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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,