenyl pyrophosphate (IPP), an activated isoprenoid molecule.
What leaves in the third reaction of step 2 of stage 1 of cholesterol biosynthesis?
Both the phosphate and the nearby carboxyl group leave producing a double bond (IPP).
What is the third step in stage 1 of cholesterol synthesis?
Isopentenyl pyrophosphate (IPP) is isomerized to dimethylallyl pyrophosphate (DMAPP). Basically, the double bond moves and is catalyzed by isopentenyl pyrophosphate isomerase.
Is step 3 in stage 1 of cholesterol synthesis reversible? Explain.
The reaction occurs in equilibrium, so we have some of both molecules. Both are needed to continue with cholesterol synthesis.
What is the enzyme that catalyzes the isomerization of IPP to DMAPP?
Isopentenyl pyrophosphate isomerase.
Isopentenyl pyrophosphate is the first of several compounds in the pathway that are referred to as _______, by reference to the compound ______.
Isoprenoids; isoprene
What happens in stage 2 of cholesterol synthesis?
Condensation of 6 molecules of isopentenyl pyrophosphate (C5) to form squalene (C30).
What enzyme catalyzes the 2 head to tail isopentenyl pyrophosphate condensation reactions?
Farnesyl diphosphate synthase
The first two reactions in stage 2 are what form of condensation rxn? What is the product and how many carbons does it contain?
2 head to tail isopentenyl pyrophosphate (IPP) (C5) reactions to DMAPP (C5). Intermediate geranyl pyrophosphate and product farnesyl pyrophosphate (15C) with PPi byproduct in both condensation reactions.
What happens after the two head-to-tail IPP condensation reactions in stage 2? How many carbons are in the final product?
A tail-to-tail (nonpolar end to nonpolar end) coupling of two molecules of farnesyl pyrophosphate (C15) with NADPH to yield squalene (C30).
The tail to tail condensation reaction in Stage 2 is also what kind of reaction?
A reductive, tail-to-tail condensation of C15 units.
The tail-to-tail coupling of farnesyl pyrophosphate is catalyzed by what enzyme? Where is this enzyme found?
ER enzyme squalene synthase
What intermediate product in the synthesis of cholesterol contains all of the carbons which will be in cholesterol?
Squalene
What is the full reaction for the tail to tail condensation reaction in stage 2 of cholesterol synthesis?
2 farnesyl pyrophosphate (C15) + NADPH —> squalene (C30) + 2PPi + NADP+ + H+
Squalene cyclizes via _____ ______ into _______.
squalene epoxide; lanosterol
What is the first fully cyclic molecule in the synthesis of cholesterol?
Lanosterol
Which enzyme adds one oxygen to the end of the squalene chain? what reduces the other oxygen atom of O2 to H2O? What is the end result of these two reactions?
Squalene monoxygenase; NADPH; squalene 2,3-epoxide
What is the first reaction in the cyclization of squalene? What enzyme is involved and what is the final product called?
Squalene monooxygenase adds one oxygen to the end of the squalene chain; NADPH reduces the other oxygen atom of O2 to H2O to make squalene 2,3-epoxide.
What initiates squalene oxidocyclase to catalyze a series of electron shifts?
Protonation of squalene 2,3-epoxide
After squalene 2,3-epoxide is formed, what reaction(s) occur to complete cyclization?
Squalene oxidocyclase catalyzes a series of electron shifts, initiated by protonation of the epoxide, resulting in cyclization.
How many reactions converts lanosterol to cholesterol? Where are the enzymes that catalyze this reaction?
19 reactions catalyzed by enzymes associated with ER membranes
What are the major changes that occur in the 19 reactions to get from lanosterol to cholesterol?
Removal of 3 methyl groups
Reduction of 1 double bond by NADPH
Migration of another double bond
What is the final product of “cholesterol” synthesis in animal cells, plants, and fungi?
Animal Cells: Cholesterol
Plants: Stigmasterol
Fungi: Ergosterol
What is the “common ancestor” of cholesterol (animals), stigmasterol (plants), and ergosterol (fungi)? What implications does this characteristic have?
Squalene 2,3-epoxide. Drugs can target enzymes after squalene 2,3-epoxide for specificity of the drug (and so the organism does not die).
What is the rate limiting step in cholesterol synthesis? What are the four methods by which this enzyme’s activity is controlled?
HMG-CoA reductase
Gene expression of HMG-CoA reductase (transcription)
Degradation of the enzyme
Covalent modification to prevent HMG-CoA reductase function (phosphorylation decreases its function)
Feed-back inhibition (allosteric regulation)
What does it mean on the physiological level if someone has high cholesterol?
This person’s body failed to regulate the rate-limiting step/HMG-CoA reductase activity.
Transcription of HMG-CoA reductase gene is controlled by? Where is this protein located?
sterol regulatory element-binding proteins (SREBPs) are synthesized as a transmembrane protein anchored in the ER membrane.
How is transcription of HMG-CoA reductase activated?
The transcriptional activation domain of SREBP must me liberated from the membrane by cleavage (catalyzed by proteases) and the transcriptional activation domain is transported to the nucleus where it binds the serum response element (SRE) region of the gene.
Is SREBP active or inactive with high sterol in the ER? Low sterol?
High sterol means no more cholesterol is needed, so SREBPs are inactive and the gene is not transcribed. Low sterol means more cholesterol is needed, so SREBPs are active/cleaved and bind the SRE for transcription of the gene, promoting lipid synthesis.
Describe the many ways in which molecules can be EXPORTED starting with the ER.
From ER to the Golgi. Exported out of the Golgi to either a late endosome, early endosome, secretory vesicles, or to the cell exterior directly. Late endosomes export to the lysosome and secretory vesicles export to the cell exterior.
Describe how a molecule may be IMPORTED from the cell exterior?
Cell exterior to early endosome to late endosome and to a lysosome.
Describe how molecules are RECYCLED in the secretory pathway.
From late endosomes, early endosomes, or secretory vesicles to the Golgi and to the ER. Recycling can also occur from an early endosome to the cell exterior.
Where does SREBP bind when transported to the nucleus?
To SRE response element DNA sequences at the promoters of cholesterol synthesis genes, including HMG-CoA reductase.
What protein binds to SREBP and regulates its activation?
SREBP cleavage activating protein (SCAP)
When there is high cholesterol, what is SCAP bound to? What is the purpose of insig? What does insig bind?
SCAP binds cholesterol, which promotes its binding to insig (insulin-induced gene). Insig acts as an anchor to retain SCAP-SREBP on the ER membrane. Insig binds a metabolite of cholesterol (NOT cholesterol though) called 2,5-hydroxy-cholesterol, and this binding also promotes insig binding to SCAP-SREBP.
When in high cholesterol and insig is stabilized by sterol binding, what else does insig bind? What process does it promote? What is the result of this process?
When stabilized by sterol binding, Insig binds HMG-CoA reductase and promotes its polyubiquitination and degradation. The SREBP TF is not released from the membrane and cannot enter the nucleus to activate transcription.
In low cholesterol conditions, what are SCAP and insig bound to? What happens to Insig?
SCAP and insig are not bound by sterols. Insig becomes weak and is polyubiquitinated and degraded.
In low cholesterol conditions, what happens after insig is degraded? What happens after? What is the result?
When unbound to Insig, SCAP can bind secretion escort proteins of vesicular trafficking, causing SCAP-SREBP to traffic to the Golgi membrane. With the help of SCAP, two proteolytic cleavage events in the Golgi free a portion of SREBP from the membrane. SREBP TF then travels to the nucleus, promoting the transcription of cholesterol synthesis enzymes (HMG-CoA reductase).
In what conditions are insig and SCAP bound? What are they bound to and what does this keep SCAP from doing?
Insig is bound to 2,5-hydroxy-cholesterol and SCAP is bound to cholesterol. Cholesterol bound to SCAP means SCAP cannot activated the proteases needed for cleavage of SREBP.
Once in the golgi (low cholesterol), what activates the proteases for cleavage of SREBP TF? What are the proteases?
SCAP without cholesterol and in the Golgi will activated serine protease to cleave part of SREBP. A metalloprotease cleaves the rest of the TF from the transmembrane portion of SREBP, freeing the TF to travel to the nucleus and promote transcription of enzymes involved in cholesterol synthesis.
What does SREBP upregulate once it binds SRE (sterol regulating element)?
Upregulates:
HMG-CoA reductase
other cholesterol synthesis enzymes
When cholesterol is high, what happens to HMG-CoA reductase? How is this process regulated and mediated?
HMG-CoA reductase undergoes proteosomal degradation. Sterol-regulated and insig-mediated ubiquitination.
What are the two domains of HMG-CoA reductase?
Sterol sensing domain (for sensing cholesterol) and the catalytic domain.
What starts the ubiquitination and degradation of HMG-CoA reductase? Describe the process.
When stabilized by sterol binding, Insig binds HMG-CoA reductase. With ubiquitinating enzymes, Insig promotes the degradation.
What is the function of geranylgeraniol in HMG-CoA reductase degradation?
Geranylgeraniol acts as a signal molecule. It disrupts the binding of a protective protein (UBIAD1) to the reductase, thereby exposing it to the cellular machinery responsible for protein breakdown. Essentially, it marks HMG-CoA reductase for destruction when sterol levels are high.
How is HMG-CoA reductase inactivated? In what structure is HMGR most active?
Phosphorylation by AMPK (AMP-dependent protein kinase). HMGR is most active in its dephosphorylated state!
What molecules (insulin/glucagon) promote the phosphorylation versus dephosphorylation of HMGR by AMPK? Why is this the case?
Glucagon promotes HMGR phosphorylation by AMPK, while insulin promotes dephosphorylation. Glucagon indicates low glucose/energy/resources, while insulin (like after eating) indicates high glucose/energy/resources.
In terms of ATP, when is AMPK phosphorylating HMGR?
Low ATP means no energy for cholesterol synthesis, so AMPK will phosphorylate HMG-CoA reductase to reduce its activity in creating mevalonate.
What is ACAT? What is its function?
ACAT is acyl-CoA-cholesterol acyl transferase. ACAT adds a fatty acid via acetylation to intracellular cholesterol to store excess cholesterol and regulate cholesterol levels within the cell.
What are the three main functions of cholesterol?
Membrane fluidity
Energy storage
Hormones
What molecule is responsible for feedback inhibition of cholesterol synthesis? Where is it derived from? What enzyme does it bind and on what domain? What is the result?
Oxysterol is a feedback inhibitor derived from cholesterol. Oxysterol binds directly to the sterol-sensing domain on HMGR to inhibit the synthesis and absorption of cholesterol. Oxysterol stimulates proteolysis of HMG-CoA reductase.
Why can’t cholesterol be put directly into the bloodstream?
Because cholesterol will clump together in a glob (will move in particles). Instead, they are moved around by lipoproteins.
In vertebrates, most cholesterol is synthesized where? To what locations is it exported?
In the liver, then to hepatic membranes, secreted from hepatocytes, or as steroid hormones or vitamin D to other tissues.
Once cholesterol is secreted from hepatocytes, what are its two fates (aside from steroids/vitamins)?
Secreted as cholesteryl ester and TAGs to other tissues.
As bile acids and their salts to facilitate lipid digestion in the intestine.
What is the analogy for why cholesterol is transported in lipoproteins?
Like grease on your plate, fatty acids will glob together and stick, just as they would in the bloodstream. This is why they need to travel in lipoproteins. Bile acids and their salts are the dish detergent, helping to digest lipids in the intestine for transport.
What are the two uses of cholesterol that is transported via lipoprotein particles?
To use as a fuel or for storage
Membrane synthesis
Describe the structure of a lipoprotein particle.
Unesterified “free” cholesterol is on the cell membrane with phospholipids. Cholesteryl ester is on the inside alongside TAGs. Apoprotein B-100 also lines the outside (a component of LDL or “bad cholesterol”).
What are the two roles of lipoproteins?
To solubilize the hydrophobic lipids and contain cell-targeting signals.
Where are cholesteryl esters formed and by what enzyme? What is the starting material and what reaction that occurs?
Formed in the liver through the action of acyl-CoA-cholesterol acyl transferase (ACAT). Catalyzes the transfer of a fatty acid from coenzyme A to the hydroxyl group of cholesterol.
How does transformation from cholesterol to cholesteryl ester change the properties of cholesterol?
Cholesteryl ester (CE) is super hydrophobic and will hide from water as much as possible. Must be transported in lipoproteins to other tissues or stored in the liver.
What are the four major classes of lipoprotein particles? Name five in order from least dense to most dense.
LEAST DENSE Chylomicrons —> very low-density lipoproteins (VLDL) —> intermeidate-density lipoproteins (IDL) —> low-density lipoproteins (LDL) —> high-density lipoproteins (HDL) MOST DENSE
Which of the lipoprotein molecules is the largest? What is its function?
Chylomicrons take the food out of the stomach and deliver fat through the body. Once the inside empties out, it becomes VLDL.
Which of the lipoprotein particles is the smallest?
High-density lipoproteins are the smallest and the most dense.
What does it mean for the lipoprotein to be less or more dense? What is the implication?
The lower the density of a lipoprotein, the more lipid it contains relative to protein.
What is the specific function of the lipoprotein determined by? (3)
Determined by its point of synthesis, lipid composition, and apolipoprotein content.
What is the last of the fats contained in a lipoprotein to leave?
Compared to TAGs and phospholipids, cholesterol is the last of the fat to leave the lipoprotein.
Cholesterol in the circulation will originate from what origins?
Exogenous or endogenous origin.
Exogenous: When you eat food, what is the first step that needs to happen?
Cholesterol makes bile acids in the liver to help acquire more cholesterol because bile lipids help digest dietary lipids in the small intestines.
Exogenous: After bile acids help digest dietary lipids, where do the fats go?
Chylomicron transports food out of the digestive track and goes to the capillaries where exchange occurs. Typically FFAs go first to both muscles and adipose tissue.
Exogenous: After exchange of FFAs to the muscles and adipose tissue by chylomicron, what happens next?
Chylomicron remnant (with a now reduced size) will travel to liver cells. Liver cells have an LDLR which binds the apolipoproteins on the chylomicron remnant.
Endogenous: Where do excess fats in the liver go to the rest of the organs by?
VLDL takes excess fats to the rest of the organs. Same as with chylomicrons, VLDLs in the capillaries exchange FFAs with muscle and adipose tissue.
Once VLDL FFA exchange occurs in the capillaries, what is the remnant called and where does it go?
IDL (size decreased) is what results after VLDL transports excess fats throughout the body. IDL is recycled to the liver (binds LDLR) OR becomes LDL.
Endogenous: Once IDL becomes LDL, where does LDL go?
To the liver (binding via LDLR) or to the organs and the periphery so there is fat there as well. This is of course important, but you don’t want too many LDLs.
Where are chylomicrons formed? What do they do?
Formed in intestinal villi to absorb fatty acids and cholesterol and mobilize them to the bloodstream.
What is the role of LPL (lipoprotein lipase)? How is it activated?
Activated by Apo C-II, LPL cleaves off the free fatty acid from either TAG or cholesteryl esther so that the FFAs can leave the chylomicrons and be transported to adipose and muscle tissue.
What is the primary fat composition of chylomicrons?
TAGs, which will leave first
What is the function of FA-albumin?
A protein in blood that binds anything hydrophobic and can mobilize fatty acids and lipids throughout the bloodstream and to tissues that need them.
What forms excess FAs and cholesterol? Where does excess carbohydrate in the diet go?
Cholesteryl esters and TAGs form excess FAs and cholesterol. Excess carbohydrate in the diet —> TAG in the liver —> VLDL.
How do peripheral tissues recognize LDL?
Peripheral tissues have LDLRs that recognize apoB-100 and take up cholesterol via receptor-mediated endocytosis.
Why is LDL considered the “bad” cholesterol? How can we mitigate these effects?
It’s not bad, we just really don’t want excess cholesterol in the bloodstream because that can lead to atherosclerosis. We can make cells have more LDLRs to prevent excess LDL from remaining in the bloodstream. Decreased LDLRs on peripheral tissues means decreased cholesterol levels in the cells (can make more LDL).
What is the first step in receptor-mediated endocytosis of LDL?
LDL particle binds the LDLR with the ApoB-100 protein. Endocytosis ensues via a clathrin-coated pit (with AP2 complex) that bends the membrane and forms the endosome/vesicle.
In receptor-mediated endocytosis of LDL, where does the early endosome go?
The early endosome containing the LDLR and the LDL particle fuse to other endosomes to form a late endosome. Late endosomes have a low-pH (pH 5 compared to pH 7) environment that causes the dissociation of the LDLR from the LDL particle and initiates the receptor going back to the membrane surface.
Receptor-mediated endocytosis of LDL: After the late endosome pH drops and the LDLR goes back to the surface, where does the LDL particle go? What happens to cholesterol?
To the lysosome where proteases and lipases digest LDL. Cholesterol is separated from free acids. Amino acids are also present for protein synthesis.
How are LDLs released from the LDLR?
At pH 7, LDLRs are in the open structure and are able to bind LDL. At pH 5 in the late endosome, the LDL-binding domain will bind the propeller-like structures and cause LDL to dissociate.
