1 00:00:00,500 --> 00:00:02,810 The following content is provided under a Creative 2 00:00:02,810 --> 00:00:04,380 Commons license. 3 00:00:04,380 --> 00:00:06,670 Your support will help MIT OpenCourseWare 4 00:00:06,670 --> 00:00:11,010 continue to offer high-quality educational resources for free. 5 00:00:11,010 --> 00:00:13,670 To make a donation or view additional materials 6 00:00:13,670 --> 00:00:17,600 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,600 --> 00:00:18,800 at ocw.mit.edu. 8 00:00:25,080 --> 00:00:29,250 JOANNE STUBBE: --that Brown and Goldstein carried out, 9 00:00:29,250 --> 00:00:32,700 which in conjunction with many other experiments 10 00:00:32,700 --> 00:00:35,790 and experiments by other investigators 11 00:00:35,790 --> 00:00:39,630 have led to the model that you see here. 12 00:00:39,630 --> 00:00:44,160 And so we'll just briefly go through this model, which, 13 00:00:44,160 --> 00:00:46,830 again, was the basis for thinking 14 00:00:46,830 --> 00:00:51,990 about the function of PCSK9 that you learned 15 00:00:51,990 --> 00:00:56,310 about recitation last week, as well as providing 16 00:00:56,310 --> 00:00:59,730 the foundation for thinking about the recitation. 17 00:00:59,730 --> 00:01:05,670 This week, we really care how you sense cholesterol levels 18 00:01:05,670 --> 00:01:07,710 in membranes, which is not an easy thing 19 00:01:07,710 --> 00:01:13,330 to do given that it's lipophilic and so are many other things. 20 00:01:13,330 --> 00:01:13,830 OK. 21 00:01:13,830 --> 00:01:17,790 So the LDL receptor-- 22 00:01:17,790 --> 00:01:21,570 that was their model, that there is a receptor-- 23 00:01:21,570 --> 00:01:26,370 is generated in the endoplasmic reticulum. 24 00:01:26,370 --> 00:01:28,590 If you looked at the handout, you'll 25 00:01:28,590 --> 00:01:32,920 see that it has a single transmembrane-spanning region, 26 00:01:32,920 --> 00:01:36,300 which means it's inserted into a membrane. 27 00:01:36,300 --> 00:01:40,850 And the membrane where it functions, at least 28 00:01:40,850 --> 00:01:43,750 at the start of its life, is in the plasma membrane. 29 00:01:43,750 --> 00:01:48,570 So somehow, it has to get from the ER to the plasma membrane. 30 00:01:48,570 --> 00:01:53,475 And this happens by forming coated vesicles. 31 00:01:53,475 --> 00:01:55,350 We'll see a little bit of that, but we're not 32 00:01:55,350 --> 00:01:58,830 going to talk about this methodology in any detail. 33 00:01:58,830 --> 00:02:00,810 But Schekman's lab won the Nobel Prize 34 00:02:00,810 --> 00:02:03,000 for this work, either last year or the year 35 00:02:03,000 --> 00:02:05,910 before, of how do you take proteins 36 00:02:05,910 --> 00:02:11,070 that are not very soluble and get them to the right membrane. 37 00:02:11,070 --> 00:02:13,170 And they do this through coated vesicles 38 00:02:13,170 --> 00:02:15,540 that, then, move through the Golgi stacks 39 00:02:15,540 --> 00:02:18,390 that we talked about at the very beginning. 40 00:02:18,390 --> 00:02:22,710 And then, eventually, they arrive at the plasma 41 00:02:22,710 --> 00:02:26,190 membrane and become inserted. 42 00:02:26,190 --> 00:02:30,550 So these little flags are the LDL receptor. 43 00:02:30,550 --> 00:02:31,050 OK. 44 00:02:31,050 --> 00:02:34,260 So that's the first thing that has to happen. 45 00:02:34,260 --> 00:02:39,540 And I just know that this whole process is extremely complex. 46 00:02:39,540 --> 00:02:44,720 And patient mutants are observed in almost every step 47 00:02:44,720 --> 00:02:45,930 in this overall process. 48 00:02:45,930 --> 00:02:50,520 It's not limited to the one set of types of experiments, 49 00:02:50,520 --> 00:02:54,300 where something binds and doesn't bind to LDL receptor 50 00:02:54,300 --> 00:02:56,650 that we talked about last time. 51 00:02:56,650 --> 00:02:59,220 So the next thing that has to happen-- again, 52 00:02:59,220 --> 00:03:01,440 and we haven't talked about the data for this at all, 53 00:03:01,440 --> 00:03:05,410 but not only do these receptors have to arrive at the surface, 54 00:03:05,410 --> 00:03:09,130 but they, in some way, need to cluster. 55 00:03:09,130 --> 00:03:11,610 And it's only when they cluster that they 56 00:03:11,610 --> 00:03:15,360 form the right kind of a structure that, then, 57 00:03:15,360 --> 00:03:18,990 can be recognized by the LDL particles 58 00:03:18,990 --> 00:03:20,100 that we've talked about. 59 00:03:20,100 --> 00:03:23,290 And so they bind in some way. 60 00:03:23,290 --> 00:03:26,430 And that's the first step in the overall process. 61 00:03:26,430 --> 00:03:34,170 And then, this receptor, bound to its cargo, its nutrients-- 62 00:03:34,170 --> 00:03:36,750 and, again, this is going to be a generic way 63 00:03:36,750 --> 00:03:39,300 of bringing any kinds of nutrients into cells. 64 00:03:39,300 --> 00:03:42,870 It's not limited to cholesterol-- 65 00:03:42,870 --> 00:03:48,060 undergoes what's now been called receptor-mediated endocytosis. 66 00:03:48,060 --> 00:03:53,700 And so when the LDL binds to the receptor, 67 00:03:53,700 --> 00:03:55,830 again, there's a complex sequence 68 00:03:55,830 --> 00:04:01,050 of events that leads to coding of the part that's 69 00:04:01,050 --> 00:04:04,110 going to bud off, by a protein called clathrin. 70 00:04:04,110 --> 00:04:06,450 Again, this is a universal process. 71 00:04:06,450 --> 00:04:08,880 We know quite a bit about that. 72 00:04:08,880 --> 00:04:10,860 And it buds off. 73 00:04:10,860 --> 00:04:13,320 And it gives you a vesicle. 74 00:04:13,320 --> 00:04:18,769 And these little lines along the outside are the clathrin coat. 75 00:04:18,769 --> 00:04:19,769 I'll show you a picture. 76 00:04:19,769 --> 00:04:21,644 I'm not going to talk about it in any detail, 77 00:04:21,644 --> 00:04:24,480 but I'll show you a picture of it. 78 00:04:24,480 --> 00:04:27,780 So the LDL binding, we talked about. 79 00:04:27,780 --> 00:04:30,180 We talked about binding in internalization. 80 00:04:30,180 --> 00:04:33,000 Those are the experiments we talked about last time 81 00:04:33,000 --> 00:04:39,250 in class that led, in part, to this working hypothesis. 82 00:04:39,250 --> 00:04:42,780 And so we have clathrin-coated pits. 83 00:04:42,780 --> 00:04:46,740 And it turns out that there's a zip code. 84 00:04:46,740 --> 00:04:49,530 And we'll see zip codes throughout-- we'll 85 00:04:49,530 --> 00:04:51,790 see zip codes again, in a few minutes, 86 00:04:51,790 --> 00:04:53,820 but we'll see zip codes which are simply 87 00:04:53,820 --> 00:04:59,545 short sequences of amino acids that signal to some protein 88 00:04:59,545 --> 00:05:00,670 that they're going to bind. 89 00:05:00,670 --> 00:05:06,870 So how do you target clathrin to form these coated pits? 90 00:05:06,870 --> 00:05:09,220 How do you form a pit, anyhow, in a circle? 91 00:05:09,220 --> 00:05:10,800 And how does it bud off? 92 00:05:10,800 --> 00:05:12,660 And where do you get the curvature from? 93 00:05:12,660 --> 00:05:14,700 Many people study these processes. 94 00:05:14,700 --> 00:05:17,220 All of these are interesting machines 95 00:05:17,220 --> 00:05:19,780 that we're not going to cover in class. 96 00:05:19,780 --> 00:05:26,210 So you form this coated pit, and then it's removed. 97 00:05:26,210 --> 00:05:29,120 So once it's formed, and you've got a little vesicle, 98 00:05:29,120 --> 00:05:29,810 it's removed. 99 00:05:29,810 --> 00:05:32,600 And then it can go on and do another step. 100 00:05:32,600 --> 00:05:35,810 And another step that it does is that it 101 00:05:35,810 --> 00:05:41,330 fuses with another organelle called an endosome, which 102 00:05:41,330 --> 00:05:42,320 is acidic pH. 103 00:05:42,320 --> 00:05:44,390 How it does that, how it's recognized, 104 00:05:44,390 --> 00:05:46,400 why does it go to the endosome and not directly 105 00:05:46,400 --> 00:05:49,633 to the lysosome-- all of these things, questions, 106 00:05:49,633 --> 00:05:51,050 that should be raised in your mind 107 00:05:51,050 --> 00:05:52,842 if you're thinking about the details of how 108 00:05:52,842 --> 00:05:56,270 this thing works, none of which we're going to discuss. 109 00:05:56,270 --> 00:06:00,710 But it gets into the endosome, and then what you want to do 110 00:06:00,710 --> 00:06:08,090 is separate the receptor from its cargo, the LDL. 111 00:06:08,090 --> 00:06:09,560 And we know quite a bit about that. 112 00:06:09,560 --> 00:06:11,810 If you read-- I'm not going to talk about that either, 113 00:06:11,810 --> 00:06:14,930 but if you read the end of the PowerPoint presentation, 114 00:06:14,930 --> 00:06:18,770 there's a model for actually how this can happen. 115 00:06:18,770 --> 00:06:23,720 And you can separate the receptor from the cargo. 116 00:06:23,720 --> 00:06:28,610 And the receptors bud off, and they 117 00:06:28,610 --> 00:06:32,930 are recycled in little vesicles to the surface, where 118 00:06:32,930 --> 00:06:35,450 they can be reused. 119 00:06:35,450 --> 00:06:40,970 The LDL particles can also, then-- and what's left here 120 00:06:40,970 --> 00:06:42,425 can then fuse with the lysosome. 121 00:06:42,425 --> 00:06:44,300 And that's, again-- we've talked about this-- 122 00:06:44,300 --> 00:06:48,050 it's a bag of proteases and a bag of esterases, hydrolysis, 123 00:06:48,050 --> 00:06:48,770 lipids. 124 00:06:48,770 --> 00:06:52,580 That's what we have in the LDL particle-- hydrolysis. 125 00:06:52,580 --> 00:06:55,910 We talked about ApoB being degraded 126 00:06:55,910 --> 00:06:58,130 with iodinated tyrosine, last time. 127 00:06:58,130 --> 00:07:03,350 That's where this happens and gives you amino acids 128 00:07:03,350 --> 00:07:04,740 and gives you cholesterol. 129 00:07:04,740 --> 00:07:05,240 OK. 130 00:07:05,240 --> 00:07:08,690 And then, again, depending on what's 131 00:07:08,690 --> 00:07:11,420 going on in the environment of the cell, 132 00:07:11,420 --> 00:07:14,420 the cholesterol would then be shuttled, somehow, 133 00:07:14,420 --> 00:07:16,170 to the appropriate membranes. 134 00:07:16,170 --> 00:07:16,670 OK. 135 00:07:16,670 --> 00:07:19,820 So you can see the complexity of all of this. 136 00:07:19,820 --> 00:07:24,070 If the cholesterol is present, and we don't need anymore 137 00:07:24,070 --> 00:07:27,710 in the membranes, then it can become esterified 138 00:07:27,710 --> 00:07:29,510 with long-chain fatty acids. 139 00:07:29,510 --> 00:07:31,760 Those become really insoluble, and they 140 00:07:31,760 --> 00:07:36,390 form these little globules inside the cell. 141 00:07:36,390 --> 00:07:38,940 And then the process can repeat itself. 142 00:07:38,940 --> 00:07:42,230 And the question we're going to focus on in lectures 4 and 5, 143 00:07:42,230 --> 00:07:45,090 really, are how do you control all of this. 144 00:07:45,090 --> 00:07:45,590 OK. 145 00:07:45,590 --> 00:07:46,640 So this is the model. 146 00:07:50,610 --> 00:07:52,800 And so I think what's interesting 147 00:07:52,800 --> 00:07:57,060 about it is people have studied this in a lot of detail. 148 00:07:57,060 --> 00:08:01,770 It was the first example of receptor-mediated endocytosis. 149 00:08:01,770 --> 00:08:05,110 So we know something about the lifetime of the receptor. 150 00:08:05,110 --> 00:08:09,270 We know it can make round trip from surface inside, back 151 00:08:09,270 --> 00:08:10,495 to the surface in 10 minutes. 152 00:08:10,495 --> 00:08:12,120 We also know it doesn't even have to be 153 00:08:12,120 --> 00:08:14,700 loaded to make that round trip. 154 00:08:14,700 --> 00:08:18,030 It could be one of the ones that isn't 155 00:08:18,030 --> 00:08:20,400 the clustering of the receptors, which 156 00:08:20,400 --> 00:08:26,140 is required for clathrin-coated vesicles to form. 157 00:08:26,140 --> 00:08:31,390 And so you can tell how many trips it makes in its lifetime. 158 00:08:31,390 --> 00:08:34,230 And so the question, then, what controls all of this? 159 00:08:34,230 --> 00:08:36,870 But before we go on and do that, I just 160 00:08:36,870 --> 00:08:40,710 want to briefly talk about, again, 161 00:08:40,710 --> 00:08:48,030 mutations that have been found in the LDL receptor processing. 162 00:08:48,030 --> 00:08:52,150 And they're really, basically, at every step in the pathway. 163 00:08:52,150 --> 00:08:55,260 So the initial ones we found, that we talked about, 164 00:08:55,260 --> 00:08:56,470 we'll come to in a minute. 165 00:08:56,470 --> 00:09:04,270 But we had some patients with no LDL receptor express at all. 166 00:09:04,270 --> 00:09:06,630 So somehow, it never makes it to the surface. 167 00:09:06,630 --> 00:09:07,620 OK? 168 00:09:07,620 --> 00:09:10,260 There are other examples-- and these have all been studied 169 00:09:10,260 --> 00:09:13,650 by many people over the decades-- 170 00:09:13,650 --> 00:09:17,940 that it takes a long time to go through this processing. 171 00:09:17,940 --> 00:09:21,270 And it gets stuck somewhere in the processing. 172 00:09:21,270 --> 00:09:23,430 That may or may not be surprising, 173 00:09:23,430 --> 00:09:27,300 in that you have transmembrane insoluble regions. 174 00:09:27,300 --> 00:09:30,210 And if the processing goes a little astray or some mutation 175 00:09:30,210 --> 00:09:34,610 changes, then you might be in trouble. 176 00:09:34,610 --> 00:09:36,780 So we talked about this last time. 177 00:09:36,780 --> 00:09:42,000 We talked about that they had just looked at 22 patients. 178 00:09:42,000 --> 00:09:46,110 Some of the patients had no binding of LDL 179 00:09:46,110 --> 00:09:49,140 to the surface of the fibroblast that they 180 00:09:49,140 --> 00:09:52,800 were using as a model, at all. 181 00:09:52,800 --> 00:09:54,630 Some have defective binding. 182 00:09:54,630 --> 00:09:56,370 So if they compared it to a normal, 183 00:09:56,370 --> 00:09:59,850 they had a range of dissociation constants. 184 00:09:59,850 --> 00:10:03,900 And we'll talk quite a bit about dissociation constants, 185 00:10:03,900 --> 00:10:06,540 not this week but next week, in recitation. 186 00:10:06,540 --> 00:10:09,540 It's not so easy to measure dissociation constants 187 00:10:09,540 --> 00:10:11,580 when things bind tightly. 188 00:10:11,580 --> 00:10:14,340 And thinking about how to measure them correctly, 189 00:10:14,340 --> 00:10:15,600 I think, is really important. 190 00:10:15,600 --> 00:10:19,410 And I would say, probably, I could pull out 191 00:10:19,410 --> 00:10:24,180 10 papers out of current journals, really good journals, 192 00:10:24,180 --> 00:10:26,060 where people haven't measured dissociation 193 00:10:26,060 --> 00:10:28,060 constant correctly, when you have tight binding. 194 00:10:28,060 --> 00:10:30,122 So this is something that we put in 195 00:10:30,122 --> 00:10:32,580 because I think it's important that people need to know how 196 00:10:32,580 --> 00:10:34,140 to think about this problem. 197 00:10:34,140 --> 00:10:37,400 So anyhow, let's assume that Brown and Goldstein 198 00:10:37,400 --> 00:10:41,520 did these experiments correctly, which I'm sure they did. 199 00:10:41,520 --> 00:10:43,950 And they got a range of binding. 200 00:10:43,950 --> 00:10:46,320 And we also saw that the patient we looked at, 201 00:10:46,320 --> 00:10:49,080 JD, had normal binding. 202 00:10:49,080 --> 00:10:51,600 That indicates he was the same as normal patients, 203 00:10:51,600 --> 00:10:54,150 but something else was problematic. 204 00:10:54,150 --> 00:10:57,960 And that something else wasn't that it 205 00:10:57,960 --> 00:11:03,300 failed to form coated pits, but that it failed 206 00:11:03,300 --> 00:11:05,050 to bring this into the cell. 207 00:11:05,050 --> 00:11:07,920 So it failed to internalize the LDL. 208 00:11:07,920 --> 00:11:10,740 That was JD's defect. 209 00:11:10,740 --> 00:11:13,560 We also, in recitation last week-- 210 00:11:13,560 --> 00:11:16,050 hopefully, you've had time, now, to go back and look 211 00:11:16,050 --> 00:11:17,970 at the paper a little bit. 212 00:11:17,970 --> 00:11:22,320 But LDL, in the model we were just looking at, gets recycled. 213 00:11:22,320 --> 00:11:24,540 It goes in and gets back to the surface. 214 00:11:24,540 --> 00:11:26,820 But what happens if, on occasion, instead 215 00:11:26,820 --> 00:11:31,560 of budding off into vesicles and returning to the surface, it, 216 00:11:31,560 --> 00:11:37,170 with the LDL cargo, goes to the lysosome and gets degraded? 217 00:11:37,170 --> 00:11:41,940 Well, that was the working hypothesis for what PCKS did. 218 00:11:41,940 --> 00:11:45,270 It targeted to the wrong place and degraded it. 219 00:11:45,270 --> 00:11:47,910 And the phenotypes of those patients were interesting, 220 00:11:47,910 --> 00:11:50,670 and that's why it was pursued. 221 00:11:50,670 --> 00:11:54,530 So there are many, many defects. 222 00:11:54,530 --> 00:11:57,840 And despite the fact that we have these statins, 223 00:11:57,840 --> 00:12:00,000 people are still spending a large amount 224 00:12:00,000 --> 00:12:03,240 of time thinking about this because of the prevalence 225 00:12:03,240 --> 00:12:04,830 of coronary disease. 226 00:12:04,830 --> 00:12:06,700 So I'm not going to talk about this, 227 00:12:06,700 --> 00:12:08,450 but I'm just going to show you two slides. 228 00:12:08,450 --> 00:12:10,850 And you can go back and think about this yourself. 229 00:12:10,850 --> 00:12:12,810 But this is the LDL receptor. 230 00:12:12,810 --> 00:12:15,930 We know quite a bit about it now. 231 00:12:15,930 --> 00:12:18,780 And one of the questions you can ask yourself, 232 00:12:18,780 --> 00:12:20,550 which is an interesting question we're not 233 00:12:20,550 --> 00:12:22,980 going to describe-- but you have LDL 234 00:12:22,980 --> 00:12:26,520 particles that are different sizes. 235 00:12:26,520 --> 00:12:29,470 How do you recognize all these different sizes? 236 00:12:29,470 --> 00:12:32,100 And how does the clustering do that? 237 00:12:32,100 --> 00:12:33,690 And so that's done up here. 238 00:12:33,690 --> 00:12:34,862 And there's calcium binding. 239 00:12:34,862 --> 00:12:37,320 We know quite a bit about that, but I don't think we really 240 00:12:37,320 --> 00:12:38,790 understand the details. 241 00:12:38,790 --> 00:12:44,230 You have a single transmembrane helix in the plasma membrane. 242 00:12:44,230 --> 00:12:46,470 And this is the part-- 243 00:12:46,470 --> 00:12:48,180 this part up here-- that actually 244 00:12:48,180 --> 00:12:51,630 binds the LDL particle. 245 00:12:51,630 --> 00:12:53,748 And the last thing I just want to briefly say, 246 00:12:53,748 --> 00:12:55,290 because we're going to see this again 247 00:12:55,290 --> 00:12:58,180 but without going through any details, 248 00:12:58,180 --> 00:13:02,070 remember that eventually we form what are 249 00:13:02,070 --> 00:13:04,680 called clathrin-coated pits. 250 00:13:04,680 --> 00:13:08,910 That's a picture of what the clathrin-coated pits look like. 251 00:13:08,910 --> 00:13:12,450 And the key thing-- and I just wanted to mention this briefly 252 00:13:12,450 --> 00:13:15,210 because we're going to see this again, over and over-- 253 00:13:15,210 --> 00:13:19,290 is the LDL receptor, itself, has a little zip code. 254 00:13:19,290 --> 00:13:21,600 And that's enough-- it's at the tail. 255 00:13:21,600 --> 00:13:25,200 That's enough for it to attract this green protein 256 00:13:25,200 --> 00:13:31,050 called to AP-2, which is key to starting clathrin binding, 257 00:13:31,050 --> 00:13:35,010 and formation of the curvature, and eventually 258 00:13:35,010 --> 00:13:40,770 being able to bud off these vesicles surrounded 259 00:13:40,770 --> 00:13:41,820 by clathrin. 260 00:13:41,820 --> 00:13:45,480 And when you do that, you start budding. 261 00:13:45,480 --> 00:13:47,590 And then, somehow, it turns out there's 262 00:13:47,590 --> 00:13:49,820 a little machine, a GTPase-- 263 00:13:49,820 --> 00:13:53,400 we've seen GTPases all over the place-- 264 00:13:53,400 --> 00:13:56,340 that's involved-- this is the name of it-- 265 00:13:56,340 --> 00:13:58,830 that allows you to bud off. 266 00:13:58,830 --> 00:14:02,100 And you use ATP energy to do all of this. 267 00:14:02,100 --> 00:14:05,700 We've seen this over and over again. 268 00:14:05,700 --> 00:14:07,740 And so the point I wanted to make here 269 00:14:07,740 --> 00:14:12,030 is we've seen this with these seminal experiments, 270 00:14:12,030 --> 00:14:13,440 by Brown and Goldstein. 271 00:14:13,440 --> 00:14:16,020 But in fact, we now know that this 272 00:14:16,020 --> 00:14:19,170 is sort of a generic mechanism for taking nutrients 273 00:14:19,170 --> 00:14:20,050 into the cell. 274 00:14:20,050 --> 00:14:26,790 So it's not limited to LDL receptor and LDL. 275 00:14:26,790 --> 00:14:30,570 And in fact, we're going to see, we're going to talk about, 276 00:14:30,570 --> 00:14:35,610 in module 7, Epidermal Growth Factor Receptor. 277 00:14:35,610 --> 00:14:40,170 And we're going to talk, in module 6, the receptor that 278 00:14:40,170 --> 00:14:42,900 takes iron into the cell, both of which 279 00:14:42,900 --> 00:14:44,910 do this kind of signaling. 280 00:14:44,910 --> 00:14:47,820 So this is a generic mechanism to do that. 281 00:14:47,820 --> 00:14:49,450 All of these things are interesting. 282 00:14:49,450 --> 00:14:51,173 We know quite a bit about it. 283 00:14:51,173 --> 00:14:52,590 And if you want to study that, you 284 00:14:52,590 --> 00:14:55,590 could have spent another weeks worth of lectures 285 00:14:55,590 --> 00:14:57,120 studying this. 286 00:14:57,120 --> 00:15:00,390 So the idea, then, is that we have nutrient sensing. 287 00:15:00,390 --> 00:15:04,080 And this is a general way to try to get nutrients 288 00:15:04,080 --> 00:15:06,930 into the cell, that is, you have a receptor, 289 00:15:06,930 --> 00:15:12,160 and it's undergoing receptor-mediated endocytosis. 290 00:15:12,160 --> 00:15:17,870 So that's the end of lecture 3. 291 00:15:17,870 --> 00:15:22,180 I think I'm one lecture behind, but that's not too bad. 292 00:15:22,180 --> 00:15:24,350 So what I'm going to do now is-- 293 00:15:24,350 --> 00:15:28,090 let's make sure I get this right-- 294 00:15:28,090 --> 00:15:30,480 I'm going to start on lecture 4. 295 00:15:30,480 --> 00:15:32,640 And now we're sort of into the question 296 00:15:32,640 --> 00:15:35,920 of how do we sense cholesterol. 297 00:15:35,920 --> 00:15:36,420 OK. 298 00:15:36,420 --> 00:15:39,240 So what I've done in the original handout, 299 00:15:39,240 --> 00:15:42,720 I had lecture 4 and 5 in the single PowerPoint. 300 00:15:42,720 --> 00:15:44,490 They're still in a single PowerPoint, 301 00:15:44,490 --> 00:15:46,200 but I've just split them into two. 302 00:15:46,200 --> 00:15:48,480 So I'll tell you how I've split them. 303 00:15:48,480 --> 00:15:53,700 So lecture 4 is going to be focused 304 00:15:53,700 --> 00:16:00,930 on sensing and transcriptional regulation. 305 00:16:07,860 --> 00:16:13,410 And lecture 5 will be focused on sensing and 306 00:16:13,410 --> 00:16:16,680 post-transcriptional regulation by 307 00:16:16,680 --> 00:16:18,430 a protein-mediated degradation. 308 00:16:18,430 --> 00:16:20,940 So I'm going to split that in two parts. 309 00:16:20,940 --> 00:16:24,750 And so today's lecture will be mostly focused 310 00:16:24,750 --> 00:16:29,180 on transcriptional regulation. 311 00:16:29,180 --> 00:16:35,250 And the key issue is how do we sense cholesterol-- 312 00:16:35,250 --> 00:16:38,640 what is the mechanisms by which we sense cholesterol. 313 00:16:38,640 --> 00:16:41,070 And the outline for the lecture is 314 00:16:41,070 --> 00:16:49,690 that the transcriptional regulation involves 315 00:16:49,690 --> 00:16:53,600 a sterol-responsive element. 316 00:16:53,600 --> 00:16:59,850 So this is sterol-responsive element. 317 00:16:59,850 --> 00:17:04,589 This is a DNA sequence of about 10 base pairs. 318 00:17:04,589 --> 00:17:10,560 And it also involves a transcriptional factor, so TF. 319 00:17:10,560 --> 00:17:13,800 This is a transcriptional factor-- 320 00:17:13,800 --> 00:17:16,197 transcription factor. 321 00:17:16,197 --> 00:17:17,280 And this is called SRE-BP. 322 00:17:20,910 --> 00:17:25,859 So this is Sterol-Responsive Element Binding Protein. 323 00:17:25,859 --> 00:17:28,005 So BP is Binding Protein. 324 00:17:33,480 --> 00:17:33,980 OK. 325 00:17:33,980 --> 00:17:36,920 So the first thing I'm going to talk about, then, 326 00:17:36,920 --> 00:17:40,987 is the discovery of SRE-BP. 327 00:17:40,987 --> 00:17:42,320 So that'll be the first section. 328 00:17:46,513 --> 00:17:47,930 And then what we're going to do is 329 00:17:47,930 --> 00:17:51,830 we want to know what are the players that 330 00:17:51,830 --> 00:17:56,300 allow us to understand how this transcription factor works. 331 00:17:56,300 --> 00:17:58,980 What we'll see that's sort of amazing-- 332 00:17:58,980 --> 00:18:00,950 it was amazing at the time, but now 333 00:18:00,950 --> 00:18:03,200 it's been found in a number of systems-- 334 00:18:03,200 --> 00:18:06,350 is where would you expect a transcription 335 00:18:06,350 --> 00:18:09,080 factor to be located? 336 00:18:09,080 --> 00:18:10,122 AUDIENCE: In the nucleus. 337 00:18:10,122 --> 00:18:11,372 JOANNE STUBBE: In the nucleus. 338 00:18:11,372 --> 00:18:11,930 OK. 339 00:18:11,930 --> 00:18:13,580 And what they found from their studies 340 00:18:13,580 --> 00:18:16,700 that it's located in the ER membrane. 341 00:18:16,700 --> 00:18:19,880 So this was a major discovery. 342 00:18:19,880 --> 00:18:25,460 So this protein is located in the ER membrane. 343 00:18:25,460 --> 00:18:27,180 They didn't know it at the time. 344 00:18:27,180 --> 00:18:29,180 But now, you're faced with the issue, 345 00:18:29,180 --> 00:18:32,190 transcription factors do work in the nucleus. 346 00:18:32,190 --> 00:18:35,420 So somehow, we have to get it from the ER membrane 347 00:18:35,420 --> 00:18:36,990 into the nucleus. 348 00:18:36,990 --> 00:18:46,610 And so to do that, what we need are players for SRE-BP 349 00:18:46,610 --> 00:18:51,185 to go from the ER to the nucleus. 350 00:18:53,780 --> 00:19:00,950 And we're going to see that these players are called SCAP, 351 00:19:00,950 --> 00:19:02,330 and they're called INSIG. 352 00:19:04,370 --> 00:19:05,870 And we'll come back, and we're going 353 00:19:05,870 --> 00:19:09,350 to talk about those in some detail. 354 00:19:09,350 --> 00:19:12,450 And then the last thing we'll focus on is-- 355 00:19:12,450 --> 00:19:13,520 we'll see it throughout. 356 00:19:13,520 --> 00:19:14,570 I'm going to give you-- 357 00:19:14,570 --> 00:19:16,490 what I usually do when we're talking 358 00:19:16,490 --> 00:19:20,037 about some complex mechanism, I give you the model upfront 359 00:19:20,037 --> 00:19:21,620 so you sort of see where you're going. 360 00:19:21,620 --> 00:19:23,360 Hopefully, you've all had time now-- 361 00:19:23,360 --> 00:19:25,700 we've been in this module for a long time-- 362 00:19:25,700 --> 00:19:29,280 to read the review articles. 363 00:19:29,280 --> 00:19:38,360 But we want a model for transcriptional regulation. 364 00:19:38,360 --> 00:19:40,650 So that's where we're going. 365 00:19:40,650 --> 00:19:45,080 And so what I want to do, before we get into the model, 366 00:19:45,080 --> 00:19:49,580 is come back where we started to try to keep you 367 00:19:49,580 --> 00:19:52,640 grounded on what we're doing. 368 00:19:52,640 --> 00:19:54,980 And what we're doing here is our cartoon of the cell 369 00:19:54,980 --> 00:19:57,740 that I showed you in the very beginning. 370 00:19:57,740 --> 00:20:02,210 We know that metabolism of hydrocarbons, 371 00:20:02,210 --> 00:20:07,370 fatty acids, and cholesterol all focus on a central player. 372 00:20:07,370 --> 00:20:10,590 And the central player is acetyl CoA. 373 00:20:10,590 --> 00:20:17,480 Acetyl CoA can be obtained from fatty acids in the diet. 374 00:20:17,480 --> 00:20:21,200 We've talked about the distribution of fatty acids 375 00:20:21,200 --> 00:20:26,060 using lipoproteins, including LDL. 376 00:20:26,060 --> 00:20:27,950 And we get to acetyl CoA-- this all 377 00:20:27,950 --> 00:20:30,290 happens in the mitochondria. 378 00:20:30,290 --> 00:20:34,640 But acetyl CoA cannot get across membranes. 379 00:20:34,640 --> 00:20:35,810 And that's true. 380 00:20:35,810 --> 00:20:37,400 There are a number of things that 381 00:20:37,400 --> 00:20:39,030 can't get across membranes. 382 00:20:39,030 --> 00:20:43,100 And so carriers in the mitochondrial membrane 383 00:20:43,100 --> 00:20:44,750 are key to metabolism. 384 00:20:44,750 --> 00:20:49,550 And I think once you look at it and think about metabolism 385 00:20:49,550 --> 00:20:51,410 overall, it's not so confusing. 386 00:20:51,410 --> 00:20:54,080 But you might not have chosen those. 387 00:20:54,080 --> 00:20:55,970 If you were the designer, you might not 388 00:20:55,970 --> 00:20:58,220 have chosen these to be the carriers 389 00:20:58,220 --> 00:21:00,520 to move in between organelles. 390 00:21:00,520 --> 00:21:03,080 So I think this happens quite frequently, so you 391 00:21:03,080 --> 00:21:05,760 need to pay attention to it. 392 00:21:05,760 --> 00:21:08,760 And so what happens in this case is 393 00:21:08,760 --> 00:21:11,660 acetyl CoA combines with oxaloacetic acid 394 00:21:11,660 --> 00:21:13,730 to form citrate. 395 00:21:13,730 --> 00:21:15,990 Citrate is an intermediate in the Krebs cycle. 396 00:21:15,990 --> 00:21:20,350 The TCA cycle is part of all of central metabolism. 397 00:21:20,350 --> 00:21:22,490 We're going to see citrate again. 398 00:21:22,490 --> 00:21:27,260 It plays a central role in iron homeostasis as well. 399 00:21:27,260 --> 00:21:29,480 And citrate-- there is a transporter that 400 00:21:29,480 --> 00:21:32,400 gets this into the cytoplasm. 401 00:21:32,400 --> 00:21:34,550 So here's the cytoplasm. 402 00:21:34,550 --> 00:21:36,860 There's an enzyme citrate, lyase that uses 403 00:21:36,860 --> 00:21:39,260 ATP to generate acetyl CoA. 404 00:21:39,260 --> 00:21:39,770 OK. 405 00:21:39,770 --> 00:21:42,350 So acetyl CoA is a central player. 406 00:21:42,350 --> 00:21:45,480 And really, what we're thinking about now, in general-- 407 00:21:45,480 --> 00:21:47,030 I'm going back through this-- is what 408 00:21:47,030 --> 00:21:52,070 do we expect sterol-responsive element-binding protein 409 00:21:52,070 --> 00:21:52,700 to regulate. 410 00:21:52,700 --> 00:21:55,400 And I'm going to show you it doesn't just regulate 411 00:21:55,400 --> 00:21:57,380 cholesterol homeostasis. 412 00:21:57,380 --> 00:22:00,860 There's a big picture [AUDIO OUT] all of this. 413 00:22:00,860 --> 00:22:06,440 So you can make-- you talked about this as a prelude 414 00:22:06,440 --> 00:22:11,015 to the polyketide synthases, the natural products Liz introduced 415 00:22:11,015 --> 00:22:12,400 you to. 416 00:22:12,400 --> 00:22:15,870 Anyhow, you can make fatty acids. 417 00:22:15,870 --> 00:22:18,540 Fatty acids can do a number of things. 418 00:22:18,540 --> 00:22:20,330 If you have a ton of them, then you 419 00:22:20,330 --> 00:22:24,210 can react them with glycerol to form triacylglycerol. 420 00:22:24,210 --> 00:22:26,180 And they're insoluble messes. 421 00:22:26,180 --> 00:22:29,300 If you look at the structures, they form little globules. 422 00:22:29,300 --> 00:22:32,800 So we have all these little insoluble globules 423 00:22:32,800 --> 00:22:33,790 inside the cell. 424 00:22:33,790 --> 00:22:36,848 And people are actually quite interested in studying 425 00:22:36,848 --> 00:22:37,390 these things. 426 00:22:37,390 --> 00:22:39,190 Now, we don't know that much about 427 00:22:39,190 --> 00:22:42,160 whether they are proteins or metabolic enzymes that 428 00:22:42,160 --> 00:22:44,740 could be sitting on the surface of these globules. 429 00:22:44,740 --> 00:22:47,290 A lot of people are trying to figure that out. 430 00:22:47,290 --> 00:22:49,960 But also, fatty acids are required 431 00:22:49,960 --> 00:22:52,780 in the presence of glycerol 3-phosphate, which 432 00:22:52,780 --> 00:22:55,270 comes from the glycolysis pathway, the other pathway 433 00:22:55,270 --> 00:22:58,780 that everybody learns about in an introductory course, 434 00:22:58,780 --> 00:23:03,580 to form phospholipids, which are the key component of all 435 00:23:03,580 --> 00:23:05,420 of your membranes. 436 00:23:05,420 --> 00:23:11,770 Alternatively, acetyl CoA, depending on the regulation 437 00:23:11,770 --> 00:23:14,100 of all of this-- that's the key-- 438 00:23:14,100 --> 00:23:16,870 gets converted to hydroxymethylglutaryl-CoA and 439 00:23:16,870 --> 00:23:18,215 mevalonic acid. 440 00:23:18,215 --> 00:23:22,510 Mevalonic acid-- that reduction between these two is a target 441 00:23:22,510 --> 00:23:23,650 of statins-- 442 00:23:23,650 --> 00:23:26,500 then ends up making cholesterol. 443 00:23:26,500 --> 00:23:28,720 And where does cholesterol have to go? 444 00:23:28,720 --> 00:23:33,190 So cholesterol is made, and a lot of it's 445 00:23:33,190 --> 00:23:34,630 happening in the membranes. 446 00:23:34,630 --> 00:23:36,580 A lot of it is associated with the ER, 447 00:23:36,580 --> 00:23:39,730 but only a small amount of the total cholesterol 448 00:23:39,730 --> 00:23:41,170 is in the ER membrane. 449 00:23:41,170 --> 00:23:43,090 Somehow, it's got to be transferred 450 00:23:43,090 --> 00:23:44,620 to all these other membranes. 451 00:23:44,620 --> 00:23:46,690 So that's a problem we haven't talked about. 452 00:23:46,690 --> 00:23:48,010 That's a big problem. 453 00:23:48,010 --> 00:23:51,790 Most of the cholesterol is in the plasma membrane. 454 00:23:51,790 --> 00:23:54,130 If you have excessive of cholesterol, 455 00:23:54,130 --> 00:23:56,440 you can esterify it, and, again, form 456 00:23:56,440 --> 00:23:58,750 little droplets of fats, which have 457 00:23:58,750 --> 00:24:01,390 fatty acids and cholesterol. 458 00:24:01,390 --> 00:24:04,820 So that's the big picture. 459 00:24:04,820 --> 00:24:09,910 And so this is the picture of the regulatory network. 460 00:24:09,910 --> 00:24:15,340 So I'll say this is a PowerPoint for the regulatory network. 461 00:24:15,340 --> 00:24:17,770 And it's governed by-- 462 00:24:17,770 --> 00:24:20,240 it turns out there are three SRE-BPs. 463 00:24:23,740 --> 00:24:25,660 They have a slightly-- and they're 464 00:24:25,660 --> 00:24:28,360 structurally homologous to each other, 465 00:24:28,360 --> 00:24:32,950 and they work in ways that they interact with other protein 466 00:24:32,950 --> 00:24:37,840 factors and control this whole homeostatic process 467 00:24:37,840 --> 00:24:40,590 between fatty acids and cholesterol biosynthesis. 468 00:24:40,590 --> 00:24:44,260 So I think there are two things that you need to think about. 469 00:24:44,260 --> 00:24:50,770 So we want to control basically its lipid metabolism. 470 00:24:50,770 --> 00:24:55,390 And I should say at the outset, we're focusing on SRE-BP, 471 00:24:55,390 --> 00:24:59,230 but some of you, in maybe a more advanced biology course, 472 00:24:59,230 --> 00:25:02,710 know that there are other transcription factors involved 473 00:25:02,710 --> 00:25:05,572 in regulating cholesterol homeostasis. 474 00:25:05,572 --> 00:25:07,030 This is a major one, and that's all 475 00:25:07,030 --> 00:25:10,120 we're going to talk about in this class. 476 00:25:10,120 --> 00:25:13,930 But what else do you need to make molecules, 477 00:25:13,930 --> 00:25:16,390 if you're going to make fatty acids, 478 00:25:16,390 --> 00:25:18,820 if you were going to make cholesterol? 479 00:25:18,820 --> 00:25:20,613 What you need is NADPH. 480 00:25:20,613 --> 00:25:22,030 So that's the other thing that you 481 00:25:22,030 --> 00:25:23,655 need to think about when you're looking 482 00:25:23,655 --> 00:25:26,050 at the regulatory network. 483 00:25:26,050 --> 00:25:28,780 So we need to control-- how do we make lipids? 484 00:25:28,780 --> 00:25:29,980 Where did they come from? 485 00:25:29,980 --> 00:25:34,510 They come from acetyl CoA. 486 00:25:34,510 --> 00:25:38,560 And the second thing we need to think about 487 00:25:38,560 --> 00:25:45,400 is a source of energy to actually form the molecules. 488 00:25:45,400 --> 00:25:47,465 We're after the long-chain fatty acids. 489 00:25:47,465 --> 00:25:49,840 Go back and look at that-- or cholesterol, if you go back 490 00:25:49,840 --> 00:25:51,215 and look at the pathway we talked 491 00:25:51,215 --> 00:25:53,005 about in the first couple lectures. 492 00:25:53,005 --> 00:25:56,140 So NADPH is at the center. 493 00:25:56,140 --> 00:25:59,290 And I forgot to point out before and probably many of you 494 00:25:59,290 --> 00:26:03,220 have heard of but never really thought about malic enzyme 495 00:26:03,220 --> 00:26:03,868 in the cytosol. 496 00:26:03,868 --> 00:26:05,410 You can go back and think about that, 497 00:26:05,410 --> 00:26:08,380 but that's a major source of NADPH. 498 00:26:08,380 --> 00:26:12,010 What is another source of NADPH in the cytosol. 499 00:26:12,010 --> 00:26:15,100 Anybody know? 500 00:26:15,100 --> 00:26:17,530 Where do you get most of your NADPH from? 501 00:26:17,530 --> 00:26:23,470 It's key to biosynthesis of any kind of anabolic pathways. 502 00:26:23,470 --> 00:26:25,224 Does anybody know? 503 00:26:25,224 --> 00:26:27,509 AUDIENCE: [INAUDIBLE] 504 00:26:27,509 --> 00:26:29,340 JOANNE STUBBE: No. 505 00:26:29,340 --> 00:26:30,130 OK. 506 00:26:30,130 --> 00:26:33,790 Did you ever hear of the pentose phosphate pathway? 507 00:26:33,790 --> 00:26:35,890 Well, hopefully, you've heard of it. 508 00:26:35,890 --> 00:26:38,560 Reproducing it might be challenging, 509 00:26:38,560 --> 00:26:40,420 but the pentose phosphate pathway 510 00:26:40,420 --> 00:26:43,770 is central to providing us with NADPH. 511 00:26:43,770 --> 00:26:47,740 It's central for controlling reactive oxygen species, 512 00:26:47,740 --> 00:26:49,890 which is going to be module 7. 513 00:26:49,890 --> 00:26:54,970 It's central for providing NADPH for nucleotide metabolism. 514 00:26:54,970 --> 00:27:01,540 So the pentose phosphate pathway and malic enzyme 515 00:27:01,540 --> 00:27:03,580 are the key sources of NADPH. 516 00:27:03,580 --> 00:27:06,340 And if you're becoming biochemists, 517 00:27:06,340 --> 00:27:09,350 I think, now, all of these pathways, 518 00:27:09,350 --> 00:27:12,640 these central pathways that we talked about in 5.07, 519 00:27:12,640 --> 00:27:15,250 should just-- you don't need to know all the details, 520 00:27:15,250 --> 00:27:18,300 but you need to know how things go in and out. 521 00:27:18,300 --> 00:27:20,630 And it's central to thinking about anything. 522 00:27:20,630 --> 00:27:22,900 And if you ever do any genetic studies, 523 00:27:22,900 --> 00:27:24,760 you can never figure out anything 524 00:27:24,760 --> 00:27:27,530 unless you know how all these things are connected. 525 00:27:27,530 --> 00:27:29,470 So knowing these central pathways 526 00:27:29,470 --> 00:27:31,990 and how things go in and out and connect 527 00:27:31,990 --> 00:27:35,868 is really critical in thinking about many, many kinds 528 00:27:35,868 --> 00:27:37,660 of reactions you might be doing in the lab. 529 00:27:37,660 --> 00:27:40,180 Because you might see something over here, 530 00:27:40,180 --> 00:27:43,870 but it might be way over here that you had the effects. 531 00:27:43,870 --> 00:27:48,970 And knowing these connections, I think, is why I spent another-- 532 00:27:48,970 --> 00:27:52,690 whatever-- five minutes describing the regulation. 533 00:27:52,690 --> 00:27:53,320 OK. 534 00:27:53,320 --> 00:27:55,930 So if we look at this, what we see here-- 535 00:27:55,930 --> 00:28:00,580 and this is an old slide, so this might have changed. 536 00:28:00,580 --> 00:28:05,560 But all of the enzymes in italics 537 00:28:05,560 --> 00:28:07,590 are all regulated by SRE-BP. 538 00:28:10,450 --> 00:28:12,460 So here's acetyl CoA. 539 00:28:12,460 --> 00:28:15,710 What do we see in this path, where we're making cholesterol? 540 00:28:15,710 --> 00:28:17,565 So many of the enzymes-- 541 00:28:17,565 --> 00:28:19,690 we're not going to talk about them-- that we talked 542 00:28:19,690 --> 00:28:25,510 about when we went through the pathway are all regulated 543 00:28:25,510 --> 00:28:28,630 by SRE-BP and is predominantly-- again, 544 00:28:28,630 --> 00:28:31,420 there's overlap of the regulation 545 00:28:31,420 --> 00:28:35,020 between the different forms of the sterol-responsive 546 00:28:35,020 --> 00:28:37,060 element-binding protein. 547 00:28:37,060 --> 00:28:41,977 But you can see, we have HMG CoA reductase, which 548 00:28:41,977 --> 00:28:43,060 is the rate-limiting step. 549 00:28:43,060 --> 00:28:44,410 So that might be expected. 550 00:28:44,410 --> 00:28:47,530 But many of the other enzymes that are also controlled 551 00:28:47,530 --> 00:28:49,060 by this transcription factor. 552 00:28:49,060 --> 00:28:51,910 And the one that turns out, I think, to be quite interesting 553 00:28:51,910 --> 00:28:54,010 for most recent studies is-- 554 00:28:54,010 --> 00:28:55,870 remember, we briefly talked about how 555 00:28:55,870 --> 00:28:59,590 you get from a linear chain, and then we 556 00:28:59,590 --> 00:29:03,820 had to use a monooxygenase to make the epoxide. 557 00:29:03,820 --> 00:29:07,840 That enzyme is a key regulatory enzyme, people now think. 558 00:29:07,840 --> 00:29:11,320 It wasn't thought to be so not all that long ago. 559 00:29:11,320 --> 00:29:14,500 So anyhow, all of these enzymes that we've talked about 560 00:29:14,500 --> 00:29:17,620 are regulated in some way by SRE-BP. 561 00:29:17,620 --> 00:29:19,450 But it doesn't stop there. 562 00:29:19,450 --> 00:29:22,480 If you go over here, you sort of have a partitioning 563 00:29:22,480 --> 00:29:25,570 between acetyl CoA also going into lipids 564 00:29:25,570 --> 00:29:28,570 and forming phospholipids or triacylglycerols, depending 565 00:29:28,570 --> 00:29:30,910 on whether you store or whether you're dividing 566 00:29:30,910 --> 00:29:32,590 and need more membranes. 567 00:29:32,590 --> 00:29:35,450 So all of this, again, it's about regulation. 568 00:29:35,450 --> 00:29:37,660 And if you look at this, you can see 569 00:29:37,660 --> 00:29:41,590 that many of the enzymes in this pathway, 570 00:29:41,590 --> 00:29:44,020 for formation of monoacylglycerol 571 00:29:44,020 --> 00:29:46,850 and triacylglycerols are also involved. 572 00:29:46,850 --> 00:29:47,350 OK. 573 00:29:47,350 --> 00:29:50,020 So that gives you the big picture 574 00:29:50,020 --> 00:29:51,340 that I want you to think about. 575 00:29:51,340 --> 00:29:53,830 So when you wonder where you're going, 576 00:29:53,830 --> 00:29:59,650 you should go back and take a look at the first few slides. 577 00:29:59,650 --> 00:30:01,990 So what I want to do now is really 578 00:30:01,990 --> 00:30:05,470 focus on the first thing. 579 00:30:05,470 --> 00:30:09,780 The first factor was how did they identify. 580 00:30:09,780 --> 00:30:15,480 So this is identification of SRE-BP. 581 00:30:18,390 --> 00:30:20,830 And so probably most people wouldn't talk about this, 582 00:30:20,830 --> 00:30:23,830 but I think it's sort of amazing. 583 00:30:23,830 --> 00:30:26,950 So I'm going to just show you what had to be done. 584 00:30:26,950 --> 00:30:29,680 And this is not an easy set of experiments. 585 00:30:29,680 --> 00:30:32,110 First of all, transcription factors, in general, 586 00:30:32,110 --> 00:30:35,080 aren't present in very large amounts. 587 00:30:35,080 --> 00:30:39,940 To get them out, they also stick to DNA. 588 00:30:39,940 --> 00:30:42,010 So that poses a problem. 589 00:30:42,010 --> 00:30:45,340 Unlike using his tags and all this stuff, none of that stuff 590 00:30:45,340 --> 00:30:48,100 works to isolate transcription factors. 591 00:30:48,100 --> 00:30:50,560 And this was all done before the-- 592 00:30:50,560 --> 00:30:52,230 a long time ago. 593 00:30:52,230 --> 00:30:54,700 And so this was this is quite a feat. 594 00:30:54,700 --> 00:30:57,490 And the key to this feat was that Brown and Goldstein 595 00:30:57,490 --> 00:31:08,420 recognized that in the front of the gene for HMGR-- 596 00:31:08,420 --> 00:31:10,720 Hydroxymethylglutaryl-CoA reductase-- 597 00:31:10,720 --> 00:31:14,380 in the LDL receptor, they found a 10-- 598 00:31:14,380 --> 00:31:16,330 I'm not going to write out the sequence-- 599 00:31:16,330 --> 00:31:20,670 base-pair sequence that was the same. 600 00:31:20,670 --> 00:31:22,830 So that suggested to them that there's 601 00:31:22,830 --> 00:31:26,070 a little piece of nucleic acid with 10 base pairs 602 00:31:26,070 --> 00:31:28,470 that might be recognized by a protein, which could 603 00:31:28,470 --> 00:31:30,750 be the transcription factor. 604 00:31:30,750 --> 00:31:34,450 So this was the key, this 10 base-pair sequence. 605 00:31:34,450 --> 00:31:37,680 And I'll just say, see PowerPoint. 606 00:31:37,680 --> 00:31:44,310 And this is the SRE, before the genes, again. 607 00:31:44,310 --> 00:31:47,010 And this has now been found in front of many genes. 608 00:31:47,010 --> 00:31:50,130 I just showed you that many, many genes are regulated, 609 00:31:50,130 --> 00:31:51,810 in some way, by these proteins. 610 00:31:51,810 --> 00:31:56,030 But this was an observation they made a long time ago. 611 00:31:56,030 --> 00:31:56,850 OK. 612 00:31:56,850 --> 00:32:00,717 So where would you expect-- 613 00:32:00,717 --> 00:32:01,800 we just went through this. 614 00:32:01,800 --> 00:32:05,190 Where would you expect SRE-BP, the transcription factor, 615 00:32:05,190 --> 00:32:06,480 to be located? 616 00:32:06,480 --> 00:32:08,100 You'd expect it to be in the nucleus. 617 00:32:08,100 --> 00:32:08,600 OK. 618 00:32:08,600 --> 00:32:10,510 That's a reasonable expectation. 619 00:32:10,510 --> 00:32:14,250 And so what step might you do, in the very beginning, 620 00:32:14,250 --> 00:32:17,910 to try to help you purify this protein? 621 00:32:17,910 --> 00:32:20,460 And let me just tell you at the outset that the protein had 622 00:32:20,460 --> 00:32:23,020 to be purified 38,000-fold. 623 00:32:23,020 --> 00:32:23,520 OK. 624 00:32:23,520 --> 00:32:27,030 Now, you guys, none of you have ever experienced, really, 625 00:32:27,030 --> 00:32:30,120 protein purification, starting with kilograms of anything. 626 00:32:30,120 --> 00:32:33,750 I have done that and spent three months 627 00:32:33,750 --> 00:32:36,320 purifying a microgram of protein. 628 00:32:36,320 --> 00:32:38,730 And I would argue that some people still need to do that, 629 00:32:38,730 --> 00:32:42,000 because when you do recombinant expression, lots of times, 630 00:32:42,000 --> 00:32:43,170 you miss a lot of stuff. 631 00:32:43,170 --> 00:32:45,450 So somewhere along the way, somebody 632 00:32:45,450 --> 00:32:49,530 needs to really know what the endogenous protein is like, 633 00:32:49,530 --> 00:32:54,180 and not the recombinant protein. 634 00:32:54,180 --> 00:33:02,860 So we're going to have to do a 38,000-fold purification. 635 00:33:02,860 --> 00:33:05,430 And I would say that's not uncommon. 636 00:33:05,430 --> 00:33:10,110 I've done 20 liter by 20 liter gradients 637 00:33:10,110 --> 00:33:13,440 that take three weeks to get through the gradients 638 00:33:13,440 --> 00:33:14,760 and looking for your proteins. 639 00:33:14,760 --> 00:33:17,370 So if your protein is not stable, 640 00:33:17,370 --> 00:33:19,710 even if you're in the cold room, what happens? 641 00:33:19,710 --> 00:33:22,500 Or if there are proteases, it gets degraded. 642 00:33:22,500 --> 00:33:25,330 So I'm just saying, transcription factors 643 00:33:25,330 --> 00:33:27,040 are not easy to deal with. 644 00:33:27,040 --> 00:33:29,730 And this was sort of an amazing feat. 645 00:33:29,730 --> 00:33:31,710 Anyhow, they started with-- 646 00:33:31,710 --> 00:33:38,910 over here-- 100 liters of tissue culture cells. 647 00:33:38,910 --> 00:33:43,080 So most of you have probably seen tissue culture plates. 648 00:33:43,080 --> 00:33:44,815 And that's what you work with. 649 00:33:44,815 --> 00:33:46,440 They started with 100 liter, and that's 650 00:33:46,440 --> 00:33:49,247 why they're using HeLa cells, because you can grow them 651 00:33:49,247 --> 00:33:49,830 on this scale. 652 00:33:49,830 --> 00:33:53,460 You can probably grow a lot of things on this scale, now. 653 00:33:53,460 --> 00:33:57,120 We have much better ways than-- this was a long time ago. 654 00:33:57,120 --> 00:34:00,490 So their approach was-- 655 00:34:00,490 --> 00:34:01,490 so the first thing-- 656 00:34:01,490 --> 00:34:02,680 I got sidetracked again. 657 00:34:02,680 --> 00:34:05,970 But the first thing is that if it's in the nucleus, what would 658 00:34:05,970 --> 00:34:11,768 you do to try to enrich in the transcription factor? 659 00:34:11,768 --> 00:34:14,060 What would be the first thing you might do after you've 660 00:34:14,060 --> 00:34:15,110 isolated the cells? 661 00:34:17,948 --> 00:34:21,207 AUDIENCE: [INAUDIBLE] 662 00:34:21,207 --> 00:34:22,540 JOANNE STUBBE: I can't hear you. 663 00:34:22,540 --> 00:34:24,855 AUDIENCE: Maybe, something involving nuclear-binding 664 00:34:24,855 --> 00:34:26,980 proteins that transport things into [INAUDIBLE]---- 665 00:34:26,980 --> 00:34:30,400 that have transported things into the-- 666 00:34:30,400 --> 00:34:31,150 JOANNE STUBBE: OK. 667 00:34:31,150 --> 00:34:32,550 So I still can't hear you. 668 00:34:32,550 --> 00:34:34,092 You're going to have to speak louder. 669 00:34:34,092 --> 00:34:34,907 I'm going deaf. 670 00:34:34,907 --> 00:34:37,199 And I will get a hearing aid, but I don't have one now. 671 00:34:37,199 --> 00:34:39,780 So you have to speak loud, and you have to articulate. 672 00:34:39,780 --> 00:34:40,280 Yeah? 673 00:34:40,280 --> 00:34:41,810 AUDIENCE: Wait, so just the absolute first step? 674 00:34:41,810 --> 00:34:42,199 JOANNE STUBBE: Yeah. 675 00:34:42,199 --> 00:34:44,059 AUDIENCE: How we're just lysing cells and pelleting them? 676 00:34:44,059 --> 00:34:44,822 AUDIENCE: Yeah. 677 00:34:44,822 --> 00:34:46,530 JOANNE STUBBE: But is there a certain way 678 00:34:46,530 --> 00:34:48,162 you would pellet them? 679 00:34:48,162 --> 00:34:50,370 AUDIENCE: You would have to do a sucrose gradient. 680 00:34:50,370 --> 00:34:52,412 JOANNE STUBBE: You would do some kind of gradient 681 00:34:52,412 --> 00:34:53,431 to try to separate the-- 682 00:34:53,431 --> 00:34:55,139 well, you have to pellet the cells first. 683 00:34:55,139 --> 00:34:57,510 But then, what you want to do is separate the nucleus 684 00:34:57,510 --> 00:34:59,430 from all the organelles. 685 00:34:59,430 --> 00:35:02,290 The issue is-- we already told you this-- 686 00:35:02,290 --> 00:35:04,290 most of the protein is not found in the nucleus. 687 00:35:04,290 --> 00:35:05,490 And that was part of this. 688 00:35:05,490 --> 00:35:08,410 They didn't know that at all, but that's what they did. 689 00:35:08,410 --> 00:35:11,045 They did some kind of a gradient to separate nuclei 690 00:35:11,045 --> 00:35:12,420 from the rest of it, because they 691 00:35:12,420 --> 00:35:16,260 were trying to enrich, which was a totally reasonable thing 692 00:35:16,260 --> 00:35:17,380 for them to have done. 693 00:35:17,380 --> 00:35:17,880 OK. 694 00:35:17,880 --> 00:35:19,110 So I'm not going to write that down, 695 00:35:19,110 --> 00:35:20,610 but that's the first thing they did. 696 00:35:20,610 --> 00:35:31,570 The second thing they did is they made an affinity column 697 00:35:31,570 --> 00:35:34,300 all out of the SRE. 698 00:35:37,200 --> 00:35:43,060 So this is a nucleotide affinity column. 699 00:35:43,060 --> 00:35:47,570 And they ended up using that a couple of times. 700 00:35:47,570 --> 00:35:51,700 And they ended up using a couple of other kinds of columns 701 00:35:51,700 --> 00:35:56,030 and eventually got protein out after a lot of effort. 702 00:35:56,030 --> 00:36:00,460 After a lot of effort, they got protein out. 703 00:36:00,460 --> 00:36:02,050 And the size of the protein-- 704 00:36:02,050 --> 00:36:03,520 so they went through this column. 705 00:36:03,520 --> 00:36:05,520 And they went through additional columns. 706 00:36:05,520 --> 00:36:06,895 I'm not going to go through the-- 707 00:36:11,520 --> 00:36:14,830 and they ended up with proteins that were actually smaller 708 00:36:14,830 --> 00:36:19,360 than the SRE-BP, but they still bound to the affinity column. 709 00:36:19,360 --> 00:36:21,152 So they ended up with proteins-- 710 00:36:21,152 --> 00:36:21,860 I don't remember. 711 00:36:21,860 --> 00:36:25,930 And again, the details of this really aren't so important. 712 00:36:25,930 --> 00:36:28,390 But they ended up with smaller proteins. 713 00:36:28,390 --> 00:36:32,560 Somewhere, I have the size written down. 714 00:36:32,560 --> 00:36:37,840 59 to 68 kilodaltons. 715 00:36:37,840 --> 00:36:41,020 So either protein had been degraded, 716 00:36:41,020 --> 00:36:44,560 or we will see the protein has been processed, 717 00:36:44,560 --> 00:36:46,780 or was being processed during all this workup. 718 00:36:46,780 --> 00:36:48,280 And there are many things that could 719 00:36:48,280 --> 00:36:50,030 have happened to this process. 720 00:36:50,030 --> 00:36:51,580 But what this allowed them to do-- 721 00:36:51,580 --> 00:36:55,750 and this was the key to allowing them to do this better-- 722 00:36:55,750 --> 00:36:57,940 was they could generate antibodies. 723 00:36:57,940 --> 00:37:00,880 So they took this protein that they isolated, 724 00:37:00,880 --> 00:37:07,325 and they generated antibodies. 725 00:37:07,325 --> 00:37:09,700 And we're going to be talking about antibodies this week. 726 00:37:09,700 --> 00:37:12,200 But we're going to be, also, talking about use of antibodies 727 00:37:12,200 --> 00:37:16,870 with fluorescent probes, the last recitation, as well. 728 00:37:16,870 --> 00:37:18,590 So what did this allow them to do? 729 00:37:18,590 --> 00:37:22,030 The antibodies, then, allowed them to go back into the cells 730 00:37:22,030 --> 00:37:26,940 and look for expression of SRE-BP. 731 00:37:26,940 --> 00:37:30,760 And instead of finding it in the nucleus, what they found 732 00:37:30,760 --> 00:37:35,080 was that most of it was localized in the ER membrane. 733 00:37:35,080 --> 00:37:50,080 So these antibodies revealed that SRE-BP is predominantly 734 00:37:50,080 --> 00:37:51,685 in ER membrane. 735 00:37:54,670 --> 00:37:57,110 And again, this question of antibodies-- 736 00:37:57,110 --> 00:38:01,000 which Liz brought up-- and the question of specificity, 737 00:38:01,000 --> 00:38:04,540 and, moreover, the question of sensitivity is really key. 738 00:38:04,540 --> 00:38:07,180 Because now, when you're looking at eukaryotic cells, 739 00:38:07,180 --> 00:38:09,130 we know things move around. 740 00:38:09,130 --> 00:38:10,960 They move around all over the place, 741 00:38:10,960 --> 00:38:13,790 and they move around dependent on the environment. 742 00:38:13,790 --> 00:38:17,230 So you could easily miss location. 743 00:38:17,230 --> 00:38:18,700 This might be the predominant one 744 00:38:18,700 --> 00:38:21,640 under the conditions you looked, but it could be somewhere else. 745 00:38:21,640 --> 00:38:23,320 And I think they didn't realize so much 746 00:38:23,320 --> 00:38:28,180 about that back in these days, but we now know that a lot. 747 00:38:28,180 --> 00:38:31,060 So anyhow, that was a surprise. 748 00:38:31,060 --> 00:38:33,970 And then, that provided the basis 749 00:38:33,970 --> 00:38:38,270 for them going back and thinking much more about this system. 750 00:38:40,715 --> 00:38:42,090 And so what I'm going to show you 751 00:38:42,090 --> 00:38:45,240 is the model that's resulted. 752 00:38:45,240 --> 00:38:47,190 And if some of you have started working 753 00:38:47,190 --> 00:38:50,550 on problem set 7 that's due this week-- 754 00:38:50,550 --> 00:38:53,340 the problem deals with some of the experiments-- 755 00:38:53,340 --> 00:38:55,590 then I'm going to tell you what the answer is. 756 00:38:55,590 --> 00:38:58,830 And you're supposedly looking at the primary data 757 00:38:58,830 --> 00:39:01,650 from where this model came-- 758 00:39:01,650 --> 00:39:03,450 a small amount of the primary data 759 00:39:03,450 --> 00:39:05,080 from where this model came. 760 00:39:05,080 --> 00:39:05,940 OK. 761 00:39:05,940 --> 00:39:09,732 So this is the model. 762 00:39:09,732 --> 00:39:11,190 And I'll write this down in minute. 763 00:39:11,190 --> 00:39:15,690 But the model is at low sterol concentrations. 764 00:39:15,690 --> 00:39:19,350 So at low sterol concentrations, what do we want to do? 765 00:39:19,350 --> 00:39:23,512 We want to-- this transcription factor-- 766 00:39:23,512 --> 00:39:24,970 I should write this down somewhere. 767 00:39:24,970 --> 00:39:32,113 But the transcription factor activates transcription. 768 00:39:35,070 --> 00:39:38,340 It could repress transcription, but it activates. 769 00:39:38,340 --> 00:39:42,970 So if you have low sterols, what do you want to do? 770 00:39:42,970 --> 00:39:45,620 You want to turn on the transcription factor. 771 00:39:45,620 --> 00:39:50,380 So it needs to somehow move from this location in the membrane 772 00:39:50,380 --> 00:39:52,150 to the nucleus. 773 00:39:52,150 --> 00:39:54,160 So that's where this model is coming from. 774 00:39:54,160 --> 00:39:56,230 And we'll walk through it step by step. 775 00:39:56,230 --> 00:39:59,352 So what you'll see-- these are cartoons for the factors 776 00:39:59,352 --> 00:40:00,560 we're going to be looking at. 777 00:40:00,560 --> 00:40:05,410 So this SRE-BP has two transmembrane regions. 778 00:40:05,410 --> 00:40:06,760 We'll come back to that. 779 00:40:06,760 --> 00:40:09,520 This little ball here, which turns out 780 00:40:09,520 --> 00:40:13,030 to be at the N terminus, is a helix-loop-helix, 781 00:40:13,030 --> 00:40:14,600 which is a DNA-binding motif. 782 00:40:14,600 --> 00:40:16,100 We'll come back to this in a minute. 783 00:40:16,100 --> 00:40:19,360 I'm just giving you an overview, and then we'll come back. 784 00:40:19,360 --> 00:40:20,650 There's a second protein. 785 00:40:20,650 --> 00:40:23,170 And this is the key sensor that we're 786 00:40:23,170 --> 00:40:26,580 going to see of cholesterol levels, called SCAP. 787 00:40:26,580 --> 00:40:29,780 And it also resides in the ER membrane. 788 00:40:29,780 --> 00:40:34,870 And it has a little domain on it that recognizes and interacts 789 00:40:34,870 --> 00:40:39,160 with part of SRE-BP. 790 00:40:39,160 --> 00:40:41,830 And so this is located in the lumen. 791 00:40:41,830 --> 00:40:46,990 And these guys, especially this guy, is located in the cytosol. 792 00:40:46,990 --> 00:40:50,290 And we don't want it inside the lumen, 793 00:40:50,290 --> 00:40:54,160 we want it on the outside so it can go into the nucleus 794 00:40:54,160 --> 00:40:55,450 eventually. 795 00:40:55,450 --> 00:41:00,582 So what happens is somehow, when you have low sterols-- 796 00:41:00,582 --> 00:41:03,040 and we're going to look at the model for how this happens-- 797 00:41:09,010 --> 00:41:12,550 both of these proteins, SCAP and SRE-BP, 798 00:41:12,550 --> 00:41:14,830 are transferred by coated vesicles-- 799 00:41:14,830 --> 00:41:16,900 we'll come back to this in a minute-- 800 00:41:16,900 --> 00:41:18,940 into the Golgi. 801 00:41:18,940 --> 00:41:21,100 So they go together into the Golgi. 802 00:41:21,100 --> 00:41:24,190 And I would say that, right now, a lot of people 803 00:41:24,190 --> 00:41:26,380 are asking the question, once you 804 00:41:26,380 --> 00:41:31,060 do the processing to get SRE-BP into the nucleus, what 805 00:41:31,060 --> 00:41:32,525 happens to SCAP. 806 00:41:32,525 --> 00:41:34,150 And there are lots of papers, now, that 807 00:41:34,150 --> 00:41:38,100 are focusing on the fact the SCAP can recycle from the Golgi 808 00:41:38,100 --> 00:41:39,790 back to the ER. 809 00:41:39,790 --> 00:41:41,470 So it's never as simple. 810 00:41:41,470 --> 00:41:44,020 This thing's continually going on that 811 00:41:44,020 --> 00:41:46,840 not that much is wasted. 812 00:41:46,840 --> 00:41:50,260 So this can actually recycle. 813 00:41:50,260 --> 00:41:52,790 And I'm not going to talk about that. 814 00:41:52,790 --> 00:41:57,010 And then, in the Golgi apparatus, 815 00:41:57,010 --> 00:42:02,530 there are two proteins, called S1P and S2P. 816 00:42:02,530 --> 00:42:03,910 And they're both proteases. 817 00:42:03,910 --> 00:42:05,840 We'll come back to this in a second. 818 00:42:05,840 --> 00:42:08,920 So what's unusual is that we want 819 00:42:08,920 --> 00:42:11,860 to get this guy into the nucleus. 820 00:42:11,860 --> 00:42:15,680 And one of the proteases cuts here. 821 00:42:15,680 --> 00:42:17,520 So then we get this piece. 822 00:42:17,520 --> 00:42:20,730 And then the second protease cuts here, 823 00:42:20,730 --> 00:42:22,410 and then we get a little soluble piece 824 00:42:22,410 --> 00:42:24,030 that can move into the nucleus. 825 00:42:24,030 --> 00:42:27,810 Now, this is also revolutionary, in that nobody had ever 826 00:42:27,810 --> 00:42:31,410 known there were proteases that are actually 827 00:42:31,410 --> 00:42:32,730 found in membranes. 828 00:42:32,730 --> 00:42:38,400 Now, we know there are lots of proteases found in membranes. 829 00:42:38,400 --> 00:42:40,380 And any of you work in Matt's lab? 830 00:42:43,080 --> 00:42:49,390 What is the factor that is regulated just like SRE-BP? 831 00:42:49,390 --> 00:42:52,310 Do you know? 832 00:42:52,310 --> 00:42:52,810 OK. 833 00:42:52,810 --> 00:42:55,000 So go look up the AFT4. 834 00:42:55,000 --> 00:42:59,060 Anyhow, so to me, what is common is once we found this, 835 00:42:59,060 --> 00:43:01,410 we've now discovered this in many other systems. 836 00:43:01,410 --> 00:43:04,460 So this system is a paradigm for many things 837 00:43:04,460 --> 00:43:07,760 that people have discovered since the original discovery. 838 00:43:07,760 --> 00:43:09,920 But of course, the thing that's amazing-- 839 00:43:09,920 --> 00:43:12,410 first of all, this was amazing. 840 00:43:12,410 --> 00:43:14,340 The fact that this thing is in the membrane 841 00:43:14,340 --> 00:43:16,630 and gets to the nucleus is amazing. 842 00:43:16,630 --> 00:43:19,520 And at low cholesterol, what you want to do 843 00:43:19,520 --> 00:43:21,230 is activate transcription. 844 00:43:21,230 --> 00:43:24,103 And you saw all the genes that could be activated 845 00:43:24,103 --> 00:43:25,020 in the previous slide. 846 00:43:25,020 --> 00:43:26,690 And it's complicated. 847 00:43:26,690 --> 00:43:29,520 There are many factors involved. 848 00:43:29,520 --> 00:43:33,080 And so the key question, then, is 849 00:43:33,080 --> 00:43:37,050 how do you sense this movement from one place to the other 850 00:43:37,050 --> 00:43:39,870 and what do we know about that. 851 00:43:39,870 --> 00:43:44,310 So what I'm going to do is look a little bit at the model. 852 00:43:44,310 --> 00:43:49,340 So the model will start with-- and the players. 853 00:43:49,340 --> 00:43:51,000 So this is part 2-- 854 00:43:51,000 --> 00:43:51,500 the players. 855 00:43:54,860 --> 00:43:59,540 And the players are-- so if you look at the ER membrane, 856 00:43:59,540 --> 00:44:04,550 what we have is two domains. 857 00:44:04,550 --> 00:44:07,790 And whenever you see a line through the membrane, 858 00:44:07,790 --> 00:44:11,750 that means a single trans helix membrane spanning region. 859 00:44:11,750 --> 00:44:13,022 We see that a lot. 860 00:44:13,022 --> 00:44:14,480 So I'm not going to write that out. 861 00:44:14,480 --> 00:44:21,020 But this is really sort of a single transmembrane helix. 862 00:44:21,020 --> 00:44:26,600 And the key thing is at the N terminus, 863 00:44:26,600 --> 00:44:29,300 you have the helix-loop-helix. 864 00:44:32,450 --> 00:44:35,210 And this binds to DNA. 865 00:44:35,210 --> 00:44:37,820 So this is a DNA-binding motif. 866 00:44:42,430 --> 00:44:45,010 And so this is the protein SRE-BP. 867 00:44:48,910 --> 00:44:50,660 And so the second protein-- 868 00:44:50,660 --> 00:44:54,670 and this is the protein you're focused on 869 00:44:54,670 --> 00:44:57,220 for your problem set-- 870 00:44:57,220 --> 00:45:00,210 has a SCAP. 871 00:45:00,210 --> 00:45:06,520 2, 3, 4, 5, 6, 7, 8. 872 00:45:06,520 --> 00:45:09,620 So it has eight transmembrane helices. 873 00:45:09,620 --> 00:45:12,770 And they've studied all of this using some of the methods 874 00:45:12,770 --> 00:45:17,060 that you're going to be looking at in your problem set. 875 00:45:17,060 --> 00:45:18,920 And to me, there's a couple of things 876 00:45:18,920 --> 00:45:21,170 that we're going to be talking about in detail, 877 00:45:21,170 --> 00:45:24,650 but your problem sets are focused on-- 878 00:45:24,650 --> 00:45:25,297 all right. 879 00:45:25,297 --> 00:45:27,380 So I haven't really shown you where the loops are, 880 00:45:27,380 --> 00:45:30,527 but there are a couple of loops, loop one and loop six, 881 00:45:30,527 --> 00:45:32,360 which is what the problem set is focused on. 882 00:45:32,360 --> 00:45:34,750 And how do you know these are interesting and important. 883 00:45:34,750 --> 00:45:39,000 And we'll come back to this in a little bit. 884 00:45:39,000 --> 00:45:41,010 So now, at low sterols-- 885 00:45:43,590 --> 00:45:47,900 so we want to turn on the machinery 886 00:45:47,900 --> 00:45:49,700 to make more cholesterol-- 887 00:45:49,700 --> 00:45:51,290 so that low sterols. 888 00:45:51,290 --> 00:45:53,720 And one of the key questions is what 889 00:45:53,720 --> 00:45:55,700 is the structure of the sterol. 890 00:45:55,700 --> 00:45:56,930 Can more than one do that? 891 00:45:56,930 --> 00:46:01,280 We'll see different sterols turn on different domains. 892 00:46:01,280 --> 00:46:06,790 And we'll see that there's a domain within SCAP-- 893 00:46:06,790 --> 00:46:10,235 so this protein here is called SCAP. 894 00:46:12,960 --> 00:46:19,820 And we'll see that SCAP has a sterol-sensor domain, 895 00:46:19,820 --> 00:46:21,410 as does another protein called INSIG, 896 00:46:21,410 --> 00:46:25,070 as does HMG-CoA reductase. 897 00:46:25,070 --> 00:46:28,100 So somehow, you have these transmembrane regions 898 00:46:28,100 --> 00:46:32,540 that can bind some kind of sterol, that then changes 899 00:46:32,540 --> 00:46:36,290 the conformations, that is going to allow all of this chemistry 900 00:46:36,290 --> 00:46:36,800 to happen. 901 00:46:36,800 --> 00:46:39,570 So here, for example, we're not going to talk about this now. 902 00:46:39,570 --> 00:46:41,970 We're going to talk about that in the last lecture. 903 00:46:41,970 --> 00:46:47,360 But here's SCAP with its sterol-sensing domain. 904 00:46:47,360 --> 00:46:51,090 So what happens, then, is this has to move. 905 00:46:51,090 --> 00:46:56,060 And as I said before, this can return. 906 00:46:56,060 --> 00:46:57,080 This moves to the Golgi. 907 00:47:00,560 --> 00:47:02,480 So this is the Golgi. 908 00:47:02,480 --> 00:47:05,220 And the Golgi are complicated. 909 00:47:05,220 --> 00:47:09,380 And so I haven't defined where within the Golgi this is. 910 00:47:09,380 --> 00:47:15,410 And these are transferred by COPII vesicles. 911 00:47:19,560 --> 00:47:20,060 OK. 912 00:47:20,060 --> 00:47:21,950 And so what you then have, again, 913 00:47:21,950 --> 00:47:26,300 is your 1, 2, 3, 4, 5, 6, 7, 8. 914 00:47:29,370 --> 00:47:36,330 And you have your sterol-responsive 915 00:47:36,330 --> 00:47:39,300 element-binding protein. 916 00:47:39,300 --> 00:47:40,740 And now what you see-- 917 00:47:40,740 --> 00:47:43,800 and so nothing happens in terms of processing, 918 00:47:43,800 --> 00:47:46,530 until you get into the Golgi. 919 00:47:46,530 --> 00:47:52,125 And then, there's one protein, S1P, which is a protease. 920 00:47:54,810 --> 00:47:57,300 And I'm not going to go into the details of it, 921 00:47:57,300 --> 00:47:58,770 but if you look over here, what's 922 00:47:58,770 --> 00:48:02,430 unusual about this protease? 923 00:48:02,430 --> 00:48:04,180 If I gave you this cartoon, what would you 924 00:48:04,180 --> 00:48:07,450 say about that protease? 925 00:48:07,450 --> 00:48:10,420 Is it unusual compared to, say, trypsin or chymotrypsin. 926 00:48:16,105 --> 00:48:16,730 Can you see it? 927 00:48:20,210 --> 00:48:23,180 You can pull out your handouts. 928 00:48:23,180 --> 00:48:25,028 What are the catalytic groups? 929 00:48:25,028 --> 00:48:26,780 AUDIENCE: [INAUDIBLE] 930 00:48:26,780 --> 00:48:27,935 JOANNE STUBBE: Huh? 931 00:48:27,935 --> 00:48:29,310 Where have you seen those before? 932 00:48:29,310 --> 00:48:30,520 AUDIENCE: [INAUDIBLE] 933 00:48:30,520 --> 00:48:32,895 JOANNE STUBBE: Yeah, so they're aspartic acid, histidine, 934 00:48:32,895 --> 00:48:33,780 and serine. 935 00:48:33,780 --> 00:48:36,390 You see these over and over and over again. 936 00:48:36,390 --> 00:48:41,730 There are 150 serine-type proteases. 937 00:48:41,730 --> 00:48:42,240 OK. 938 00:48:42,240 --> 00:48:45,070 But what's unusual about this? 939 00:48:45,070 --> 00:48:47,510 Huge-- huge. 940 00:48:47,510 --> 00:48:48,150 OK. 941 00:48:48,150 --> 00:48:50,910 And then, the other thing that's unusual about it 942 00:48:50,910 --> 00:48:52,710 is that you have a transmembrane region. 943 00:48:52,710 --> 00:48:54,930 So it's completely different from serine proteases, 944 00:48:54,930 --> 00:48:57,240 so there's got to be some little domain that's 945 00:48:57,240 --> 00:48:58,520 actually doing all of this. 946 00:48:58,520 --> 00:49:02,880 So I just want to note that it's huge. 947 00:49:02,880 --> 00:49:07,110 But you could still pick up D, H, S 948 00:49:07,110 --> 00:49:09,218 and know that that's the protease domain. 949 00:49:09,218 --> 00:49:10,260 And you could study that. 950 00:49:10,260 --> 00:49:17,700 You could mutate serine to alanine or something. 951 00:49:17,700 --> 00:49:19,855 And then you have S2 domains. 952 00:49:19,855 --> 00:49:20,730 So we've gotten here. 953 00:49:20,730 --> 00:49:24,480 And this protease ends up clipping. 954 00:49:24,480 --> 00:49:26,070 so within the membrane-- so somehow, 955 00:49:26,070 --> 00:49:27,800 these things got to come together. 956 00:49:27,800 --> 00:49:34,680 And the active side of this protease needs to clip SRE-BP. 957 00:49:34,680 --> 00:49:37,450 So it does that. 958 00:49:37,450 --> 00:49:39,330 And when does that, what you end up with-- 959 00:49:39,330 --> 00:49:42,910 I'm not drawing the whole thing out, but what you end up with, 960 00:49:42,910 --> 00:49:46,570 then, is your helix-loop-helix. 961 00:49:46,570 --> 00:49:50,230 So this part is still embedded in the membrane. 962 00:49:54,870 --> 00:49:56,745 And then you have your second protease. 963 00:49:58,898 --> 00:49:59,440 I don't know. 964 00:49:59,440 --> 00:50:00,890 I probably have the wrong numbers. 965 00:50:00,890 --> 00:50:04,230 So this is S2P. 966 00:50:04,230 --> 00:50:08,100 And if you look at S2P, what's unusual about it 967 00:50:08,100 --> 00:50:11,280 and what people picked up on is that it has 968 00:50:11,280 --> 00:50:13,440 another little sequence motif. 969 00:50:13,440 --> 00:50:16,140 And this is what you see over and over again, in enzymology. 970 00:50:16,140 --> 00:50:18,510 Once you sort of know something in detail, 971 00:50:18,510 --> 00:50:22,140 you know, even though there's no homology between the proteins 972 00:50:22,140 --> 00:50:24,480 at all, you can pick up little motifs, 973 00:50:24,480 --> 00:50:26,790 just like you can pick out little motifs that 974 00:50:26,790 --> 00:50:29,790 are zip codes that move things around inside the cell. 975 00:50:29,790 --> 00:50:33,180 This little motif is the key player 976 00:50:33,180 --> 00:50:34,680 that tells you that this is probably 977 00:50:34,680 --> 00:50:38,340 a zinc-dependent metalloprotease. 978 00:50:38,340 --> 00:50:47,520 So this turns out to be a zinc metalloprotease. 979 00:50:47,520 --> 00:50:52,890 And this, then, does cleavage. 980 00:50:52,890 --> 00:50:56,330 But now, we actually-- it's pretty close to the membrane. 981 00:50:56,330 --> 00:50:57,180 OK. 982 00:50:57,180 --> 00:50:58,740 It does cleavage. 983 00:50:58,740 --> 00:51:02,700 And now what you've done is you've released this thing. 984 00:51:02,700 --> 00:51:05,290 It pulls itself out of the membrane. 985 00:51:05,290 --> 00:51:08,370 And what you can do, then-- 986 00:51:08,370 --> 00:51:10,420 I'll just put this in here for a second. 987 00:51:10,420 --> 00:51:13,650 But what you can do now is we now move to the nucleus. 988 00:51:17,320 --> 00:51:19,565 And we have our pieces of DNA. 989 00:51:22,230 --> 00:51:25,740 And we have our SRE. 990 00:51:25,740 --> 00:51:30,120 And now we have this helix-loop-helix 991 00:51:30,120 --> 00:51:33,625 that activates transcription. 992 00:51:36,880 --> 00:51:37,380 OK. 993 00:51:37,380 --> 00:51:38,940 So this is really sort of what I just 994 00:51:38,940 --> 00:51:41,970 told you in the other cartoon. 995 00:51:41,970 --> 00:51:43,860 And I just want to repoint out again 996 00:51:43,860 --> 00:51:47,340 that we now believe that these SCAP proteins can recycle back 997 00:51:47,340 --> 00:51:49,410 into the ER and be used again. 998 00:51:49,410 --> 00:51:51,800 And so controlling the levels of all these things-- 999 00:51:51,800 --> 00:51:54,050 we're going to see at the very end-- 1000 00:51:54,050 --> 00:51:56,910 is also related to protein-mediated degradation 1001 00:51:56,910 --> 00:51:59,700 that we're just now beginning to appreciate. 1002 00:51:59,700 --> 00:52:00,330 OK. 1003 00:52:00,330 --> 00:52:02,190 So here's the model. 1004 00:52:02,190 --> 00:52:04,920 This now sets the stage for you to solve 1005 00:52:04,920 --> 00:52:07,110 problem set 7 that's due. 1006 00:52:07,110 --> 00:52:08,490 Because the key question you want 1007 00:52:08,490 --> 00:52:14,520 to ask yourself is how do we know 1008 00:52:14,520 --> 00:52:17,190 about the structure of SCAP. 1009 00:52:17,190 --> 00:52:19,920 And so problem set-- sorry, I'm over again. 1010 00:52:19,920 --> 00:52:22,530 But problem set 7 is focused on how 1011 00:52:22,530 --> 00:52:26,340 do you know that this little loop here, this little loop 1012 00:52:26,340 --> 00:52:29,070 here, and this little zip code plays 1013 00:52:29,070 --> 00:52:33,420 a key role in this whole process of moving from the ER 1014 00:52:33,420 --> 00:52:34,310 into the Golgi. 1015 00:52:34,310 --> 00:52:35,370 OK. 1016 00:52:35,370 --> 00:52:39,030 And we'll come back and talk about this briefly. 1017 00:52:39,030 --> 00:52:41,460 We're not going to talk in detail about the experiments. 1018 00:52:41,460 --> 00:52:43,320 And then we're going to move on and look 1019 00:52:43,320 --> 00:52:48,120 at the post-transcriptional regulation of cholesterol 1020 00:52:48,120 --> 00:52:50,270 homeostasis.