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  • JOANNE STUBBE: So what we were doing last time is we were

  • still focused the first two lectures were trying

  • to understand the biosynthetic pathway for cholesterol bio--

  • it's good, thanks--

  • for cholesterol biosynthesis.

  • And we almost got to where we wanted to go,

  • but we didn't quite get there.

  • So what we've been focusing on is a new way

  • of forming carbon-carbon bonds using

  • C5 units, isopentenyl pyrophosphate

  • and dimethylallyl pyrophosphate.

  • And to do that, we had an initiation

  • process where these molecules were generated from acetyl CoA.

  • And then the last lecture we were

  • focused on how we did the elongation process where

  • we took a bunch of these IPP units,

  • strung them together to make farnesyl pyrophosphate, which

  • is C15, and I showed you that C15

  • could be linear or cyclized.

  • And we went through the general rules

  • of what you're going to see with all turpine chemistry, which

  • is quite diverse, given that there

  • are estimated to be 70,000 natural products

  • in the terpenome.

  • So we had gotten to production of farnesyl pryophosphate

  • and now the next step--

  • remember, cholesterol, if you look at its structure--

  • this is a precursor to its structure--

  • is a C30.

  • And so the next step is quite an interesting enzymatic reaction

  • which we're not going to talk about in any detail,

  • but those of you who are interested can go look it up.

  • But how do you take two C15s and form a C30

  • so you lose your pyrophosphates?

  • And you can see when you generate this,

  • now you have a linear c30, which,

  • of course, is a complete hydrocarbon and is insoluble.

  • So this now sort of defines that you

  • need to be in the membrane to be able to do

  • any further chemistry.

  • So those of you who are interested in mechanisms

  • of how things work, that's really

  • sort of a fascinating system it's really pretty well

  • worked out at this stage.

  • But today what I mean to do is focus on

  • the next step is, how do we get from C30, which

  • is this linear squalene hydrocarbon, into lanosterol,

  • which is then the precursor to steroids

  • but also the precursor to cholesterol,

  • which is what we're focusing on in this particular module.

  • So what we're going to be looking at

  • is how we went from two FPPs--

  • we're still doing chain elongation-- to a C30.

  • And then the question is, how do you

  • get from C30, which is linear, to a linear epoxide.

  • And I'm not going to draw the whole structure out,

  • but we're still linear.

  • And then the next step is the step I want to talk about.

  • So this is when lanosterol synthase.

  • So that's where we're going in the next few minutes

  • to get to our final product.

  • So if you look at this reaction, remember,

  • we're going to do a cyclization.

  • And what do you need to do to do cyclizations?

  • What was the general rule that I gave you last time?

  • Does anybody remember?

  • If you want to cyclize something, we talked about it.

  • We looked at a number of examples.

  • What did we do in those examples?

  • Anybody remember?

  • So here's a second example.

  • I gave you two rules.

  • If you go back and you look at your notes,

  • we protonated the olefin and that triggered off

  • the cyclization.

  • And here, perhaps you could have protonated the olefin

  • to trigger off the cyclization, but in the end,

  • cholesterol has a hydroxyl group in the C3 position.

  • So the next step in the pathway, which also will involve,

  • ultimately, protonation and ring cyclization,

  • so those are the two rules I gave you

  • during the last lecture, to get to this epoxide,

  • we have to do some chemistry.

  • Does anybody know what cofactors you

  • would use to do this reaction?

  • Anybody got any ideas from introductory biochemistry?

  • You have a vitamin bottle.

  • What vitamin would be involved in doing this transformation

  • or could be involved with doing this kind of a transformation?

  • It's an oxidation.

  • Requires oxygen gas.

  • So what are the possibilities?

  • AUDIENCE: NAD.

  • JOANNE STUBBE: So NAD.

  • Does NAD-- this is a good teaching point.

  • Does NAD react with oxygen?

  • Who suggested NAD?

  • Why doesn't it make you react with oxygen?

  • That's one of the things you learn

  • in any introductory course.

  • NAD does not react with oxygen. Why?

  • What is the chemistry of NAD/NADH?

  • Whoa.

  • Maybe I should be teaching 5.07.

  • So NAD/NADH, we just went through this

  • with conversion of acetyl CoA moiety of mevalonic acid

  • to the alcohol.

  • It involves hydride transfer.

  • And if you tried to do this chemistry instead of two

  • electrons at a time, one electron at a time,

  • and you looked at the reduction potentials,

  • it would be way uphill, thermodynamically.

  • So NAD/NADH never does one electron in chemistry.

  • So that's not going to be a possibility.

  • Yeah?

  • AUDIENCE: You could use something that's like iron?

  • JOANNE STUBBE: So that would be one thing.

  • And we're going to see iron--

  • heme irons play a key role in all of this process.

  • This turns out to be a flavoprotein.

  • That's the other redox active cofactor.

  • So this is a flavin monooxygenase.

  • You don't need to remember this.

  • We understand the details.

  • I'm not going to talk about the detailed mechanism,

  • but flavin cofactors are extremely well understood.

  • The chemistry of them is extremely well understood now.

  • So we've gotten to our oxidosqualene

  • and now we've finally gotten to this really cool step.

  • So how do we go from this step--

  • so this is this molecule here.

  • And what I'm emphasizing again is

  • we're going from a linear step into the cyclic product.

  • So remember, triggering off cyclization,

  • there were two rules-- protonation,

  • protonation of an olefin.

  • In this case, you have some kind of protonation of the epoxide.

  • Epoxides are not very good leaving groups.

  • You need to protonate it.

  • And that is then going to trigger off

  • this cascade of reactions to allow you to generate

  • a molecule with four rings.

  • And this all occurs with a single enzymatic step.

  • And so the way you can visualize this happening--

  • and again, you don't need to copy this down.

  • It's all-- if you look at your handouts ahead of time,

  • there's some things that are written down

  • that would take you 10 minutes to copy

  • and then you probably get it written down incorrectly

  • because you're looking like this is.

  • The hard things that are hard to write down

  • are all given to you in your handouts.

  • You can write it down if you want, that's fine.

  • So what we want to do is we want a ring open,

  • so we need to protonate the epoxide,

  • and that generates what?

  • A carbocation.

  • And then now what happens?

  • We generate another carbocation.

  • And now what happens?

  • We generate another carbocation.

  • And now what happens?

  • We generate another carbocation and we end up

  • with a carbocation at this position.

  • So I'm going to draw the structure of this.

  • So we have ring opened, and let me also

  • emphasize that the key to this process occurring

  • to give us lanosterol is the conformation

  • of the linear molecule.

  • So what do we see here?

  • What does this look like?

  • In terms of cyclohexanes, what does this look like?

  • If you have cyclohexyl rings, what kinds of conformations

  • do they have?

  • AUDIENCE: Chair?

  • JOANNE STUBBE: Chair and chair and boat.

  • So the key here is that you have a chair conformation here.

  • You have a chair conformation here,

  • but here you have a boat conformation.

  • And one of the general rules I told you

  • last time about terpene chemistry

  • in general was, what do the enzymes do in the active site

  • to transform something that's linear into something that's

  • cyclic?

  • They need to fold the molecule into the right conformation.

  • And that can, in part, be done, the fact

  • is the active site is very hydrophobic.

  • We talked about that.

  • And you can also have aromatics that could potentially--

  • I'm not drawing out all these intermediates,

  • but could potentially facilitate not only the conformation

  • but stabilization somewhat of the intermediates

  • that you observe along the reaction pathway.

  • So here's another example of the importance

  • of shape to defining the chemistry that's

  • actually going to happen.

  • And in contrast to the enzymes we

  • talked about last time, which were type I. You probably

  • don't remember that.

  • But this is, again, a different super family

  • involved that you observe, and it's observed quite frequently.

  • So these are type II.

  • So if you look up the structures, and in the article

  • you had to read by Christiansen, the second type of structure.

  • There are two general types of structure.

  • This is the second type of structure involved in making

  • interesting terpene molecules.

  • So what I'm doing now is showing you

  • how we've cyclized this to leave us with a carbocation.

  • And remember, if you have just a stick as opposed to a stick

  • with a hydrogen, that's a methyl group.

  • So here at the ring juncture, we have a hydrogen.

  • We have a trans ring juncture.

  • And again, if we have a stick with nothing on it,

  • it's a methyl group.

  • And we're into a chair conformation again,

  • and then we need to attach the last ring

  • so we have three six-membered rings and a five-membered ring.

  • And in the end, what have we generated?

  • We've generated a carbocation.

  • So I've written this as a single step.

  • Nobody has seen the intermediates.

  • You could write it is multiple steps.

  • I mean, the fact is it would be--

  • it's pretty hard to trap any of these carbocations,

  • and people have spent a lot of time trapping them.

  • So what you see, I think, is quite amazing,

  • but we aren't finished yet because we have a carbocation

  • and we need to get rid of that.

  • And what you need to do and this is-- you

  • will have one of these problems on the problem set

  • that will be due next week.

  • You'll be given something simple,

  • not as complicated as cholesterol.

  • But what you need to think about is

  • where do all these methyl groups end up in.

  • What's the stereochemistry of the reaction?

  • So then this geometry becomes critical

  • if you're thinking-- you need to think

  • about the stereo electronic control of hydride and methyl

  • anion equivalent migrations.

  • So what you have in this particular reaction

  • is you're going to have--

  • and I like this example because, again, I gave you

  • a set of rules that you can see that are associated, typically,

  • with carbocation reactions in general,

  • and this one does all of them.

  • So one of the rules was that you have

  • hydrogen migrate with a pair of electrons, so that's a hydride.

  • Again, you have a second hydrogen migrate

  • with a pair of electrons.

  • So I'm not drawing out all the intermediates.

  • Now what we have is a methyl group

  • migrate with its electrons.

  • We now have a second methyl group

  • migrate with its electrons.

  • And in the end, we're left with a cation here,

  • and the last step in many of these reactions

  • is loss of a proton.

  • So here we would have loss of a proton.

  • And if you look at the chemistry and you

  • look at the final product, which I'm not going to draw out,

  • you end up with this molecule.

  • So this is a flat rendition of what I've actually

  • drawn on the board.

  • So this is an example of all of the chemistry I talked about

  • as being general in all of these 70,000 terpenes.

  • You'll find most of them don't do all of the chemistry.

  • This one does all the different kind

  • of chemistries associated with carbocation type chemistries

  • that hopefully some of you have learned

  • about in introductory organic chemistry classes.

  • So again, to me, this is the most amazing reaction

  • I think I've ever seen.

  • I told you already that I heard about this in 1969

  • when they'd just figured out that this

  • could happen enzymatically.

  • And this became the basis, for those of you,

  • if there are any synthetic people here,

  • people doing cascade reactions.

  • Kim Jameson's lab does this, but back in those days,

  • they were using this approach, trying to define the folding

  • to do all these steps, just like nature had figured out

  • how to do this.

  • And if you look at the number of asymmetric centers,

  • you end up with seven asymmetric centers and no other products

  • that people could detect.

  • So this is quite an amazing feat.

  • So this is the model that I just drew on the board.

  • And so we still aren't quite there yet

  • because if you look at this structure

  • and you look at the final structure of cholesterol,

  • you have a methyl group here, here,

  • and you're going to have a methyl group here.

  • So we have 1, 2, 3 methyl groups.

  • And if you look at the final product, cholesterol,

  • they're all gone.

  • So you need 19 more steps to get to cholesterol.

  • This is not a simple biosynthetic pathway.

  • So to get from cholesterol--

  • so this is lanosterol.

  • So we've gotten to the precursor to steroids and cholesterol.

  • And when we start talking about regulation,

  • you'll see that lanosterol is, again,

  • a central player because it can partition

  • between different kinds of natural products

  • that we aren't going to be talking about,

  • other kinds of natural products we aren't

  • going to be talking about.

  • But to get to cholesterol, which I'm abbreviating from now

  • on as Ch, it's 19 steps.

  • So let's go over here.

  • My goal is not to teach you about the chemistry of all

  • this.

  • I'm not sure how easily you can see it.

  • Hopefully, you have the handouts with you,

  • but we have this methyl group, this methyl group,

  • and this methyl group that need to be removed over here.

  • So that methyl group is gone and these two methyl groups

  • are gone.

  • So how do we do that?

  • And so all of this reaction-- so we

  • have loss of three methyl groups.

  • And all of these reactions are catalyzed

  • by one kind of enzyme, which is a cytochrome P450

  • monooxygenase.

  • So we're going to see that all the reactions are catalyzed

  • by a cytochrome P450 monooxygenase, not a flavin

  • monooxygenase.

  • And if you look at the chemistry,

  • flavins are not anywhere near as strong are

  • oxidants as heme-dependent oxidation.

  • So if you have something really hard to oxidize,

  • you're never going to use a flavin.

  • You're going to use a heme.

  • And what do we know about all these enzymes?

  • I'm not going to talk about this in detail,

  • but you have an iron 3 heme.

  • And for those of you who don't remember what heme is,

  • we're going to be talking about this

  • in more detail in the section on reactive oxygen species.

  • It's a protoporphyrin IX.

  • That's what you see in hemoglobin.

  • It's the exact same co-factor you see in hemoglobin,

  • but what's distinct about this is

  • that instead of having a histadine ligand,

  • it has a thiolate ligand.

  • And that's key to why P450s can catalyze

  • these inactivated hydroxylations--

  • can catalyze hydroxylations of unactivated bonds

  • where this hemoglobin reversibly binds oxygen.

  • So these P450s use this heme system in an oxygen system.

  • And what did they do?

  • And so what I do is refer you over here to-- let's

  • look simply at 7 through 10 and we're

  • removing this methyl group.

  • So we're removing this little methyl group in the A ring.

  • The first ring is the A ring.

  • Sorry.

  • So stereo specific, and so I'm not drawing

  • the rest of the structure.

  • And our goal is if we go through 9 through 10

  • and then 11 through 13, we want to get

  • rid of both of these methyl groups.

  • And it's thought that one enzyme, but they don't know,

  • can catalyze multiple oxidations.

  • And why don't they know?

  • Where do you think all was chemistry happens?

  • You have cholesterol.

  • What do we know about the structure of cholesterol?

  • It's a grease ball.

  • So where do you think the chemistry happens?

  • AUDIENCE: In the membranes.

  • JOANNE STUBBE: In the membranes, yeah.

  • And so that's been--

  • P450s, you go to meetings, thousands of people

  • still go to P450 meetings on the major targets

  • of all kinds of therapeutics, and they're

  • almost all membrane-associated, which

  • has been problematic in terms of isolation and characterization.

  • And here, despite a lot of effort,

  • people really still don't know the sequence of events

  • or have isolated and purified the enzymes.

  • They're all in the ER, which is what

  • we're going to come back to, and there

  • are a membrane-associated.

  • So what happens in these reactions

  • is you take a methyl group and then you

  • oxidize it with one P450.

  • So we somehow use oxygen-iron chemistry

  • to do a hydroxylation reaction.

  • Have you seen that before, in the first part of the semester?

  • Anybody remember seeing it?

  • Maybe you didn't see it.

  • I missed a couple of lectures.

  • Do you remember seeing hydroxylation reactions

  • anywhere?

  • Liz, do you talk--

  • was that in any of the natural products?

  • AUDIENCE: Sometimes [INAUDIBLE] P450s [INAUDIBLE]..

  • JOANNE STUBBE: But what you'll see--

  • I think this would be, like, a decorating

  • module that you saw in the non-ribosomal peptide

  • synthetases.

  • But here these things, as in the non-ribosomal peptide

  • synthetases, are absolutely specific.

  • And so you have one hydroxylation,

  • you have a second hydroxylation, you

  • have a third hydroxylation, which is chemically distinct.

  • And then the question is, how do you get rid of this altogether?

  • Because our goal is to remove the methyl.

  • That's what our goal is.

  • So we've gone hydroxymethyl, the aldehyde, the acid.

  • So now you have an acid next to the alcohol.

  • How do you get rid of that?

  • Has anybody-- what kind of chemistry

  • could you do to allow you to lose the CO2?

  • AUDIENCE: [INAUDIBLE].

  • JOANNE STUBBE: You need to speak louder.

  • Don't be-- I mean, just tell me what you think.

  • AUDIENCE: Decarboxylation.

  • JOANNE STUBBE: The what?

  • Decarboxylation.

  • But can you decarboxylate--

  • so you're right.

  • We want to decarboxylate.

  • AUDIENCE: [INAUDIBLE].

  • JOANNE STUBBE: What do you have to do to decarboxylate?

  • AUDIENCE: You reduce the alcohol [INAUDIBLE]..

  • JOANNE STUBBE: Reduce the alcohol?

  • AUDIENCE: [INAUDIBLE].

  • JOANNE STUBBE: What?

  • What are you going to do?

  • These are the kinds of--

  • you'll see these reactions happen over and over

  • again in biochemical pathways.

  • AUDIENCE: Oxidize [INAUDIBLE].

  • JOANNE STUBBE: Right.

  • You want to oxidize it.

  • So what happens, if you look at this pathway over here,

  • in this step, you use-- it should be NADP,

  • so you use NADP.

  • And what does that do?

  • I'm not going to write this out, but it oxidizes this

  • to a ketone.

  • And now what do you have?

  • You have a beta-ketoacid.

  • And beta-ketoacids rapidly undergo

  • decarboxylation reactions.

  • So this is a strategy that nature uses over and over again

  • in many biosynthetic pathways.

  • And the thing that's interesting,

  • if you look at that pathway in detail-- and again,

  • you're not responsible for that--

  • but then it does the same thing on the next methyl.

  • So in the end, you end up with a carbon with two hydrogens here.

  • But it's not straightforward, but this kind sequence

  • of events you actually see a lot in metabolic pathways.

  • So I don't want to really say much more about this.

  • In 19 steps, you need to remove three methyl groups.

  • All the enzymes are ER bound, making

  • it difficult to study the individual enzymatic reactions.

  • And we would like to know the order,

  • but we don't know it at this stage.

  • What we know is what we see at the end.

  • So finally, I wanted to get here at the end of lecture 2.

  • We've gotten here a little later.

  • We've started with acetyl CoA.

  • We've made the major building blocks for all terpenes, IPP

  • and dimethyl APP.

  • And we've gotten to form this very complicated molecule.

  • Everything starts with acetyl CoA and you can--

  • this was classic work by Konrad Bloch,

  • who won the Nobel Prize for this work, who

  • then by doing label chasing, which you learned about,

  • hopefully, in introductory chemistry,

  • helped them to figure out this complex biosynthetic pathway,

  • which isn't so easy because things are membrane

  • bound and very lipophilic.

  • So we've gotten to cholesterol.

  • So this module is on cholesterol and we've

  • been able to biosynthesize it through an amazing sequence

  • of reactions that have been studied over the decades.

  • But we can also get cholesterol--

  • we want to ask the question, first of all,

  • why are we interested in cholesterol?

  • I think you've already seen hints

  • of that with the statins inhibiting HMG-CoA reductase.

  • We have issues when cholesterol levels are too high or too low.

  • We need to control the levels of cholesterol.

  • And the second way we can get cholesterol besides making it

  • is we get it from our diet.

  • So if we get it from the diet, the molecule we'll see

  • is not very soluble.

  • How is it distributed into the tissues?

  • And then if you've distributed a lot of cholesterol

  • from your diet, you certainly don't want

  • to keep making cholesterol.

  • So the question is, how do you control those two events?

  • What are the general mechanisms of regulation

  • of the levels of cholesterol?

  • And we're going to at the end look

  • at some of the classic experiments

  • that Brown and Goldstein did to understand how cholesterol,

  • which from the diet can get into the bloodstream,

  • can get transferred into cells.

  • And so that's where we're going.

  • And again, the reading is a reading

  • I've already given you before.

  • So why do we care about this?

  • We have a 30-step synthesis.

  • We're getting it from the diet.

  • We have key issues in homeostasis, which is

  • what our focus is going to be.

  • So why do we care about cholesterol?

  • We care about cholesterol because it's

  • associated with human health and coronary artery disease.

  • Probably many of people who have had heart attacks.

  • And so elevated cholesterol levels

  • have been known for some time to be associated with plaques,

  • artherosclerotic plaques, which can lead to heart attacks

  • and strokes.

  • So what happens is the cholesterol deposits,

  • you try to remove the cholesterol,

  • you generate a lot of scar tissue,

  • which then inhibits blood flow.

  • And then you're in trouble if you can't figure out

  • how to unblock the blood flow.

  • So that's the main motivator and we'll

  • see another main motivator is related to young children dying

  • of heart attacks, which is what got

  • Brown and Goldstein into the area of cholesterol

  • homeostasis.

  • So there have been three Nobel Prizes given for work

  • on cholesterol over the years.

  • This is a classic paper.

  • Some of the classic papers are actually

  • quite interesting to read, and often the original papers

  • get things wrong.

  • So it was mostly right, but not completely right.

  • But anyhow, I think if you put it into the context, 1928,

  • how would you do experiments like that?

  • We had no IR.

  • We had no MR. We had no mass spec.

  • What did we have?

  • We had ways of degrading things.

  • People don't do that anymore.

  • If you go back and you look at the discoveries

  • before 1970 or something, these feats

  • of pulling out the structures with the right stereochemistry

  • is really, I think, quite astonishing.

  • And I think what's most amazing to

  • me is this old literature is actually

  • much more reproducible than anything

  • in the current literature.

  • The current literature, we're spewing out papers,

  • a lot of which will never get reproduced

  • so we won't know if it's reproducible.

  • But if you go back and you do anything, in the old days,

  • you had to learn German because a lot of the original papers,

  • all of the chemical papers, were in German.

  • They did seminal experiments back in those days.

  • And most of the time it was correct.

  • So anyhow, these guys figured out the structure almost.

  • And then Konrad Bloch figured out, along with Fritz Lynen,

  • figured out how you make cholesterol

  • by labeling experiments.

  • Now, many of you-- how many did label

  • chasing in an introductory biochemistry course?

  • Any of you have problem sets with label chasing?

  • So it's quite distinct.

  • I taught with John Essigmann.

  • All those problems were label chasing

  • and I used to say, oh, no.

  • Who wants to do label chasing?

  • But the fact is now if you read any

  • of the current papers in the literature, everybody

  • is label chasing.

  • And now we have much better ways of actually chasing labels

  • using mass spec methods.

  • So you can hardly pick up a journal nowadays

  • without thinking about label chasing.

  • So these guys who were way ahead of their time, but it

  • was much harder in those days.

  • And then here are Brown and Goldstein.

  • They won the Nobel Prize for the discovery of low-density

  • lipoprotein and may still win another Nobel Prize

  • for the regulatory mechanisms that we'll talk

  • about at the end of lecture 5.

  • So the first thing I want to talk about in lecture 3

  • is focused on--

  • let's see.

  • What do I want to do?

  • Is focused on the properties of cholesterol.

  • So we want to look at the properties of cholesterol.

  • Then we're going to ask the question,

  • how does cholesterol get from the diet to the bloodstream?

  • And then we're going to ask the question,

  • how does cholesterol get from the bloodstream

  • into the tissues where it's essential for membrane

  • controlling membrane fluidity?

  • So what do we know about cholesterol itself?

  • If you look at the structure, what do we have?

  • We have a grease ball and a little hydrophilic head.

  • And so this cholesterol moiety, if you look at the structure

  • up there, is really pretty rigid.

  • And it, in fact, rigidifies.

  • So this is rigid--

  • and in fact rigidifies membranes.

  • And so you can see this if you go back and look at this.

  • Hard to see these little things, but those are cholesterols

  • stuck within the phospholipid bilayers.

  • And this is key since this is something

  • that I think a lot of people are spending a lot more time on

  • and we're getting much better at this now.

  • People have stayed away from membranes because it's so--

  • and membrane proteins because it's so hard to work with

  • and they stick to everything.

  • How do you control all of this?

  • And Brown and Goldstein really did

  • some of the classic experiments that

  • taught us how to deal with these type of really

  • hydrophobic molecules.

  • And so cholesterol is pretty important.

  • 10% of the membranes actually have--

  • of the lipids in the membranes are from cholesterol.

  • So if you look at this, you would think

  • it wouldn't be very soluble.

  • And in fact, the solubility of this--

  • solubility is about five micromolar.

  • So it isn't very soluble, but in fact, as an adult,

  • we have 35 to 50 grams--

  • we each have 35 to 50 grams of cholesterol.

  • And we know that per day 1 gram is derived from synthesis

  • in the liver, so the predominant organ

  • where cholesterol is made, like we just were describing,

  • is the liver.

  • But we also have--

  • and I don't know how good these numbers are.

  • I got them out of some book.

  • So I'm not an expert in this, but anyhow,

  • these are all rough numbers and you'll

  • see these in other nutrient uptake systems.

  • You want to have some vague idea of the contributions

  • to the two distinct processes.

  • We get from the diet, say, 200 to 300 milligrams

  • from the diet.

  • So then if you think about this, cholesterol we're going to see

  • is transported in the blood, and we'll see how that happens.

  • Whoops.

  • Transported in blood.

  • And we know something about the amounts.

  • And if you do a calculation, that

  • says that you would have five millimolar cholesterol.

  • So that's impossible.

  • The number is squishy, but it's impossible.

  • So you'd have this insoluble mess.

  • So the question is, how do you deal with it?

  • And so that's what we need to think about.

  • So how does cholesterol move--

  • I think [INAUDIBLE].

  • So how does cholesterol go from the blood

  • to tissues, given the solubility problems?

  • So here is again the structure of cholesterol.

  • Again, it's pretty rigid and it inserts itself into membranes.

  • Where do you get cholesterol from?

  • You all know you get cholesterol from beef and chicken and eggs.

  • I guess there aren't very many--

  • do any of you eat at McDonald's?

  • Or is that a passe thing?

  • I love McDonald's anyhow.

  • That was my favorite when I was in Wisconsin.

  • There was only one restaurant near where

  • the biochemistry department was and I went there every day,

  • and my favorite thing was like two of those things slathered

  • in cheese with French fries.

  • Anyhow, fortunately, I have very low cholesterol.

  • But anyhow, you get that our diet is

  • a major source of cholesterol and what

  • you eat can, in fact, be problematic and part of it

  • really sort of depends on how lucky you are genetically,

  • right?

  • That's sort of the key thing.

  • So what do we do?

  • We have this insoluble molecule and the question is,

  • how do how are we going to get this insoluble molecule

  • into the tissues where it's needed to control

  • the fluidity of the membranes?

  • That's the issue.

  • So the second thing I'm just going to introduce you to,

  • and this is taken from Voet and Voet.

  • So many of you may have read that if you

  • had 705 or something, but in 507 we don't cover this reaction.

  • So I'm going to spend a few minutes going over it.

  • So what has to happen is cholesterol

  • is found in lipoprotein particles.

  • And we know a lot about the composition

  • of these lipoprotein particles, which--

  • this is taken from Voet Voet.

  • And what you can see is-- and I think,

  • again, the relative amounts isn't all that important.

  • But you can see you have--

  • and we're going to be focused on low-density lipoprotein, which

  • is the major deliverer of cholesterol to the tissues.

  • And why is that true?

  • So if we look at free cholesterol,

  • we see we see 7% to 10%.

  • I'm going to tell you about the structure of the lipoproteins

  • in a minute.

  • But most of the cholesterol is actually

  • esterified with fatty acids, and you

  • can see that the cholesterol esters are 35% to 40%.

  • So if you look at the total amount of cholesterol

  • in the LDL particles compared to all the other particles,

  • it's much higher.

  • So what do you see and what do you

  • have to worry about if you're getting this from the diet?

  • So what would you expect to see?

  • You would expect to see proteins.

  • You would see phospholipids.

  • So this is a phospholipid.

  • You would expect to see triacylglycerol.

  • Everybody know what triacylglycerol is?

  • We expect to see fatty acids.

  • We expect to see cholesterol.

  • And if you look over here, what people have done,

  • have isolated different particles.

  • How do they isolate the particles?

  • The particles are isolated based on density differences.

  • And if you look at all of these different compositions,

  • they vary between very low-density lipoprotein,

  • intermediate-density lipoprotein,

  • high-density lipoprotein.

  • They have different amounts of these different species.

  • And in fact, most of them have very hydrophobic stuff

  • in the outside and more hydrophilic stuff--

  • on the inside and more hydrophilic

  • filled stuff on the exterior, which is more dense.

  • And then that tells you something

  • about how these things sediment by a subterfugation

  • method and a density gradient.

  • So these lipoprotein particles--

  • and we're going to see this kind of method

  • in next week's recitations--

  • are separated by the centrifugation

  • due to density differences.

  • So let's just briefly look at LDL.

  • That's what we're going to be dealing with today.

  • And it's important because LDL is

  • what we're going to try to take into the cell,

  • and the composition of the LDL is

  • key to thinking about how to studying

  • that process in the classic Brown and Goldstein

  • experiments.

  • So if we look at the cartoon of LDL

  • that you see up there, what you see is a particle.

  • They're sort of circular.

  • The LDL particles, which is what we're going to be focusing on,

  • low-density lipoprotein particles,

  • have only a single protein and this protein

  • is called the ApoB.

  • It's a huge protein and it covers about 50%

  • of the surface of the particle itself.

  • Again, these things change in size.

  • We'll see when we actually look at the transport process.

  • What do we know?

  • It's on the exterior.

  • On the exterior what you see all over the exterior,

  • these things that are phospholipids.

  • So the phosphate is on the outside,

  • the fatty acids are on the inside.

  • What else do you see on the exterior?

  • You see a lot of cholesterol molecules,

  • which I'm indicating like this.

  • And then it turns out that the predominant-- and that maybe

  • covers, I don't know, 20 Angstroms,

  • but the particles are 200 Angstroms, 220 Angstroms.

  • So what's in the center?

  • And what's in the center, so this is the interior.

  • You basically have triacylglycerols

  • and then you have cholesterol.

  • And remember, cholesterol has one lone 3 prime hydroxyl

  • group, and this is a esterified with a fatty acid.

  • And so this is also in the interior.

  • So that's the composition of the LDL particles.

  • And the question is, again, where

  • did they come from starting with stuff we get from the diet?

  • So that's what we're going to be focused on

  • and that's what we're going to try--

  • the cholesterol is stuck on the surface and in the interior.

  • Yeah?

  • AUDIENCE: I don't understand.

  • Aren't there different splice variants ApoB

  • and which one is the one that's involved now?

  • JOANNE STUBBE: There could be.

  • There could be We're not talking about this in detail at all.

  • I don't know how many splice variances there are.

  • And I don't really know that much about all

  • of these different proteins.

  • You'd have to go read about them in detail.

  • So I'm giving you sort of a cartoon general overview

  • of what you need to think about.

  • There are splice variance of almost any protein.

  • And in humans, you have in the PCSK that we talked about,

  • there were nine isozymes.

  • So isozymes and eukaryotic systems

  • are something you don't have to worry

  • about a lot for this lecture and for what I want to say.

  • You don't have to worry about that.

  • And if you want to read about it, go for it.

  • So we have LDL particles and they

  • are distinct from all these other particles which

  • have different densities, different proteins.

  • And there are two cartoons I want to use.

  • This cartoon was taken from Voet and Voet.

  • I think it was the third issue.

  • The one from the fourth issue, which

  • I'll show you in a minute, I think, is much better.

  • So I'm going to change the handout.

  • But I just want to very briefly walk you through this.

  • This is a really complicated process and, from my reading,

  • is really not completely understood.

  • But you have diet.

  • And what do you have in your diet?

  • Triacylglycerols, phospholipids, proteins.

  • They get taken-- in the diet, they get into the intestine

  • and somehow in that process they need

  • to get packaged into one of these lipoprotein particles.

  • And the lipoprotein particle that

  • really is the predominant one that comes out of the intestine

  • are these things called chylomicrons.

  • And you can see they have a lot of proteins.

  • They have a lot of triacylglycerol.

  • They have a lot of phospholipids.

  • Anyhow, the composition varies and the sizes also vary.

  • So these chylomicrons come from the diet.

  • So how do these lipoproteins deliver LDL

  • to the extrahepatic tissues?

  • That's what we're really after.

  • And so these chylomicrons-- let me just

  • show you the next slide for a second.

  • I think this is probably a better one, anyhow.

  • So these chylomicrons, somehow they

  • have to package all this stuff into these lipoproteins.

  • You know how that happens?

  • I don't really know very much about it.

  • Maybe somebody does.

  • I don't know that much about it.

  • So anyhow, it gets packaged into these little particles

  • and then it goes into the intestinal lymph, which

  • then goes into the bloodstream and then it

  • needs to start circulating.

  • So everything comes from the diet

  • comes from these chylomicron particles.

  • So what happens when you go adjacent to adipose

  • tissue or muscles?

  • So what you need, if you're going

  • to be involved with fat metabolism

  • or you need energy to run down the street,

  • you need fatty acids.

  • So where do the fatty acids come from?

  • They come from the triacylglycerol.

  • So what you have are lipases and all of these chylomicrons

  • in the lipases.

  • Then when you get near the tissue--

  • let's see.

  • Here we get near the tissue, the muscle or the adipose tissue,

  • a li-- does everybody know what a lipase is

  • or do you want me to write that reaction on the board?

  • Does everybody know what a lipase is?

  • No?

  • OK.

  • So a lipase-- so here is your triacylglycerol

  • with different fatty acids.

  • So this is a TAG.

  • This is glycerol.

  • It's stereo-- this is a chiral center.

  • And so what happens then is lipase

  • is simply an esterase that hydrolyzes the bond.

  • So I'm not going to draw the reaction

  • out, but a lipase catalyzes, release--

  • this is a fatty acid.

  • And it actually cuts off two of them, and so most of the time

  • you have monoacylated fatty acids.

  • But again, from what we're talking about,

  • this is not really important because really

  • what we want to do is get to the low-density lipoproteins.

  • So what you see when you start doing

  • this is that if you drop off triacylglycerols

  • or monoacylglycerols here, and you drop off

  • something else to the other tissues along the way,

  • the sizes of your particles change size.

  • So they call these things, then, the remnants

  • from your starting material.

  • And it turns out there is a receptor that

  • takes up chylomicron remnants into the liver.

  • So the central player in all of this is the liver.

  • So you got to take stuff--

  • we get it from the diet, but we got to get it into the liver

  • and that's where everything is controlled.

  • And that's predominantly where cholesterol is actually

  • biosynthesized.

  • So if you start--

  • you bring in-- what are you bringing in?

  • You're bringing in cholesterol because you've dropped off

  • triacylglycerols and lipids and phospholipids to the tissues.

  • So what's predominantly left?

  • What's predominantly left is cholesterol.

  • So from these chylomicrons you drop off fatty acids

  • and monoacylglycerols to adipocytes or muscle,

  • and then what you have left is cholesterol.

  • You have a lot of stuff left, but cholesterol

  • is a major thing, which is then going to go to the liver.

  • And so then once this gets into the liver,

  • the liver has all this machinery to repackage things

  • and they can make very low-density lipoproteins again.

  • So you can go back and look at this.

  • It's very complicated.

  • They then in the bloodstream can drop off stuff along the way

  • to tissues as well.

  • And then they change into intermediate-density

  • lipoproteins, which then can change

  • into low-density lipoproteins.

  • And it's these low-density lipoproteins

  • that are going to then deliver cholesterol, that

  • has more cholesterol than any of these other particles,

  • either two extrahepatic tissues or back into the liver.

  • So you have a complicated set of transport systems

  • that we're not going to spend any time on,

  • but it's all related to the fact that cholesterol is basically

  • not a happy camper in water.

  • And so we've got to figure out how to move cholesterol around.

  • So that just summarizes--

  • what I didn't show you over here,

  • you all have heard about high-density cholesterol,

  • low-density cholesterol.

  • And high-density cholesterol is distinct from all

  • these other lipoprotein particles.

  • And it sort of scavenges excess cholesterol

  • from these extrahepatic cells and returns it to the liver.

  • But unlike the look the receptor-mediated endocytosis

  • we're going to talk about with the LDL receptor,

  • this receptor is completely distinct.

  • I'm not going to talk about it, but the mechanism

  • is distinct from these other receptors

  • that people have also studied in some detail.

  • So the other thing that I wanted to briefly say

  • is that in addition to cholesterol what you see--

  • and I'm not going to spend much time on this either,

  • but I think it's worth mentioning--

  • is when you get cholesterol back into the liver,

  • what can you do with that excess cholesterol?

  • If you have too much of it, how do you control the levels?

  • That's the key thing we're going to be trying to focus on.

  • What have we learned, at least to some level,

  • in control of cholesterol levels?

  • But it turns out in the liver-- so the key organ in all of this

  • is the liver--

  • cholesterol can be metabolized to form molecules

  • that have four rings just like cholesterol

  • that are called bile acids.

  • And these are multiple steps.

  • I'm not going to draw out the steps,

  • but if you look at a bile acid--

  • and I have cartoons of bile acids over here.

  • So here's cholesterol and if you look at this--

  • it's hard to see it, but if you look at it,

  • it really sort of it looks a lot like cholesterol.

  • The only differences are you add additional hydroxyl groups.

  • So in cholesterol we have a 3-alpha hydroxyl group.

  • In the bile acid you have two additional hydroxyl groups put

  • on again by cythochrome P450s.

  • So you have a hydroxyl group at C7.

  • You have a hydroxyl group at 12 alpha,

  • simply means the stereochemistry.

  • So the stereo chemistry of the hydroxyl group

  • is on the same face.

  • So that's what I mean here.

  • So what you have then is hydrophobic and hydrophilic.

  • And in addition, if you look at the very end,

  • it turns out you have molecules glycine or taurine,

  • which are on the handout, which has a negative charge.

  • And again, it's on the same face.

  • So we have a bunch of hydroxyls, something charge,

  • and they act as emulsifying agents,

  • and that's all you really need to know.

  • So these become emulsifying and they really

  • play sort of key role in also helping to take things back

  • into the cell.

  • And this is a really complicated process.

  • And in fact, I think it was 15 years ago,

  • something, people used to try to remove

  • bile acids as a mechanism of controlling cholesterol levels.

  • And what you did was actually-- boy, I'm way over again.

  • What you actually did was eat--

  • have any of you ever worked with Dowex in an exchange?

  • You used to eat the resin you have in the lab

  • called Dowex because it would bind the negatively

  • charged materials.

  • And so, really, it was very hard for people to stomach this,

  • but that was before we had really sort of wonder drugs--

  • Dowex, eating Dowex in these little grainy resins.

  • You should go look in the lab if you're doing a UROP.

  • That's how we used to treat high levels of cholesterol.

  • So anyhow, bile acids also play a key role.

  • We're not talking about this in detail.

  • So the next time we're going to come back

  • and we now sort of see what the properties of cholesterol

  • are, that they're in lipoproteins.

  • And we want to focus on the key experiments that showed

  • how LDL is taken into cells.

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