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  • JOANNE STUBBE: This is the second recitation

  • on cholesterol, and it's really focused

  • on this question of how do you sense cholesterol

  • in a membrane?

  • So that's really a tough problem.

  • And they've developed new tools, and that's

  • what we're going to be talking about-- what the tools are,

  • and whether you would think they were

  • adequate to be able to address this question about what kinds

  • of changes in concentration of cholesterol.

  • Number one, can you measure them?

  • And number two, what effects do they have,

  • in terms of whether you're going to turn on cholesterol

  • biosynthesis and uptake, because you need more cholesterol,

  • or you're going to turn the whole thing off?

  • So we've been focusing, as we've described

  • in the last few lectures, in the endoplasmic reticulum.

  • And what would the cholesterol--

  • what kinds of changes in cholesterols

  • did they see in the experiments they were doing in this paper?

  • What were the range of changes that they saw?

  • AUDIENCE: 3% to 10%?

  • JOANNE STUBBE: Yeah, so see, something low.

  • Say they were trying to do this same experiment in the plasma

  • membrane-- how do we know it's the ER membrane that

  • does this sensing?

  • That's what the whole paper is focused on,

  • that's what everything we've focused on in class.

  • Say you wanted to do a similar kind of experiment

  • in the plasma membrane, do you remember

  • what I said about the levels of cholesterol?

  • So they distributed throughout the cell, in all membranes.

  • Where is the most cholesterol?

  • So if you don't remember, it's the plasma membrane.

  • So say, instead of having 7% or 8% of the lipids cholesterol,

  • say you had 40%--

  • that's an over-exaggeration-- do you

  • think this kind of an experiment would be hard to do,

  • that they've talked about in this paper?

  • So you would want to do this-- if you

  • tried to do the same experiment with the plasma membrane?

  • So the key issue that you need to think about,

  • is go back and look at the changes--

  • they did a whole bunch of different experiments.

  • The numbers are squishy, but they came up

  • with numbers that reproduced themselves, I thought,

  • in an amazing way.

  • But now say you wanted to do this

  • in the plasma membrane, where the levels of cholesterol

  • are much higher.

  • Do you think it would be easy to do?

  • Using the same tech techniques that

  • are described, that we're going to discuss, or not?

  • And what would the issues be?

  • Yeah?

  • AUDIENCE: So they had to deplete the cholesterol

  • from the membrane, and so that would probably

  • be hard to deplete it to a level that's low enough, so that you

  • don't get the activity.

  • Right?

  • JOANNE STUBBE: So, I don't know.

  • So that's an interesting question.

  • So you'd have to deplete--

  • so that's going to be it, we're going

  • to have to control the cholesterol levels.

  • But what change-- if you looked at the changes

  • in levels of cholesterol in the ER, how much did they change?

  • They change from what to what?

  • From-- 2% to 7%.

  • Say that you were in that same range of change that

  • was going to turn on a switch in the plasma membrane.

  • And say you could control the levels.

  • Do you think it would be easy to see that?

  • So you start with 40%, say, that's the norm.

  • Say the change was very similar to what

  • you see in the change in the ER--

  • do you think that would be easy to detect?

  • No, because now you have two big numbers,

  • and there's a huge amount of error

  • in this method of analysis.

  • So those are the kinds of things I'm trying

  • to get you to think about.

  • I don't know why it's the ER--

  • I mean, everybody's focused on the ER.

  • Could cholesterol and other organelles

  • have a different regulatory mechanism?

  • Or somehow be connected, still, to what's going on in the ER?

  • Could be-- I mean, you start out with the simplest model

  • you can get and you test it, but then as you learn more,

  • or we have more and more technology,

  • we learn new things, you go back and you revisit and rethink

  • about what's going on.

  • So the key question is, it's really

  • this switch of having cholesterol

  • that keeps it in the membrane, or not having cholesterol.

  • And the question is, what are the differences

  • in the levels that allow turn on of cholesterol-- biosynthesis

  • and LDL biosynthesis, which then allows uptake of cholesterol

  • from the diet?

  • OK, so that's the question.

  • And what does this look like?

  • And people hadn't measured this by any method,

  • and this model I've gone through a number of times in class

  • today, so I'm not going to go through it again.

  • Hopefully you all know that in some form in your head,

  • or you have the picture in front of you so you can remember it.

  • So these are the questions I want to pose,

  • and I want you guys to do the talking today.

  • And what I'm going to do is, I have most of the figures

  • on my PowerPoint, so we can bring them up and look at them.

  • And you can tell me what you see.

  • And then everybody might be seeing something different--

  • and so we're thinking about this differently,

  • and maybe we come to some kind of consensus about

  • whether these experiments were carried out well or not.

  • So one of the first things-- so these

  • will be the general things, and then we'll step through them.

  • But they wanted to perturb the cellular cholesterol levels.

  • And how did they end up doing that?

  • Did that make sense?

  • We talked a little bit about this already.

  • I mean, what did they use as tools to do that?

  • AUDIENCE: [INAUDIBLE]

  • JOANNE STUBBE: So you need to speak louder,

  • because I really am deaf.

  • Sorry.

  • AUDIENCE: So just, right here, they

  • were careful of the amount of cholesterol present in this?

  • JOANNE STUBBE: So that's one place,

  • so they can deplete cholesterol for the media.

  • But then what did they do?

  • So the whole paper is about this-- how did they

  • control the [INAUDIBLE]?

  • Let's assume that they can do that,

  • and they got good at that.

  • I think a lot of people have used that method,

  • and so they can deplete media.

  • So how did they deplete cholesterol?

  • There was some unusual ways to deplete cholesterol

  • in this paper.

  • Did any of you pick up on that?

  • AUDIENCE: A chemical that could bind to cholesterol.

  • JOANNE STUBBE: So did you think that was unusual?

  • Did any of you look up what that was?

  • AUDIENCE: It was a kind of carbohydrate

  • that can bind to cholesterol.

  • JOANNE STUBBE: Yeah, so but what was interesting about it,

  • it was hydroxypropyl--

  • remember HP, cyclodextrin.

  • We're going to look at this in a minute.

  • But what do we know--

  • what was the other molecule they used to add cholesterol back?

  • AUDIENCE: Another form of that molecule is--

  • JOANNE STUBBE: So methyl-cyclodextrin--

  • I'm going to show you the structure,

  • but they aren't very different.

  • So have any of you ever heard of cyclodextrin before?

  • People won the Nobel Prize for that, Don Cram won it,

  • Breslow spent his whole life studying host guest

  • interactions.

  • So you guys, I don't know what you teach you now anymore,

  • but that used to be something that was taught a lot,

  • host guest interactions, trying to understand

  • weak non-covalent interactions as the basis for understanding

  • catalysis.

  • But to me, that was--

  • immediately when I saw this, what the heck's going on?

  • So then I Googled it, and immediately--

  • and I don't know anything about hydroxypropyl-- you Google it,

  • you look it up.

  • And then you look at it, and if you were a chemist

  • and you were really interested in the molecular interactions,

  • you might make a model of it.

  • And then see, what is the difference between that one

  • little group, when you look at the structure, it's amazing.

  • And that's the basis of most of the experiments.

  • So you need to believe that they figured that out.

  • And that's not in this paper, so if you really cared about it

  • you would have to go back and read earlier papers,

  • and see what are the experiments that led them

  • to focus on these molecules?

  • How else did they end up getting cholesterol levels back

  • into the cell?

  • Do you remember what the other method was?

  • So we'll come back and we'll talk about this in a minute--

  • so that was one of the methods.

  • AUDIENCE: They added two kind of sterols.

  • JOANNE STUBBE: OK, so they did add two kind of sterols--

  • and they tried to figure out, this

  • is another unknown, what was the difference between the sterols?

  • Simply a hydroxyl group.

  • OK, so if you looked at this, cholesterol is this guy.

  • And then they had something like this guy--

  • 25, and remember where [INAUDIBLE] the side chain,

  • hanging out of the little [? cheer ?] system you have.

  • I don't think they learned very much from that.

  • And in fact, in your problem set,

  • you had all of these different cholesterol analogs.

  • I mean, I think we still really don't get it.

  • That's complicated-- we talked about this in class.

  • You have these transmembrane helices--

  • what is it that's actually the signaling agent?

  • So people are still asking that question,

  • and we haven't quite gotten that far.

  • But if you've read the reading, for HMG CoA reductase

  • degradation, which is what we we're

  • going to be talking about in class,

  • the signaler is not the sterile, it's lanosterol.

  • OK, and where have you seen lanosterol?

  • The biosynthetic pathway has lanosterol

  • sitting in the middle.

  • It's not all that different, structurally, from cholesterol.

  • You need to go back in, they all have four-membered rings,

  • they have different extra methyl groups.

  • So people are trying to sort that out.

  • I don't think we really know.

  • But how well?

  • So you're right, they use sterols.

  • They didn't use that, they didn't see very much difference

  • with the sterols.

  • What was the other way, which is sort of unusual,

  • that they added cholesterol back into the system.

  • So they could add it back with the methyl cyclodextrin--

  • they told you that that worked, and if you believe that--

  • and you look at the data-- it looked like that was happening.

  • Nobody remembers?

  • OK, well, we'll get to that in a little bit.

  • OK, so the question we're focusing on

  • is what are the changes in concentrations

  • of cholesterol in the ER?

  • So what method did they use to try

  • to separate the ER membranes from all the other membranes?

  • AUDIENCE: They first separated the [INAUDIBLE]----

  • JOANNE STUBBE: They separated the what?

  • AUDIENCE: The sterols and the nucleus in the [INAUDIBLE]..

  • JOANNE STUBBE: OK, so that's good.

  • You can separate out the nucleus,

  • and you could do that by ultracentrifugation-- we've

  • seen that used in different kinds of ultracentrifugation.

  • We've seen the different particles,

  • the lipoproteins in the diet, how do we separate those?

  • We talked about that in class briefly,

  • you haven't had any papers to read.

  • But what was the method of separation?

  • If you look at all those particles--

  • remember we had a little cartoon of all the particles,