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  • JOANNE STUBBE: Where we were at at the end of the last lecture

  • was trying to figure out what do we

  • do with the fact that cholesterol--

  • its solubility is five micromolar.

  • Yet if you look inside your blood,

  • the levels would be 5 millimolar.

  • And so the question is, how does it gets transported?

  • And it gets transported in a complex fashion.

  • We need to deal with that with any kind of very

  • insoluble lipophilic materials.

  • And I briefly introduced you to lipoproteins,

  • which are mixtures of different kinds of lipids,

  • triacylglycerols, phospholipids, cholesterol,

  • cholesterol esters.

  • And the key question we learned in the first couple

  • lectures that cholesterol could be biosynthesized.

  • And what we started focusing on in the last lecture

  • was that it can be taken up by the diet.

  • That's what we're focusing on now.

  • And then after we do a little more background,

  • then how is it taken up and then how is this all regulated?

  • How do you control biosynthesis versus cholesterol

  • from the diet.

  • What are the sort of major mechanisms?

  • So at the end of the last lecture

  • I'd given you a second picture.

  • And the PowerPoint-- the original PowerPoint

  • didn't have this figure.

  • This is taken out of a new Voet and Voet--

  • the newest Voet and Voet-- which I think better

  • describes what's going on.

  • But really sort of what you need to know

  • is you form these particles, chylomicrons,

  • if you look at the handout I gave

  • you have lots of proteins, all kinds of lipids, cholesterol.

  • And they get into the bloodstream

  • and they pass off as they go through adipocytes

  • or as they go through muscle.

  • The surface of these cells have lipases, phospholipases

  • that can clip off the fatty acids

  • that you need for metabolism at most cells.

  • And what happens is the size of these particles just change.

  • And so in the end, you remove the triacylglycerols

  • and you remove phospholipids.

  • And what you're left with is more of a cholesterol.

  • And that-- and so what happens is the chylomicrons change

  • size.

  • They call them the remnants.

  • And there are receptors on liver cells, which

  • can take up these remnants, these lipoprotein remnants.

  • And then they repackage them into other lipoproteins.

  • And again, the differences in the lipoproteins

  • we talked about very briefly, we have an outline.

  • Somebody measured these with a--

  • again, they're variable, but they're based on density.

  • And so the liver repackages these things

  • to a particle that's very low density, lipoprotein.

  • And then again, they can dump off components

  • into the tissues where you can use the lipids to do

  • metabolism, changing the size, intermediate density,

  • eventually low density lipoprotein which

  • is what we're focused on now.

  • And then today what we're focused

  • on is how does the low density lipoprotein get taken up

  • by the liver?

  • And also, can it get taken up by other kinds of cells?

  • And if you have excess cholesterol produced

  • in any of these extrahepatic cells,

  • it can be taken up to form particles called high density

  • lipoproteins.

  • And they can come back.

  • So they act as cholesterol scavengers,

  • come back and deliver it back into the liver

  • by a mechanism that is really different from what we're going

  • to be talking about today.

  • So that's the overview picture.

  • And so what I want to do now is focus

  • on the question, why do we care about cholesterol

  • and what was the motivator for Brown

  • and Goldstein's discovery of the low density lipoprotein

  • receptor.

  • So this is the motivator.

  • They were seeing when they were at medical school,

  • a number of children that presented at an early age.

  • These guys were six and eight.

  • And the way they present, if they turn out

  • to have both genes, both copies of the gene

  • are messed up for low density lipoprotein

  • receptor, that's called familial hypercholesterolemia.

  • The way they present is they have these little xanthomases

  • that are apparently yellow.

  • And what they are is they're full of cholesterol.

  • OK, and so if you have someone that's heterozygous

  • rather than homozygous--

  • these guys are homozygous--

  • you still see these but you see it

  • at a much later time in their life.

  • And so again, what it is, it's a function of the fact

  • that you have too much cholesterol

  • and this is the way--

  • one of the ways-- it manifests itself.

  • The second way it manifests itself

  • is if you look at the concentration of low density

  • lipoprotein and the plasma, which

  • is given in milligrams for 100 mils, what you see

  • is the concentrations of cholesterol

  • are actually 5 to 10 times higher.

  • So that's the manifestation.

  • And children that manifest at this early age

  • die of heart attacks by the time they're 30.

  • And so this was the motivator.

  • They were trying to figure out what is the basis

  • or bases for this disease.

  • So that's what I said.

  • This is a dominant effect.

  • At the time, the gene or genes responsible for this

  • were not known.

  • It turns out from the data that I've gotten from some paper,

  • one in 500 people are heterozygotes.

  • That's quite prevalent, actually.

  • But the ones that manifest themselves

  • in this really terrible way early on

  • is something like one in a million.

  • And so-- but even the heterozygotes,

  • Brown and Goldstein study all of these people,

  • also manifest in this way.

  • They have elevated cholesterol levels.

  • And so this was is a huge problem.

  • And so they decided they wanted to really devote

  • their life to it.

  • And I think they didn't know this in the beginning,

  • but it's really associated with one gene.

  • Most diseases are much more complicated than that.

  • And so I think because of the, quote, "simplicity" unquote,

  • you'll see it's not so simple, they

  • were able to make progress.

  • And these experiments were carried out really

  • sort of in the--

  • started in the 1970s.

  • So I think Brown and Goldstein-- we talked about the cholesterol

  • biosynthetic pathway.

  • And we talked about what was rate limiting.

  • So hopefully you all know that the rate limiting step

  • is the reduction of hydroxymethylglutaryl CoA down.

  • So the CoA is reduced all the way down to an alcohol

  • and that product is mevalonic acid.

  • And if you can't remember this, you

  • should pull out the biosynthetic pathway.

  • And that was proposed to be by other people working

  • in this field to be the rate limiting

  • step in this overall process.

  • And when you take an introductory course

  • in biochemistry, you talk about regulation.

  • I guess it depends on who's teaching it,

  • how much you talk about regulation.

  • But of course, one of the major mechanisms of regulation

  • that's sort of easy to understand in some fashion,

  • is that oftentimes the end product of a pathway

  • can come back way at the beginning

  • and inhibit the pathway.

  • So that's called feedback inhibition.

  • We saw that cholesterol biosynthesis was 30 steps.

  • And if you go back and you look at the pathway, you know,

  • I think this is step four or five.

  • I can't remember which one it is.

  • And so the model was--

  • and there was some evidence that suggested that

  • from what had been done in the literature--

  • that cholesterol was potentially acting as a feedback inhibitor.

  • And that's what their original working hypothesis was.

  • So the hypothesis was--

  • this is how they started it out.

  • And what we'll do is just look at a few experiments

  • of how they were trying to test their hypothesis

  • and then how they change their hypothesis to come up

  • with a new model for cholesterol regulation.

  • So you start out with acetyl CoA and you

  • go through mevalonic acid.

  • And then we get to cholesterol.

  • And so the model was that--

  • this is HMG reductase-- that this was a feedback inhibitor.

  • And that it inhibited by allosteric regulation.

  • And that's true of many pathways.

  • And often, that's one out of many mechanisms

  • that are involved in regulation.

  • So the first problem they faced--

  • and for those of you who want to read

  • about this in more detail, the original experiments,

  • I'm just going to present a few simple experiments

  • and I'm going to present them in a simple way.

  • OK, everything with human cells is more complicated

  • than the way I'm presenting it.

  • But for those of you would like to read a little bit more

  • about the actual experiments, there

  • are two papers that I think are particularly compelling.

  • And in previous years, I've actually used these papers

  • in recitation.

  • OK, so this is one of them.

  • I'll put the other one up later on so

  • that you can look at the detail, more

  • about the experimental details.

  • And I think in these particular experiments, what you're

  • being introduced to, which most students don't experience,

  • is the fact that you have--

  • all you do with these insoluble membrane-like proteins

  • and how do you deal with membrane proteins.

  • Most of us-- I haven't had any experience with this at all.

  • So this week's recitation, for example,

  • sort of shows you what they had to go through

  • to be able to answer these questions.

  • And it's complicated.

  • And I think reading the experimental details

  • in the end, if you're going to do something like this,

  • this provides a nice blueprint of how you try--

  • how you try to design experiments.

  • And you'll see some of the complexity

  • from the few experiments I'm just going to briefly describe.

  • OK, so what they needed was a model system.

  • And of course, you can't do experiments on humans.

  • So what they wanted to do was have some kind

  • of tissue culture system.

  • So they wanted a model system.

  • And there was some evidence in the literature

  • that human fibroblast skin cells were actually

  • able to biosynthesize cholesterol.

  • So they wanted to ask the question,

  • do these skin cells recapitulate what

  • people had seen from the biological studies in humans?

  • And so the first experiments I'll show you,

  • does recapitulate that.

  • It didn't have to.

  • But then this became their model,

  • human fibroblast cells became the model

  • for which they're carrying out all of these experiments

  • that we're going to very briefly look at.

  • OK, so the experiments, I think, are simple,

  • at least on the surface.

  • Although I think it wasn't so easy to figure out

  • how to do these experiments.

  • So what they wanted to do, they had patients--

  • whoops.

  • I didn't want to do that.

  • Anyhow, sorry I'm wasting time.

  • OK, this patient is JD.

  • And all of the experiments I'm going to show you is JD.

  • But they had 25 other patients.

  • And what you'll see is they all manifest themselves

  • in different ways.

  • And we're going to see that that, in the end,

  • becomes important in sorting out really what was going on.

  • OK, so the first set of experiments they did

  • was the following.

  • So they had some kind of normal control.

  • And then so we have a normal--

  • so we have skin cells from a normal person.

  • And this is the control.

  • And then you have the FH patient, JD.

  • And in the two papers I'm going to reference,

  • they did a lot of experiments on JD's fibroblasts.

  • And so they did some simple experiments.

  • And remember, the rate limiting step

  • is proposed to be hydroxymethyl--

  • HMG CoA reductase.

  • And so they wanted to first ask what

  • happens if you treat the cells, so you have them growing.

  • OK, and you let them grow for a certain period of days.

  • And then what you do is you take the media,

  • change it, and remove low density lipoproteins

  • from the media.

  • I don't know whether they removed them all.

  • They said they removed 5%.

  • I don't know what the percent that was there.

  • And so we're going to do that for both

  • the experiment and the control.

  • So this is the experiment.

  • This is the normal person.

  • This is the experiment, the FH patient.

  • And if you look at the axis in measuring HMG CoA

  • reductase activity.

  • So what they're going to do is look at plus or minus LDL.

  • So in this panel, they've removed the LDL, OK?

  • And if we remove the LDL, you remove the cholesterol,

  • what might you expect to happen to the normal HMG CoA reductase

  • levels or activities?

  • If you remove the cholesterol from the plasma,

  • what might you expect to happen to the activity?

  • What would you want to do?

  • Would you want to turn it on?

  • Would you want to turn it off?

  • STUDENT: Turn on.

  • JOANNE STUBBE: Turn it on, right.

  • So that's what they're going to be assaying.

  • They remove it and if you look at the normal patient,

  • the normal control, what's going to happen

  • is the biosynthesis is turned on.

  • So it'll look at this, then, you need to have--

  • and this goes back to the things we've talked about a little bit

  • about in class--

  • and in fact, the original recitation

  • that we had on radioactivity was completely

  • focused on Brown and Goldstein's work.

  • So we're going to see that they use a lot of radioactivity

  • and all the assays I'm going to be describing today.

  • So what we're going to be doing is revisiting radioisotopes.

  • They couldn't have done that without these radioisotopes.

  • And this is converted to this.

  • OK, what's the cofactor for this reaction?

  • So, I'm not going to draw up the rest of this.

  • This is mevalonic acid.

  • What's the cofactor required for this process?

  • Any DPH.

  • So you have any DPH.

  • OK, so how would you assay this?

  • So we're doing this now in tissue culture systems.

  • That's what-- we are doing this in fibroblast cells in tissue

  • culture.

  • So we don't have very much material.

  • You might have a plateful of cells.

  • How would you do the assay?

  • So this is the first thing you have to figure out.

  • And I would say, almost everything in this class,

  • when you're studying the biology,

  • first thing you have to do is figure out a robust assay.

  • This case, I think it turned out to be quite easy.

  • But it's not necessarily easy in many cases.

  • So this is something, as a chemist,

  • you bring a lot to the table.

  • Yeah?

  • STUDENT: You would measure the change

  • in the absorption at 340.

  • JOANNE STUBBE: 340.

  • So that's the way chemists would do that.

  • Why can't you do that here?

  • STUDENT: You have to isolate the HMG CoA reductase

  • or somehow be able to parse it from everything--

  • JOANNE STUBBE: Well, you might be

  • able to do it in crude extracts if you had a lot of it.

  • But it's tough.

  • NADPH is used in hundreds of reactions.

  • It's a great assay because the absorption change is removed

  • from where most of the material absorbs,

  • which is, you know, 280, 260, 280.

  • It's not that sensitive.

  • The extinction coefficient is 6,300 molar inverse centimeter

  • inverse.

  • And the bottom line is if you look at it,

  • it's nowhere near sensitive enough.

  • So if it's not sensitive enough, then what do you need to go to?

  • That's what-- the radioactivity.

  • So what you're going to be doing here is--

  • so you could use either 14c--

  • hopefully you remember that's a beta emitter, which then gets

  • converted into mevalonic acid.

  • And then you need a way of separating starting material

  • from products.

  • And there are many ways that one could do that.

  • But in the original paper, they use TLC.

  • And that's how they monitored their reactions.

  • And you need to have material that's

  • of hot enough radioactivity so you can see these

  • into conversion.

  • So that's the assay that they used.

  • And so in the PowerPoint, I decided not to draw out.

  • So if you PowerPoint, you look at the data, what do you see?

  • What you see is that if you look at the experiment

  • where they removed the low density

  • lipoprotein from the media--

  • so they've taken it out.

  • They've grown the cells they have HMG CoA

  • reductase activity.

  • What do you see immediately--

  • and the control and the patient's cells

  • are growing exactly the same way.

  • What do you see immediately?

  • You see a huge difference in the amount of activity.

  • So this is 2.

  • This 150 or something.

  • And so there could be a number of reasons for all of that.

  • And so the question is, what is the basis for this increase

  • in activity due to increased huge amount,

  • the amount of HMG CoA reductase.

  • Has the activity changed?

  • Is there a mutation that changes the activity?

  • There are lots of explanations.

  • And so what they then did, when they remove this,

  • they started doing assays over 24 hours.

  • And they crack open the cells and do this radioactive assay.

  • And then they looked at the rate of formation of mevalonic acid.

  • And so what do you see with the normal control?

  • You see exactly what you might predict.

  • So if the cholesterol levels become low,

  • you might want to biosynthesize it.

  • But then what do you see with a homozygote, the JD patient?

  • What you see is the levels start out

  • high you have complete absence of regulation

  • by changing the concentration of cholesterol.

  • That's what you're seeing.

  • So it seems like a simple experiment.

  • It is a simple experiment.

  • The basis for these observations is still open to debate.

  • But the experiment turned out to be straightforward.

  • Then what they did is at 24 hours,

  • they then started adding low density lipoprotein back

  • into the media.

  • So they start over here, they removed it.

  • They add it back.

  • Here's with non.

  • Here's with two micrograms per mL.

  • Here's with 20 micrograms per mL.

  • And what do you see with a normal patient?

  • With a normal control?

  • What you see with the normal control is a loss of activity.

  • So that's exactly what you would expect that cholesterol-- you

  • have a lot of cholesterol, you don't need to make it anymore.

  • So this data, then, this simple data told you--

  • the control told you that minus LDL,

  • you increased HMGR activity.

  • And plus LDL, you decreased activity.

  • And what about the patient?

  • The FH JD patient?

  • So here what you see is that removing cholesterol

  • from the plasma has no effect.

  • What about adding it back?

  • Has no effect.

  • So somehow the patient is--

  • the patient's cells is oblivious to the presence or absence

  • of cholesterol.

  • So in this case, plus or minus LDL had no effect.

  • So we say loss of cholesterol regulation, which

  • could be due to feedback inhibition,

  • it could be due to something else.

  • We'll see it is due to something else.

  • And so this was consistent with what they predicted.

  • And they furthermore learned that these fibroblast cells

  • might be a good model for actually studying

  • what's going on in the liver.

  • I mean, you always have this issue.

  • You have to figure out what you can study as a model system

  • since we don't work on the humans.

  • And so you always have to worry about how

  • that extrapolates to humans.

  • So basically, you're looking at cholesterol in the media.

  • You're looking at cholesterol not in the media.

  • And these are the experiments we just described.

  • And so one of the questions you can ask,

  • then, is what happens now?

  • Another thing that can happen is what if cholesterol

  • can't get into the cell?

  • So what they did is another experiment where they--

  • they did two things to look at the HMG CoA reductase

  • activity in the normal control and in the FH patient.

  • And one of them was they repeated this experiment

  • in the presence of ethanol, where

  • they dissolved the cholesterol.

  • And apparently that allows the cholesterol

  • to get across the membrane.

  • OK, so we're bypassing what we now

  • know is going to be a receptor.

  • So they did a second experiment and they

  • used ethanol cholesterol.

  • And it goes across membrane.

  • And then they looked at the HMG CoA reductase activity.

  • And the activity of both the patient and the normal controls

  • was the same.

  • OK, so the activity, HMGR activity the same.

  • They don't report the details of this experiment.

  • But another way you could do this is you

  • could pull out the protein or partially purify

  • the protein in crude extracts and try to measure

  • the activity using this assay.

  • And if you have a good measure of the amount of protein, which

  • is key, so you can measure specific activity,

  • micromoles of product or nanomoles of product produced

  • per minute per milligram of protein,

  • you could actually see that the HMG CoA reductase

  • activity was the same in the wild type in the normal

  • and in the patient.

  • So you could also measure this using assay.

  • And again, the result was that they

  • were the same in both the normal and the patient.

  • So then the elevated levels could be--

  • elevated levels, you saw in the very beginning of the HMG CoA

  • reductase activity, could be due to the fact,

  • they had a huge amount of protein,

  • more so than you do with the fibroblasts.

  • And so, there's no reason to think a prior

  • if you looked at that previous slide, that the control,

  • that normal control in the wild type--

  • I don't know what the scatter is in the data for HMG reductase

  • activities, but that's something you need to think about.

  • But a 60-fold change is a huge change.

  • So this data, the initial set of data

  • said that, yeah, cholesterol may be

  • acting as a feedback inhibitor.

  • But here, we can get cholesterol into the cell

  • and the activities are the same.

  • So they needed to come up with an alternative hypothesis.

  • OK, so they then, using these two sets of data,

  • came up with an alternative hypothesis.

  • So they concluded that it's not cholesterol feed back

  • regulated.

  • And so then they set out to do a second set of experiments

  • based on a new hypothesis.

  • And the new hypothesis is that there

  • would be some protein that might be involved

  • in taking up the LDL particle, which has

  • a cholesterol into the cell.

  • So the new hypothesis was there is an LDL receptor,

  • so r is receptor.

  • That's how I'm going to abbreviate it.

  • That's key to taking up LDL.

  • And so that's what's shown here.

  • And so then the question is, what sets of experiments

  • do they do next.

  • So this is a second set of experiments

  • that was done in a paper that's also quite interesting.

  • And so, for those of you who want

  • to look at the details of this, this was published in 1976.

  • And so this is where the data that I'm going to show you

  • on this slide came from.

  • Because I think they actually put it in one of the two review

  • articles I gave you to read.

  • But if you want to read the original data,

  • the papers aren't that long.

  • And they go through the details of the rationale of how

  • they design their experiments.

  • OK, so what we want to do now is test

  • the idea that to get cholesterol into the cell,

  • there is an LDL receptor.

  • And that that's going to play a key role in controlling

  • cholesterol levels.

  • That was the working hypothesis.

  • OK, so how would you go about testing this experimentally?

  • So these are the results of the experiments.

  • And the question is, how would you

  • go about testing this experimentally

  • if this were your hypothesis?

  • And so if you think about it, you might like to know,

  • does the LDL particle bind to the surface of the cell?

  • Does it bind?

  • OK, so that would be one thing you could do.

  • And in fact, Brown and Goldstein were

  • treating many, many patients.

  • So they had fibroblasts for many patients, 20 to 25 patients.

  • They all had different phenotypes.

  • And again, these were differences

  • in the phenotypes actually helped them

  • to try to dissect this process.

  • And so could it bind?

  • And so we can ask the question, how would we look at binding?

  • I'm going to ask you that question.

  • We're going to have a recitation on binding,

  • I think, not this week, but next week.

  • Then it gets into the cell.

  • OK, so how do you know it gets into the cell?

  • And so that's another question.

  • Inside, outside.

  • And then the next question is, what is LDL?

  • Hopefully you remember it's a lipoprotein that has

  • a single protein on it, apoB.

  • And then it's full with cholesterol, cholesterol

  • esters, and phospholipids.

  • What happens to that stuff once it's inside the cell?

  • OK, so those are the questions in this experiment

  • that they set out to ask.

  • OK, so what I want to do--

  • so binding, internalization, and then

  • the fate of LDL inside the cell.

  • So that's what they were focused on.

  • So what I want to do is show you the tools

  • that they developed to try to answer these questions.

  • OK, I'm going to show you a few things because this

  • isn't such an easy set of experiments to carry out.

  • And then what they observed on the normal cells

  • and the patient cells.

  • OK, so the tools that I want to talk about are the following.

  • OK, so we just talked about the fact that, to do the assay,

  • we needed radioactivity.

  • We needed to be sensitive enough.

  • If you're going to be looking at binding on the surface,

  • how do you do that?

  • Do you think there are a lot of receptors?

  • Are there a few receptors?

  • So you might not know that.

  • But in general, there aren't huge numbers of receptors.

  • So measuring binding to the surface of the cell usually

  • requires a very sensitive assay.

  • So the first thing they needed to do

  • was they decided that they needed

  • to make the LDL radial labels.

  • And if you go back and you look through your notes

  • in recitation three where we talked about radioactivity,

  • we saw that we have beta, c14 beta, which

  • is what they used up there.

  • But they also used i125, which is a gamma, which

  • is much more sensitive.

  • And so what they decided they needed to make

  • was i125 labeled LDL.

  • So if you haven't radio labeled, can you

  • somehow see it sitting on the surface of the cell?

  • So the question is, how can you do that?

  • Well, we talked about the composition

  • of the LDL particle.

  • There is cholesterol.

  • There's cholesterol esters, phospholipids, and one protein.

  • And so what they're doing to put the iodine in

  • is putting it into only the protein.

  • OK, so what they use is a method called

  • Bolton Hunter, which uses radial level iodide and a reagent.

  • I'm not going to go through--

  • you can look it up if you're interested--

  • I'm not going to go through the details.

  • And what it does is it takes a protein--

  • this is still actually widely used.

  • So this would be apoB.

  • And it iodinates at the ortho position.

  • So what you end up with, then, is iodinated apoB.

  • So that's going to be your handle.

  • You can make this a very high, specific activity.

  • OK, so that's one thing that they needed to do.

  • OK, the second thing that they needed to do

  • is if they're going to look for binding to the surface,

  • how would you design that experiment?

  • What might you need to do to figure out

  • how you're going to look at binding only and not

  • binding and uptake?

  • What parameter could you change that would help you do that?

  • Temperature.

  • So everything-- and you'll see this also

  • in experiments this week-- the temperature is really critical.

  • Why?

  • Because hopefully you all know lipid bilayers are very fluid.

  • And if you cool the temperature, you

  • prevent uptake and other things.

  • You have to test all this out.

  • They did a huge number of controls.

  • So the second thing that they wanted to do

  • is they used temperature.

  • So four degrees, they're going to use to look at binding.

  • Or if they're looking at a time course and they

  • want to stop the reaction, and the reaction is normally

  • done at 37 degrees--

  • so uptake experiments would be at 37 degrees.

  • OK, so again, temperature is the key parameter.

  • You could, if you wanted to a time course

  • and stop the reaction, you could cool with down to four degrees.

  • I mean, this was a hypothesis they had.

  • And so that's the second tool that they're going to use.

  • And the third tool, which I think isn't necessarily so

  • intuitive, is if you're looking at something binding

  • on the surface, you have to always worry

  • about non-specific binding.

  • You'll talk about that in the recitation.

  • On this that's always a problem.

  • You're using really hot, iodine-labeled materials,

  • so you could get neuron specific binding.

  • And so how do you-- so you need to wash it.

  • So if the LDL particle bound loosely to the LDL receptor,

  • that makes the problem extremely challenging

  • because when you're trying to wash away the excess as you

  • change the concentration of the LDL,

  • you're going to start to lose--

  • you're going to have an equilibrium

  • and you're going to start to lose binding.

  • It binds really tightly so they had

  • to have some kind of a wash.

  • So they figured out and optimized a wash.

  • So you need to have a wash.

  • So if you have a wash and then you're

  • still looking at the receptor with the particle bound--

  • so that's the LDL-LDL receptor-- then the question is--

  • and it's tight binding--

  • how do you get that off?

  • And remember, you're also going to have

  • LDL that's been internalized.

  • So the creative approach they used

  • was to use the molecule heparin.

  • OK, so heparin--

  • I'm not going to draw out the structure--

  • but this is a third tool and this was key.

  • And so they have heparin-sensitive

  • and heparin-resistant.

  • And what does this mean?

  • Heparin turns out to-- it's a sugar.

  • Many of you have probably heard about it.

  • It plays a key role in blood coagulation.

  • But anyhow, from the point of view of today's lecture,

  • you just need to know it's a sugar

  • and it's got sulfates all over the outside of it.

  • So it's negatively charged.

  • So heparin is a sulfated sugar.

  • So basically, you have something like this with SO3 minuses

  • on the outside.

  • And so what happens is if--

  • what you want to do is release the LDL particle

  • from the receptor.

  • And apparently, treatment with heparin at certain levels--

  • I think they tried a lot of things--

  • was able to release the surface LDL.

  • So this is involved in release of surface bound.

  • So then what you have left after you release this,

  • is you could still have radio label that's been internalized.

  • So that then becomes heparin-resistant.

  • And so you can count that.

  • And so then you have bound and internalized.

  • Now, if you're studying this as a function of time,

  • what can happen to-- once you internalize the LDL particle,

  • what can happen to the iodinated LDL particle?

  • What can happen?

  • So this is something else you need

  • to think about in these assays.

  • So now we have internalized LDL, i125 label.

  • What can happen-- if you remember from recitation

  • this past week, you remember what happened to the LDL?

  • So you got protein.

  • You got lipids.

  • What's going to happen?

  • You might not know the details.

  • That's what this whole--

  • that's what Brian and Goldstein uncovered,

  • which we're going to talk about in the next few minutes.

  • But LDL, you have a protein.

  • What can happen to proteins?

  • They can get degraded.

  • So if you have the apoB, what can happen is inside

  • the cells-- so this is inside--

  • you could have proteases that degrade this down to peptides.

  • This happens in a lysosol where you still

  • have iodinated tyrosine.

  • Or it can be broken down all the way to just iodinated tyrosine.

  • So if you're breaking this down all the way here,

  • the iodinated tyrosine could likely exit the cell.

  • So you need to really think-- so what

  • do you do to control for this aspect of the metabolism?

  • What happens to the LDL inside the cell?

  • And so to do this, how would you distinguish LDL itself from,

  • say--

  • as a chemist, what could you do to distinguish LDL protein

  • from LDL on small peptides or LDL as an amino acid?

  • So the key question was, what sort of bulk method

  • do you use to try to distinguish between these two things.

  • So then you can incorporate that into the analysis, which

  • is what's on the slide here.

  • So what happens if you treat proteins in general with acid?

  • They what?

  • They hydrolize?

  • So peptide bonds are really strong.

  • If you want to break a peptide bond,

  • you have to heat it for 16 hours at 100 degrees.

  • So that's not going to happen.

  • So that's not an option.

  • But what else happens?

  • What do you do when you put a protein into acid?

  • What happens to the protein?

  • It what?

  • Yeah, it crashes out.

  • So proteins in general, not all proteins, most proteins

  • precipitate, but these kinds of things would be soluble.

  • So they've been able to take advantage--

  • so you have to, again, treat the cells in a certain way so

  • that you can look at what's still

  • retained in the LDL versus what's undergone degradation.

  • OK, we're going to see that's key to the model we're

  • going to come up with.

  • OK, so those are the tools that they needed to develop.

  • And so the question is, then, what did they observe?

  • OK, so we're doing these same experiments.

  • We're looking for binding on the outside, internalization,

  • and breakdown.

  • That's what we're looking for.

  • And so here is the patient and here is the control.

  • So if we look at here, these guys are the binding.

  • So Brown and Goldstein, in this particular paper,

  • which is underneath here but in the cell paper,

  • looked at 22 patients.

  • And out of the 22 patients, most of them were binding deficient.

  • They could see no binding at all.

  • Some of them were binding modified.

  • That is, they had lower levels of binding.

  • And this one patient, JD, had normal binding.

  • So in this experiment, we're looking at here-- so

  • in the PowerPoint, this is one of 22 patients

  • they had normal binding.

  • And the others-- and that's because we'll

  • see that there are multiple ways you can have

  • defects in your LDL receptor.

  • We'll come back to that in a minute.

  • But you can have deficient binding

  • or you could have no binding.

  • Or you could have normal binding.

  • So those are all possible.

  • And the one that we've taken the data for here

  • and that's described in the paper, is normal binding.

  • And they did a lot of experiments

  • I'm not describing to try to show you

  • that this experiment, which suggests normal binding,

  • is in fact normal binding.

  • They looked at off rates.

  • They looked at competition with HDL and LDL.

  • And so if you look at that, if you

  • look at the levels of binding, they really

  • aren't very different between the experiment and the control.

  • And so now what happens, if you look

  • at the normal, what happens is with time,

  • the LDL on the surface goes away.

  • And that's because it's becoming internalized.

  • Whereas down here, what happens?

  • You started out the same, but now you can see over--

  • this is hours down here, it really

  • hasn't changed very much.

  • It's not becoming internalized.

  • And so then they wanted to use their method

  • to look at internalized LDL.

  • And so internalized LDL, using the heparin-resistant

  • versus heparin-sensitive, that's the assay they used,

  • what you see is as the surface binding at least early

  • on decreases, the amount internalized increases.

  • But what happens over here to the patient?

  • With the patient, you get nothing internalized.

  • And the other question is, what happens to the LDL--

  • and it's labeled on the protein--

  • does that get degraded?

  • And so using a method with TCA, they

  • used a couple of different methods, what they see

  • is that you slowly degrade the protein into small pieces.

  • And again, with the patient, it's not internalized

  • so you can't get degradation.

  • So this type of experiment with this particular patient

  • and also with the other patients that I talked about, one

  • through 21, they drew a strong conclusion

  • that there are two things that have to happen for cholesterol

  • to get into the cell.

  • Number one, it has to bind.

  • And number two, there's got to be some mechanism

  • for internalization.

  • So the conclusions from this is we need binding,

  • which is consistent with the LDL receptor.

  • And then we need, in some way, internalization.

  • And of course, JD was the only one out of all of these

  • patients where they can study internalization

  • because in the other patients they didn't-- they had really

  • poor binding or no binding at all.

  • So they needed to have this spectrum of patients

  • to be able to start to sort out what was going on

  • in these experiments.

  • So I think on the surface, the experiments look pretty--

  • you'll look at them, they look like they're really simple.

  • But technically, they're not so simple.

  • And if you care about the technical details, which we'll

  • see again in this week's recitation dealing

  • with these membrane proteins and stickiness,

  • becomes the key how creative you can be.

  • And usually, we're not really plugged into that.

  • And you usually don't do experiments like that

  • unless you work in a lab that is focused

  • on membrane-bound proteins.

  • So this resulted in the model.

  • So this kind of experiment and many other experiments

  • resulted in the model for receptor mediated endocytosis.

  • And you've seen this before.

  • You saw this in recitation last week

  • because we saw interference with the PCSK9

  • with receptor mediated endocytosis.

  • So we're back where we started last week.

  • And the first slide I showed you was this slide.

  • And so what is the model?

  • So there are many, many more experiments

  • that have gone into coming up with this model.

  • And the model is really still incomplete.

  • I have a cartoon here, the whole process,

  • every step along the pathway, how you go here and there

  • and what the kinetics are, it's all complicated.

  • But this is the working hypothesis.

  • And so the first thing is you make the LDL receptor.

  • It's a membrane protein, has a single transmembrane spanning

  • region.

  • Is made in the ER.

  • And because of this transmembrane spanning region,

  • it's got to be transported to the surface.

  • And it's done so in little coded vesicles,

  • which keeps things soluble.

  • And it does this by passing through the Golgi stacks, which

  • we talked about at the very beginning.

  • Eventually, it gets to the surface.

  • These little things here are the LDL receptors.

  • You can go home and sleep on this

  • and look at it again because I'm over.

  • And it just seems like I just started and it's already over.

  • I'm sorry.

  • OK, I must have spent too much time

  • talking about something I wasn't supposed to talk about.

  • But anyhow, hopefully you now all

  • can go back and look at this and think

  • about this, because we're going to be talking about this

  • again in recitation this week.

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