R9. Cholesterol Homeostasis and Sensing
18
September

By Adem Lewis / in , , , , , , , , , , , /


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visit MIT OpenCourseWare at ocw.mit.edu. 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, and we focused on LDL,
which is the particle that has the most cholesterol. So that’s why everybody
is focusing on that. What was the basis
of the separation? AUDIENCE: Was it
sucrose screening? JOANNE STUBBE: Was the what? AUDIENCE: Was it a
sucrose screening– the ultracentrifugation? JOANNE STUBBE: You need to– AUDIENCE: Did they use
a sucrose screening, like ultracentrifugation? JOANNE STUBBE: Yeah,
ultracentrifugation. But how did the– AUDIENCE: For the
sucrose screening? JOANNE STUBBE: Yeah, OK, so
they have different density gradients. , OK so that’s
going to be a key thing, and that’s because if you
look at the composition, they have different
amounts of proteins, different amounts of fats. And they have different–
they float differently. So that’s the method that
they’re going to use here. Is that a good method? Can you think of
a better method? So in order to understand
the switch for cholesterol, you’ve got to be able to measure
the changes in cholesterol. Not an easy problem,
because cholesterol is really insoluble in everything. And so how much is
really in there, and how does it change under
different sets of conditions? So is this a good method? What do you think? We’ll look at the method in
a little more detail, when I pull up the figures,
but what did you think when you read the paper? AUDIENCE: Seems a
pretty good method, other than that they’re
slightly different any other like properties
different from the membrane than say, press on
golgi bodies and ER. So it’s like the only
one I can think of. JOANNE STUBBE: Yeah,
so the question is, you could you separate? Even separating the nucleus from
the cytosol is not so trivial. But these methods are
really gross methods, and during the centrifugation,
things diffuse. So if you’re having
close separations, it’s a equlibrating
down this thing. And so you’re getting
your proteins, or your lipids
are spreading out. Is there anything else any of
you experience with insoluble– this is what we’re dealing
with, is an insoluble mess, and how do you how do you
separate things in a way that you have control
over it so that you can address the key
questions in this paper? Nobody thought
about anything else? Did you like this method? Were you convinced by the data? AUDIENCE: I mean, like
I couldn’t necessarily think of something better. I don’t know, I
guess the thing that sketches me out the most
about it just like how– I’m not really familiar
with the method. I haven’t done this
myself, so I don’t know how that process affects
the membrane integrity. JOANNE STUBBE: So that’s an
incredibly important question, because lipids confuse. They can mix. The question is, what are the
rate constants for all of that? And we don’t really
teach very much in the introductory
courses about lipids, and they’re partitioning between
other membranes and fusion, and all that stuff. But if you think about it,
that’s what the cell is, right? How do you get a
plasma membrane, and all these membranes around
all these little organelles– that’s an amazing observation. And we’ve seen in
class already, what have we seen to get LDL
receptor from here to the plasma membrane? How do we have to do that? We had to use these
little vesicles. So you’re generating
something over here, it goes through the Golgi stack. Again, another set
of membranes has got to come out the different
levels of the Golgi stack. And then it’s still got to
get into the plasma membrane, and fuse, and dump its cargo. So I think it’s an
amazing process. And people interested
in evolution, this is one of the major
things people are focused on is, how can you make
cells, little fake cells, artificial cells, that
can replicate themselves. You can make it,
and they’re going to have to divide and fuse. And it’s exactly the
same problem here. And so this question of fluidity
is an extremely important question. And a lot of people
that focus on lipids– which is not a popular thing to
study, because it’s so hard– it’s incredibly important. And people that look
at membrane proteins, they almost always
have lipids on them. And when you do
them yourself, you have a detergent, which
is not a real lipid– does that change the property? So all of these
questions, I think, are really central
to what happens in the membranes, which is a
lot of stuff inside the cell. So I think it’s good to
question what they did. I think their results turned
out to be quite interesting. But we’ll come back– I think that was a hard problem. And so we’ll come back
and we’ll look at this. And so then, let’s say that we
could end up separating things. Then the question is, what was
the key type of measurement they made, where they
could correlate the changes in cholesterol levels– we talked about, you can
control perhaps the cholesterol levels with the cyclodextrin. But then, how did they
correlate the changes in the cholesterol
levels in the membrane with this transcriptional
regulation? Which, that is what happens
with the steroid-responsive element-binding protein,
the transcription factor. So what happens in that process? What are the changes
in the SRE BP dependent on the concentrations
of the cholesterol? And how did they take
advantage of that in answering this question
about what the cholesterol levels were that allowed
you to turn on transcription of LDL receptor, and
HMG CoA reductase. So what’s the major assay? We’ll look at that, as well. So if you go back and
you look at the model, what happens in this model? All right, here we go– what happens in this model? What’s happening to SREBP? AUDIENCE: It has
completely changed and exposed [INAUDIBLE]. JOANNE STUBBE: No, that’s SCAP– SCAP, that’s this guy. OK? So SCAP, that’s a key player. That’s what we talked about. I know the names
are all confusing. You’re going to need to
write these down to remember. The names are very confusing. Yeah? AUDIENCE: So the SCAP
SREBP, whatever you call it, complex move signal g-apperatus
then part of it’s cleaved and moves to the nucleus? JOANNE STUBBE: Right,
so how could you take advantage of that? This is the key observation that
they’re taking advantage of, to ask the question– since this whole process is
dependent on the concentration of cholesterol. If you have high
cholesterol, there’s no way you want this to happen–
you want to shut it off. If you have low cholesterol,
you want to turn these guys on. So this movement is the key. And what do we see,
if we look at what happens to this protein,
SREBP, what happens to it during this process? It gets cleaved. And how could you
monitor that cleavage? How do they do it in the paper? AUDIENCE: They used a– was it a [? florifor– ?]
or is that the homework? JOANNE STUBBE: They could
use a [? florifor, ?] they didn’t do that. They did a what? AUDIENCE: They were able to
separate the [INAUDIBLE] gel? JOANNE STUBBE: So it can
be operated by a gel. So to me, this is
quite an easy assay. Because if you look at this– I don’t remember what
the molecular weight is, but it’s a lot
smaller over here. And so, that turns out
to be a great assay. So that part of their
analysis, I think, was a really smart
part of the analysis. And so then the
question becomes, can you quantitate all of this? So if you have a
lot of cholesterol, this doesn’t happen. And so everything is bigger,
and resides in the membrane. You could even
probably look at that. Whereas, when the cholesterol is
really lower, things go there. And it’s everything in between. The question is,
what is the concept– can you measure if you have
X% cholesterol in the ER, how much do you have to decrease
it to see a change or a switch in where this protein goes? So I think the experimental
design is actually amazingly creative. But then you see the
data of the other side. And what I want to do now is
focus on what the issues are. So we’re going to
come back and look at, how did they look at SREBP? So you could look
at this a number of ways– you could look at
this by protein gel directly. How else do people look at
proteins using westerns? What’s a western? Anybody know what a
western analysis is? Didn’t I ask you that at
the beginning of class? How else do you detect proteins? You’ve seen this in the first
half of the semester a lot. Yeah, antibodies. So if you have
antibodies to this– and we’ll talk about this,
because the western analysis, which people use all the
time, and there are so many issues with
it, that I think I want you to think about
what the issues are. And then you correlate the two– changing the levels
of cholesterol. Which they measure by
mass spec after separation and purification of
lipids, and the cleavage. And they plot the
data, and that’s where they got
the analysis from. So the first thing that you
want to do– the first thing, and the key to everything, is
separation of the membranes. And so, this is a cartoon
of when you put something, you load something on the
top, and you have a gradient, and the gradient could be
made of a number of things. Have any of you ever run
these kinds of gradients? OK, so you can make
them out of glycerol, you can make them
out of sucrose– did anybody look at how
these gradients were made? Did you read the
experimental carefully enough to look at that? Yeah, how do you make
a sucrose gradient? You have no idea? But yeah, so layering. So what you really like to do
is have a continuous gradient, or something. But sucrose is
incredibly viscous. So if you were trying to
make a linear gradient, which you could do
by mixing two things of different concentrations–
if you could get them to stir really well, and then
add it in, and you could generate a gradient. But it’s so hard to
do, that what happens is they end up layering it. So they make X%, Y%,
Z%, they put it down. And then they try to layer
something on top of it. And then they put whatever
the interest in at the top, and then they centrifuge it. So what are the issues? Do you think this is what
the gradient would look like? So what are the
issues when you’re doing this, when you layer it? And this is why the data– which we’ll talk
about in a minute– is the data, or part of
the issue is this method. That’s why you need to
think about the method. And there are better
ways to do this. And it really depends on what
you’re trying to separate. So if this band– say these were two bands,
you wouldn’t really get very much separation at all. If there were two
separate things that sedimented under these
conditions very close together. So what would happen when
you’re sedimenting this? Does anybody have any
idea how long it takes? Do you think you’d do
this in a centrifuge, you spin it for three
minutes, and then– so sometimes you sediment
these things for 16, 20 hours. So what happens during
the sedimentation? That might make this
more challenging, in terms of separating
what you want to separate? AUDIENCE: I’m not sure, but
it [INAUDIBLE] diffusion. JOANNE STUBBE: Yeah, so
exactly, you have diffusion. And even when you’ve layered
things on top of each other like that, you start
to have diffusion. And if you shake up the tube
a little bit, it’s all over. So how do you prepare
these things is not– so people still
use these methods, but I would like to
see better methods. And so they tried one
method with sucrose, and then that
wasn’t good enough. We’ll look at the data. So they went to a second method. And where did they
come up with this? I have no idea where
they came up with this, but there was an MD PhD
student in our class who had seen this and
one of his classes, and they use it and
some blood test. So I think that’s probably
where these guys got it from, because Brown and
Goldstein are both MDs. But again, it’s just another
way to make a gradient. And I’m not sure why
this gradient works as effectively as it does. But the first gradient
didn’t work so great, and we’ll look at that data. So then they added
on a few more steps, because they weren’t happy
with the level of separation. So looking at
membranes, I think this is going to be more and
more looking at membranes, because membranes, you
have two leaflets– the lipids and the
leaflets are different. Do you think that
affects the biology? I guarantee you it affects
the biology in ways that we would really like
to understand that I don’t think we understand very well. When you isolate a membrane
protein, have any of you ever isolated a
membrane protein? So you have an insoluble– it’s in this lipid system. How do you think you
get it out, so you can go through the steps,
a protein purification that you’ve talked about,
or you have probably done in an introductory lab course? What is the first
thing you need to do? Yeah, solubalize it. And how do you solubalize it? AUDIENCE: With a detergent. JOANNE STUBBE: Yeah, with
some kind of detergent. It’s like what you saw with a
kilo microns, or the bile acids that we talked about. So you can use different–
and people have their own favorite detergents. But again, that changes things. But otherwise, you
can’t purify anything unless you happen to
have a membrane where the only protein
in the membrane is the one you’re interested
in, which, of course, doesn’t exist. So anyhow, they
went through that. And then what did
they end up seeing? So they went through
different steps, and they separate them into
different– the supernate, or the light and the
heavy membrane fractions. And then they have
to analyze it. And so the question
is, how do they analyze to tell how well these
separations actually worked? What was the method
that they did to determine whether
they separated the ER from the plasma
membrane, from the Golgi stacks, from the lisosomes,
from the peroxisomes. So they have all we have
all these little organelles in there. What did they do to test
each one of these fractions? Let me ask you this question–
how do you think they got the– how do you how did they get the
material out of these gradients to do the experiments that
I was just talking about. So they want to analyze
what’s in each of these bands. How did they get it
out of this tube? AUDIENCE: Would they
use a Pasteur filter? JOANNE STUBBE: So
what do you think? You just stick it down
in and suck it out? Well, I mean, yes,
so what do you think? You could do that– you open the top, you stick it
in, you carefully stick it in. If you can see it. Lots of times you can see these
lipids, because they’re opaque, or something. So you can see. Or, if you still hope your
sucrose layers, lots of times they layer in between the
different concentrations of the sucrose, and you see
white stuff precipitating. So you could conceivably
stick a pipe head from the top and suck it out. AUDIENCE: But that would
perturb all the other layers. JOANNE STUBBE:
Absolutely it would perturb all the other layers. So here you’re doing
something– it’s already a very hard experiment, because
they’re all being perturbed anyhow, because of diffusion. So is there any other
way you could think about separating these things? And so, the hint is that
they use plastic tubes. So these things are not glass. Most centrifuges– AUDIENCE: Freeze it? Cut it? JOANNE STUBBE: Well, so you
don’t do that, that could be– OK, so you could. But you then have to,
if you were cutting it, you still have to get
it out of the tube. Unless you had a saw that
didn’t have any vibrations when you were cutting it, of
course, which would not happen. But if you look here
in this cartoon, so I gave you this, what
are they doing here? They’re sticking a syringe in
through the side of the tube. And that’s still
what people use. So you can suck out– if
you can see something. So you have to be able
to see in some way to know where to suck it out, so
you might have a way, actually, in doing ultracentrifugations. I think with the lipids you
can see them by eyeball, but you might look
at absorption. If they have proteins, you
could monitor absorption through the gradient,
and that might tell you how to fractionate things. But anyhow, that’s
also an issue. Because before they can do
the next step in the analysis, they’ve got to get
the material out. So they’ve got the material
out in each of these steps, and then, how do
they look at this? They can pull it out. So what are they looking for? To tell them how
effective this method is. AUDIENCE: Maybe some specific
markers for each protein. JOANNE STUBBE: Exactly. So what are they– to do that, what they’re
going to have to do is, before we look at the
details of the method, I want to go through
a western blot. So what do we know
about a western blot? AUDIENCE: I have a quick
question about the method here. JOANNE STUBBE: About
the which method? AUDIENCE: The lysis
method [INAUDIBLE] ball bearing homogenizer. So they’re literally putting
these cells in something like a bunch of ball bearings? JOANNE STUBBE: Yeah,
you could do that. There’s a lot of ways
to crack open cells. I don’t know which
one’s the best– mammalian cells are
really easy to open. Sometimes what I
like to do is freeze and thaw them–
sometimes you have like a little mortar and
pestle, or something like that. But that’s– I mean, yeast
cells, you roll them. You have to have enough cells
so you can do something. If you only have a
tiny amount of cells, it makes it really
challenging with beads, because it covers the beads. AUDIENCE: Do you have any issues
with any of the different types of membranes that– JOANNE STUBBE: Sticking to that? Absolutely. I’m sure you have to look at
all of that kind of stuff. So how you choose,
that’s an important thing to look at, how you choose
to crack open the cells. And it’s the same
with bacterial cells– there are three or four ways
to crack open the cells. And I can tell you only one of
them really works efficiently. And a lot of people, when
they use some of the others, they do something and
they assume it works, but they never check
to see whether the cell walls have been cracked open. A lot of times they haven’t,
and so what you get out is very, very low
levels of protein, because you haven’t
cracked open the cell. So figuring out– mammalian
cells are apparently, I haven’t worked
with those myself, but they’re
apparently much easier to disrupt than bacteria. Or if you look at fungi– fungi are really hard
to crack open, yeast. So anyhow, that’s an
important thing to look at. So every one of these
things, again, the devil is in the details. But when you’re doing
your own research, it doesn’t matter what
method you’re looking at. The first time around, you
need to look at it in detail, and convince yourself
that this is a good way to chase this down. And you look at it in detail
the first time around. And when you convince
yourself it’s working really well, and
doing what you want to do, then you just use it. And that’s the end of it. You don’t have to go back and
keep thinking about this over and over again. So the method we’re going
to use is a western blot. So we’ve got this stuff
out, and have you all run SDS page shells? OK, so SDS page shells
separate proteins how? AUDIENCE: Based on size… JOANNE STUBBE: By the what? AUDIENCE: It separates into a
a charge gradient, and then– not a charge gradient, but– JOANNE STUBBE: Not charge. AUDIENCE: That’s what
drives the protein, but… JOANNE STUBBE: Right,
but it’s based on size, because it’s coded– every protein ratio is coded
with this detergent, sodium dodecyl sulfate, which
makes them migrate pretty much like the molecular weight. But if you’ve done
these, it’s not exactly like the molecular weight. You can do standards where
you know the molecular weight, you can do a standard
curve, and then you see where your
protein migrates. And sometimes they migrate
a little faster, sometimes a little slower, but it’s OK. So you run this, and
then what do you do? Does anybody know what you
do next, to do a western? AUDIENCE: You need to
use the membrane to… JOANNE STUBBE: Right,
so the next thing they did was they used– I’m going to put all of these
up– so they transferred it to a membrane. And why did they have to
transfer it to a membrane to do this analysis? This is an extra step. And it turns out– we’re going to look at
an antibody interacting with a protein. Why don’t we just look at
the antibody interacting with the protein to start with? AUDIENCE: It doesn’t have
access to the protein. JOANNE STUBBE: Right, it
doesn’t have very good access. It’s really not very efficient. So people found, pretty
much by trial and error, that you needed to transfer
this to a membrane. I mean, we have hundreds
of kinds of membranes. How did they choose
nitrocellulose? If any of you have
one run westerns, you remember what kind
of a membrane you used? Did you use nitrocellulose? You do this in undergraduate
class, don’t you? You don’t do a western? We used to do– AUDIENCE: Did it once
in undergrad class. JOANNE STUBBE: Yeah, in
what kind of a membrane? Was it in biology? AUDIENCE: Yes, biology. JOANNE STUBBE: So what membrane? Do you remember what
the membrane was? AUDIENCE: I think it was–
it was not nitrocellulose. JOANNE STUBBE: It’s
not nitrocellulose. So this PVDF, polyvinyl
difluoride is the standard one that people use now. It works much better than
nitrocellulose– this paper is really old, and so they’re
looking at nitrocellulose. So then they do this. And then, what do they do next? They have an antibody– we’ll look at the details
of this in a minute– that can recognize
the protein, that can find it on the membrane. And then what we’re
going to see is– you still can’t see
anything really, because you don’t have
very much material there. And you can’t observe– you don’t have enough to stain,
oftentimes, by Coomassie, so you’re going to have
to amplify the signal. So then you’re going to make
an antibody to an antibody. And then you have to
figure out how to, then, amplify the signal. And we’ll look at
that in a second. Is this what– you
ran a western, is this what westerns look like? AUDIENCE: I remember,
we first [INAUDIBLE] non-specific proteins
to occupy the sites. JOANNE STUBBE: Yeah,
so that’s good, you have to block everything,
if you’re using crude extract. So in this case, we would
be using the crude mixture– well, not a crude
mixture, it’s been fractured by the
ultracentrifugation that’s been fractionated. But you still have mixtures
of proteins in there. Have any of you ever looked
at westerns in a paper? Or even the papers
you had to read? The paper on the PC– go look at the PCK– PCSK9 paper, that
had westerns in it. What do you see? Do people show you something
that looks like this? And if they did show you
that, what would it look like? So you have an antibody that’s
specific for the protein of interest, whatever that
is– supposedly specific. What do you see? What do you think you see? Do you think antibodies
are specific? I think I have an example
of a typical western. AUDIENCE: I don’t think they’re
as specific as [INAUDIBLE] JOANNE STUBBE: Yeah. Yeah. So when you look at a paper,
you should pay attention to this when you read a paper,
if you’re doing anything in biology, what do you see? You never see a gel, ever. What you see is a slice of
a gel where they cut off this– the way they cut up all
this stuff and all this stuff. The reason they do that is
because it’s a hell of a mess. So let me just show
you a typical– I don’t care what
kind of an antibody you’re using, in crude
extracts, it’s a mess. Because you have
non-specific interactions. We’ll just look at that. So that would be something
like you might see– depending on how much antibody you have. So when you see this,
the reason everybody reports data like that now. So it looks like it’s really
clean, but in reality– I think if it is dirty as
that, then in my opinion, I would make you
publish the whole gel. But people don’t do that. They just cut off
the little band they’re interested in– they can
see it change in concentration using this method. But you should be aware of the
fact that antibodies in general aren’t as specific as you
think they’re going to be. Yeah? AUDIENCE: Are they required
to report the whole gel in supplementals? JOANNE STUBBE: I mean,
I think, it probably depends on the journal,
and it probably depends on the reviewer. But I would say, we’re
going away from data– is something that is
a pet peeve for me. And all the data, which
I think is all right, is published in
supplementary information, as opposed to the paper. I think if you have
something really dirty, you should publish in the paper,
in the main body of the paper. If you have something
that’s really clean, and it looks like that,
it’s fine with me. You don’t even
have to publish it, if you could believe
what people were saying. Because people know
what this looks like, a lot of people–
everybody uses westerns. But if it’s a real
mess, then you need to let your reader
know that this is not such an easy experiment,
and it’s not so clear-cut. That’s what your objective
is, is to show people the data from which you
drew your conclusions. And then they can draw
their own conclusions, which may be different. So let’s look at the
apparatus to do this. So how do you get
from here to here? So you have a gel, you run the
gel, a polyacrylamide gel– what do you do? AUDIENCE: Put the
membrane on the gel. JOANNE STUBBE: So you put
the membrane on the gel. And what do you do? AUDIENCE: [INAUDIBLE]
applying charges to. JOANNE STUBBE: Yeah, so
you’re transferring it based on applying a
voltage across this system. So here’s your gel. And here’s your membrane,
nitrocellulose membrane. And then they have filter
paper above the gel, and below the membrane. Why do you think they have
the filter paper there? When you ran the gel, did
you have filter paper? AUDIENCE: Yes. JOANNE STUBBE: Yeah. How do you think they decide
how to do this transfer? Do you think is a
straightforward? Do you run it for an hour,
do you run it for five hours, do you run it for 15 minutes? What is the voltage you
use to do the transfer? Do you think any of that
is hard to figure out? So how do you figure that out? Somebody told you that this
is a good way to do it? Yeah, so that might
be a place you start. So you do it because
somebody gave you a recipe. But then what do you
need to do to make sure this recipe is correct? AUDIENCE: Find out
what conditions that work for what
you’re working on. JOANNE STUBBE: Right, and
then how do you do that? So that’s true, every protein
is going to be different. And if you have a protein– if you have a clean protein,
versus a mess of proteins, and you try to do this transfer,
the transfer conditions will be different. So for example,
if you really want to look at the concentration
of something inside the cell, in the crude extracts,
you never compare it to a standard with
clean protein, because this transfer
is different. So you need– in the
back of your mind, if you care about
quantitating this, you need to understand
the basis of the transfer. So why do you think they have
these filter papers here? So this goes back
to what controls you would do to see whether
your transfer was working. So what would you look for? Did you do this? What did you do? What did you do with the
filter papers in your– AUDIENCE: You want to filter
all to the SDS molecules… JOANNE STUBBE: You did what? AUDIENCE: You want
to filter all– JOANNE STUBBE: No,
that’s not what you do. I mean, you might want
to do some of that, too, but in terms of thinking
about whether your transfer is successful– figuring out the conditions
to blot from the gel to a piece of paper
is not trivial. And there is a standard way that
you do this, initially, to try. But then you have to make sure
that that method is working. And lots of times
it doesn’t work. So it’s something that’s
going to be experimentally determined. So the question
is, what would you think would happen if you did
this for six or seven hours? Whereas, a normal blot
would take two hours? AUDIENCE: Would be transferred
onto the filter paper? JOANNE STUBBE: Right, it would
go right into the filter paper, or even off the filter paper. So what you do is you
take the filter paper out, you look for
protein being bound. What about the gel? What do you do with the gel
after your experiment’s over? AUDIENCE: Make sure a
protein’s not on it? JOANNE STUBBE: Right, make sure
that the protein is not on it. So these are simple
controls, but these are the controls you always
do until you work out the conditions to
make sure this works. And it’s pretty critical to make
sure you have good transfer. So then, so this is the
antibody thing that they do. Has anybody thought about
these kinds of assays? You’ve seen them, I
think, already in class. But what’s wrong
with this picture? The target protein, what’s
wrong with this picture in the target? So here’s your
nitrocellulose filter paper. What’s wrong with this cartoon? Should be unfolded, yeah. So you’re doing SDS
page, it’s unfolded. So then we react it
with an antibody. Presumably we have
a good antibody, but you’ve already
learned in the first half of this course that having
really good antibodies is not so trivial– you can get them,
but most of the time they are not specific if you’re
looking at crude extracts. They have little
epitopes they recognize, if you’re using
monoclonals that could be present in other proteins. And furthermore, how are
you detecting something? An antibody as a protein,
it has absorption of 280. Again, this is too low to see,
so putting an antibody on it is still going to be
too low to detect. So how do you
detect your signal? So have you done this? I’m surprised they don’t do this
in your introductory class– they don’t do westerns, at all. So what you’re looking at is
an antibody to an antibody. So you put your
antibody on, that’s specific for your protein. And then you make an
antibody in another organism that can specifically recognize
antibodies in general. So if this is to a mouse, you
make it to go and isolate that. And then what you do is
derivatize the second antibody with what? A protein? That can function as a catalyst. AUDIENCE: Why can’t you just
derivatize the first antibody? JOANNE STUBBE: Well, what? What did you say? AUDIENCE: It’s more expensive? JOANNE STUBBE: Well, no,
I don’t know whether it’s more expensive or not. But– AUDIENCE: Well, because
you’d have to derivatize every primary antibody. JOANNE STUBBE: So you’d
have the derivatize every primary antibody, and so
this is a standard procedure. You could derivatize
the primary antibody. So that’s not a bad question. And so what you’re
doing now, you can buy these commercially,
so they have rabbit, rabbit, mouse, whatever, antibodies. And the key is the
amplification of the signal, and you use enzymes
to amplify the signal. Does anybody know
what the enzymes are, what the enzymes do
to amplify the signal? AUDIENCE: You can covert the
molecule to a blue molecule… JOANNE STUBBE: To
something that’s colored. So does anybody know what
that horseradish peroxidase– have you ever heard of
horseradish peroxidase? So that’s a heme
iron– we’re going to be talking about
heme irons pretty soon, and hydrogen peroxide. It makes a chemically
very reactive iron oxide species, that can oxidize
a dye that changes color. And it has extremely high
extinction coefficients. So you can see it, and
it does it catalytically and the lifetime of
the dye is long enough. So it accumulates,
and you can get really amplification of your signal. Or you can use a
phosphatase that liberates something that’s
highly colored, again, and you can see it. So this is a standard
method that everybody uses. And so, that’s our gel. So now we’re
looking at sort of– at the end already– but
we’re looking at these gels, and what do you see through
the different steps? So if we look through
the first gradient, through the sucrose gradient,
that gets us through DNE. And if you look,
say, at lane E– our goal is to
separate proteins that are specifically localized in
each one of these membranes. So you need to
believe that’s true, that people have selected
the right group of proteins to look for. And you notice they
do more than one. So they look at
multiple proteins. Why do you think– do you think it’s easy to
select the proteins to look for? And why or why not? So, they obviously have
selected a group of proteins, and I think most people would
agree that they’ve selected a good group of proteins. But what do we know
now about proteins, do they stay in one place? No, they move around. But some might be present
in very low amounts, sometimes in much
higher amounts. And so you need to have more
than one protein as a control to make sure you’re looking
in the right region. And what do you see in E? If you look over here, it tells
you what the organelle is. And if you look at
this protein, this is localized to the lisosomes–
we talked about that in class. If you looked at
this protein, it’s localized to the peroxisomes. So in addition to the ones we
care about, the ER proteins, we’re also getting
proteins that are localized in other membranes. So that’s when they
went to the next method, and they added on
another gradient to try to separate out,
again, the lysosomal and the peroxisomal proteins. And you can see they were
pretty successful at this. There’s none of these proteins
left in this gradient. So that’s good. And they took it a step further. Do you remember what this is? What are they looking
for down here, in this? AUDIENCE: Enzymatic activity. JOANNE STUBBE: Yeah,
so enzymatic activity is localized in
certain organelles. So they again did
a second experiment to look at all of that. So they were very
careful in this, they figured out
how to separate. And that’s the
key thing for them to analyzing the
concentration of cholesterol in these membranes. And what they looked
at– we’re over time– but is the concentration
of cholesterol compared to the total amount of lipids. And how did they
do that analysis? Gene Kennedy, who’s at
Harvard Medical School– he’s in his 90s, now–
really trained all the lipid chemists in the whole country. And they figured
out many years ago how to separate lipid fractions
with methanol, chloroform extract, something that you
guys probably haven’t though about at all. But we’re really pretty
good at separating things, and it’s nothing more
than an extraction like you do as organic chemist
to purify and separate things. We’ve figured that out. And so then they use mass spec
to allow them to quantitate the amount of glycerol. And then in the end, so they use
mass spec, these western blots, and they can change
the concentration of the cholesterol and do
the experiments over and over again, to see what happens. And when they do that, this is
the picture of cyclodextrin. So you can see the only
difference is this group here versus that with a methyl. And one, so this
is hydroxypropryl– hydroxypropionyl
cyclodextrin– so it’s like a cavity like this. And the other only other
change here is a methyl group, removing that. And they have very
different properties about binding and releasing
cholesterol, which somebody had to do a lot of studying on
to be able to ensure that they can use it to
remove cholesterol, and then to add it
back to the media. And so you have to think
about the exchange kinetics, you have to think
about a lot of things. This is not trivial
to set this up, to figure out how to control
the levels of cholesterol. And then what they do is,
this is like a typical assay, and this is the end. What you can do is this,
removes cholesterol, and you can see it change. This reports on low
levels of cholesterol, which is happening over
here, allows the protein to move to the nucleus
where it’s smaller. And that’s how they do the
correlation– the correlation between the levels
in the nucleus and the levels of cholesterol. So I thought this was
a pretty cool paper. And these kinds of
methods, I think, will be applicable to
a wide range of things if people ever do
biochemistry, looking at the function of membranes. So, OK, guys.


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