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  • JOANNE STUBBE: --that Brown and Goldstein carried out,

  • which in conjunction with many other experiments

  • and experiments by other investigators

  • have led to the model that you see here.

  • And so we'll just briefly go through this model, which,

  • again, was the basis for thinking

  • about the function of PCSK9 that you learned

  • about recitation last week, as well as providing

  • the foundation for thinking about the recitation.

  • This week, we really care how you sense cholesterol levels

  • in membranes, which is not an easy thing

  • to do given that it's lipophilic and so are many other things.

  • OK.

  • So the LDL receptor--

  • that was their model, that there is a receptor--

  • is generated in the endoplasmic reticulum.

  • If you looked at the handout, you'll

  • see that it has a single transmembrane-spanning region,

  • which means it's inserted into a membrane.

  • And the membrane where it functions, at least

  • at the start of its life, is in the plasma membrane.

  • So somehow, it has to get from the ER to the plasma membrane.

  • And this happens by forming coated vesicles.

  • We'll see a little bit of that, but we're not

  • going to talk about this methodology in any detail.

  • But Schekman's lab won the Nobel Prize

  • for this work, either last year or the year

  • before, of how do you take proteins

  • that are not very soluble and get them to the right membrane.

  • And they do this through coated vesicles

  • that, then, move through the Golgi stacks

  • that we talked about at the very beginning.

  • And then, eventually, they arrive at the plasma

  • membrane and become inserted.

  • So these little flags are the LDL receptor.

  • OK.

  • So that's the first thing that has to happen.

  • And I just know that this whole process is extremely complex.

  • And patient mutants are observed in almost every step

  • in this overall process.

  • It's not limited to the one set of types of experiments,

  • where something binds and doesn't bind to LDL receptor

  • that we talked about last time.

  • So the next thing that has to happen-- again,

  • and we haven't talked about the data for this at all,

  • but not only do these receptors have to arrive at the surface,

  • but they, in some way, need to cluster.

  • And it's only when they cluster that they

  • form the right kind of a structure that, then,

  • can be recognized by the LDL particles

  • that we've talked about.

  • And so they bind in some way.

  • And that's the first step in the overall process.

  • And then, this receptor, bound to its cargo, its nutrients--

  • and, again, this is going to be a generic way

  • of bringing any kinds of nutrients into cells.

  • It's not limited to cholesterol--

  • undergoes what's now been called receptor-mediated endocytosis.

  • And so when the LDL binds to the receptor,

  • again, there's a complex sequence

  • of events that leads to coding of the part that's

  • going to bud off, by a protein called clathrin.

  • Again, this is a universal process.

  • We know quite a bit about that.

  • And it buds off.

  • And it gives you a vesicle.

  • And these little lines along the outside are the clathrin coat.

  • I'll show you a picture.

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

  • but I'll show you a picture of it.

  • So the LDL binding, we talked about.

  • We talked about binding in internalization.

  • Those are the experiments we talked about last time

  • in class that led, in part, to this working hypothesis.

  • And so we have clathrin-coated pits.

  • And it turns out that there's a zip code.

  • And we'll see zip codes throughout-- we'll

  • see zip codes again, in a few minutes,

  • but we'll see zip codes which are simply

  • short sequences of amino acids that signal to some protein

  • that they're going to bind.

  • So how do you target clathrin to form these coated pits?

  • How do you form a pit, anyhow, in a circle?

  • And how does it bud off?

  • And where do you get the curvature from?

  • Many people study these processes.

  • All of these are interesting machines

  • that we're not going to cover in class.

  • So you form this coated pit, and then it's removed.

  • So once it's formed, and you've got a little vesicle,

  • it's removed.

  • And then it can go on and do another step.

  • And another step that it does is that it

  • fuses with another organelle called an endosome, which

  • is acidic pH.

  • How it does that, how it's recognized,

  • why does it go to the endosome and not directly

  • to the lysosome-- all of these things, questions,

  • that should be raised in your mind

  • if you're thinking about the details of how

  • this thing works, none of which we're going to discuss.

  • But it gets into the endosome, and then what you want to do

  • is separate the receptor from its cargo, the LDL.

  • And we know quite a bit about that.

  • If you read-- I'm not going to talk about that either,

  • but if you read the end of the PowerPoint presentation,

  • there's a model for actually how this can happen.

  • And you can separate the receptor from the cargo.

  • And the receptors bud off, and they

  • are recycled in little vesicles to the surface, where

  • they can be reused.

  • The LDL particles can also, then-- and what's left here

  • can then fuse with the lysosome.

  • And that's, again-- we've talked about this--

  • it's a bag of proteases and a bag of esterases, hydrolysis,

  • lipids.

  • That's what we have in the LDL particle-- hydrolysis.

  • We talked about ApoB being degraded

  • with iodinated tyrosine, last time.

  • That's where this happens and gives you amino acids

  • and gives you cholesterol.

  • OK.

  • And then, again, depending on what's

  • going on in the environment of the cell,

  • the cholesterol would then be shuttled, somehow,

  • to the appropriate membranes.

  • OK.

  • So you can see the complexity of all of this.

  • If the cholesterol is present, and we don't need anymore

  • in the membranes, then it can become esterified

  • with long-chain fatty acids.

  • Those become really insoluble, and they

  • form these little globules inside the cell.

  • And then the process can repeat itself.

  • And the question we're going to focus on in lectures 4 and 5,

  • really, are how do you control all of this.

  • OK.

  • So this is the model.

  • And so I think what's interesting

  • about it is people have studied this in a lot of detail.

  • It was the first example of receptor-mediated endocytosis.

  • So we know something about the lifetime of the receptor.

  • We know it can make round trip from surface inside, back

  • to the surface in 10 minutes.

  • We also know it doesn't even have to be

  • loaded to make that round trip.

  • It could be one of the ones that isn't

  • the clustering of the receptors, which

  • is required for clathrin-coated vesicles to form.

  • And so you can tell how many trips it makes in its lifetime.

  • And so the question, then, what controls all of this?

  • But before we go on and do that, I just

  • want to briefly talk about, again,

  • mutations that have been found in the LDL receptor processing.

  • And they're really, basically, at every step in the pathway.

  • So the initial ones we found, that we talked about,

  • we'll come to in a minute.

  • But we had some patients with no LDL receptor express at all.

  • So somehow, it never makes it to the surface.

  • OK?

  • There are other examples-- and these have all been studied

  • by many people over the decades--

  • that it takes a long time to go through this processing.

  • And it gets stuck somewhere in the processing.

  • That may or may not be surprising,

  • in that you have transmembrane insoluble regions.

  • And if the processing goes a little astray or some mutation

  • changes, then you might be in trouble.

  • So we talked about this last time.

  • We talked about that they had just looked at 22 patients.

  • Some of the patients had no binding of LDL

  • to the surface of the fibroblast that they

  • were using as a model, at all.

  • Some have defective binding.

  • So if they compared it to a normal,

  • they had a range of dissociation constants.

  • And we'll talk quite a bit about dissociation constants,

  • not this week but next week, in recitation.

  • It's not so easy to measure dissociation constants

  • when things bind tightly.

  • And thinking about how to measure them correctly,

  • I think, is really important.

  • And I would say, probably, I could pull out

  • 10 papers out of current journals, really good journals,

  • where people haven't measured dissociation

  • constant correctly, when you have tight binding.

  • So this is something that we put in

  • because I think it's important that people need to know how

  • to think about this problem.

  • So anyhow, let's assume that Brown and Goldstein

  • did these experiments correctly, which I'm sure they did.

  • And they got a range of binding.

  • And we also saw that the patient we looked at,

  • JD, had normal binding.

  • That indicates he was the same as normal patients,

  • but something else was problematic.

  • And that something else wasn't that it

  • failed to form coated pits, but that it failed

  • to bring this into the cell.

  • So it failed to internalize the LDL.

  • That was JD's defect.

  • We also, in recitation last week--

  • hopefully, you've had time, now, to go back and look

  • at the paper a little bit.

  • But LDL, in the model we were just looking at, gets recycled.

  • It goes in and gets back to the surface.

  • But what happens if, on occasion, instead

  • of budding off into vesicles and returning to the surface, it,

  • with the LDL cargo, goes to the lysosome and gets degraded?

  • Well, that was the working hypothesis for what PCKS did.

  • It targeted to the wrong place and degraded it.

  • And the phenotypes of those patients were interesting,

  • and that's why it was pursued.

  • So there are many, many defects.

  • And despite the fact that we have these statins,

  • people are still spending a large amount

  • of time thinking about this because of the prevalence

  • of coronary disease.

  • So I'm not going to talk about this,

  • but I'm just going to show you two slides.

  • And you can go back and think about this yourself.

  • But this is the LDL receptor.

  • We know quite a bit about it now.

  • And one of the questions you can ask yourself,

  • which is an interesting question we're not

  • going to describe-- but you have LDL

  • particles that are different sizes.

  • How do you recognize all these different sizes?

  • And how does the clustering do that?

  • And so that's done up here.

  • And there's calcium binding.

  • We know quite a bit about that, but I don't think we really

  • understand the details.

  • You have a single transmembrane helix in the plasma membrane.

  • And this is the part--

  • this part up here-- that actually

  • binds the LDL particle.

  • And the last thing I just want to briefly say,

  • because we're going to see this again

  • but without going through any details,

  • remember that eventually we form what are

  • called clathrin-coated pits.

  • That's a picture of what the clathrin-coated pits look like.

  • And the key thing-- and I just wanted to mention this briefly

  • because we're going to see this again, over and over--

  • is the LDL receptor, itself, has a little zip code.