1 00:00:00,500 --> 00:00:02,640 The following content is provided under a Creative 2 00:00:02,640 --> 00:00:04,210 Commons license. 3 00:00:04,210 --> 00:00:06,510 Your support will help MIT OpenCourseWare 4 00:00:06,510 --> 00:00:10,840 continue to offer high-quality educational resources for free. 5 00:00:10,840 --> 00:00:13,500 To make a donation or view additional materials 6 00:00:13,500 --> 00:00:17,440 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,440 --> 00:00:18,410 at ocw.mit.edu. 8 00:00:25,975 --> 00:00:28,350 JOANNE STUBBE: So what we were doing last time is we were 9 00:00:28,350 --> 00:00:31,050 still focused the first two lectures were trying 10 00:00:31,050 --> 00:00:34,260 to understand the biosynthetic pathway for cholesterol bio-- 11 00:00:34,260 --> 00:00:36,110 it's good, thanks-- 12 00:00:36,110 --> 00:00:38,160 for cholesterol biosynthesis. 13 00:00:38,160 --> 00:00:42,450 And we almost got to where we wanted to go, 14 00:00:42,450 --> 00:00:43,870 but we didn't quite get there. 15 00:00:43,870 --> 00:00:48,780 So what we've been focusing on is a new way 16 00:00:48,780 --> 00:00:50,670 of forming carbon-carbon bonds using 17 00:00:50,670 --> 00:00:53,610 C5 units, isopentenyl pyrophosphate 18 00:00:53,610 --> 00:00:56,760 and dimethylallyl pyrophosphate. 19 00:00:56,760 --> 00:01:00,720 And to do that, we had an initiation 20 00:01:00,720 --> 00:01:07,440 process where these molecules were generated from acetyl CoA. 21 00:01:07,440 --> 00:01:09,630 And then the last lecture we were 22 00:01:09,630 --> 00:01:14,250 focused on how we did the elongation process where 23 00:01:14,250 --> 00:01:17,700 we took a bunch of these IPP units, 24 00:01:17,700 --> 00:01:21,600 strung them together to make farnesyl pyrophosphate, which 25 00:01:21,600 --> 00:01:25,080 is C15, and I showed you that C15 26 00:01:25,080 --> 00:01:26,760 could be linear or cyclized. 27 00:01:26,760 --> 00:01:28,980 And we went through the general rules 28 00:01:28,980 --> 00:01:32,940 of what you're going to see with all turpine chemistry, which 29 00:01:32,940 --> 00:01:34,680 is quite diverse, given that there 30 00:01:34,680 --> 00:01:39,120 are estimated to be 70,000 natural products 31 00:01:39,120 --> 00:01:40,700 in the terpenome. 32 00:01:40,700 --> 00:01:46,170 So we had gotten to production of farnesyl pryophosphate 33 00:01:46,170 --> 00:01:48,210 and now the next step-- 34 00:01:48,210 --> 00:01:51,420 remember, cholesterol, if you look at its structure-- 35 00:01:51,420 --> 00:01:54,480 this is a precursor to its structure-- 36 00:01:54,480 --> 00:01:55,980 is a C30. 37 00:01:55,980 --> 00:01:59,610 And so the next step is quite an interesting enzymatic reaction 38 00:01:59,610 --> 00:02:01,840 which we're not going to talk about in any detail, 39 00:02:01,840 --> 00:02:04,740 but those of you who are interested can go look it up. 40 00:02:04,740 --> 00:02:08,699 But how do you take two C15s and form a C30 41 00:02:08,699 --> 00:02:12,090 so you lose your pyrophosphates? 42 00:02:12,090 --> 00:02:13,890 And you can see when you generate this, 43 00:02:13,890 --> 00:02:16,440 now you have a linear c30, which, 44 00:02:16,440 --> 00:02:19,500 of course, is a complete hydrocarbon and is insoluble. 45 00:02:19,500 --> 00:02:22,350 So this now sort of defines that you 46 00:02:22,350 --> 00:02:26,670 need to be in the membrane to be able to do 47 00:02:26,670 --> 00:02:28,230 any further chemistry. 48 00:02:28,230 --> 00:02:31,470 So those of you who are interested in mechanisms 49 00:02:31,470 --> 00:02:33,060 of how things work, that's really 50 00:02:33,060 --> 00:02:36,120 sort of a fascinating system it's really pretty well 51 00:02:36,120 --> 00:02:38,400 worked out at this stage. 52 00:02:38,400 --> 00:02:40,830 But today what I mean to do is focus on 53 00:02:40,830 --> 00:02:45,720 the next step is, how do we get from C30, which 54 00:02:45,720 --> 00:02:52,020 is this linear squalene hydrocarbon, into lanosterol, 55 00:02:52,020 --> 00:02:54,480 which is then the precursor to steroids 56 00:02:54,480 --> 00:02:57,490 but also the precursor to cholesterol, 57 00:02:57,490 --> 00:03:01,680 which is what we're focusing on in this particular module. 58 00:03:01,680 --> 00:03:04,230 So what we're going to be looking at 59 00:03:04,230 --> 00:03:08,980 is how we went from two FPPs-- 60 00:03:08,980 --> 00:03:13,620 we're still doing chain elongation-- to a C30. 61 00:03:13,620 --> 00:03:15,120 And then the question is, how do you 62 00:03:15,120 --> 00:03:27,810 get from C30, which is linear, to a linear epoxide. 63 00:03:30,924 --> 00:03:33,075 And I'm not going to draw the whole structure out, 64 00:03:33,075 --> 00:03:35,820 but we're still linear. 65 00:03:35,820 --> 00:03:39,820 And then the next step is the step I want to talk about. 66 00:03:39,820 --> 00:03:45,265 So this is when lanosterol synthase. 67 00:03:48,630 --> 00:03:51,210 So that's where we're going in the next few minutes 68 00:03:51,210 --> 00:03:52,780 to get to our final product. 69 00:03:52,780 --> 00:03:56,850 So if you look at this reaction, remember, 70 00:03:56,850 --> 00:03:58,320 we're going to do a cyclization. 71 00:03:58,320 --> 00:04:00,540 And what do you need to do to do cyclizations? 72 00:04:00,540 --> 00:04:03,260 What was the general rule that I gave you last time? 73 00:04:03,260 --> 00:04:05,740 Does anybody remember? 74 00:04:05,740 --> 00:04:08,460 If you want to cyclize something, we talked about it. 75 00:04:08,460 --> 00:04:10,350 We looked at a number of examples. 76 00:04:10,350 --> 00:04:12,270 What did we do in those examples? 77 00:04:12,270 --> 00:04:13,110 Anybody remember? 78 00:04:16,220 --> 00:04:17,839 So here's a second example. 79 00:04:17,839 --> 00:04:20,269 I gave you two rules. 80 00:04:20,269 --> 00:04:22,019 If you go back and you look at your notes, 81 00:04:22,019 --> 00:04:25,170 we protonated the olefin and that triggered off 82 00:04:25,170 --> 00:04:27,450 the cyclization. 83 00:04:27,450 --> 00:04:31,890 And here, perhaps you could have protonated the olefin 84 00:04:31,890 --> 00:04:34,680 to trigger off the cyclization, but in the end, 85 00:04:34,680 --> 00:04:38,820 cholesterol has a hydroxyl group in the C3 position. 86 00:04:38,820 --> 00:04:44,670 So the next step in the pathway, which also will involve, 87 00:04:44,670 --> 00:04:47,560 ultimately, protonation and ring cyclization, 88 00:04:47,560 --> 00:04:49,590 so those are the two rules I gave you 89 00:04:49,590 --> 00:04:53,122 during the last lecture, to get to this epoxide, 90 00:04:53,122 --> 00:04:54,330 we have to do some chemistry. 91 00:04:54,330 --> 00:04:57,060 Does anybody know what cofactors you 92 00:04:57,060 --> 00:05:01,500 would use to do this reaction? 93 00:05:01,500 --> 00:05:04,590 Anybody got any ideas from introductory biochemistry? 94 00:05:04,590 --> 00:05:06,210 You have a vitamin bottle. 95 00:05:06,210 --> 00:05:09,150 What vitamin would be involved in doing this transformation 96 00:05:09,150 --> 00:05:13,202 or could be involved with doing this kind of a transformation? 97 00:05:17,060 --> 00:05:20,260 It's an oxidation. 98 00:05:20,260 --> 00:05:23,030 Requires oxygen gas. 99 00:05:23,030 --> 00:05:26,995 So what are the possibilities? 100 00:05:26,995 --> 00:05:29,590 AUDIENCE: NAD. 101 00:05:29,590 --> 00:05:30,670 JOANNE STUBBE: So NAD. 102 00:05:30,670 --> 00:05:32,960 Does NAD-- this is a good teaching point. 103 00:05:32,960 --> 00:05:35,440 Does NAD react with oxygen? 104 00:05:35,440 --> 00:05:36,280 Who suggested NAD? 105 00:05:41,280 --> 00:05:43,350 Why doesn't it make you react with oxygen? 106 00:05:43,350 --> 00:05:44,790 That's one of the things you learn 107 00:05:44,790 --> 00:05:47,660 in any introductory course. 108 00:05:47,660 --> 00:05:50,003 NAD does not react with oxygen. Why? 109 00:05:50,003 --> 00:05:51,420 What is the chemistry of NAD/NADH? 110 00:05:55,870 --> 00:05:58,620 Whoa. 111 00:05:58,620 --> 00:06:01,440 Maybe I should be teaching 5.07. 112 00:06:01,440 --> 00:06:04,500 So NAD/NADH, we just went through this 113 00:06:04,500 --> 00:06:10,200 with conversion of acetyl CoA moiety of mevalonic acid 114 00:06:10,200 --> 00:06:11,190 to the alcohol. 115 00:06:11,190 --> 00:06:13,320 It involves hydride transfer. 116 00:06:13,320 --> 00:06:16,560 And if you tried to do this chemistry instead of two 117 00:06:16,560 --> 00:06:19,200 electrons at a time, one electron at a time, 118 00:06:19,200 --> 00:06:21,480 and you looked at the reduction potentials, 119 00:06:21,480 --> 00:06:24,030 it would be way uphill, thermodynamically. 120 00:06:24,030 --> 00:06:27,900 So NAD/NADH never does one electron in chemistry. 121 00:06:27,900 --> 00:06:29,840 So that's not going to be a possibility. 122 00:06:29,840 --> 00:06:30,340 Yeah? 123 00:06:30,340 --> 00:06:32,465 AUDIENCE: You could use something that's like iron? 124 00:06:32,465 --> 00:06:34,215 JOANNE STUBBE: So that would be one thing. 125 00:06:34,215 --> 00:06:35,500 And we're going to see iron-- 126 00:06:35,500 --> 00:06:38,685 heme irons play a key role in all of this process. 127 00:06:38,685 --> 00:06:40,990 This turns out to be a flavoprotein. 128 00:06:40,990 --> 00:06:43,830 That's the other redox active cofactor. 129 00:06:43,830 --> 00:06:51,150 So this is a flavin monooxygenase. 130 00:06:51,150 --> 00:06:53,010 You don't need to remember this. 131 00:06:53,010 --> 00:06:54,360 We understand the details. 132 00:06:54,360 --> 00:06:56,730 I'm not going to talk about the detailed mechanism, 133 00:06:56,730 --> 00:07:00,050 but flavin cofactors are extremely well understood. 134 00:07:00,050 --> 00:07:03,430 The chemistry of them is extremely well understood now. 135 00:07:03,430 --> 00:07:05,970 So we've gotten to our oxidosqualene 136 00:07:05,970 --> 00:07:09,810 and now we've finally gotten to this really cool step. 137 00:07:09,810 --> 00:07:13,140 So how do we go from this step-- 138 00:07:13,140 --> 00:07:15,510 so this is this molecule here. 139 00:07:15,510 --> 00:07:19,740 And what I'm emphasizing again is 140 00:07:19,740 --> 00:07:24,570 we're going from a linear step into the cyclic product. 141 00:07:24,570 --> 00:07:28,230 So remember, triggering off cyclization, 142 00:07:28,230 --> 00:07:30,510 there were two rules-- protonation, 143 00:07:30,510 --> 00:07:32,070 protonation of an olefin. 144 00:07:32,070 --> 00:07:37,430 In this case, you have some kind of protonation of the epoxide. 145 00:07:37,430 --> 00:07:39,360 Epoxides are not very good leaving groups. 146 00:07:39,360 --> 00:07:41,550 You need to protonate it. 147 00:07:41,550 --> 00:07:44,670 And that is then going to trigger off 148 00:07:44,670 --> 00:07:50,550 this cascade of reactions to allow you to generate 149 00:07:50,550 --> 00:07:52,670 a molecule with four rings. 150 00:07:52,670 --> 00:07:58,950 And this all occurs with a single enzymatic step. 151 00:07:58,950 --> 00:08:01,980 And so the way you can visualize this happening-- 152 00:08:01,980 --> 00:08:04,680 and again, you don't need to copy this down. 153 00:08:04,680 --> 00:08:06,930 It's all-- if you look at your handouts ahead of time, 154 00:08:06,930 --> 00:08:08,638 there's some things that are written down 155 00:08:08,638 --> 00:08:10,950 that would take you 10 minutes to copy 156 00:08:10,950 --> 00:08:14,280 and then you probably get it written down incorrectly 157 00:08:14,280 --> 00:08:16,140 because you're looking like this is. 158 00:08:16,140 --> 00:08:18,180 The hard things that are hard to write down 159 00:08:18,180 --> 00:08:20,310 are all given to you in your handouts. 160 00:08:20,310 --> 00:08:23,270 You can write it down if you want, that's fine. 161 00:08:23,270 --> 00:08:25,590 So what we want to do is we want a ring open, 162 00:08:25,590 --> 00:08:28,320 so we need to protonate the epoxide, 163 00:08:28,320 --> 00:08:29,580 and that generates what? 164 00:08:29,580 --> 00:08:32,669 A carbocation. 165 00:08:32,669 --> 00:08:34,470 And then now what happens? 166 00:08:34,470 --> 00:08:38,039 We generate another carbocation. 167 00:08:38,039 --> 00:08:39,179 And now what happens? 168 00:08:39,179 --> 00:08:42,012 We generate another carbocation. 169 00:08:42,012 --> 00:08:43,679 And now what happens? 170 00:08:43,679 --> 00:08:48,420 We generate another carbocation and we end up 171 00:08:48,420 --> 00:08:51,720 with a carbocation at this position. 172 00:08:51,720 --> 00:08:55,270 So I'm going to draw the structure of this. 173 00:08:55,270 --> 00:08:59,190 So we have ring opened, and let me also 174 00:08:59,190 --> 00:09:05,160 emphasize that the key to this process occurring 175 00:09:05,160 --> 00:09:09,822 to give us lanosterol is the conformation 176 00:09:09,822 --> 00:09:12,170 of the linear molecule. 177 00:09:12,170 --> 00:09:13,320 So what do we see here? 178 00:09:13,320 --> 00:09:15,910 What does this look like? 179 00:09:15,910 --> 00:09:19,930 In terms of cyclohexanes, what does this look like? 180 00:09:19,930 --> 00:09:23,298 If you have cyclohexyl rings, what kinds of conformations 181 00:09:23,298 --> 00:09:23,840 do they have? 182 00:09:23,840 --> 00:09:24,650 AUDIENCE: Chair? 183 00:09:24,650 --> 00:09:26,510 JOANNE STUBBE: Chair and chair and boat. 184 00:09:26,510 --> 00:09:30,590 So the key here is that you have a chair conformation here. 185 00:09:30,590 --> 00:09:32,690 You have a chair conformation here, 186 00:09:32,690 --> 00:09:35,630 but here you have a boat conformation. 187 00:09:35,630 --> 00:09:37,640 And one of the general rules I told you 188 00:09:37,640 --> 00:09:41,060 last time about terpene chemistry 189 00:09:41,060 --> 00:09:46,940 in general was, what do the enzymes do in the active site 190 00:09:46,940 --> 00:09:50,630 to transform something that's linear into something that's 191 00:09:50,630 --> 00:09:51,980 cyclic? 192 00:09:51,980 --> 00:09:56,240 They need to fold the molecule into the right conformation. 193 00:09:56,240 --> 00:09:58,230 And that can, in part, be done, the fact 194 00:09:58,230 --> 00:10:00,830 is the active site is very hydrophobic. 195 00:10:00,830 --> 00:10:02,330 We talked about that. 196 00:10:02,330 --> 00:10:06,240 And you can also have aromatics that could potentially-- 197 00:10:06,240 --> 00:10:08,450 I'm not drawing out all these intermediates, 198 00:10:08,450 --> 00:10:13,250 but could potentially facilitate not only the conformation 199 00:10:13,250 --> 00:10:16,670 but stabilization somewhat of the intermediates 200 00:10:16,670 --> 00:10:20,400 that you observe along the reaction pathway. 201 00:10:20,400 --> 00:10:23,480 So here's another example of the importance 202 00:10:23,480 --> 00:10:27,020 of shape to defining the chemistry that's 203 00:10:27,020 --> 00:10:28,290 actually going to happen. 204 00:10:28,290 --> 00:10:30,848 And in contrast to the enzymes we 205 00:10:30,848 --> 00:10:33,140 talked about last time, which were type I. You probably 206 00:10:33,140 --> 00:10:33,973 don't remember that. 207 00:10:33,973 --> 00:10:37,250 But this is, again, a different super family 208 00:10:37,250 --> 00:10:40,490 involved that you observe, and it's observed quite frequently. 209 00:10:40,490 --> 00:10:42,440 So these are type II. 210 00:10:42,440 --> 00:10:45,050 So if you look up the structures, and in the article 211 00:10:45,050 --> 00:10:49,040 you had to read by Christiansen, the second type of structure. 212 00:10:49,040 --> 00:10:50,780 There are two general types of structure. 213 00:10:50,780 --> 00:10:56,060 This is the second type of structure involved in making 214 00:10:56,060 --> 00:10:58,500 interesting terpene molecules. 215 00:10:58,500 --> 00:11:00,680 So what I'm doing now is showing you 216 00:11:00,680 --> 00:11:08,060 how we've cyclized this to leave us with a carbocation. 217 00:11:08,060 --> 00:11:11,870 And remember, if you have just a stick as opposed to a stick 218 00:11:11,870 --> 00:11:15,270 with a hydrogen, that's a methyl group. 219 00:11:15,270 --> 00:11:20,460 So here at the ring juncture, we have a hydrogen. 220 00:11:20,460 --> 00:11:21,725 We have a trans ring juncture. 221 00:11:24,290 --> 00:11:28,340 And again, if we have a stick with nothing on it, 222 00:11:28,340 --> 00:11:29,330 it's a methyl group. 223 00:11:32,500 --> 00:11:37,220 And we're into a chair conformation again, 224 00:11:37,220 --> 00:11:41,470 and then we need to attach the last ring 225 00:11:41,470 --> 00:11:45,710 so we have three six-membered rings and a five-membered ring. 226 00:11:49,220 --> 00:11:57,900 And in the end, what have we generated? 227 00:11:57,900 --> 00:11:59,480 We've generated a carbocation. 228 00:11:59,480 --> 00:12:03,050 So I've written this as a single step. 229 00:12:03,050 --> 00:12:04,580 Nobody has seen the intermediates. 230 00:12:04,580 --> 00:12:06,950 You could write it is multiple steps. 231 00:12:06,950 --> 00:12:08,630 I mean, the fact is it would be-- 232 00:12:08,630 --> 00:12:11,750 it's pretty hard to trap any of these carbocations, 233 00:12:11,750 --> 00:12:14,600 and people have spent a lot of time trapping them. 234 00:12:14,600 --> 00:12:18,290 So what you see, I think, is quite amazing, 235 00:12:18,290 --> 00:12:22,370 but we aren't finished yet because we have a carbocation 236 00:12:22,370 --> 00:12:24,020 and we need to get rid of that. 237 00:12:24,020 --> 00:12:26,240 And what you need to do and this is-- you 238 00:12:26,240 --> 00:12:28,580 will have one of these problems on the problem set 239 00:12:28,580 --> 00:12:29,875 that will be due next week. 240 00:12:29,875 --> 00:12:31,250 You'll be given something simple, 241 00:12:31,250 --> 00:12:34,190 not as complicated as cholesterol. 242 00:12:34,190 --> 00:12:38,000 But what you need to think about is 243 00:12:38,000 --> 00:12:41,600 where do all these methyl groups end up in. 244 00:12:41,600 --> 00:12:44,840 What's the stereochemistry of the reaction? 245 00:12:44,840 --> 00:12:48,387 So then this geometry becomes critical 246 00:12:48,387 --> 00:12:49,970 if you're thinking-- you need to think 247 00:12:49,970 --> 00:12:53,930 about the stereo electronic control of hydride and methyl 248 00:12:53,930 --> 00:12:56,870 anion equivalent migrations. 249 00:12:56,870 --> 00:12:59,450 So what you have in this particular reaction 250 00:12:59,450 --> 00:13:01,240 is you're going to have-- 251 00:13:01,240 --> 00:13:04,150 and I like this example because, again, I gave you 252 00:13:04,150 --> 00:13:09,200 a set of rules that you can see that are associated, typically, 253 00:13:09,200 --> 00:13:11,530 with carbocation reactions in general, 254 00:13:11,530 --> 00:13:13,820 and this one does all of them. 255 00:13:13,820 --> 00:13:19,350 So one of the rules was that you have 256 00:13:19,350 --> 00:13:23,630 hydrogen migrate with a pair of electrons, so that's a hydride. 257 00:13:23,630 --> 00:13:27,800 Again, you have a second hydrogen migrate 258 00:13:27,800 --> 00:13:29,670 with a pair of electrons. 259 00:13:29,670 --> 00:13:32,780 So I'm not drawing out all the intermediates. 260 00:13:32,780 --> 00:13:35,990 Now what we have is a methyl group 261 00:13:35,990 --> 00:13:39,230 migrate with its electrons. 262 00:13:39,230 --> 00:13:43,850 We now have a second methyl group 263 00:13:43,850 --> 00:13:46,430 migrate with its electrons. 264 00:13:46,430 --> 00:13:49,620 And in the end, we're left with a cation here, 265 00:13:49,620 --> 00:13:52,460 and the last step in many of these reactions 266 00:13:52,460 --> 00:13:54,650 is loss of a proton. 267 00:13:54,650 --> 00:13:59,450 So here we would have loss of a proton. 268 00:13:59,450 --> 00:14:02,000 And if you look at the chemistry and you 269 00:14:02,000 --> 00:14:05,570 look at the final product, which I'm not going to draw out, 270 00:14:05,570 --> 00:14:06,980 you end up with this molecule. 271 00:14:06,980 --> 00:14:11,540 So this is a flat rendition of what I've actually 272 00:14:11,540 --> 00:14:12,860 drawn on the board. 273 00:14:12,860 --> 00:14:17,630 So this is an example of all of the chemistry I talked about 274 00:14:17,630 --> 00:14:22,010 as being general in all of these 70,000 terpenes. 275 00:14:22,010 --> 00:14:24,350 You'll find most of them don't do all of the chemistry. 276 00:14:24,350 --> 00:14:26,180 This one does all the different kind 277 00:14:26,180 --> 00:14:32,240 of chemistries associated with carbocation type chemistries 278 00:14:32,240 --> 00:14:34,100 that hopefully some of you have learned 279 00:14:34,100 --> 00:14:38,070 about in introductory organic chemistry classes. 280 00:14:38,070 --> 00:14:41,390 So again, to me, this is the most amazing reaction 281 00:14:41,390 --> 00:14:42,740 I think I've ever seen. 282 00:14:42,740 --> 00:14:46,970 I told you already that I heard about this in 1969 283 00:14:46,970 --> 00:14:49,100 when they'd just figured out that this 284 00:14:49,100 --> 00:14:50,660 could happen enzymatically. 285 00:14:50,660 --> 00:14:53,360 And this became the basis, for those of you, 286 00:14:53,360 --> 00:14:55,490 if there are any synthetic people here, 287 00:14:55,490 --> 00:14:59,490 people doing cascade reactions. 288 00:14:59,490 --> 00:15:03,050 Kim Jameson's lab does this, but back in those days, 289 00:15:03,050 --> 00:15:07,610 they were using this approach, trying to define the folding 290 00:15:07,610 --> 00:15:11,330 to do all these steps, just like nature had figured out 291 00:15:11,330 --> 00:15:12,043 how to do this. 292 00:15:12,043 --> 00:15:14,210 And if you look at the number of asymmetric centers, 293 00:15:14,210 --> 00:15:17,750 you end up with seven asymmetric centers and no other products 294 00:15:17,750 --> 00:15:19,370 that people could detect. 295 00:15:19,370 --> 00:15:25,220 So this is quite an amazing feat. 296 00:15:25,220 --> 00:15:29,000 So this is the model that I just drew on the board. 297 00:15:29,000 --> 00:15:33,170 And so we still aren't quite there yet 298 00:15:33,170 --> 00:15:37,400 because if you look at this structure 299 00:15:37,400 --> 00:15:40,910 and you look at the final structure of cholesterol, 300 00:15:40,910 --> 00:15:43,250 you have a methyl group here, here, 301 00:15:43,250 --> 00:15:47,180 and you're going to have a methyl group here. 302 00:15:47,180 --> 00:15:51,577 So we have 1, 2, 3 methyl groups. 303 00:15:51,577 --> 00:15:53,660 And if you look at the final product, cholesterol, 304 00:15:53,660 --> 00:15:55,690 they're all gone. 305 00:15:55,690 --> 00:16:00,040 So you need 19 more steps to get to cholesterol. 306 00:16:00,040 --> 00:16:03,740 This is not a simple biosynthetic pathway. 307 00:16:03,740 --> 00:16:07,130 So to get from cholesterol-- 308 00:16:07,130 --> 00:16:08,200 so this is lanosterol. 309 00:16:08,200 --> 00:16:15,910 So we've gotten to the precursor to steroids and cholesterol. 310 00:16:15,910 --> 00:16:18,160 And when we start talking about regulation, 311 00:16:18,160 --> 00:16:20,140 you'll see that lanosterol is, again, 312 00:16:20,140 --> 00:16:23,020 a central player because it can partition 313 00:16:23,020 --> 00:16:25,450 between different kinds of natural products 314 00:16:25,450 --> 00:16:27,190 that we aren't going to be talking about, 315 00:16:27,190 --> 00:16:28,898 other kinds of natural products we aren't 316 00:16:28,898 --> 00:16:30,250 going to be talking about. 317 00:16:30,250 --> 00:16:34,780 But to get to cholesterol, which I'm abbreviating from now 318 00:16:34,780 --> 00:16:38,110 on as Ch, it's 19 steps. 319 00:16:40,870 --> 00:16:42,880 So let's go over here. 320 00:16:42,880 --> 00:16:45,290 My goal is not to teach you about the chemistry of all 321 00:16:45,290 --> 00:16:45,790 this. 322 00:16:45,790 --> 00:16:47,415 I'm not sure how easily you can see it. 323 00:16:47,415 --> 00:16:50,570 Hopefully, you have the handouts with you, 324 00:16:50,570 --> 00:16:55,910 but we have this methyl group, this methyl group, 325 00:16:55,910 --> 00:16:59,650 and this methyl group that need to be removed over here. 326 00:16:59,650 --> 00:17:02,200 So that methyl group is gone and these two methyl groups 327 00:17:02,200 --> 00:17:03,730 are gone. 328 00:17:03,730 --> 00:17:04,940 So how do we do that? 329 00:17:04,940 --> 00:17:08,560 And so all of this reaction-- so we 330 00:17:08,560 --> 00:17:15,220 have loss of three methyl groups. 331 00:17:19,089 --> 00:17:21,520 And all of these reactions are catalyzed 332 00:17:21,520 --> 00:17:28,349 by one kind of enzyme, which is a cytochrome P450 333 00:17:28,349 --> 00:17:29,290 monooxygenase. 334 00:17:29,290 --> 00:17:33,040 So we're going to see that all the reactions are catalyzed 335 00:17:33,040 --> 00:17:45,700 by a cytochrome P450 monooxygenase, not a flavin 336 00:17:45,700 --> 00:17:46,765 monooxygenase. 337 00:17:46,765 --> 00:17:48,140 And if you look at the chemistry, 338 00:17:48,140 --> 00:17:51,050 flavins are not anywhere near as strong are 339 00:17:51,050 --> 00:17:54,140 oxidants as heme-dependent oxidation. 340 00:17:54,140 --> 00:17:56,810 So if you have something really hard to oxidize, 341 00:17:56,810 --> 00:17:58,340 you're never going to use a flavin. 342 00:17:58,340 --> 00:18:00,767 You're going to use a heme. 343 00:18:00,767 --> 00:18:02,600 And what do we know about all these enzymes? 344 00:18:02,600 --> 00:18:05,120 I'm not going to talk about this in detail, 345 00:18:05,120 --> 00:18:10,170 but you have an iron 3 heme. 346 00:18:10,170 --> 00:18:14,390 And for those of you who don't remember what heme is, 347 00:18:14,390 --> 00:18:15,890 we're going to be talking about this 348 00:18:15,890 --> 00:18:20,360 in more detail in the section on reactive oxygen species. 349 00:18:20,360 --> 00:18:21,510 It's a protoporphyrin IX. 350 00:18:21,510 --> 00:18:22,927 That's what you see in hemoglobin. 351 00:18:22,927 --> 00:18:26,630 It's the exact same co-factor you see in hemoglobin, 352 00:18:26,630 --> 00:18:30,650 but what's distinct about this is 353 00:18:30,650 --> 00:18:33,180 that instead of having a histadine ligand, 354 00:18:33,180 --> 00:18:34,790 it has a thiolate ligand. 355 00:18:34,790 --> 00:18:38,670 And that's key to why P450s can catalyze 356 00:18:38,670 --> 00:18:42,410 these inactivated hydroxylations-- 357 00:18:42,410 --> 00:18:45,740 can catalyze hydroxylations of unactivated bonds 358 00:18:45,740 --> 00:18:49,220 where this hemoglobin reversibly binds oxygen. 359 00:18:49,220 --> 00:18:54,470 So these P450s use this heme system in an oxygen system. 360 00:18:54,470 --> 00:18:55,700 And what did they do? 361 00:18:55,700 --> 00:18:59,390 And so what I do is refer you over here to-- let's 362 00:18:59,390 --> 00:19:04,120 look simply at 7 through 10 and we're 363 00:19:04,120 --> 00:19:07,060 removing this methyl group. 364 00:19:07,060 --> 00:19:10,750 So we're removing this little methyl group in the A ring. 365 00:19:10,750 --> 00:19:12,010 The first ring is the A ring. 366 00:19:14,590 --> 00:19:15,090 Sorry. 367 00:19:22,946 --> 00:19:26,200 So stereo specific, and so I'm not drawing 368 00:19:26,200 --> 00:19:27,490 the rest of the structure. 369 00:19:27,490 --> 00:19:31,390 And our goal is if we go through 9 through 10 370 00:19:31,390 --> 00:19:34,270 and then 11 through 13, we want to get 371 00:19:34,270 --> 00:19:37,030 rid of both of these methyl groups. 372 00:19:37,030 --> 00:19:41,620 And it's thought that one enzyme, but they don't know, 373 00:19:41,620 --> 00:19:43,720 can catalyze multiple oxidations. 374 00:19:43,720 --> 00:19:44,950 And why don't they know? 375 00:19:44,950 --> 00:19:47,350 Where do you think all was chemistry happens? 376 00:19:47,350 --> 00:19:48,250 You have cholesterol. 377 00:19:48,250 --> 00:19:50,375 What do we know about the structure of cholesterol? 378 00:19:50,375 --> 00:19:51,202 It's a grease ball. 379 00:19:51,202 --> 00:19:53,035 So where do you think the chemistry happens? 380 00:19:53,035 --> 00:19:54,250 AUDIENCE: In the membranes. 381 00:19:54,250 --> 00:19:55,833 JOANNE STUBBE: In the membranes, yeah. 382 00:19:55,833 --> 00:19:57,520 And so that's been-- 383 00:19:57,520 --> 00:20:01,090 P450s, you go to meetings, thousands of people 384 00:20:01,090 --> 00:20:04,300 still go to P450 meetings on the major targets 385 00:20:04,300 --> 00:20:07,090 of all kinds of therapeutics, and they're 386 00:20:07,090 --> 00:20:09,760 almost all membrane-associated, which 387 00:20:09,760 --> 00:20:13,580 has been problematic in terms of isolation and characterization. 388 00:20:13,580 --> 00:20:15,640 And here, despite a lot of effort, 389 00:20:15,640 --> 00:20:19,120 people really still don't know the sequence of events 390 00:20:19,120 --> 00:20:21,610 or have isolated and purified the enzymes. 391 00:20:21,610 --> 00:20:24,870 They're all in the ER, which is what 392 00:20:24,870 --> 00:20:26,890 we're going to come back to, and there 393 00:20:26,890 --> 00:20:28,810 are a membrane-associated. 394 00:20:28,810 --> 00:20:30,790 So what happens in these reactions 395 00:20:30,790 --> 00:20:36,040 is you take a methyl group and then you 396 00:20:36,040 --> 00:20:41,020 oxidize it with one P450. 397 00:20:41,020 --> 00:20:44,860 So we somehow use oxygen-iron chemistry 398 00:20:44,860 --> 00:20:46,840 to do a hydroxylation reaction. 399 00:20:46,840 --> 00:20:52,380 Have you seen that before, in the first part of the semester? 400 00:20:52,380 --> 00:20:53,540 Anybody remember seeing it? 401 00:20:53,540 --> 00:20:54,775 Maybe you didn't see it. 402 00:20:54,775 --> 00:20:56,025 I missed a couple of lectures. 403 00:20:59,060 --> 00:21:01,150 Do you remember seeing hydroxylation reactions 404 00:21:01,150 --> 00:21:03,110 anywhere? 405 00:21:03,110 --> 00:21:04,580 Liz, do you talk-- 406 00:21:04,580 --> 00:21:06,350 was that in any of the natural products? 407 00:21:06,350 --> 00:21:08,760 AUDIENCE: Sometimes [INAUDIBLE] P450s [INAUDIBLE].. 408 00:21:10,950 --> 00:21:12,450 JOANNE STUBBE: But what you'll see-- 409 00:21:12,450 --> 00:21:15,120 I think this would be, like, a decorating 410 00:21:15,120 --> 00:21:17,760 module that you saw in the non-ribosomal peptide 411 00:21:17,760 --> 00:21:19,590 synthetases. 412 00:21:19,590 --> 00:21:23,760 But here these things, as in the non-ribosomal peptide 413 00:21:23,760 --> 00:21:26,580 synthetases, are absolutely specific. 414 00:21:26,580 --> 00:21:29,490 And so you have one hydroxylation, 415 00:21:29,490 --> 00:21:33,180 you have a second hydroxylation, you 416 00:21:33,180 --> 00:21:39,330 have a third hydroxylation, which is chemically distinct. 417 00:21:39,330 --> 00:21:44,940 And then the question is, how do you get rid of this altogether? 418 00:21:44,940 --> 00:21:48,360 Because our goal is to remove the methyl. 419 00:21:48,360 --> 00:21:50,280 That's what our goal is. 420 00:21:50,280 --> 00:21:55,470 So we've gone hydroxymethyl, the aldehyde, the acid. 421 00:21:55,470 --> 00:21:58,350 So now you have an acid next to the alcohol. 422 00:21:58,350 --> 00:21:59,530 How do you get rid of that? 423 00:21:59,530 --> 00:22:01,770 Has anybody-- what kind of chemistry 424 00:22:01,770 --> 00:22:07,122 could you do to allow you to lose the CO2? 425 00:22:07,122 --> 00:22:10,543 AUDIENCE: [INAUDIBLE]. 426 00:22:10,543 --> 00:22:12,210 JOANNE STUBBE: You need to speak louder. 427 00:22:12,210 --> 00:22:15,420 Don't be-- I mean, just tell me what you think. 428 00:22:15,420 --> 00:22:16,550 AUDIENCE: Decarboxylation. 429 00:22:16,550 --> 00:22:17,550 JOANNE STUBBE: The what? 430 00:22:17,550 --> 00:22:18,510 Decarboxylation. 431 00:22:18,510 --> 00:22:21,570 But can you decarboxylate-- 432 00:22:21,570 --> 00:22:22,380 so you're right. 433 00:22:22,380 --> 00:22:23,760 We want to decarboxylate. 434 00:22:26,182 --> 00:22:27,099 AUDIENCE: [INAUDIBLE]. 435 00:22:27,099 --> 00:22:31,230 JOANNE STUBBE: What do you have to do to decarboxylate? 436 00:22:31,230 --> 00:22:35,070 AUDIENCE: You reduce the alcohol [INAUDIBLE].. 437 00:22:35,070 --> 00:22:38,332 JOANNE STUBBE: Reduce the alcohol? 438 00:22:38,332 --> 00:22:42,280 AUDIENCE: [INAUDIBLE]. 439 00:22:42,280 --> 00:22:43,880 JOANNE STUBBE: What? 440 00:22:43,880 --> 00:22:46,540 What are you going to do? 441 00:22:46,540 --> 00:22:47,540 These are the kinds of-- 442 00:22:47,540 --> 00:22:49,970 you'll see these reactions happen over and over 443 00:22:49,970 --> 00:22:52,520 again in biochemical pathways. 444 00:22:52,520 --> 00:22:53,770 AUDIENCE: Oxidize [INAUDIBLE]. 445 00:22:53,770 --> 00:22:54,645 JOANNE STUBBE: Right. 446 00:22:54,645 --> 00:22:55,670 You want to oxidize it. 447 00:22:55,670 --> 00:22:59,450 So what happens, if you look at this pathway over here, 448 00:22:59,450 --> 00:23:02,690 in this step, you use-- it should be NADP, 449 00:23:02,690 --> 00:23:04,800 so you use NADP. 450 00:23:04,800 --> 00:23:05,780 And what does that do? 451 00:23:05,780 --> 00:23:09,380 I'm not going to write this out, but it oxidizes this 452 00:23:09,380 --> 00:23:11,300 to a ketone. 453 00:23:11,300 --> 00:23:12,590 And now what do you have? 454 00:23:12,590 --> 00:23:14,570 You have a beta-ketoacid. 455 00:23:14,570 --> 00:23:17,570 And beta-ketoacids rapidly undergo 456 00:23:17,570 --> 00:23:19,080 decarboxylation reactions. 457 00:23:19,080 --> 00:23:23,450 So this is a strategy that nature uses over and over again 458 00:23:23,450 --> 00:23:25,730 in many biosynthetic pathways. 459 00:23:25,730 --> 00:23:27,107 And the thing that's interesting, 460 00:23:27,107 --> 00:23:29,190 if you look at that pathway in detail-- and again, 461 00:23:29,190 --> 00:23:30,650 you're not responsible for that-- 462 00:23:30,650 --> 00:23:33,860 but then it does the same thing on the next methyl. 463 00:23:33,860 --> 00:23:39,050 So in the end, you end up with a carbon with two hydrogens here. 464 00:23:39,050 --> 00:23:42,350 But it's not straightforward, but this kind sequence 465 00:23:42,350 --> 00:23:47,330 of events you actually see a lot in metabolic pathways. 466 00:23:47,330 --> 00:23:50,800 So I don't want to really say much more about this. 467 00:23:50,800 --> 00:23:53,960 In 19 steps, you need to remove three methyl groups. 468 00:23:53,960 --> 00:23:56,150 All the enzymes are ER bound, making 469 00:23:56,150 --> 00:24:02,300 it difficult to study the individual enzymatic reactions. 470 00:24:02,300 --> 00:24:04,400 And we would like to know the order, 471 00:24:04,400 --> 00:24:06,240 but we don't know it at this stage. 472 00:24:06,240 --> 00:24:09,110 What we know is what we see at the end. 473 00:24:09,110 --> 00:24:13,540 So finally, I wanted to get here at the end of lecture 2. 474 00:24:13,540 --> 00:24:16,400 We've gotten here a little later. 475 00:24:16,400 --> 00:24:18,380 We've started with acetyl CoA. 476 00:24:18,380 --> 00:24:21,680 We've made the major building blocks for all terpenes, IPP 477 00:24:21,680 --> 00:24:24,470 and dimethyl APP. 478 00:24:24,470 --> 00:24:28,940 And we've gotten to form this very complicated molecule. 479 00:24:28,940 --> 00:24:32,840 Everything starts with acetyl CoA and you can-- 480 00:24:32,840 --> 00:24:35,180 this was classic work by Konrad Bloch, 481 00:24:35,180 --> 00:24:38,150 who won the Nobel Prize for this work, who 482 00:24:38,150 --> 00:24:41,090 then by doing label chasing, which you learned about, 483 00:24:41,090 --> 00:24:44,690 hopefully, in introductory chemistry, 484 00:24:44,690 --> 00:24:48,110 helped them to figure out this complex biosynthetic pathway, 485 00:24:48,110 --> 00:24:50,840 which isn't so easy because things are membrane 486 00:24:50,840 --> 00:24:55,310 bound and very lipophilic. 487 00:24:55,310 --> 00:24:58,640 So we've gotten to cholesterol. 488 00:24:58,640 --> 00:25:03,020 So this module is on cholesterol and we've 489 00:25:03,020 --> 00:25:08,390 been able to biosynthesize it through an amazing sequence 490 00:25:08,390 --> 00:25:14,720 of reactions that have been studied over the decades. 491 00:25:14,720 --> 00:25:17,260 But we can also get cholesterol-- 492 00:25:17,260 --> 00:25:19,010 we want to ask the question, first of all, 493 00:25:19,010 --> 00:25:20,510 why are we interested in cholesterol? 494 00:25:20,510 --> 00:25:21,885 I think you've already seen hints 495 00:25:21,885 --> 00:25:25,850 of that with the statins inhibiting HMG-CoA reductase. 496 00:25:25,850 --> 00:25:31,740 We have issues when cholesterol levels are too high or too low. 497 00:25:31,740 --> 00:25:35,120 We need to control the levels of cholesterol. 498 00:25:35,120 --> 00:25:38,600 And the second way we can get cholesterol besides making it 499 00:25:38,600 --> 00:25:40,790 is we get it from our diet. 500 00:25:40,790 --> 00:25:43,040 So if we get it from the diet, the molecule we'll see 501 00:25:43,040 --> 00:25:44,000 is not very soluble. 502 00:25:44,000 --> 00:25:47,250 How is it distributed into the tissues? 503 00:25:47,250 --> 00:25:50,030 And then if you've distributed a lot of cholesterol 504 00:25:50,030 --> 00:25:51,830 from your diet, you certainly don't want 505 00:25:51,830 --> 00:25:53,820 to keep making cholesterol. 506 00:25:53,820 --> 00:25:58,190 So the question is, how do you control those two events? 507 00:25:58,190 --> 00:26:00,620 What are the general mechanisms of regulation 508 00:26:00,620 --> 00:26:03,050 of the levels of cholesterol? 509 00:26:03,050 --> 00:26:05,960 And we're going to at the end look 510 00:26:05,960 --> 00:26:08,690 at some of the classic experiments 511 00:26:08,690 --> 00:26:12,890 that Brown and Goldstein did to understand how cholesterol, 512 00:26:12,890 --> 00:26:16,220 which from the diet can get into the bloodstream, 513 00:26:16,220 --> 00:26:20,120 can get transferred into cells. 514 00:26:20,120 --> 00:26:22,370 And so that's where we're going. 515 00:26:22,370 --> 00:26:23,840 And again, the reading is a reading 516 00:26:23,840 --> 00:26:26,420 I've already given you before. 517 00:26:26,420 --> 00:26:28,070 So why do we care about this? 518 00:26:28,070 --> 00:26:29,670 We have a 30-step synthesis. 519 00:26:29,670 --> 00:26:31,250 We're getting it from the diet. 520 00:26:31,250 --> 00:26:35,030 We have key issues in homeostasis, which is 521 00:26:35,030 --> 00:26:37,130 what our focus is going to be. 522 00:26:37,130 --> 00:26:38,840 So why do we care about cholesterol? 523 00:26:38,840 --> 00:26:41,480 We care about cholesterol because it's 524 00:26:41,480 --> 00:26:47,240 associated with human health and coronary artery disease. 525 00:26:47,240 --> 00:26:51,170 Probably many of people who have had heart attacks. 526 00:26:51,170 --> 00:26:53,570 And so elevated cholesterol levels 527 00:26:53,570 --> 00:27:00,110 have been known for some time to be associated with plaques, 528 00:27:00,110 --> 00:27:06,980 artherosclerotic plaques, which can lead to heart attacks 529 00:27:06,980 --> 00:27:07,670 and strokes. 530 00:27:07,670 --> 00:27:11,990 So what happens is the cholesterol deposits, 531 00:27:11,990 --> 00:27:14,420 you try to remove the cholesterol, 532 00:27:14,420 --> 00:27:16,490 you generate a lot of scar tissue, 533 00:27:16,490 --> 00:27:18,097 which then inhibits blood flow. 534 00:27:18,097 --> 00:27:20,180 And then you're in trouble if you can't figure out 535 00:27:20,180 --> 00:27:23,660 how to unblock the blood flow. 536 00:27:23,660 --> 00:27:25,640 So that's the main motivator and we'll 537 00:27:25,640 --> 00:27:31,880 see another main motivator is related to young children dying 538 00:27:31,880 --> 00:27:33,950 of heart attacks, which is what got 539 00:27:33,950 --> 00:27:37,430 Brown and Goldstein into the area of cholesterol 540 00:27:37,430 --> 00:27:38,360 homeostasis. 541 00:27:38,360 --> 00:27:45,170 So there have been three Nobel Prizes given for work 542 00:27:45,170 --> 00:27:47,400 on cholesterol over the years. 543 00:27:47,400 --> 00:27:49,095 This is a classic paper. 544 00:27:49,095 --> 00:27:50,720 Some of the classic papers are actually 545 00:27:50,720 --> 00:27:54,500 quite interesting to read, and often the original papers 546 00:27:54,500 --> 00:27:56,270 get things wrong. 547 00:27:56,270 --> 00:27:58,700 So it was mostly right, but not completely right. 548 00:27:58,700 --> 00:28:04,680 But anyhow, I think if you put it into the context, 1928, 549 00:28:04,680 --> 00:28:06,440 how would you do experiments like that? 550 00:28:06,440 --> 00:28:07,140 We had no IR. 551 00:28:07,140 --> 00:28:10,330 We had no MR. We had no mass spec. 552 00:28:10,330 --> 00:28:11,150 What did we have? 553 00:28:11,150 --> 00:28:15,100 We had ways of degrading things. 554 00:28:15,100 --> 00:28:16,873 People don't do that anymore. 555 00:28:16,873 --> 00:28:18,790 If you go back and you look at the discoveries 556 00:28:18,790 --> 00:28:22,540 before 1970 or something, these feats 557 00:28:22,540 --> 00:28:26,350 of pulling out the structures with the right stereochemistry 558 00:28:26,350 --> 00:28:28,930 is really, I think, quite astonishing. 559 00:28:28,930 --> 00:28:31,540 And I think what's most amazing to 560 00:28:31,540 --> 00:28:35,740 me is this old literature is actually 561 00:28:35,740 --> 00:28:37,420 much more reproducible than anything 562 00:28:37,420 --> 00:28:39,700 in the current literature. 563 00:28:39,700 --> 00:28:43,583 The current literature, we're spewing out papers, 564 00:28:43,583 --> 00:28:45,250 a lot of which will never get reproduced 565 00:28:45,250 --> 00:28:47,350 so we won't know if it's reproducible. 566 00:28:47,350 --> 00:28:52,270 But if you go back and you do anything, in the old days, 567 00:28:52,270 --> 00:28:55,758 you had to learn German because a lot of the original papers, 568 00:28:55,758 --> 00:28:57,550 all of the chemical papers, were in German. 569 00:28:57,550 --> 00:29:00,690 They did seminal experiments back in those days. 570 00:29:00,690 --> 00:29:03,280 And most of the time it was correct. 571 00:29:03,280 --> 00:29:09,400 So anyhow, these guys figured out the structure almost. 572 00:29:09,400 --> 00:29:14,440 And then Konrad Bloch figured out, along with Fritz Lynen, 573 00:29:14,440 --> 00:29:17,500 figured out how you make cholesterol 574 00:29:17,500 --> 00:29:18,940 by labeling experiments. 575 00:29:18,940 --> 00:29:21,100 Now, many of you-- how many did label 576 00:29:21,100 --> 00:29:24,470 chasing in an introductory biochemistry course? 577 00:29:24,470 --> 00:29:29,380 Any of you have problem sets with label chasing? 578 00:29:29,380 --> 00:29:31,510 So it's quite distinct. 579 00:29:31,510 --> 00:29:32,800 I taught with John Essigmann. 580 00:29:32,800 --> 00:29:34,450 All those problems were label chasing 581 00:29:34,450 --> 00:29:35,550 and I used to say, oh, no. 582 00:29:35,550 --> 00:29:37,270 Who wants to do label chasing? 583 00:29:37,270 --> 00:29:38,770 But the fact is now if you read any 584 00:29:38,770 --> 00:29:40,853 of the current papers in the literature, everybody 585 00:29:40,853 --> 00:29:42,040 is label chasing. 586 00:29:42,040 --> 00:29:46,210 And now we have much better ways of actually chasing labels 587 00:29:46,210 --> 00:29:47,800 using mass spec methods. 588 00:29:47,800 --> 00:29:50,110 So you can hardly pick up a journal nowadays 589 00:29:50,110 --> 00:29:52,450 without thinking about label chasing. 590 00:29:52,450 --> 00:29:54,910 So these guys who were way ahead of their time, but it 591 00:29:54,910 --> 00:29:56,800 was much harder in those days. 592 00:29:56,800 --> 00:29:59,740 And then here are Brown and Goldstein. 593 00:29:59,740 --> 00:30:03,460 They won the Nobel Prize for the discovery of low-density 594 00:30:03,460 --> 00:30:06,940 lipoprotein and may still win another Nobel Prize 595 00:30:06,940 --> 00:30:09,460 for the regulatory mechanisms that we'll talk 596 00:30:09,460 --> 00:30:12,960 about at the end of lecture 5. 597 00:30:12,960 --> 00:30:17,100 So the first thing I want to talk about in lecture 3 598 00:30:17,100 --> 00:30:18,420 is focused on-- 599 00:30:18,420 --> 00:30:18,920 let's see. 600 00:30:18,920 --> 00:30:20,430 What do I want to do? 601 00:30:20,430 --> 00:30:22,860 Is focused on the properties of cholesterol. 602 00:30:30,600 --> 00:30:34,650 So we want to look at the properties of cholesterol. 603 00:30:37,360 --> 00:30:41,130 Then we're going to ask the question, 604 00:30:41,130 --> 00:30:43,592 how does cholesterol get from the diet to the bloodstream? 605 00:30:43,592 --> 00:30:45,300 And then we're going to ask the question, 606 00:30:45,300 --> 00:30:47,550 how does cholesterol get from the bloodstream 607 00:30:47,550 --> 00:30:52,200 into the tissues where it's essential for membrane 608 00:30:52,200 --> 00:30:54,520 controlling membrane fluidity? 609 00:30:54,520 --> 00:30:58,020 So what do we know about cholesterol itself? 610 00:30:58,020 --> 00:31:00,850 If you look at the structure, what do we have? 611 00:31:00,850 --> 00:31:06,600 We have a grease ball and a little hydrophilic head. 612 00:31:06,600 --> 00:31:11,880 And so this cholesterol moiety, if you look at the structure 613 00:31:11,880 --> 00:31:14,220 up there, is really pretty rigid. 614 00:31:14,220 --> 00:31:16,430 And it, in fact, rigidifies. 615 00:31:16,430 --> 00:31:18,300 So this is rigid-- 616 00:31:18,300 --> 00:31:21,600 and in fact rigidifies membranes. 617 00:31:21,600 --> 00:31:24,180 And so you can see this if you go back and look at this. 618 00:31:24,180 --> 00:31:26,970 Hard to see these little things, but those are cholesterols 619 00:31:26,970 --> 00:31:30,600 stuck within the phospholipid bilayers. 620 00:31:30,600 --> 00:31:33,090 And this is key since this is something 621 00:31:33,090 --> 00:31:35,710 that I think a lot of people are spending a lot more time on 622 00:31:35,710 --> 00:31:37,980 and we're getting much better at this now. 623 00:31:37,980 --> 00:31:40,485 People have stayed away from membranes because it's so-- 624 00:31:40,485 --> 00:31:42,852 and membrane proteins because it's so hard to work with 625 00:31:42,852 --> 00:31:44,060 and they stick to everything. 626 00:31:44,060 --> 00:31:45,780 How do you control all of this? 627 00:31:45,780 --> 00:31:47,340 And Brown and Goldstein really did 628 00:31:47,340 --> 00:31:49,530 some of the classic experiments that 629 00:31:49,530 --> 00:31:52,260 taught us how to deal with these type of really 630 00:31:52,260 --> 00:31:55,110 hydrophobic molecules. 631 00:31:55,110 --> 00:31:58,590 And so cholesterol is pretty important. 632 00:31:58,590 --> 00:32:02,340 10% of the membranes actually have-- 633 00:32:02,340 --> 00:32:05,890 of the lipids in the membranes are from cholesterol. 634 00:32:05,890 --> 00:32:08,970 So if you look at this, you would think 635 00:32:08,970 --> 00:32:10,770 it wouldn't be very soluble. 636 00:32:10,770 --> 00:32:14,490 And in fact, the solubility of this-- 637 00:32:14,490 --> 00:32:18,600 solubility is about five micromolar. 638 00:32:18,600 --> 00:32:30,690 So it isn't very soluble, but in fact, as an adult, 639 00:32:30,690 --> 00:32:34,380 we have 35 to 50 grams-- 640 00:32:34,380 --> 00:32:38,340 we each have 35 to 50 grams of cholesterol. 641 00:32:38,340 --> 00:32:43,920 And we know that per day 1 gram is derived from synthesis 642 00:32:43,920 --> 00:32:47,210 in the liver, so the predominant organ 643 00:32:47,210 --> 00:32:50,490 where cholesterol is made, like we just were describing, 644 00:32:50,490 --> 00:32:52,050 is the liver. 645 00:32:52,050 --> 00:32:54,726 But we also have-- 646 00:32:54,726 --> 00:32:57,000 and I don't know how good these numbers are. 647 00:32:57,000 --> 00:32:58,800 I got them out of some book. 648 00:32:58,800 --> 00:33:00,847 So I'm not an expert in this, but anyhow, 649 00:33:00,847 --> 00:33:02,430 these are all rough numbers and you'll 650 00:33:02,430 --> 00:33:06,720 see these in other nutrient uptake systems. 651 00:33:06,720 --> 00:33:10,230 You want to have some vague idea of the contributions 652 00:33:10,230 --> 00:33:12,600 to the two distinct processes. 653 00:33:12,600 --> 00:33:19,380 We get from the diet, say, 200 to 300 milligrams 654 00:33:19,380 --> 00:33:21,000 from the diet. 655 00:33:21,000 --> 00:33:25,770 So then if you think about this, cholesterol we're going to see 656 00:33:25,770 --> 00:33:30,690 is transported in the blood, and we'll see how that happens. 657 00:33:34,948 --> 00:33:35,448 Whoops. 658 00:33:38,410 --> 00:33:44,410 Transported in blood. 659 00:33:44,410 --> 00:33:46,990 And we know something about the amounts. 660 00:33:46,990 --> 00:33:49,930 And if you do a calculation, that 661 00:33:49,930 --> 00:33:55,840 says that you would have five millimolar cholesterol. 662 00:33:55,840 --> 00:33:56,830 So that's impossible. 663 00:33:56,830 --> 00:33:59,060 The number is squishy, but it's impossible. 664 00:33:59,060 --> 00:34:01,450 So you'd have this insoluble mess. 665 00:34:01,450 --> 00:34:04,030 So the question is, how do you deal with it? 666 00:34:04,030 --> 00:34:06,670 And so that's what we need to think about. 667 00:34:06,670 --> 00:34:09,810 So how does cholesterol move-- 668 00:34:09,810 --> 00:34:11,228 I think [INAUDIBLE]. 669 00:34:15,550 --> 00:34:25,489 So how does cholesterol go from the blood 670 00:34:25,489 --> 00:34:31,280 to tissues, given the solubility problems? 671 00:34:31,280 --> 00:34:34,070 So here is again the structure of cholesterol. 672 00:34:34,070 --> 00:34:41,380 Again, it's pretty rigid and it inserts itself into membranes. 673 00:34:41,380 --> 00:34:44,340 Where do you get cholesterol from? 674 00:34:44,340 --> 00:34:49,179 You all know you get cholesterol from beef and chicken and eggs. 675 00:34:49,179 --> 00:34:50,739 I guess there aren't very many-- 676 00:34:50,739 --> 00:34:52,480 do any of you eat at McDonald's? 677 00:34:52,480 --> 00:34:53,739 Or is that a passe thing? 678 00:34:53,739 --> 00:34:55,645 I love McDonald's anyhow. 679 00:34:55,645 --> 00:34:57,520 That was my favorite when I was in Wisconsin. 680 00:34:57,520 --> 00:34:59,892 There was only one restaurant near where 681 00:34:59,892 --> 00:35:02,350 the biochemistry department was and I went there every day, 682 00:35:02,350 --> 00:35:05,730 and my favorite thing was like two of those things slathered 683 00:35:05,730 --> 00:35:08,470 in cheese with French fries. 684 00:35:08,470 --> 00:35:12,230 Anyhow, fortunately, I have very low cholesterol. 685 00:35:12,230 --> 00:35:15,430 But anyhow, you get that our diet is 686 00:35:15,430 --> 00:35:17,260 a major source of cholesterol and what 687 00:35:17,260 --> 00:35:20,770 you eat can, in fact, be problematic and part of it 688 00:35:20,770 --> 00:35:23,230 really sort of depends on how lucky you are genetically, 689 00:35:23,230 --> 00:35:23,870 right? 690 00:35:23,870 --> 00:35:25,930 That's sort of the key thing. 691 00:35:25,930 --> 00:35:28,600 So what do we do? 692 00:35:28,600 --> 00:35:32,730 We have this insoluble molecule and the question is, 693 00:35:32,730 --> 00:35:35,290 how do how are we going to get this insoluble molecule 694 00:35:35,290 --> 00:35:37,750 into the tissues where it's needed to control 695 00:35:37,750 --> 00:35:39,890 the fluidity of the membranes? 696 00:35:39,890 --> 00:35:40,720 That's the issue. 697 00:35:40,720 --> 00:35:43,420 So the second thing I'm just going to introduce you to, 698 00:35:43,420 --> 00:35:47,500 and this is taken from Voet and Voet. 699 00:35:47,500 --> 00:35:49,630 So many of you may have read that if you 700 00:35:49,630 --> 00:35:55,340 had 705 or something, but in 507 we don't cover this reaction. 701 00:35:55,340 --> 00:35:58,060 So I'm going to spend a few minutes going over it. 702 00:35:58,060 --> 00:36:00,940 So what has to happen is cholesterol 703 00:36:00,940 --> 00:36:06,220 is found in lipoprotein particles. 704 00:36:13,830 --> 00:36:16,380 And we know a lot about the composition 705 00:36:16,380 --> 00:36:19,290 of these lipoprotein particles, which-- 706 00:36:19,290 --> 00:36:21,780 this is taken from Voet Voet. 707 00:36:21,780 --> 00:36:24,000 And what you can see is-- and I think, 708 00:36:24,000 --> 00:36:27,610 again, the relative amounts isn't all that important. 709 00:36:27,610 --> 00:36:29,280 But you can see you have-- 710 00:36:29,280 --> 00:36:33,750 and we're going to be focused on low-density lipoprotein, which 711 00:36:33,750 --> 00:36:38,910 is the major deliverer of cholesterol to the tissues. 712 00:36:38,910 --> 00:36:40,300 And why is that true? 713 00:36:40,300 --> 00:36:43,590 So if we look at free cholesterol, 714 00:36:43,590 --> 00:36:46,290 we see we see 7% to 10%. 715 00:36:46,290 --> 00:36:49,080 I'm going to tell you about the structure of the lipoproteins 716 00:36:49,080 --> 00:36:50,200 in a minute. 717 00:36:50,200 --> 00:36:52,650 But most of the cholesterol is actually 718 00:36:52,650 --> 00:36:55,320 esterified with fatty acids, and you 719 00:36:55,320 --> 00:37:00,298 can see that the cholesterol esters are 35% to 40%. 720 00:37:00,298 --> 00:37:02,340 So if you look at the total amount of cholesterol 721 00:37:02,340 --> 00:37:06,090 in the LDL particles compared to all the other particles, 722 00:37:06,090 --> 00:37:08,010 it's much higher. 723 00:37:08,010 --> 00:37:10,590 So what do you see and what do you 724 00:37:10,590 --> 00:37:14,470 have to worry about if you're getting this from the diet? 725 00:37:14,470 --> 00:37:15,870 So what would you expect to see? 726 00:37:15,870 --> 00:37:17,280 You would expect to see proteins. 727 00:37:20,400 --> 00:37:23,100 You would see phospholipids. 728 00:37:23,100 --> 00:37:25,212 So this is a phospholipid. 729 00:37:28,710 --> 00:37:31,600 You would expect to see triacylglycerol. 730 00:37:31,600 --> 00:37:36,270 Everybody know what triacylglycerol is? 731 00:37:36,270 --> 00:37:39,000 We expect to see fatty acids. 732 00:37:39,000 --> 00:37:42,720 We expect to see cholesterol. 733 00:37:42,720 --> 00:37:46,230 And if you look over here, what people have done, 734 00:37:46,230 --> 00:37:48,720 have isolated different particles. 735 00:37:48,720 --> 00:37:50,970 How do they isolate the particles? 736 00:37:50,970 --> 00:37:55,260 The particles are isolated based on density differences. 737 00:37:55,260 --> 00:38:00,510 And if you look at all of these different compositions, 738 00:38:00,510 --> 00:38:05,640 they vary between very low-density lipoprotein, 739 00:38:05,640 --> 00:38:07,830 intermediate-density lipoprotein, 740 00:38:07,830 --> 00:38:09,810 high-density lipoprotein. 741 00:38:09,810 --> 00:38:13,060 They have different amounts of these different species. 742 00:38:13,060 --> 00:38:19,200 And in fact, most of them have very hydrophobic stuff 743 00:38:19,200 --> 00:38:23,130 in the outside and more hydrophilic stuff-- 744 00:38:23,130 --> 00:38:24,660 on the inside and more hydrophilic 745 00:38:24,660 --> 00:38:27,455 filled stuff on the exterior, which is more dense. 746 00:38:27,455 --> 00:38:28,830 And then that tells you something 747 00:38:28,830 --> 00:38:33,780 about how these things sediment by a subterfugation 748 00:38:33,780 --> 00:38:35,920 method and a density gradient. 749 00:38:35,920 --> 00:38:40,090 So these lipoprotein particles-- 750 00:38:40,090 --> 00:38:42,120 and we're going to see this kind of method 751 00:38:42,120 --> 00:38:45,130 in next week's recitations-- 752 00:38:45,130 --> 00:38:52,020 are separated by the centrifugation 753 00:38:52,020 --> 00:38:55,320 due to density differences. 754 00:39:00,660 --> 00:39:03,068 So let's just briefly look at LDL. 755 00:39:03,068 --> 00:39:05,110 That's what we're going to be dealing with today. 756 00:39:05,110 --> 00:39:07,140 And it's important because LDL is 757 00:39:07,140 --> 00:39:09,540 what we're going to try to take into the cell, 758 00:39:09,540 --> 00:39:12,180 and the composition of the LDL is 759 00:39:12,180 --> 00:39:13,920 key to thinking about how to studying 760 00:39:13,920 --> 00:39:16,560 that process in the classic Brown and Goldstein 761 00:39:16,560 --> 00:39:17,770 experiments. 762 00:39:17,770 --> 00:39:21,780 So if we look at the cartoon of LDL 763 00:39:21,780 --> 00:39:26,198 that you see up there, what you see is a particle. 764 00:39:26,198 --> 00:39:27,240 They're sort of circular. 765 00:39:29,760 --> 00:39:33,270 The LDL particles, which is what we're going to be focusing on, 766 00:39:33,270 --> 00:39:36,540 low-density lipoprotein particles, 767 00:39:36,540 --> 00:39:41,730 have only a single protein and this protein 768 00:39:41,730 --> 00:39:44,700 is called the ApoB. 769 00:39:44,700 --> 00:39:53,110 It's a huge protein and it covers about 50% 770 00:39:53,110 --> 00:39:57,850 of the surface of the particle itself. 771 00:39:57,850 --> 00:39:59,680 Again, these things change in size. 772 00:39:59,680 --> 00:40:03,250 We'll see when we actually look at the transport process. 773 00:40:03,250 --> 00:40:04,100 What do we know? 774 00:40:04,100 --> 00:40:06,100 It's on the exterior. 775 00:40:06,100 --> 00:40:13,180 On the exterior what you see all over the exterior, 776 00:40:13,180 --> 00:40:16,470 these things that are phospholipids. 777 00:40:16,470 --> 00:40:19,120 So the phosphate is on the outside, 778 00:40:19,120 --> 00:40:22,120 the fatty acids are on the inside. 779 00:40:22,120 --> 00:40:24,040 What else do you see on the exterior? 780 00:40:24,040 --> 00:40:29,960 You see a lot of cholesterol molecules, 781 00:40:29,960 --> 00:40:33,950 which I'm indicating like this. 782 00:40:33,950 --> 00:40:37,670 And then it turns out that the predominant-- and that maybe 783 00:40:37,670 --> 00:40:40,760 covers, I don't know, 20 Angstroms, 784 00:40:40,760 --> 00:40:44,690 but the particles are 200 Angstroms, 220 Angstroms. 785 00:40:44,690 --> 00:40:46,760 So what's in the center? 786 00:40:46,760 --> 00:40:52,610 And what's in the center, so this is the interior. 787 00:40:52,610 --> 00:40:56,900 You basically have triacylglycerols 788 00:40:56,900 --> 00:40:58,670 and then you have cholesterol. 789 00:40:58,670 --> 00:41:02,840 And remember, cholesterol has one lone 3 prime hydroxyl 790 00:41:02,840 --> 00:41:08,600 group, and this is a esterified with a fatty acid. 791 00:41:11,300 --> 00:41:16,330 And so this is also in the interior. 792 00:41:16,330 --> 00:41:19,940 So that's the composition of the LDL particles. 793 00:41:19,940 --> 00:41:21,350 And the question is, again, where 794 00:41:21,350 --> 00:41:25,778 did they come from starting with stuff we get from the diet? 795 00:41:25,778 --> 00:41:27,570 So that's what we're going to be focused on 796 00:41:27,570 --> 00:41:29,240 and that's what we're going to try-- 797 00:41:29,240 --> 00:41:34,450 the cholesterol is stuck on the surface and in the interior. 798 00:41:34,450 --> 00:41:34,950 Yeah? 799 00:41:34,950 --> 00:41:36,158 AUDIENCE: I don't understand. 800 00:41:36,158 --> 00:41:38,300 Aren't there different splice variants ApoB 801 00:41:38,300 --> 00:41:40,630 and which one is the one that's involved now? 802 00:41:40,630 --> 00:41:41,880 JOANNE STUBBE: There could be. 803 00:41:41,880 --> 00:41:44,870 There could be We're not talking about this in detail at all. 804 00:41:44,870 --> 00:41:48,140 I don't know how many splice variances there are. 805 00:41:48,140 --> 00:41:50,660 And I don't really know that much about all 806 00:41:50,660 --> 00:41:52,010 of these different proteins. 807 00:41:52,010 --> 00:41:54,800 You'd have to go read about them in detail. 808 00:41:54,800 --> 00:41:58,280 So I'm giving you sort of a cartoon general overview 809 00:41:58,280 --> 00:42:00,410 of what you need to think about. 810 00:42:00,410 --> 00:42:02,700 There are splice variance of almost any protein. 811 00:42:02,700 --> 00:42:08,390 And in humans, you have in the PCSK that we talked about, 812 00:42:08,390 --> 00:42:10,730 there were nine isozymes. 813 00:42:10,730 --> 00:42:12,488 So isozymes and eukaryotic systems 814 00:42:12,488 --> 00:42:14,030 are something you don't have to worry 815 00:42:14,030 --> 00:42:17,540 about a lot for this lecture and for what I want to say. 816 00:42:17,540 --> 00:42:19,310 You don't have to worry about that. 817 00:42:19,310 --> 00:42:22,730 And if you want to read about it, go for it. 818 00:42:22,730 --> 00:42:26,700 So we have LDL particles and they 819 00:42:26,700 --> 00:42:29,480 are distinct from all these other particles which 820 00:42:29,480 --> 00:42:32,750 have different densities, different proteins. 821 00:42:32,750 --> 00:42:35,180 And there are two cartoons I want to use. 822 00:42:35,180 --> 00:42:37,580 This cartoon was taken from Voet and Voet. 823 00:42:37,580 --> 00:42:39,500 I think it was the third issue. 824 00:42:39,500 --> 00:42:41,000 The one from the fourth issue, which 825 00:42:41,000 --> 00:42:43,970 I'll show you in a minute, I think, is much better. 826 00:42:43,970 --> 00:42:46,910 So I'm going to change the handout. 827 00:42:46,910 --> 00:42:50,630 But I just want to very briefly walk you through this. 828 00:42:50,630 --> 00:42:54,620 This is a really complicated process and, from my reading, 829 00:42:54,620 --> 00:42:56,870 is really not completely understood. 830 00:42:56,870 --> 00:42:57,860 But you have diet. 831 00:42:57,860 --> 00:42:59,277 And what do you have in your diet? 832 00:42:59,277 --> 00:43:03,980 Triacylglycerols, phospholipids, proteins. 833 00:43:03,980 --> 00:43:07,760 They get taken-- in the diet, they get into the intestine 834 00:43:07,760 --> 00:43:10,370 and somehow in that process they need 835 00:43:10,370 --> 00:43:14,960 to get packaged into one of these lipoprotein particles. 836 00:43:14,960 --> 00:43:17,150 And the lipoprotein particle that 837 00:43:17,150 --> 00:43:21,830 really is the predominant one that comes out of the intestine 838 00:43:21,830 --> 00:43:25,340 are these things called chylomicrons. 839 00:43:25,340 --> 00:43:27,510 And you can see they have a lot of proteins. 840 00:43:27,510 --> 00:43:29,720 They have a lot of triacylglycerol. 841 00:43:29,720 --> 00:43:31,250 They have a lot of phospholipids. 842 00:43:31,250 --> 00:43:35,660 Anyhow, the composition varies and the sizes also vary. 843 00:43:35,660 --> 00:43:39,080 So these chylomicrons come from the diet. 844 00:43:39,080 --> 00:43:48,410 So how do these lipoproteins deliver LDL 845 00:43:48,410 --> 00:43:51,980 to the extrahepatic tissues? 846 00:43:51,980 --> 00:43:55,220 That's what we're really after. 847 00:43:55,220 --> 00:43:58,250 And so these chylomicrons-- let me just 848 00:43:58,250 --> 00:43:59,930 show you the next slide for a second. 849 00:43:59,930 --> 00:44:02,870 I think this is probably a better one, anyhow. 850 00:44:02,870 --> 00:44:04,760 So these chylomicrons, somehow they 851 00:44:04,760 --> 00:44:09,387 have to package all this stuff into these lipoproteins. 852 00:44:09,387 --> 00:44:10,470 You know how that happens? 853 00:44:10,470 --> 00:44:12,140 I don't really know very much about it. 854 00:44:12,140 --> 00:44:13,130 Maybe somebody does. 855 00:44:13,130 --> 00:44:14,690 I don't know that much about it. 856 00:44:14,690 --> 00:44:18,230 So anyhow, it gets packaged into these little particles 857 00:44:18,230 --> 00:44:21,530 and then it goes into the intestinal lymph, which 858 00:44:21,530 --> 00:44:24,710 then goes into the bloodstream and then it 859 00:44:24,710 --> 00:44:26,120 needs to start circulating. 860 00:44:26,120 --> 00:44:28,280 So everything comes from the diet 861 00:44:28,280 --> 00:44:34,310 comes from these chylomicron particles. 862 00:44:34,310 --> 00:44:38,780 So what happens when you go adjacent to adipose 863 00:44:38,780 --> 00:44:40,880 tissue or muscles? 864 00:44:40,880 --> 00:44:43,320 So what you need, if you're going 865 00:44:43,320 --> 00:44:45,560 to be involved with fat metabolism 866 00:44:45,560 --> 00:44:47,930 or you need energy to run down the street, 867 00:44:47,930 --> 00:44:49,520 you need fatty acids. 868 00:44:49,520 --> 00:44:51,660 So where do the fatty acids come from? 869 00:44:51,660 --> 00:44:54,140 They come from the triacylglycerol. 870 00:44:54,140 --> 00:44:59,000 So what you have are lipases and all of these chylomicrons 871 00:44:59,000 --> 00:45:00,950 in the lipases. 872 00:45:00,950 --> 00:45:03,660 Then when you get near the tissue-- 873 00:45:03,660 --> 00:45:04,400 let's see. 874 00:45:04,400 --> 00:45:09,200 Here we get near the tissue, the muscle or the adipose tissue, 875 00:45:09,200 --> 00:45:11,870 a li-- does everybody know what a lipase is 876 00:45:11,870 --> 00:45:14,120 or do you want me to write that reaction on the board? 877 00:45:14,120 --> 00:45:16,340 Does everybody know what a lipase is? 878 00:45:16,340 --> 00:45:17,030 No? 879 00:45:17,030 --> 00:45:17,530 OK. 880 00:45:17,530 --> 00:45:32,100 So a lipase-- so here is your triacylglycerol 881 00:45:32,100 --> 00:45:33,610 with different fatty acids. 882 00:45:33,610 --> 00:45:36,570 So this is a TAG. 883 00:45:36,570 --> 00:45:38,070 This is glycerol. 884 00:45:38,070 --> 00:45:42,090 It's stereo-- this is a chiral center. 885 00:45:42,090 --> 00:45:44,370 And so what happens then is lipase 886 00:45:44,370 --> 00:45:47,700 is simply an esterase that hydrolyzes the bond. 887 00:45:47,700 --> 00:45:49,290 So I'm not going to draw the reaction 888 00:45:49,290 --> 00:45:55,100 out, but a lipase catalyzes, release-- 889 00:45:55,100 --> 00:45:57,540 this is a fatty acid. 890 00:45:57,540 --> 00:46:00,420 And it actually cuts off two of them, and so most of the time 891 00:46:00,420 --> 00:46:02,142 you have monoacylated fatty acids. 892 00:46:02,142 --> 00:46:03,850 But again, from what we're talking about, 893 00:46:03,850 --> 00:46:06,300 this is not really important because really 894 00:46:06,300 --> 00:46:11,100 what we want to do is get to the low-density lipoproteins. 895 00:46:11,100 --> 00:46:12,810 So what you see when you start doing 896 00:46:12,810 --> 00:46:17,160 this is that if you drop off triacylglycerols 897 00:46:17,160 --> 00:46:19,650 or monoacylglycerols here, and you drop off 898 00:46:19,650 --> 00:46:22,710 something else to the other tissues along the way, 899 00:46:22,710 --> 00:46:25,620 the sizes of your particles change size. 900 00:46:25,620 --> 00:46:29,250 So they call these things, then, the remnants 901 00:46:29,250 --> 00:46:30,820 from your starting material. 902 00:46:30,820 --> 00:46:33,930 And it turns out there is a receptor that 903 00:46:33,930 --> 00:46:38,670 takes up chylomicron remnants into the liver. 904 00:46:38,670 --> 00:46:43,200 So the central player in all of this is the liver. 905 00:46:43,200 --> 00:46:44,480 So you got to take stuff-- 906 00:46:44,480 --> 00:46:46,980 we get it from the diet, but we got to get it into the liver 907 00:46:46,980 --> 00:46:48,970 and that's where everything is controlled. 908 00:46:48,970 --> 00:46:52,200 And that's predominantly where cholesterol is actually 909 00:46:52,200 --> 00:46:53,820 biosynthesized. 910 00:46:53,820 --> 00:46:54,810 So if you start-- 911 00:46:57,510 --> 00:46:59,280 you bring in-- what are you bringing in? 912 00:46:59,280 --> 00:47:02,190 You're bringing in cholesterol because you've dropped off 913 00:47:02,190 --> 00:47:06,150 triacylglycerols and lipids and phospholipids to the tissues. 914 00:47:06,150 --> 00:47:08,400 So what's predominantly left? 915 00:47:08,400 --> 00:47:10,840 What's predominantly left is cholesterol. 916 00:47:10,840 --> 00:47:21,880 So from these chylomicrons you drop off fatty acids 917 00:47:21,880 --> 00:47:32,400 and monoacylglycerols to adipocytes or muscle, 918 00:47:32,400 --> 00:47:35,070 and then what you have left is cholesterol. 919 00:47:35,070 --> 00:47:37,200 You have a lot of stuff left, but cholesterol 920 00:47:37,200 --> 00:47:41,580 is a major thing, which is then going to go to the liver. 921 00:47:41,580 --> 00:47:43,860 And so then once this gets into the liver, 922 00:47:43,860 --> 00:47:47,520 the liver has all this machinery to repackage things 923 00:47:47,520 --> 00:47:51,320 and they can make very low-density lipoproteins again. 924 00:47:51,320 --> 00:47:52,820 So you can go back and look at this. 925 00:47:52,820 --> 00:47:54,760 It's very complicated. 926 00:47:54,760 --> 00:47:59,280 They then in the bloodstream can drop off stuff along the way 927 00:47:59,280 --> 00:48:01,620 to tissues as well. 928 00:48:01,620 --> 00:48:05,520 And then they change into intermediate-density 929 00:48:05,520 --> 00:48:09,390 lipoproteins, which then can change 930 00:48:09,390 --> 00:48:12,350 into low-density lipoproteins. 931 00:48:12,350 --> 00:48:14,700 And it's these low-density lipoproteins 932 00:48:14,700 --> 00:48:17,550 that are going to then deliver cholesterol, that 933 00:48:17,550 --> 00:48:20,700 has more cholesterol than any of these other particles, 934 00:48:20,700 --> 00:48:26,880 either two extrahepatic tissues or back into the liver. 935 00:48:26,880 --> 00:48:31,500 So you have a complicated set of transport systems 936 00:48:31,500 --> 00:48:33,330 that we're not going to spend any time on, 937 00:48:33,330 --> 00:48:37,770 but it's all related to the fact that cholesterol is basically 938 00:48:37,770 --> 00:48:40,390 not a happy camper in water. 939 00:48:40,390 --> 00:48:45,240 And so we've got to figure out how to move cholesterol around. 940 00:48:48,690 --> 00:48:52,680 So that just summarizes-- 941 00:48:52,680 --> 00:48:54,390 what I didn't show you over here, 942 00:48:54,390 --> 00:48:57,690 you all have heard about high-density cholesterol, 943 00:48:57,690 --> 00:49:00,090 low-density cholesterol. 944 00:49:00,090 --> 00:49:05,040 And high-density cholesterol is distinct from all 945 00:49:05,040 --> 00:49:09,150 these other lipoprotein particles. 946 00:49:09,150 --> 00:49:14,250 And it sort of scavenges excess cholesterol 947 00:49:14,250 --> 00:49:23,880 from these extrahepatic cells and returns it to the liver. 948 00:49:23,880 --> 00:49:28,140 But unlike the look the receptor-mediated endocytosis 949 00:49:28,140 --> 00:49:31,770 we're going to talk about with the LDL receptor, 950 00:49:31,770 --> 00:49:33,810 this receptor is completely distinct. 951 00:49:33,810 --> 00:49:36,300 I'm not going to talk about it, but the mechanism 952 00:49:36,300 --> 00:49:39,450 is distinct from these other receptors 953 00:49:39,450 --> 00:49:42,120 that people have also studied in some detail. 954 00:49:44,740 --> 00:49:47,110 So the other thing that I wanted to briefly say 955 00:49:47,110 --> 00:49:51,733 is that in addition to cholesterol what you see-- 956 00:49:51,733 --> 00:49:53,900 and I'm not going to spend much time on this either, 957 00:49:53,900 --> 00:49:56,170 but I think it's worth mentioning-- 958 00:49:56,170 --> 00:50:01,270 is when you get cholesterol back into the liver, 959 00:50:01,270 --> 00:50:04,570 what can you do with that excess cholesterol? 960 00:50:04,570 --> 00:50:08,230 If you have too much of it, how do you control the levels? 961 00:50:08,230 --> 00:50:10,870 That's the key thing we're going to be trying to focus on. 962 00:50:10,870 --> 00:50:13,540 What have we learned, at least to some level, 963 00:50:13,540 --> 00:50:16,600 in control of cholesterol levels? 964 00:50:16,600 --> 00:50:20,710 But it turns out in the liver-- so the key organ in all of this 965 00:50:20,710 --> 00:50:21,820 is the liver-- 966 00:50:21,820 --> 00:50:26,800 cholesterol can be metabolized to form molecules 967 00:50:26,800 --> 00:50:29,080 that have four rings just like cholesterol 968 00:50:29,080 --> 00:50:30,430 that are called bile acids. 969 00:50:34,130 --> 00:50:35,680 And these are multiple steps. 970 00:50:35,680 --> 00:50:39,100 I'm not going to draw out the steps, 971 00:50:39,100 --> 00:50:42,210 but if you look at a bile acid-- 972 00:50:42,210 --> 00:50:46,010 and I have cartoons of bile acids over here. 973 00:50:46,010 --> 00:50:48,768 So here's cholesterol and if you look at this-- 974 00:50:48,768 --> 00:50:50,560 it's hard to see it, but if you look at it, 975 00:50:50,560 --> 00:50:55,240 it really sort of it looks a lot like cholesterol. 976 00:50:55,240 --> 00:51:01,210 The only differences are you add additional hydroxyl groups. 977 00:51:01,210 --> 00:51:08,360 So in cholesterol we have a 3-alpha hydroxyl group. 978 00:51:08,360 --> 00:51:12,220 In the bile acid you have two additional hydroxyl groups put 979 00:51:12,220 --> 00:51:15,040 on again by cythochrome P450s. 980 00:51:15,040 --> 00:51:20,810 So you have a hydroxyl group at C7. 981 00:51:20,810 --> 00:51:24,820 You have a hydroxyl group at 12 alpha, 982 00:51:24,820 --> 00:51:26,770 simply means the stereochemistry. 983 00:51:26,770 --> 00:51:29,860 So the stereo chemistry of the hydroxyl group 984 00:51:29,860 --> 00:51:31,390 is on the same face. 985 00:51:31,390 --> 00:51:32,600 So that's what I mean here. 986 00:51:32,600 --> 00:51:37,240 So what you have then is hydrophobic and hydrophilic. 987 00:51:40,580 --> 00:51:43,170 And in addition, if you look at the very end, 988 00:51:43,170 --> 00:51:54,360 it turns out you have molecules glycine or taurine, 989 00:51:54,360 --> 00:51:56,985 which are on the handout, which has a negative charge. 990 00:51:59,490 --> 00:52:02,460 And again, it's on the same face. 991 00:52:02,460 --> 00:52:05,040 So we have a bunch of hydroxyls, something charge, 992 00:52:05,040 --> 00:52:06,750 and they act as emulsifying agents, 993 00:52:06,750 --> 00:52:09,300 and that's all you really need to know. 994 00:52:09,300 --> 00:52:15,210 So these become emulsifying and they really 995 00:52:15,210 --> 00:52:19,740 play sort of key role in also helping to take things back 996 00:52:19,740 --> 00:52:20,480 into the cell. 997 00:52:20,480 --> 00:52:22,960 And this is a really complicated process. 998 00:52:22,960 --> 00:52:25,860 And in fact, I think it was 15 years ago, 999 00:52:25,860 --> 00:52:28,410 something, people used to try to remove 1000 00:52:28,410 --> 00:52:32,910 bile acids as a mechanism of controlling cholesterol levels. 1001 00:52:32,910 --> 00:52:36,360 And what you did was actually-- boy, I'm way over again. 1002 00:52:36,360 --> 00:52:38,190 What you actually did was eat-- 1003 00:52:38,190 --> 00:52:41,385 have any of you ever worked with Dowex in an exchange? 1004 00:52:41,385 --> 00:52:43,800 You used to eat the resin you have in the lab 1005 00:52:43,800 --> 00:52:46,680 called Dowex because it would bind the negatively 1006 00:52:46,680 --> 00:52:48,030 charged materials. 1007 00:52:48,030 --> 00:52:51,270 And so, really, it was very hard for people to stomach this, 1008 00:52:51,270 --> 00:52:54,240 but that was before we had really sort of wonder drugs-- 1009 00:52:54,240 --> 00:52:58,110 Dowex, eating Dowex in these little grainy resins. 1010 00:52:58,110 --> 00:53:01,990 You should go look in the lab if you're doing a UROP. 1011 00:53:01,990 --> 00:53:05,130 That's how we used to treat high levels of cholesterol. 1012 00:53:05,130 --> 00:53:07,235 So anyhow, bile acids also play a key role. 1013 00:53:07,235 --> 00:53:08,860 We're not talking about this in detail. 1014 00:53:08,860 --> 00:53:10,700 So the next time we're going to come back 1015 00:53:10,700 --> 00:53:13,560 and we now sort of see what the properties of cholesterol 1016 00:53:13,560 --> 00:53:16,440 are, that they're in lipoproteins. 1017 00:53:16,440 --> 00:53:19,740 And we want to focus on the key experiments that showed 1018 00:53:19,740 --> 00:53:23,060 how LDL is taken into cells.