WEBVTT 00:00:00.500 --> 00:00:02.820 The following content is provided under a Creative 00:00:02.820 --> 00:00:04.360 Commons license. 00:00:04.360 --> 00:00:06.660 Your support will help MIT OpenCourseWare 00:00:06.660 --> 00:00:11.020 continue to offer high quality educational resources for free. 00:00:11.020 --> 00:00:13.650 To make a donation or view additional materials 00:00:13.650 --> 00:00:17.600 from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:17.600 --> 00:00:18.550 at ocw.mit.edu. 00:00:25.740 --> 00:00:29.160 JOANNE STUBBE: Where we were at at the end of the last lecture 00:00:29.160 --> 00:00:30.810 was trying to figure out what do we 00:00:30.810 --> 00:00:34.580 do with the fact that cholesterol-- 00:00:34.580 --> 00:00:37.530 its solubility is five micromolar. 00:00:37.530 --> 00:00:42.670 Yet if you look inside your blood, 00:00:42.670 --> 00:00:45.120 the levels would be 5 millimolar. 00:00:45.120 --> 00:00:47.580 And so the question is, how does it gets transported? 00:00:47.580 --> 00:00:53.280 And it gets transported in a complex fashion. 00:00:53.280 --> 00:00:56.130 We need to deal with that with any kind of very 00:00:56.130 --> 00:00:59.430 insoluble lipophilic materials. 00:00:59.430 --> 00:01:04.709 And I briefly introduced you to lipoproteins, 00:01:04.709 --> 00:01:08.060 which are mixtures of different kinds of lipids, 00:01:08.060 --> 00:01:11.700 triacylglycerols, phospholipids, cholesterol, 00:01:11.700 --> 00:01:14.310 cholesterol esters. 00:01:14.310 --> 00:01:17.670 And the key question we learned in the first couple 00:01:17.670 --> 00:01:20.780 lectures that cholesterol could be biosynthesized. 00:01:20.780 --> 00:01:23.760 And what we started focusing on in the last lecture 00:01:23.760 --> 00:01:25.703 was that it can be taken up by the diet. 00:01:25.703 --> 00:01:27.120 That's what we're focusing on now. 00:01:27.120 --> 00:01:29.970 And then after we do a little more background, 00:01:29.970 --> 00:01:33.480 then how is it taken up and then how is this all regulated? 00:01:33.480 --> 00:01:37.560 How do you control biosynthesis versus cholesterol 00:01:37.560 --> 00:01:38.190 from the diet. 00:01:38.190 --> 00:01:39.930 What are the sort of major mechanisms? 00:01:39.930 --> 00:01:42.810 So at the end of the last lecture 00:01:42.810 --> 00:01:44.670 I'd given you a second picture. 00:01:44.670 --> 00:01:47.600 And the PowerPoint-- the original PowerPoint 00:01:47.600 --> 00:01:48.600 didn't have this figure. 00:01:48.600 --> 00:01:50.910 This is taken out of a new Voet and Voet-- 00:01:50.910 --> 00:01:53.220 the newest Voet and Voet-- which I think better 00:01:53.220 --> 00:01:55.003 describes what's going on. 00:01:55.003 --> 00:01:56.670 But really sort of what you need to know 00:01:56.670 --> 00:01:58.860 is you form these particles, chylomicrons, 00:01:58.860 --> 00:02:00.270 if you look at the handout I gave 00:02:00.270 --> 00:02:06.390 you have lots of proteins, all kinds of lipids, cholesterol. 00:02:06.390 --> 00:02:11.220 And they get into the bloodstream 00:02:11.220 --> 00:02:16.170 and they pass off as they go through adipocytes 00:02:16.170 --> 00:02:18.780 or as they go through muscle. 00:02:18.780 --> 00:02:23.010 The surface of these cells have lipases, phospholipases 00:02:23.010 --> 00:02:25.080 that can clip off the fatty acids 00:02:25.080 --> 00:02:28.390 that you need for metabolism at most cells. 00:02:28.390 --> 00:02:32.610 And what happens is the size of these particles just change. 00:02:32.610 --> 00:02:36.870 And so in the end, you remove the triacylglycerols 00:02:36.870 --> 00:02:38.880 and you remove phospholipids. 00:02:38.880 --> 00:02:42.600 And what you're left with is more of a cholesterol. 00:02:42.600 --> 00:02:47.350 And that-- and so what happens is the chylomicrons change 00:02:47.350 --> 00:02:47.850 size. 00:02:47.850 --> 00:02:49.770 They call them the remnants. 00:02:49.770 --> 00:02:53.700 And there are receptors on liver cells, which 00:02:53.700 --> 00:02:58.020 can take up these remnants, these lipoprotein remnants. 00:02:58.020 --> 00:03:03.080 And then they repackage them into other lipoproteins. 00:03:03.080 --> 00:03:05.310 And again, the differences in the lipoproteins 00:03:05.310 --> 00:03:08.480 we talked about very briefly, we have an outline. 00:03:08.480 --> 00:03:10.580 Somebody measured these with a-- 00:03:10.580 --> 00:03:14.640 again, they're variable, but they're based on density. 00:03:14.640 --> 00:03:19.980 And so the liver repackages these things 00:03:19.980 --> 00:03:24.090 to a particle that's very low density, lipoprotein. 00:03:24.090 --> 00:03:28.260 And then again, they can dump off components 00:03:28.260 --> 00:03:33.540 into the tissues where you can use the lipids to do 00:03:33.540 --> 00:03:37.590 metabolism, changing the size, intermediate density, 00:03:37.590 --> 00:03:40.260 eventually low density lipoprotein which 00:03:40.260 --> 00:03:41.610 is what we're focused on now. 00:03:41.610 --> 00:03:43.500 And then today what we're focused 00:03:43.500 --> 00:03:48.540 on is how does the low density lipoprotein get taken up 00:03:48.540 --> 00:03:50.100 by the liver? 00:03:50.100 --> 00:03:54.640 And also, can it get taken up by other kinds of cells? 00:03:54.640 --> 00:03:58.200 And if you have excess cholesterol produced 00:03:58.200 --> 00:04:01.350 in any of these extrahepatic cells, 00:04:01.350 --> 00:04:05.110 it can be taken up to form particles called high density 00:04:05.110 --> 00:04:06.600 lipoproteins. 00:04:06.600 --> 00:04:08.590 And they can come back. 00:04:08.590 --> 00:04:10.830 So they act as cholesterol scavengers, 00:04:10.830 --> 00:04:15.030 come back and deliver it back into the liver 00:04:15.030 --> 00:04:18.006 by a mechanism that is really different from what we're going 00:04:18.006 --> 00:04:19.089 to be talking about today. 00:04:19.089 --> 00:04:21.209 So that's the overview picture. 00:04:21.209 --> 00:04:26.700 And so what I want to do now is focus 00:04:26.700 --> 00:04:28.950 on the question, why do we care about cholesterol 00:04:28.950 --> 00:04:31.650 and what was the motivator for Brown 00:04:31.650 --> 00:04:38.130 and Goldstein's discovery of the low density lipoprotein 00:04:38.130 --> 00:04:39.130 receptor. 00:04:39.130 --> 00:04:44.160 So this is the motivator. 00:04:44.160 --> 00:04:47.550 They were seeing when they were at medical school, 00:04:47.550 --> 00:04:52.850 a number of children that presented at an early age. 00:04:52.850 --> 00:04:55.170 These guys were six and eight. 00:04:55.170 --> 00:04:58.020 And the way they present, if they turn out 00:04:58.020 --> 00:05:01.110 to have both genes, both copies of the gene 00:05:01.110 --> 00:05:04.200 are messed up for low density lipoprotein 00:05:04.200 --> 00:05:09.780 receptor, that's called familial hypercholesterolemia. 00:05:09.780 --> 00:05:14.730 The way they present is they have these little xanthomases 00:05:14.730 --> 00:05:16.620 that are apparently yellow. 00:05:16.620 --> 00:05:20.010 And what they are is they're full of cholesterol. 00:05:20.010 --> 00:05:24.600 OK, and so if you have someone that's heterozygous 00:05:24.600 --> 00:05:26.350 rather than homozygous-- 00:05:26.350 --> 00:05:28.420 these guys are homozygous-- 00:05:28.420 --> 00:05:30.250 you still see these but you see it 00:05:30.250 --> 00:05:34.940 at a much later time in their life. 00:05:34.940 --> 00:05:38.750 And so again, what it is, it's a function of the fact 00:05:38.750 --> 00:05:40.400 that you have too much cholesterol 00:05:40.400 --> 00:05:41.420 and this is the way-- 00:05:41.420 --> 00:05:44.010 one of the ways-- it manifests itself. 00:05:44.010 --> 00:05:45.740 The second way it manifests itself 00:05:45.740 --> 00:05:49.970 is if you look at the concentration of low density 00:05:49.970 --> 00:05:51.830 lipoprotein and the plasma, which 00:05:51.830 --> 00:05:56.900 is given in milligrams for 100 mils, what you see 00:05:56.900 --> 00:05:59.240 is the concentrations of cholesterol 00:05:59.240 --> 00:06:02.300 are actually 5 to 10 times higher. 00:06:02.300 --> 00:06:03.980 So that's the manifestation. 00:06:03.980 --> 00:06:08.540 And children that manifest at this early age 00:06:08.540 --> 00:06:13.400 die of heart attacks by the time they're 30. 00:06:13.400 --> 00:06:16.040 And so this was the motivator. 00:06:16.040 --> 00:06:19.190 They were trying to figure out what is the basis 00:06:19.190 --> 00:06:22.690 or bases for this disease. 00:06:22.690 --> 00:06:25.910 So that's what I said. 00:06:25.910 --> 00:06:30.410 This is a dominant effect. 00:06:30.410 --> 00:06:34.160 At the time, the gene or genes responsible for this 00:06:34.160 --> 00:06:36.320 were not known. 00:06:36.320 --> 00:06:40.610 It turns out from the data that I've gotten from some paper, 00:06:40.610 --> 00:06:43.970 one in 500 people are heterozygotes. 00:06:43.970 --> 00:06:46.160 That's quite prevalent, actually. 00:06:46.160 --> 00:06:48.200 But the ones that manifest themselves 00:06:48.200 --> 00:06:51.410 in this really terrible way early on 00:06:51.410 --> 00:06:54.650 is something like one in a million. 00:06:54.650 --> 00:06:58.523 And so-- but even the heterozygotes, 00:06:58.523 --> 00:07:00.440 Brown and Goldstein study all of these people, 00:07:00.440 --> 00:07:02.450 also manifest in this way. 00:07:02.450 --> 00:07:05.150 They have elevated cholesterol levels. 00:07:05.150 --> 00:07:09.350 And so this was is a huge problem. 00:07:09.350 --> 00:07:12.110 And so they decided they wanted to really devote 00:07:12.110 --> 00:07:12.980 their life to it. 00:07:12.980 --> 00:07:15.690 And I think they didn't know this in the beginning, 00:07:15.690 --> 00:07:19.850 but it's really associated with one gene. 00:07:19.850 --> 00:07:23.270 Most diseases are much more complicated than that. 00:07:23.270 --> 00:07:27.020 And so I think because of the, quote, "simplicity" unquote, 00:07:27.020 --> 00:07:30.140 you'll see it's not so simple, they 00:07:30.140 --> 00:07:31.490 were able to make progress. 00:07:31.490 --> 00:07:34.340 And these experiments were carried out really 00:07:34.340 --> 00:07:35.480 sort of in the-- 00:07:35.480 --> 00:07:37.920 started in the 1970s. 00:07:37.920 --> 00:07:43.640 So I think Brown and Goldstein-- we talked about the cholesterol 00:07:43.640 --> 00:07:45.800 biosynthetic pathway. 00:07:45.800 --> 00:07:47.630 And we talked about what was rate limiting. 00:07:47.630 --> 00:07:50.990 So hopefully you all know that the rate limiting step 00:07:50.990 --> 00:07:57.410 is the reduction of hydroxymethylglutaryl CoA down. 00:07:57.410 --> 00:08:00.790 So the CoA is reduced all the way down to an alcohol 00:08:00.790 --> 00:08:02.882 and that product is mevalonic acid. 00:08:02.882 --> 00:08:04.340 And if you can't remember this, you 00:08:04.340 --> 00:08:08.540 should pull out the biosynthetic pathway. 00:08:08.540 --> 00:08:11.180 And that was proposed to be by other people working 00:08:11.180 --> 00:08:14.030 in this field to be the rate limiting 00:08:14.030 --> 00:08:16.460 step in this overall process. 00:08:16.460 --> 00:08:19.160 And when you take an introductory course 00:08:19.160 --> 00:08:22.323 in biochemistry, you talk about regulation. 00:08:22.323 --> 00:08:23.990 I guess it depends on who's teaching it, 00:08:23.990 --> 00:08:26.090 how much you talk about regulation. 00:08:26.090 --> 00:08:29.390 But of course, one of the major mechanisms of regulation 00:08:29.390 --> 00:08:32.450 that's sort of easy to understand in some fashion, 00:08:32.450 --> 00:08:37.039 is that oftentimes the end product of a pathway 00:08:37.039 --> 00:08:39.830 can come back way at the beginning 00:08:39.830 --> 00:08:42.000 and inhibit the pathway. 00:08:42.000 --> 00:08:45.650 So that's called feedback inhibition. 00:08:45.650 --> 00:08:50.355 We saw that cholesterol biosynthesis was 30 steps. 00:08:50.355 --> 00:08:52.730 And if you go back and you look at the pathway, you know, 00:08:52.730 --> 00:08:54.250 I think this is step four or five. 00:08:54.250 --> 00:08:56.180 I can't remember which one it is. 00:08:56.180 --> 00:08:58.610 And so the model was-- 00:08:58.610 --> 00:09:01.528 and there was some evidence that suggested that 00:09:01.528 --> 00:09:03.320 from what had been done in the literature-- 00:09:03.320 --> 00:09:08.630 that cholesterol was potentially acting as a feedback inhibitor. 00:09:08.630 --> 00:09:12.800 And that's what their original working hypothesis was. 00:09:12.800 --> 00:09:16.430 So the hypothesis was-- 00:09:16.430 --> 00:09:18.470 this is how they started it out. 00:09:18.470 --> 00:09:22.150 And what we'll do is just look at a few experiments 00:09:22.150 --> 00:09:24.710 of how they were trying to test their hypothesis 00:09:24.710 --> 00:09:28.400 and then how they change their hypothesis to come up 00:09:28.400 --> 00:09:32.330 with a new model for cholesterol regulation. 00:09:32.330 --> 00:09:36.410 So you start out with acetyl CoA and you 00:09:36.410 --> 00:09:41.390 go through mevalonic acid. 00:09:41.390 --> 00:09:42.740 And then we get to cholesterol. 00:09:42.740 --> 00:09:45.740 And so the model was that-- 00:09:45.740 --> 00:09:51.931 this is HMG reductase-- that this was a feedback inhibitor. 00:09:55.160 --> 00:09:58.350 And that it inhibited by allosteric regulation. 00:09:58.350 --> 00:10:00.290 And that's true of many pathways. 00:10:00.290 --> 00:10:03.590 And often, that's one out of many mechanisms 00:10:03.590 --> 00:10:08.160 that are involved in regulation. 00:10:08.160 --> 00:10:12.110 So the first problem they faced-- 00:10:12.110 --> 00:10:14.030 and for those of you who want to read 00:10:14.030 --> 00:10:16.970 about this in more detail, the original experiments, 00:10:16.970 --> 00:10:21.485 I'm just going to present a few simple experiments 00:10:21.485 --> 00:10:23.610 and I'm going to present them in a simple way. 00:10:23.610 --> 00:10:26.870 OK, everything with human cells is more complicated 00:10:26.870 --> 00:10:28.820 than the way I'm presenting it. 00:10:28.820 --> 00:10:34.190 But for those of you would like to read a little bit more 00:10:34.190 --> 00:10:36.200 about the actual experiments, there 00:10:36.200 --> 00:10:40.220 are two papers that I think are particularly compelling. 00:10:40.220 --> 00:10:43.520 And in previous years, I've actually used these papers 00:10:43.520 --> 00:10:45.450 in recitation. 00:10:45.450 --> 00:10:46.670 OK, so this is one of them. 00:10:46.670 --> 00:10:49.640 I'll put the other one up later on so 00:10:49.640 --> 00:10:51.620 that you can look at the detail, more 00:10:51.620 --> 00:10:52.940 about the experimental details. 00:10:52.940 --> 00:10:56.420 And I think in these particular experiments, what you're 00:10:56.420 --> 00:11:02.690 being introduced to, which most students don't experience, 00:11:02.690 --> 00:11:04.700 is the fact that you have-- 00:11:04.700 --> 00:11:08.570 all you do with these insoluble membrane-like proteins 00:11:08.570 --> 00:11:10.460 and how do you deal with membrane proteins. 00:11:10.460 --> 00:11:13.400 Most of us-- I haven't had any experience with this at all. 00:11:13.400 --> 00:11:16.380 So this week's recitation, for example, 00:11:16.380 --> 00:11:19.580 sort of shows you what they had to go through 00:11:19.580 --> 00:11:22.310 to be able to answer these questions. 00:11:22.310 --> 00:11:23.750 And it's complicated. 00:11:23.750 --> 00:11:26.220 And I think reading the experimental details 00:11:26.220 --> 00:11:28.470 in the end, if you're going to do something like this, 00:11:28.470 --> 00:11:32.180 this provides a nice blueprint of how you try-- 00:11:32.180 --> 00:11:34.910 how you try to design experiments. 00:11:34.910 --> 00:11:36.620 And you'll see some of the complexity 00:11:36.620 --> 00:11:40.510 from the few experiments I'm just going to briefly describe. 00:11:40.510 --> 00:11:45.260 OK, so what they needed was a model system. 00:11:48.230 --> 00:11:51.860 And of course, you can't do experiments on humans. 00:11:51.860 --> 00:11:54.220 So what they wanted to do was have some kind 00:11:54.220 --> 00:11:56.680 of tissue culture system. 00:11:56.680 --> 00:11:58.735 So they wanted a model system. 00:12:01.630 --> 00:12:06.250 And there was some evidence in the literature 00:12:06.250 --> 00:12:13.870 that human fibroblast skin cells were actually 00:12:13.870 --> 00:12:16.970 able to biosynthesize cholesterol. 00:12:16.970 --> 00:12:19.690 So they wanted to ask the question, 00:12:19.690 --> 00:12:22.690 do these skin cells recapitulate what 00:12:22.690 --> 00:12:28.480 people had seen from the biological studies in humans? 00:12:28.480 --> 00:12:31.030 And so the first experiments I'll show you, 00:12:31.030 --> 00:12:33.280 does recapitulate that. 00:12:33.280 --> 00:12:34.810 It didn't have to. 00:12:34.810 --> 00:12:37.000 But then this became their model, 00:12:37.000 --> 00:12:39.640 human fibroblast cells became the model 00:12:39.640 --> 00:12:42.730 for which they're carrying out all of these experiments 00:12:42.730 --> 00:12:46.300 that we're going to very briefly look at. 00:12:46.300 --> 00:12:50.440 OK, so the experiments, I think, are simple, 00:12:50.440 --> 00:12:51.550 at least on the surface. 00:12:51.550 --> 00:12:53.590 Although I think it wasn't so easy to figure out 00:12:53.590 --> 00:12:55.810 how to do these experiments. 00:12:55.810 --> 00:13:00.890 So what they wanted to do, they had patients-- 00:13:00.890 --> 00:13:01.390 whoops. 00:13:01.390 --> 00:13:02.490 I didn't want to do that. 00:13:02.490 --> 00:13:05.960 Anyhow, sorry I'm wasting time. 00:13:05.960 --> 00:13:08.650 OK, this patient is JD. 00:13:08.650 --> 00:13:11.390 And all of the experiments I'm going to show you is JD. 00:13:11.390 --> 00:13:14.650 But they had 25 other patients. 00:13:14.650 --> 00:13:17.830 And what you'll see is they all manifest themselves 00:13:17.830 --> 00:13:18.955 in different ways. 00:13:18.955 --> 00:13:20.830 And we're going to see that that, in the end, 00:13:20.830 --> 00:13:25.120 becomes important in sorting out really what was going on. 00:13:29.050 --> 00:13:31.580 OK, so the first set of experiments they did 00:13:31.580 --> 00:13:32.690 was the following. 00:13:32.690 --> 00:13:35.690 So they had some kind of normal control. 00:13:35.690 --> 00:13:37.310 And then so we have a normal-- 00:13:39.860 --> 00:13:42.440 so we have skin cells from a normal person. 00:13:42.440 --> 00:13:45.620 And this is the control. 00:13:45.620 --> 00:13:53.070 And then you have the FH patient, JD. 00:13:53.070 --> 00:13:55.170 And in the two papers I'm going to reference, 00:13:55.170 --> 00:14:01.950 they did a lot of experiments on JD's fibroblasts. 00:14:01.950 --> 00:14:05.960 And so they did some simple experiments. 00:14:05.960 --> 00:14:08.280 And remember, the rate limiting step 00:14:08.280 --> 00:14:13.620 is proposed to be hydroxymethyl-- 00:14:13.620 --> 00:14:16.320 HMG CoA reductase. 00:14:16.320 --> 00:14:18.480 And so they wanted to first ask what 00:14:18.480 --> 00:14:22.470 happens if you treat the cells, so you have them growing. 00:14:22.470 --> 00:14:28.320 OK, and you let them grow for a certain period of days. 00:14:28.320 --> 00:14:31.020 And then what you do is you take the media, 00:14:31.020 --> 00:14:35.460 change it, and remove low density lipoproteins 00:14:35.460 --> 00:14:36.278 from the media. 00:14:36.278 --> 00:14:38.070 I don't know whether they removed them all. 00:14:38.070 --> 00:14:39.600 They said they removed 5%. 00:14:39.600 --> 00:14:42.277 I don't know what the percent that was there. 00:14:42.277 --> 00:14:43.860 And so we're going to do that for both 00:14:43.860 --> 00:14:45.960 the experiment and the control. 00:14:45.960 --> 00:14:48.880 So this is the experiment. 00:14:48.880 --> 00:14:50.940 This is the normal person. 00:14:50.940 --> 00:14:55.080 This is the experiment, the FH patient. 00:14:55.080 --> 00:14:59.040 And if you look at the axis in measuring HMG CoA 00:14:59.040 --> 00:15:00.660 reductase activity. 00:15:00.660 --> 00:15:06.240 So what they're going to do is look at plus or minus LDL. 00:15:06.240 --> 00:15:11.130 So in this panel, they've removed the LDL, OK? 00:15:11.130 --> 00:15:15.180 And if we remove the LDL, you remove the cholesterol, 00:15:15.180 --> 00:15:21.180 what might you expect to happen to the normal HMG CoA reductase 00:15:21.180 --> 00:15:23.890 levels or activities? 00:15:23.890 --> 00:15:28.240 If you remove the cholesterol from the plasma, 00:15:28.240 --> 00:15:31.907 what might you expect to happen to the activity? 00:15:31.907 --> 00:15:32.990 What would you want to do? 00:15:32.990 --> 00:15:34.198 Would you want to turn it on? 00:15:34.198 --> 00:15:35.570 Would you want to turn it off? 00:15:35.570 --> 00:15:35.990 STUDENT: Turn on. 00:15:35.990 --> 00:15:37.400 JOANNE STUBBE: Turn it on, right. 00:15:37.400 --> 00:15:40.530 So that's what they're going to be assaying. 00:15:40.530 --> 00:15:44.440 They remove it and if you look at the normal patient, 00:15:44.440 --> 00:15:46.190 the normal control, what's going to happen 00:15:46.190 --> 00:15:50.000 is the biosynthesis is turned on. 00:15:50.000 --> 00:15:53.630 So it'll look at this, then, you need to have-- 00:15:53.630 --> 00:16:01.250 and this goes back to the things we've talked about a little bit 00:16:01.250 --> 00:16:02.600 about in class-- 00:16:02.600 --> 00:16:04.790 and in fact, the original recitation 00:16:04.790 --> 00:16:10.120 that we had on radioactivity was completely 00:16:10.120 --> 00:16:13.330 focused on Brown and Goldstein's work. 00:16:13.330 --> 00:16:16.330 So we're going to see that they use a lot of radioactivity 00:16:16.330 --> 00:16:19.030 and all the assays I'm going to be describing today. 00:16:19.030 --> 00:16:23.680 So what we're going to be doing is revisiting radioisotopes. 00:16:23.680 --> 00:16:27.130 They couldn't have done that without these radioisotopes. 00:16:27.130 --> 00:16:32.620 And this is converted to this. 00:16:32.620 --> 00:16:36.740 OK, what's the cofactor for this reaction? 00:16:36.740 --> 00:16:38.670 So, I'm not going to draw up the rest of this. 00:16:38.670 --> 00:16:41.700 This is mevalonic acid. 00:16:41.700 --> 00:16:43.635 What's the cofactor required for this process? 00:16:52.300 --> 00:16:54.340 Any DPH. 00:16:54.340 --> 00:16:56.810 So you have any DPH. 00:16:56.810 --> 00:16:58.420 OK, so how would you assay this? 00:17:02.540 --> 00:17:05.050 So we're doing this now in tissue culture systems. 00:17:05.050 --> 00:17:07.750 That's what-- we are doing this in fibroblast cells in tissue 00:17:07.750 --> 00:17:09.099 culture. 00:17:09.099 --> 00:17:10.630 So we don't have very much material. 00:17:10.630 --> 00:17:12.730 You might have a plateful of cells. 00:17:12.730 --> 00:17:15.026 How would you do the assay? 00:17:15.026 --> 00:17:17.109 So this is the first thing you have to figure out. 00:17:17.109 --> 00:17:19.359 And I would say, almost everything in this class, 00:17:19.359 --> 00:17:20.859 when you're studying the biology, 00:17:20.859 --> 00:17:24.339 first thing you have to do is figure out a robust assay. 00:17:24.339 --> 00:17:26.630 This case, I think it turned out to be quite easy. 00:17:26.630 --> 00:17:29.482 But it's not necessarily easy in many cases. 00:17:29.482 --> 00:17:30.940 So this is something, as a chemist, 00:17:30.940 --> 00:17:33.080 you bring a lot to the table. 00:17:33.080 --> 00:17:33.628 Yeah? 00:17:33.628 --> 00:17:35.170 STUDENT: You would measure the change 00:17:35.170 --> 00:17:38.740 in the absorption at 340. 00:17:38.740 --> 00:17:39.820 JOANNE STUBBE: 340. 00:17:39.820 --> 00:17:41.890 So that's the way chemists would do that. 00:17:41.890 --> 00:17:43.560 Why can't you do that here? 00:17:43.560 --> 00:17:47.180 STUDENT: You have to isolate the HMG CoA reductase 00:17:47.180 --> 00:17:49.270 or somehow be able to parse it from everything-- 00:17:49.270 --> 00:17:50.645 JOANNE STUBBE: Well, you might be 00:17:50.645 --> 00:17:53.380 able to do it in crude extracts if you had a lot of it. 00:17:53.380 --> 00:17:54.520 But it's tough. 00:17:54.520 --> 00:17:59.070 NADPH is used in hundreds of reactions. 00:17:59.070 --> 00:18:04.210 It's a great assay because the absorption change is removed 00:18:04.210 --> 00:18:06.190 from where most of the material absorbs, 00:18:06.190 --> 00:18:10.540 which is, you know, 280, 260, 280. 00:18:10.540 --> 00:18:11.620 It's not that sensitive. 00:18:11.620 --> 00:18:16.690 The extinction coefficient is 6,300 molar inverse centimeter 00:18:16.690 --> 00:18:17.622 inverse. 00:18:17.622 --> 00:18:19.330 And the bottom line is if you look at it, 00:18:19.330 --> 00:18:21.130 it's nowhere near sensitive enough. 00:18:21.130 --> 00:18:24.250 So if it's not sensitive enough, then what do you need to go to? 00:18:27.150 --> 00:18:28.680 That's what-- the radioactivity. 00:18:28.680 --> 00:18:31.340 So what you're going to be doing here is-- 00:18:31.340 --> 00:18:34.110 so you could use either 14c-- 00:18:34.110 --> 00:18:38.130 hopefully you remember that's a beta emitter, which then gets 00:18:38.130 --> 00:18:40.710 converted into mevalonic acid. 00:18:40.710 --> 00:18:44.400 And then you need a way of separating starting material 00:18:44.400 --> 00:18:46.350 from products. 00:18:46.350 --> 00:18:48.340 And there are many ways that one could do that. 00:18:48.340 --> 00:18:52.890 But in the original paper, they use TLC. 00:18:52.890 --> 00:18:55.620 And that's how they monitored their reactions. 00:18:55.620 --> 00:18:58.550 And you need to have material that's 00:18:58.550 --> 00:19:02.693 of hot enough radioactivity so you can see these 00:19:02.693 --> 00:19:03.360 into conversion. 00:19:03.360 --> 00:19:07.920 So that's the assay that they used. 00:19:07.920 --> 00:19:15.090 And so in the PowerPoint, I decided not to draw out. 00:19:15.090 --> 00:19:21.000 So if you PowerPoint, you look at the data, what do you see? 00:19:21.000 --> 00:19:25.800 What you see is that if you look at the experiment 00:19:25.800 --> 00:19:28.980 where they removed the low density 00:19:28.980 --> 00:19:31.300 lipoprotein from the media-- 00:19:31.300 --> 00:19:32.560 so they've taken it out. 00:19:32.560 --> 00:19:35.260 They've grown the cells they have HMG CoA 00:19:35.260 --> 00:19:36.870 reductase activity. 00:19:36.870 --> 00:19:39.250 What do you see immediately-- 00:19:39.250 --> 00:19:44.930 and the control and the patient's cells 00:19:44.930 --> 00:19:47.740 are growing exactly the same way. 00:19:47.740 --> 00:19:50.230 What do you see immediately? 00:19:50.230 --> 00:19:53.490 You see a huge difference in the amount of activity. 00:19:53.490 --> 00:19:55.430 So this is 2. 00:19:55.430 --> 00:19:57.720 This 150 or something. 00:19:57.720 --> 00:20:02.370 And so there could be a number of reasons for all of that. 00:20:02.370 --> 00:20:05.370 And so the question is, what is the basis for this increase 00:20:05.370 --> 00:20:08.130 in activity due to increased huge amount, 00:20:08.130 --> 00:20:10.100 the amount of HMG CoA reductase. 00:20:10.100 --> 00:20:11.150 Has the activity changed? 00:20:11.150 --> 00:20:13.830 Is there a mutation that changes the activity? 00:20:13.830 --> 00:20:16.680 There are lots of explanations. 00:20:16.680 --> 00:20:19.920 And so what they then did, when they remove this, 00:20:19.920 --> 00:20:23.580 they started doing assays over 24 hours. 00:20:23.580 --> 00:20:28.140 And they crack open the cells and do this radioactive assay. 00:20:28.140 --> 00:20:33.180 And then they looked at the rate of formation of mevalonic acid. 00:20:33.180 --> 00:20:38.860 And so what do you see with the normal control? 00:20:38.860 --> 00:20:40.920 You see exactly what you might predict. 00:20:40.920 --> 00:20:42.990 So if the cholesterol levels become low, 00:20:42.990 --> 00:20:46.620 you might want to biosynthesize it. 00:20:46.620 --> 00:20:53.070 But then what do you see with a homozygote, the JD patient? 00:20:53.070 --> 00:20:55.710 What you see is the levels start out 00:20:55.710 --> 00:21:00.450 high you have complete absence of regulation 00:21:00.450 --> 00:21:02.427 by changing the concentration of cholesterol. 00:21:02.427 --> 00:21:03.510 That's what you're seeing. 00:21:03.510 --> 00:21:05.520 So it seems like a simple experiment. 00:21:05.520 --> 00:21:08.410 It is a simple experiment. 00:21:08.410 --> 00:21:12.390 The basis for these observations is still open to debate. 00:21:12.390 --> 00:21:16.030 But the experiment turned out to be straightforward. 00:21:16.030 --> 00:21:19.810 Then what they did is at 24 hours, 00:21:19.810 --> 00:21:25.240 they then started adding low density lipoprotein back 00:21:25.240 --> 00:21:28.210 into the media. 00:21:28.210 --> 00:21:31.060 So they start over here, they removed it. 00:21:31.060 --> 00:21:32.580 They add it back. 00:21:32.580 --> 00:21:33.850 Here's with non. 00:21:33.850 --> 00:21:36.070 Here's with two micrograms per mL. 00:21:36.070 --> 00:21:38.170 Here's with 20 micrograms per mL. 00:21:38.170 --> 00:21:41.500 And what do you see with a normal patient? 00:21:41.500 --> 00:21:42.610 With a normal control? 00:21:42.610 --> 00:21:47.040 What you see with the normal control is a loss of activity. 00:21:47.040 --> 00:21:51.600 So that's exactly what you would expect that cholesterol-- you 00:21:51.600 --> 00:21:54.930 have a lot of cholesterol, you don't need to make it anymore. 00:21:54.930 --> 00:21:58.410 So this data, then, this simple data told you-- 00:21:58.410 --> 00:22:05.890 the control told you that minus LDL, 00:22:05.890 --> 00:22:13.210 you increased HMGR activity. 00:22:16.630 --> 00:22:24.390 And plus LDL, you decreased activity. 00:22:24.390 --> 00:22:26.710 And what about the patient? 00:22:26.710 --> 00:22:30.400 The FH JD patient? 00:22:30.400 --> 00:22:36.310 So here what you see is that removing cholesterol 00:22:36.310 --> 00:22:38.600 from the plasma has no effect. 00:22:38.600 --> 00:22:40.570 What about adding it back? 00:22:40.570 --> 00:22:42.400 Has no effect. 00:22:42.400 --> 00:22:46.470 So somehow the patient is-- 00:22:46.470 --> 00:22:50.200 the patient's cells is oblivious to the presence or absence 00:22:50.200 --> 00:22:51.800 of cholesterol. 00:22:51.800 --> 00:22:59.140 So in this case, plus or minus LDL had no effect. 00:23:02.460 --> 00:23:09.380 So we say loss of cholesterol regulation, which 00:23:09.380 --> 00:23:12.890 could be due to feedback inhibition, 00:23:12.890 --> 00:23:14.390 it could be due to something else. 00:23:14.390 --> 00:23:18.060 We'll see it is due to something else. 00:23:18.060 --> 00:23:21.050 And so this was consistent with what they predicted. 00:23:21.050 --> 00:23:25.700 And they furthermore learned that these fibroblast cells 00:23:25.700 --> 00:23:28.550 might be a good model for actually studying 00:23:28.550 --> 00:23:29.810 what's going on in the liver. 00:23:29.810 --> 00:23:31.268 I mean, you always have this issue. 00:23:31.268 --> 00:23:34.130 You have to figure out what you can study as a model system 00:23:34.130 --> 00:23:36.320 since we don't work on the humans. 00:23:36.320 --> 00:23:38.630 And so you always have to worry about how 00:23:38.630 --> 00:23:42.110 that extrapolates to humans. 00:23:42.110 --> 00:23:51.390 So basically, you're looking at cholesterol in the media. 00:23:51.390 --> 00:23:55.050 You're looking at cholesterol not in the media. 00:23:55.050 --> 00:23:58.720 And these are the experiments we just described. 00:23:58.720 --> 00:24:01.350 And so one of the questions you can ask, 00:24:01.350 --> 00:24:06.990 then, is what happens now? 00:24:06.990 --> 00:24:09.900 Another thing that can happen is what if cholesterol 00:24:09.900 --> 00:24:11.590 can't get into the cell? 00:24:11.590 --> 00:24:14.850 So what they did is another experiment where they-- 00:24:14.850 --> 00:24:18.300 they did two things to look at the HMG CoA reductase 00:24:18.300 --> 00:24:25.620 activity in the normal control and in the FH patient. 00:24:25.620 --> 00:24:29.010 And one of them was they repeated this experiment 00:24:29.010 --> 00:24:30.810 in the presence of ethanol, where 00:24:30.810 --> 00:24:32.550 they dissolved the cholesterol. 00:24:32.550 --> 00:24:35.700 And apparently that allows the cholesterol 00:24:35.700 --> 00:24:37.730 to get across the membrane. 00:24:37.730 --> 00:24:39.900 OK, so we're bypassing what we now 00:24:39.900 --> 00:24:42.030 know is going to be a receptor. 00:24:42.030 --> 00:24:47.640 So they did a second experiment and they 00:24:47.640 --> 00:24:52.330 used ethanol cholesterol. 00:24:52.330 --> 00:24:54.100 And it goes across membrane. 00:24:58.260 --> 00:25:02.340 And then they looked at the HMG CoA reductase activity. 00:25:02.340 --> 00:25:06.660 And the activity of both the patient and the normal controls 00:25:06.660 --> 00:25:07.980 was the same. 00:25:07.980 --> 00:25:18.360 OK, so the activity, HMGR activity the same. 00:25:18.360 --> 00:25:21.030 They don't report the details of this experiment. 00:25:21.030 --> 00:25:23.160 But another way you could do this is you 00:25:23.160 --> 00:25:26.610 could pull out the protein or partially purify 00:25:26.610 --> 00:25:28.920 the protein in crude extracts and try to measure 00:25:28.920 --> 00:25:31.800 the activity using this assay. 00:25:31.800 --> 00:25:35.520 And if you have a good measure of the amount of protein, which 00:25:35.520 --> 00:25:38.780 is key, so you can measure specific activity, 00:25:38.780 --> 00:25:41.160 micromoles of product or nanomoles of product produced 00:25:41.160 --> 00:25:43.890 per minute per milligram of protein, 00:25:43.890 --> 00:25:49.140 you could actually see that the HMG CoA reductase 00:25:49.140 --> 00:25:55.850 activity was the same in the wild type in the normal 00:25:55.850 --> 00:25:56.700 and in the patient. 00:25:56.700 --> 00:26:02.770 So you could also measure this using assay. 00:26:02.770 --> 00:26:06.690 And again, the result was that they 00:26:06.690 --> 00:26:10.110 were the same in both the normal and the patient. 00:26:10.110 --> 00:26:12.960 So then the elevated levels could be-- 00:26:12.960 --> 00:26:15.895 elevated levels, you saw in the very beginning of the HMG CoA 00:26:15.895 --> 00:26:17.770 reductase activity, could be due to the fact, 00:26:17.770 --> 00:26:20.040 they had a huge amount of protein, 00:26:20.040 --> 00:26:22.440 more so than you do with the fibroblasts. 00:26:22.440 --> 00:26:25.120 And so, there's no reason to think a prior 00:26:25.120 --> 00:26:29.430 if you looked at that previous slide, that the control, 00:26:29.430 --> 00:26:31.080 that normal control in the wild type-- 00:26:31.080 --> 00:26:34.200 I don't know what the scatter is in the data for HMG reductase 00:26:34.200 --> 00:26:36.730 activities, but that's something you need to think about. 00:26:36.730 --> 00:26:40.320 But a 60-fold change is a huge change. 00:26:40.320 --> 00:26:43.290 So this data, the initial set of data 00:26:43.290 --> 00:26:46.320 said that, yeah, cholesterol may be 00:26:46.320 --> 00:26:48.450 acting as a feedback inhibitor. 00:26:48.450 --> 00:26:50.820 But here, we can get cholesterol into the cell 00:26:50.820 --> 00:26:52.680 and the activities are the same. 00:26:52.680 --> 00:26:57.980 So they needed to come up with an alternative hypothesis. 00:26:57.980 --> 00:27:03.120 OK, so they then, using these two sets of data, 00:27:03.120 --> 00:27:07.780 came up with an alternative hypothesis. 00:27:07.780 --> 00:27:18.250 So they concluded that it's not cholesterol feed back 00:27:18.250 --> 00:27:19.030 regulated. 00:27:22.310 --> 00:27:27.000 And so then they set out to do a second set of experiments 00:27:27.000 --> 00:27:28.810 based on a new hypothesis. 00:27:34.220 --> 00:27:36.900 And the new hypothesis is that there 00:27:36.900 --> 00:27:40.530 would be some protein that might be involved 00:27:40.530 --> 00:27:44.540 in taking up the LDL particle, which has 00:27:44.540 --> 00:27:46.420 a cholesterol into the cell. 00:27:46.420 --> 00:27:51.900 So the new hypothesis was there is an LDL receptor, 00:27:51.900 --> 00:27:53.340 so r is receptor. 00:27:53.340 --> 00:27:56.760 That's how I'm going to abbreviate it. 00:27:56.760 --> 00:28:07.980 That's key to taking up LDL. 00:28:07.980 --> 00:28:12.120 And so that's what's shown here. 00:28:12.120 --> 00:28:16.200 And so then the question is, what sets of experiments 00:28:16.200 --> 00:28:17.110 do they do next. 00:28:17.110 --> 00:28:20.880 So this is a second set of experiments 00:28:20.880 --> 00:28:25.620 that was done in a paper that's also quite interesting. 00:28:25.620 --> 00:28:27.180 And so, for those of you who want 00:28:27.180 --> 00:28:39.150 to look at the details of this, this was published in 1976. 00:28:39.150 --> 00:28:41.730 And so this is where the data that I'm going to show you 00:28:41.730 --> 00:28:43.260 on this slide came from. 00:28:43.260 --> 00:28:46.590 Because I think they actually put it in one of the two review 00:28:46.590 --> 00:28:48.230 articles I gave you to read. 00:28:48.230 --> 00:28:49.980 But if you want to read the original data, 00:28:49.980 --> 00:28:51.510 the papers aren't that long. 00:28:51.510 --> 00:28:54.150 And they go through the details of the rationale of how 00:28:54.150 --> 00:28:57.030 they design their experiments. 00:28:57.030 --> 00:28:59.910 OK, so what we want to do now is test 00:28:59.910 --> 00:29:05.840 the idea that to get cholesterol into the cell, 00:29:05.840 --> 00:29:07.965 there is an LDL receptor. 00:29:12.290 --> 00:29:15.380 And that that's going to play a key role in controlling 00:29:15.380 --> 00:29:16.400 cholesterol levels. 00:29:16.400 --> 00:29:18.340 That was the working hypothesis. 00:29:18.340 --> 00:29:22.350 OK, so how would you go about testing this experimentally? 00:29:22.350 --> 00:29:26.180 So these are the results of the experiments. 00:29:26.180 --> 00:29:28.460 And the question is, how would you 00:29:28.460 --> 00:29:30.650 go about testing this experimentally 00:29:30.650 --> 00:29:33.030 if this were your hypothesis? 00:29:33.030 --> 00:29:38.150 And so if you think about it, you might like to know, 00:29:38.150 --> 00:29:42.050 does the LDL particle bind to the surface of the cell? 00:29:42.050 --> 00:29:42.980 Does it bind? 00:29:42.980 --> 00:29:46.130 OK, so that would be one thing you could do. 00:29:46.130 --> 00:29:48.920 And in fact, Brown and Goldstein were 00:29:48.920 --> 00:29:50.330 treating many, many patients. 00:29:50.330 --> 00:29:54.320 So they had fibroblasts for many patients, 20 to 25 patients. 00:29:54.320 --> 00:29:56.300 They all had different phenotypes. 00:29:56.300 --> 00:29:57.710 And again, these were differences 00:29:57.710 --> 00:29:59.870 in the phenotypes actually helped them 00:29:59.870 --> 00:30:03.380 to try to dissect this process. 00:30:03.380 --> 00:30:06.290 And so could it bind? 00:30:06.290 --> 00:30:08.922 And so we can ask the question, how would we look at binding? 00:30:08.922 --> 00:30:10.380 I'm going to ask you that question. 00:30:10.380 --> 00:30:12.380 We're going to have a recitation on binding, 00:30:12.380 --> 00:30:16.550 I think, not this week, but next week. 00:30:16.550 --> 00:30:19.520 Then it gets into the cell. 00:30:19.520 --> 00:30:21.530 OK, so how do you know it gets into the cell? 00:30:24.270 --> 00:30:26.190 And so that's another question. 00:30:26.190 --> 00:30:27.720 Inside, outside. 00:30:27.720 --> 00:30:30.240 And then the next question is, what is LDL? 00:30:30.240 --> 00:30:33.360 Hopefully you remember it's a lipoprotein that has 00:30:33.360 --> 00:30:36.360 a single protein on it, apoB. 00:30:36.360 --> 00:30:39.320 And then it's full with cholesterol, cholesterol 00:30:39.320 --> 00:30:41.400 esters, and phospholipids. 00:30:41.400 --> 00:30:44.610 What happens to that stuff once it's inside the cell? 00:30:44.610 --> 00:30:47.760 OK, so those are the questions in this experiment 00:30:47.760 --> 00:30:49.650 that they set out to ask. 00:30:49.650 --> 00:30:53.070 OK, so what I want to do-- 00:30:53.070 --> 00:31:10.160 so binding, internalization, and then 00:31:10.160 --> 00:31:15.600 the fate of LDL inside the cell. 00:31:15.600 --> 00:31:18.050 So that's what they were focused on. 00:31:18.050 --> 00:31:23.270 So what I want to do is show you the tools 00:31:23.270 --> 00:31:26.630 that they developed to try to answer these questions. 00:31:26.630 --> 00:31:28.760 OK, I'm going to show you a few things because this 00:31:28.760 --> 00:31:32.100 isn't such an easy set of experiments to carry out. 00:31:32.100 --> 00:31:34.970 And then what they observed on the normal cells 00:31:34.970 --> 00:31:37.110 and the patient cells. 00:31:37.110 --> 00:31:44.660 OK, so the tools that I want to talk about are the following. 00:31:44.660 --> 00:31:48.440 OK, so we just talked about the fact that, to do the assay, 00:31:48.440 --> 00:31:50.240 we needed radioactivity. 00:31:50.240 --> 00:31:52.910 We needed to be sensitive enough. 00:31:52.910 --> 00:31:56.108 If you're going to be looking at binding on the surface, 00:31:56.108 --> 00:31:56.900 how do you do that? 00:31:56.900 --> 00:31:58.670 Do you think there are a lot of receptors? 00:31:58.670 --> 00:32:00.980 Are there a few receptors? 00:32:00.980 --> 00:32:02.370 So you might not know that. 00:32:02.370 --> 00:32:05.700 But in general, there aren't huge numbers of receptors. 00:32:05.700 --> 00:32:08.840 So measuring binding to the surface of the cell usually 00:32:08.840 --> 00:32:11.510 requires a very sensitive assay. 00:32:11.510 --> 00:32:14.060 So the first thing they needed to do 00:32:14.060 --> 00:32:16.340 was they decided that they needed 00:32:16.340 --> 00:32:19.910 to make the LDL radial labels. 00:32:19.910 --> 00:32:22.220 And if you go back and you look through your notes 00:32:22.220 --> 00:32:26.660 in recitation three where we talked about radioactivity, 00:32:26.660 --> 00:32:29.780 we saw that we have beta, c14 beta, which 00:32:29.780 --> 00:32:31.070 is what they used up there. 00:32:31.070 --> 00:32:35.810 But they also used i125, which is a gamma, which 00:32:35.810 --> 00:32:37.590 is much more sensitive. 00:32:37.590 --> 00:32:40.670 And so what they decided they needed to make 00:32:40.670 --> 00:32:44.630 was i125 labeled LDL. 00:32:44.630 --> 00:32:46.970 So if you haven't radio labeled, can you 00:32:46.970 --> 00:32:50.240 somehow see it sitting on the surface of the cell? 00:32:50.240 --> 00:32:53.150 So the question is, how can you do that? 00:32:53.150 --> 00:32:54.980 Well, we talked about the composition 00:32:54.980 --> 00:32:58.070 of the LDL particle. 00:32:58.070 --> 00:32:59.710 There is cholesterol. 00:32:59.710 --> 00:33:04.010 There's cholesterol esters, phospholipids, and one protein. 00:33:04.010 --> 00:33:06.830 And so what they're doing to put the iodine in 00:33:06.830 --> 00:33:09.970 is putting it into only the protein. 00:33:09.970 --> 00:33:15.110 OK, so what they use is a method called 00:33:15.110 --> 00:33:23.120 Bolton Hunter, which uses radial level iodide and a reagent. 00:33:23.120 --> 00:33:24.502 I'm not going to go through-- 00:33:24.502 --> 00:33:26.210 you can look it up if you're interested-- 00:33:26.210 --> 00:33:28.310 I'm not going to go through the details. 00:33:28.310 --> 00:33:32.430 And what it does is it takes a protein-- 00:33:32.430 --> 00:33:34.950 this is still actually widely used. 00:33:34.950 --> 00:33:35.890 So this would be apoB. 00:33:38.910 --> 00:33:43.070 And it iodinates at the ortho position. 00:33:43.070 --> 00:33:53.960 So what you end up with, then, is iodinated apoB. 00:33:53.960 --> 00:33:56.120 So that's going to be your handle. 00:33:56.120 --> 00:34:00.560 You can make this a very high, specific activity. 00:34:00.560 --> 00:34:03.140 OK, so that's one thing that they needed to do. 00:34:03.140 --> 00:34:06.860 OK, the second thing that they needed to do 00:34:06.860 --> 00:34:11.588 is if they're going to look for binding to the surface, 00:34:11.588 --> 00:34:13.130 how would you design that experiment? 00:34:13.130 --> 00:34:15.830 What might you need to do to figure out 00:34:15.830 --> 00:34:19.280 how you're going to look at binding only and not 00:34:19.280 --> 00:34:22.710 binding and uptake? 00:34:22.710 --> 00:34:26.580 What parameter could you change that would help you do that? 00:34:26.580 --> 00:34:27.150 Temperature. 00:34:27.150 --> 00:34:29.850 So everything-- and you'll see this also 00:34:29.850 --> 00:34:34.420 in experiments this week-- the temperature is really critical. 00:34:34.420 --> 00:34:34.920 Why? 00:34:34.920 --> 00:34:39.389 Because hopefully you all know lipid bilayers are very fluid. 00:34:39.389 --> 00:34:40.889 And if you cool the temperature, you 00:34:40.889 --> 00:34:42.940 prevent uptake and other things. 00:34:42.940 --> 00:34:44.190 You have to test all this out. 00:34:44.190 --> 00:34:46.750 They did a huge number of controls. 00:34:46.750 --> 00:34:49.400 So the second thing that they wanted to do 00:34:49.400 --> 00:34:52.900 is they used temperature. 00:34:52.900 --> 00:34:58.110 So four degrees, they're going to use to look at binding. 00:34:58.110 --> 00:35:00.750 Or if they're looking at a time course and they 00:35:00.750 --> 00:35:03.720 want to stop the reaction, and the reaction is normally 00:35:03.720 --> 00:35:05.420 done at 37 degrees-- 00:35:05.420 --> 00:35:14.280 so uptake experiments would be at 37 degrees. 00:35:14.280 --> 00:35:17.700 OK, so again, temperature is the key parameter. 00:35:17.700 --> 00:35:19.440 You could, if you wanted to a time course 00:35:19.440 --> 00:35:22.620 and stop the reaction, you could cool with down to four degrees. 00:35:22.620 --> 00:35:27.720 I mean, this was a hypothesis they had. 00:35:27.720 --> 00:35:33.210 And so that's the second tool that they're going to use. 00:35:33.210 --> 00:35:37.380 And the third tool, which I think isn't necessarily so 00:35:37.380 --> 00:35:40.440 intuitive, is if you're looking at something binding 00:35:40.440 --> 00:35:42.540 on the surface, you have to always worry 00:35:42.540 --> 00:35:44.430 about non-specific binding. 00:35:44.430 --> 00:35:46.710 You'll talk about that in the recitation. 00:35:46.710 --> 00:35:48.560 On this that's always a problem. 00:35:48.560 --> 00:35:51.130 You're using really hot, iodine-labeled materials, 00:35:51.130 --> 00:35:53.670 so you could get neuron specific binding. 00:35:53.670 --> 00:35:56.280 And so how do you-- so you need to wash it. 00:35:56.280 --> 00:36:03.300 So if the LDL particle bound loosely to the LDL receptor, 00:36:03.300 --> 00:36:05.220 that makes the problem extremely challenging 00:36:05.220 --> 00:36:08.250 because when you're trying to wash away the excess as you 00:36:08.250 --> 00:36:10.860 change the concentration of the LDL, 00:36:10.860 --> 00:36:12.152 you're going to start to lose-- 00:36:12.152 --> 00:36:13.610 you're going to have an equilibrium 00:36:13.610 --> 00:36:15.830 and you're going to start to lose binding. 00:36:15.830 --> 00:36:17.960 It binds really tightly so they had 00:36:17.960 --> 00:36:19.330 to have some kind of a wash. 00:36:19.330 --> 00:36:21.670 So they figured out and optimized a wash. 00:36:21.670 --> 00:36:22.795 So you need to have a wash. 00:36:25.470 --> 00:36:28.950 So if you have a wash and then you're 00:36:28.950 --> 00:36:34.290 still looking at the receptor with the particle bound-- 00:36:34.290 --> 00:36:37.890 so that's the LDL-LDL receptor-- then the question is-- 00:36:37.890 --> 00:36:39.360 and it's tight binding-- 00:36:39.360 --> 00:36:42.180 how do you get that off? 00:36:42.180 --> 00:36:46.590 And remember, you're also going to have 00:36:46.590 --> 00:36:50.100 LDL that's been internalized. 00:36:50.100 --> 00:36:53.430 So the creative approach they used 00:36:53.430 --> 00:36:55.790 was to use the molecule heparin. 00:36:55.790 --> 00:36:57.212 OK, so heparin-- 00:36:57.212 --> 00:36:58.920 I'm not going to draw out the structure-- 00:36:58.920 --> 00:37:01.485 but this is a third tool and this was key. 00:37:05.280 --> 00:37:09.480 And so they have heparin-sensitive 00:37:09.480 --> 00:37:11.020 and heparin-resistant. 00:37:14.130 --> 00:37:15.130 And what does this mean? 00:37:15.130 --> 00:37:19.630 Heparin turns out to-- it's a sugar. 00:37:19.630 --> 00:37:22.960 Many of you have probably heard about it. 00:37:22.960 --> 00:37:25.240 It plays a key role in blood coagulation. 00:37:25.240 --> 00:37:27.490 But anyhow, from the point of view of today's lecture, 00:37:27.490 --> 00:37:29.160 you just need to know it's a sugar 00:37:29.160 --> 00:37:31.460 and it's got sulfates all over the outside of it. 00:37:31.460 --> 00:37:33.130 So it's negatively charged. 00:37:33.130 --> 00:37:36.910 So heparin is a sulfated sugar. 00:37:42.270 --> 00:37:46.680 So basically, you have something like this with SO3 minuses 00:37:46.680 --> 00:37:48.600 on the outside. 00:37:48.600 --> 00:37:51.930 And so what happens is if-- 00:37:51.930 --> 00:37:55.170 what you want to do is release the LDL particle 00:37:55.170 --> 00:37:57.780 from the receptor. 00:37:57.780 --> 00:38:01.590 And apparently, treatment with heparin at certain levels-- 00:38:01.590 --> 00:38:03.510 I think they tried a lot of things-- 00:38:03.510 --> 00:38:10.020 was able to release the surface LDL. 00:38:10.020 --> 00:38:16.760 So this is involved in release of surface bound. 00:38:23.170 --> 00:38:26.230 So then what you have left after you release this, 00:38:26.230 --> 00:38:31.000 is you could still have radio label that's been internalized. 00:38:31.000 --> 00:38:35.260 So that then becomes heparin-resistant. 00:38:35.260 --> 00:38:36.450 And so you can count that. 00:38:39.220 --> 00:38:42.850 And so then you have bound and internalized. 00:38:42.850 --> 00:38:46.990 Now, if you're studying this as a function of time, 00:38:46.990 --> 00:38:51.010 what can happen to-- once you internalize the LDL particle, 00:38:51.010 --> 00:38:56.050 what can happen to the iodinated LDL particle? 00:38:56.050 --> 00:38:56.770 What can happen? 00:39:00.343 --> 00:39:01.760 So this is something else you need 00:39:01.760 --> 00:39:04.010 to think about in these assays. 00:39:04.010 --> 00:39:14.500 So now we have internalized LDL, i125 label. 00:39:14.500 --> 00:39:18.775 What can happen-- if you remember from recitation 00:39:18.775 --> 00:39:21.025 this past week, you remember what happened to the LDL? 00:39:24.840 --> 00:39:25.720 So you got protein. 00:39:25.720 --> 00:39:26.470 You got lipids. 00:39:26.470 --> 00:39:27.260 What's going to happen? 00:39:27.260 --> 00:39:28.552 You might not know the details. 00:39:28.552 --> 00:39:30.040 That's what this whole-- 00:39:30.040 --> 00:39:31.870 that's what Brian and Goldstein uncovered, 00:39:31.870 --> 00:39:36.130 which we're going to talk about in the next few minutes. 00:39:36.130 --> 00:39:37.760 But LDL, you have a protein. 00:39:37.760 --> 00:39:39.850 What can happen to proteins? 00:39:39.850 --> 00:39:41.290 They can get degraded. 00:39:41.290 --> 00:39:47.350 So if you have the apoB, what can happen is inside 00:39:47.350 --> 00:39:51.010 the cells-- so this is inside-- 00:39:51.010 --> 00:39:56.170 you could have proteases that degrade this down to peptides. 00:39:56.170 --> 00:40:00.340 This happens in a lysosol where you still 00:40:00.340 --> 00:40:03.810 have iodinated tyrosine. 00:40:03.810 --> 00:40:10.050 Or it can be broken down all the way to just iodinated tyrosine. 00:40:10.050 --> 00:40:12.140 So if you're breaking this down all the way here, 00:40:12.140 --> 00:40:16.728 the iodinated tyrosine could likely exit the cell. 00:40:16.728 --> 00:40:18.270 So you need to really think-- so what 00:40:18.270 --> 00:40:23.490 do you do to control for this aspect of the metabolism? 00:40:23.490 --> 00:40:27.970 What happens to the LDL inside the cell? 00:40:27.970 --> 00:40:33.660 And so to do this, how would you distinguish LDL itself from, 00:40:33.660 --> 00:40:36.240 say-- 00:40:36.240 --> 00:40:39.840 as a chemist, what could you do to distinguish LDL protein 00:40:39.840 --> 00:40:45.220 from LDL on small peptides or LDL as an amino acid? 00:40:45.220 --> 00:40:48.930 So the key question was, what sort of bulk method 00:40:48.930 --> 00:40:52.470 do you use to try to distinguish between these two things. 00:40:52.470 --> 00:40:55.350 So then you can incorporate that into the analysis, which 00:40:55.350 --> 00:40:57.660 is what's on the slide here. 00:40:57.660 --> 00:41:01.980 So what happens if you treat proteins in general with acid? 00:41:06.630 --> 00:41:07.140 They what? 00:41:07.140 --> 00:41:08.970 They hydrolize? 00:41:08.970 --> 00:41:11.640 So peptide bonds are really strong. 00:41:11.640 --> 00:41:14.190 If you want to break a peptide bond, 00:41:14.190 --> 00:41:17.880 you have to heat it for 16 hours at 100 degrees. 00:41:17.880 --> 00:41:19.350 So that's not going to happen. 00:41:19.350 --> 00:41:21.750 So that's not an option. 00:41:21.750 --> 00:41:22.800 But what else happens? 00:41:22.800 --> 00:41:27.210 What do you do when you put a protein into acid? 00:41:27.210 --> 00:41:30.070 What happens to the protein? 00:41:30.070 --> 00:41:31.900 It what? 00:41:31.900 --> 00:41:33.160 Yeah, it crashes out. 00:41:33.160 --> 00:41:36.490 So proteins in general, not all proteins, most proteins 00:41:36.490 --> 00:41:41.350 precipitate, but these kinds of things would be soluble. 00:41:44.020 --> 00:41:46.160 So they've been able to take advantage-- 00:41:46.160 --> 00:41:49.120 so you have to, again, treat the cells in a certain way so 00:41:49.120 --> 00:41:51.010 that you can look at what's still 00:41:51.010 --> 00:41:58.300 retained in the LDL versus what's undergone degradation. 00:41:58.300 --> 00:42:00.930 OK, we're going to see that's key to the model we're 00:42:00.930 --> 00:42:01.870 going to come up with. 00:42:01.870 --> 00:42:06.200 OK, so those are the tools that they needed to develop. 00:42:06.200 --> 00:42:10.710 And so the question is, then, what did they observe? 00:42:10.710 --> 00:42:12.630 OK, so we're doing these same experiments. 00:42:12.630 --> 00:42:15.840 We're looking for binding on the outside, internalization, 00:42:15.840 --> 00:42:16.490 and breakdown. 00:42:16.490 --> 00:42:18.690 That's what we're looking for. 00:42:18.690 --> 00:42:25.290 And so here is the patient and here is the control. 00:42:25.290 --> 00:42:30.400 So if we look at here, these guys are the binding. 00:42:30.400 --> 00:42:33.970 So Brown and Goldstein, in this particular paper, 00:42:33.970 --> 00:42:37.300 which is underneath here but in the cell paper, 00:42:37.300 --> 00:42:38.960 looked at 22 patients. 00:42:38.960 --> 00:42:44.950 And out of the 22 patients, most of them were binding deficient. 00:42:44.950 --> 00:42:47.240 They could see no binding at all. 00:42:47.240 --> 00:42:51.220 Some of them were binding modified. 00:42:51.220 --> 00:42:55.150 That is, they had lower levels of binding. 00:42:55.150 --> 00:43:00.040 And this one patient, JD, had normal binding. 00:43:00.040 --> 00:43:02.770 So in this experiment, we're looking at here-- so 00:43:02.770 --> 00:43:11.380 in the PowerPoint, this is one of 22 patients 00:43:11.380 --> 00:43:14.450 they had normal binding. 00:43:17.060 --> 00:43:18.890 And the others-- and that's because we'll 00:43:18.890 --> 00:43:22.280 see that there are multiple ways you can have 00:43:22.280 --> 00:43:24.380 defects in your LDL receptor. 00:43:24.380 --> 00:43:26.110 We'll come back to that in a minute. 00:43:26.110 --> 00:43:32.300 But you can have deficient binding 00:43:32.300 --> 00:43:33.540 or you could have no binding. 00:43:37.020 --> 00:43:39.780 Or you could have normal binding. 00:43:39.780 --> 00:43:41.610 So those are all possible. 00:43:41.610 --> 00:43:44.850 And the one that we've taken the data for here 00:43:44.850 --> 00:43:48.115 and that's described in the paper, is normal binding. 00:43:48.115 --> 00:43:49.490 And they did a lot of experiments 00:43:49.490 --> 00:43:52.260 I'm not describing to try to show you 00:43:52.260 --> 00:43:55.650 that this experiment, which suggests normal binding, 00:43:55.650 --> 00:43:56.910 is in fact normal binding. 00:43:56.910 --> 00:43:57.990 They looked at off rates. 00:43:57.990 --> 00:44:02.513 They looked at competition with HDL and LDL. 00:44:02.513 --> 00:44:03.930 And so if you look at that, if you 00:44:03.930 --> 00:44:06.570 look at the levels of binding, they really 00:44:06.570 --> 00:44:11.500 aren't very different between the experiment and the control. 00:44:11.500 --> 00:44:14.970 And so now what happens, if you look 00:44:14.970 --> 00:44:19.200 at the normal, what happens is with time, 00:44:19.200 --> 00:44:21.700 the LDL on the surface goes away. 00:44:21.700 --> 00:44:24.840 And that's because it's becoming internalized. 00:44:24.840 --> 00:44:26.970 Whereas down here, what happens? 00:44:26.970 --> 00:44:30.750 You started out the same, but now you can see over-- 00:44:30.750 --> 00:44:33.510 this is hours down here, it really 00:44:33.510 --> 00:44:35.700 hasn't changed very much. 00:44:35.700 --> 00:44:37.830 It's not becoming internalized. 00:44:37.830 --> 00:44:39.870 And so then they wanted to use their method 00:44:39.870 --> 00:44:43.620 to look at internalized LDL. 00:44:43.620 --> 00:44:48.540 And so internalized LDL, using the heparin-resistant 00:44:48.540 --> 00:44:52.230 versus heparin-sensitive, that's the assay they used, 00:44:52.230 --> 00:44:55.920 what you see is as the surface binding at least early 00:44:55.920 --> 00:45:00.480 on decreases, the amount internalized increases. 00:45:00.480 --> 00:45:04.120 But what happens over here to the patient? 00:45:04.120 --> 00:45:07.460 With the patient, you get nothing internalized. 00:45:07.460 --> 00:45:11.120 And the other question is, what happens to the LDL-- 00:45:11.120 --> 00:45:13.250 and it's labeled on the protein-- 00:45:13.250 --> 00:45:14.520 does that get degraded? 00:45:14.520 --> 00:45:16.880 And so using a method with TCA, they 00:45:16.880 --> 00:45:19.760 used a couple of different methods, what they see 00:45:19.760 --> 00:45:26.510 is that you slowly degrade the protein into small pieces. 00:45:26.510 --> 00:45:30.030 And again, with the patient, it's not internalized 00:45:30.030 --> 00:45:34.650 so you can't get degradation. 00:45:34.650 --> 00:45:38.660 So this type of experiment with this particular patient 00:45:38.660 --> 00:45:45.950 and also with the other patients that I talked about, one 00:45:45.950 --> 00:45:50.030 through 21, they drew a strong conclusion 00:45:50.030 --> 00:45:54.590 that there are two things that have to happen for cholesterol 00:45:54.590 --> 00:45:55.520 to get into the cell. 00:45:55.520 --> 00:45:57.320 Number one, it has to bind. 00:45:57.320 --> 00:45:59.480 And number two, there's got to be some mechanism 00:45:59.480 --> 00:46:01.010 for internalization. 00:46:01.010 --> 00:46:08.980 So the conclusions from this is we need binding, 00:46:08.980 --> 00:46:11.760 which is consistent with the LDL receptor. 00:46:11.760 --> 00:46:17.310 And then we need, in some way, internalization. 00:46:17.310 --> 00:46:22.200 And of course, JD was the only one out of all of these 00:46:22.200 --> 00:46:25.670 patients where they can study internalization 00:46:25.670 --> 00:46:28.320 because in the other patients they didn't-- they had really 00:46:28.320 --> 00:46:31.560 poor binding or no binding at all. 00:46:31.560 --> 00:46:34.080 So they needed to have this spectrum of patients 00:46:34.080 --> 00:46:38.210 to be able to start to sort out what was going on 00:46:38.210 --> 00:46:39.210 in these experiments. 00:46:39.210 --> 00:46:42.433 So I think on the surface, the experiments look pretty-- 00:46:42.433 --> 00:46:44.850 you'll look at them, they look like they're really simple. 00:46:44.850 --> 00:46:47.580 But technically, they're not so simple. 00:46:47.580 --> 00:46:50.460 And if you care about the technical details, which we'll 00:46:50.460 --> 00:46:53.040 see again in this week's recitation dealing 00:46:53.040 --> 00:46:55.860 with these membrane proteins and stickiness, 00:46:55.860 --> 00:46:58.890 becomes the key how creative you can be. 00:46:58.890 --> 00:47:03.363 And usually, we're not really plugged into that. 00:47:03.363 --> 00:47:05.280 And you usually don't do experiments like that 00:47:05.280 --> 00:47:07.080 unless you work in a lab that is focused 00:47:07.080 --> 00:47:11.940 on membrane-bound proteins. 00:47:11.940 --> 00:47:14.960 So this resulted in the model. 00:47:14.960 --> 00:47:19.980 So this kind of experiment and many other experiments 00:47:19.980 --> 00:47:31.890 resulted in the model for receptor mediated endocytosis. 00:47:31.890 --> 00:47:34.140 And you've seen this before. 00:47:34.140 --> 00:47:37.170 You saw this in recitation last week 00:47:37.170 --> 00:47:42.360 because we saw interference with the PCSK9 00:47:42.360 --> 00:47:44.850 with receptor mediated endocytosis. 00:47:44.850 --> 00:47:46.860 So we're back where we started last week. 00:47:46.860 --> 00:47:49.980 And the first slide I showed you was this slide. 00:47:49.980 --> 00:47:52.110 And so what is the model? 00:47:52.110 --> 00:47:54.690 So there are many, many more experiments 00:47:54.690 --> 00:47:56.610 that have gone into coming up with this model. 00:47:56.610 --> 00:47:59.670 And the model is really still incomplete. 00:47:59.670 --> 00:48:02.640 I have a cartoon here, the whole process, 00:48:02.640 --> 00:48:06.300 every step along the pathway, how you go here and there 00:48:06.300 --> 00:48:09.420 and what the kinetics are, it's all complicated. 00:48:09.420 --> 00:48:12.730 But this is the working hypothesis. 00:48:12.730 --> 00:48:17.580 And so the first thing is you make the LDL receptor. 00:48:17.580 --> 00:48:21.010 It's a membrane protein, has a single transmembrane spanning 00:48:21.010 --> 00:48:21.510 region. 00:48:21.510 --> 00:48:23.670 Is made in the ER. 00:48:23.670 --> 00:48:26.450 And because of this transmembrane spanning region, 00:48:26.450 --> 00:48:29.850 it's got to be transported to the surface. 00:48:29.850 --> 00:48:34.170 And it's done so in little coded vesicles, 00:48:34.170 --> 00:48:36.000 which keeps things soluble. 00:48:36.000 --> 00:48:38.670 And it does this by passing through the Golgi stacks, which 00:48:38.670 --> 00:48:41.550 we talked about at the very beginning. 00:48:41.550 --> 00:48:43.980 Eventually, it gets to the surface. 00:48:43.980 --> 00:48:46.650 These little things here are the LDL receptors. 00:48:49.620 --> 00:48:51.480 You can go home and sleep on this 00:48:51.480 --> 00:48:53.500 and look at it again because I'm over. 00:48:53.500 --> 00:48:57.150 And it just seems like I just started and it's already over. 00:48:57.150 --> 00:48:58.070 I'm sorry. 00:48:58.070 --> 00:48:59.617 OK, I must have spent too much time 00:48:59.617 --> 00:49:01.950 talking about something I wasn't supposed to talk about. 00:49:01.950 --> 00:49:04.380 But anyhow, hopefully you now all 00:49:04.380 --> 00:49:06.697 can go back and look at this and think 00:49:06.697 --> 00:49:09.030 about this, because we're going to be talking about this 00:49:09.030 --> 00:49:12.770 again in recitation this week.