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 --> 00:00:07,190 Your support will help in MIT OpenCourseWare continue 4 00:00:07,190 --> 00:00:11,010 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 00:00:25,575 --> 00:00:27,200 JOANNE STUBBE: So anyhow, this is where 9 00:00:27,200 --> 00:00:31,220 we ended the lecture last time. 10 00:00:31,220 --> 00:00:32,580 We're finishing up. 11 00:00:32,580 --> 00:00:35,880 We were talking about two kinds of regulation 12 00:00:35,880 --> 00:00:37,330 for cholesterol sensing. 13 00:00:37,330 --> 00:00:42,110 One using the transcription factor sterol responsive 14 00:00:42,110 --> 00:00:47,300 element binding protein, and how we can use that when 15 00:00:47,300 --> 00:00:50,020 cholesterol levels-- or sterol levels 16 00:00:50,020 --> 00:00:54,200 are low to upregulate the amount of sterol 17 00:00:54,200 --> 00:00:58,323 by turning on the genes for the biosynthetic pathway. 18 00:00:58,323 --> 00:00:59,990 And so that's what we were talking about 19 00:00:59,990 --> 00:01:04,790 at the end, and this week in recitation as well. 20 00:01:04,790 --> 00:01:10,920 And also you can turn on the gene for the LDL receptor, 21 00:01:10,920 --> 00:01:12,380 which then allows you to take more 22 00:01:12,380 --> 00:01:14,780 cholesterol in from the diet. 23 00:01:14,780 --> 00:01:20,290 And so what I wanted to do is in this week's problem set 7, 24 00:01:20,290 --> 00:01:24,240 you were focused on looking at the SCAP protein 25 00:01:24,240 --> 00:01:27,590 and how do you know what the SCAP protein is doing. 26 00:01:27,590 --> 00:01:31,640 And what you were seeing in the data 27 00:01:31,640 --> 00:01:35,120 you were give given, which was taken from one paper. 28 00:01:35,120 --> 00:01:37,520 There are many papers trying to study 29 00:01:37,520 --> 00:01:40,010 these proteins to understand what's 30 00:01:40,010 --> 00:01:45,620 going on with these complex membrane proteins. 31 00:01:45,620 --> 00:01:47,540 Where does the sterol bind? 32 00:01:47,540 --> 00:01:50,210 How does the sterol bind? 33 00:01:50,210 --> 00:01:53,810 What is it that causes this chemistry 34 00:01:53,810 --> 00:01:57,260 to happen where this complex migrates 35 00:01:57,260 --> 00:02:01,040 from the endoplasmic reticulum to the Golgi, 36 00:02:01,040 --> 00:02:03,420 allows cleavage chemistry to happen, 37 00:02:03,420 --> 00:02:07,670 and ultimately a little piece of DNA which 38 00:02:07,670 --> 00:02:10,850 binds to the sterol responsive element 39 00:02:10,850 --> 00:02:14,030 to actually turn on a bunch of genes 40 00:02:14,030 --> 00:02:16,400 that we just talked about? 41 00:02:16,400 --> 00:02:22,730 So I just wanted to say one or two words about the players. 42 00:02:22,730 --> 00:02:24,980 And you've all thought about the players by now. 43 00:02:24,980 --> 00:02:27,890 We're going to come back and look at some of them 44 00:02:27,890 --> 00:02:32,820 in a few minutes, but the key player in the scheme 45 00:02:32,820 --> 00:02:35,460 I just showed you was SCAP and that 46 00:02:35,460 --> 00:02:39,030 was the focus of what you guys had to do in your problem set. 47 00:02:39,030 --> 00:02:44,940 And what you notice again is a sterol sensing domain. 48 00:02:44,940 --> 00:02:46,740 And there is also-- 49 00:02:46,740 --> 00:02:51,470 I point out we'll come back to that at the end of today's 50 00:02:51,470 --> 00:02:53,930 lecture-- there's a sterol sensing domain 51 00:02:53,930 --> 00:02:56,660 in HMG-CoA reductase, which we will 52 00:02:56,660 --> 00:02:59,790 see is involved in post-transcriptional 53 00:02:59,790 --> 00:03:00,560 regulation. 54 00:03:00,560 --> 00:03:04,670 So we're still looking at transcriptional regulation now. 55 00:03:04,670 --> 00:03:06,050 And the question is-- 56 00:03:06,050 --> 00:03:11,960 and then you have a bunch of other transhelices, 57 00:03:11,960 --> 00:03:16,250 single transmembrane helices probably 58 00:03:16,250 --> 00:03:18,050 helical within the membrane. 59 00:03:18,050 --> 00:03:20,360 And the question is, how does this guy 60 00:03:20,360 --> 00:03:27,150 work to allow the model we showed on the previous slide? 61 00:03:27,150 --> 00:03:29,450 So that defines all those terms. 62 00:03:29,450 --> 00:03:31,640 Hopefully, you're now familiar with all those terms. 63 00:03:31,640 --> 00:03:34,880 It's written down in a place we can go read about it again. 64 00:03:34,880 --> 00:03:36,830 But this is the cartoon that you were dealing 65 00:03:36,830 --> 00:03:40,160 with in the problem set. 66 00:03:40,160 --> 00:03:42,860 And so the key question is-- 67 00:03:42,860 --> 00:03:45,620 most of these things are defined. 68 00:03:45,620 --> 00:03:47,510 Whether you have a transmembrane helix 69 00:03:47,510 --> 00:03:50,870 is defined by some kind of sequence gazing, 70 00:03:50,870 --> 00:03:52,670 and then you have to do experiments to test 71 00:03:52,670 --> 00:03:55,070 whether the model is correct. 72 00:03:55,070 --> 00:04:00,080 And we don't have any pictures of the SCAP protein 73 00:04:00,080 --> 00:04:01,670 at all at this stage. 74 00:04:01,670 --> 00:04:03,803 So the kinds of experiments that you 75 00:04:03,803 --> 00:04:05,720 were looking at your problem set are the kinds 76 00:04:05,720 --> 00:04:07,220 of experiments that people are still 77 00:04:07,220 --> 00:04:10,340 doing to try to figure out how all this information is 78 00:04:10,340 --> 00:04:14,390 coordinated to allow the chemistry to happen, 79 00:04:14,390 --> 00:04:17,360 or that migration from the ER to the Golgi. 80 00:04:17,360 --> 00:04:20,600 And we talked about last time-- 81 00:04:20,600 --> 00:04:23,480 we talked about a zip code. 82 00:04:23,480 --> 00:04:26,540 So if we look at SCAP, so we have 83 00:04:26,540 --> 00:04:29,110 eight transmembrane helices. 84 00:04:32,020 --> 00:04:35,260 And the key to the way this works 85 00:04:35,260 --> 00:04:39,640 is that there's a little zip code. 86 00:04:39,640 --> 00:04:42,670 And you've seen a zip code before transiently 87 00:04:42,670 --> 00:04:45,490 when we were looking at a zip code 88 00:04:45,490 --> 00:04:48,970 on the LDL receptor, which targeted AP2 to then 89 00:04:48,970 --> 00:04:50,760 bring in the clathrin coats to make 90 00:04:50,760 --> 00:04:52,955 the clathrin-coated vesicles. 91 00:04:52,955 --> 00:04:54,580 I think what you'll see over the course 92 00:04:54,580 --> 00:04:56,170 of the rest of the semester, there 93 00:04:56,170 --> 00:05:01,180 are lots of times three or four amino acid sequences that 94 00:05:01,180 --> 00:05:05,140 are the key that allow some kind of confirmational change 95 00:05:05,140 --> 00:05:08,080 to occur, which can trigger off a sequence of events 96 00:05:08,080 --> 00:05:11,410 that people have found by doing a lot of studies on the system. 97 00:05:11,410 --> 00:05:13,960 So the zip code here, and that's what you were focused 98 00:05:13,960 --> 00:05:17,040 on in your problem set, again. 99 00:05:17,040 --> 00:05:19,300 And I don't expect you to remember any of this, 100 00:05:19,300 --> 00:05:23,410 except to sort of know that these little zip codes play 101 00:05:23,410 --> 00:05:29,260 a role quite frequently in biological transformations 102 00:05:29,260 --> 00:05:31,270 of these complex systems. 103 00:05:31,270 --> 00:05:34,270 And so here is the little zip code. 104 00:05:34,270 --> 00:05:36,940 And we've been talking about so far what 105 00:05:36,940 --> 00:05:40,990 happens under low sterol levels, where 106 00:05:40,990 --> 00:05:45,670 we want to make cholesterol or get more in from the diet. 107 00:05:45,670 --> 00:05:50,620 And under these conditions, if we 108 00:05:50,620 --> 00:05:52,360 want to make more cholesterol, we 109 00:05:52,360 --> 00:05:55,990 have to turn on the biosynthetic machinery, HMG-CoA reductase, 110 00:05:55,990 --> 00:06:01,870 or turn on LDL receptor that allows you to take things 111 00:06:01,870 --> 00:06:02,690 into the cell. 112 00:06:02,690 --> 00:06:09,040 And so this is proposed to be a key player in loop 6. 113 00:06:09,040 --> 00:06:11,518 And this loop 6, which is pretty big-- 114 00:06:11,518 --> 00:06:13,810 and you might ask yourself the question, where did they 115 00:06:13,810 --> 00:06:14,680 get this loop? 116 00:06:14,680 --> 00:06:16,450 So that's something that you have 117 00:06:16,450 --> 00:06:20,320 to design experiments to figure out what is cytoplasmic, 118 00:06:20,320 --> 00:06:24,070 what faces the lumen, how big are these loops? 119 00:06:24,070 --> 00:06:27,190 All of that plays a role in thinking about how this works 120 00:06:27,190 --> 00:06:29,950 from a molecular perspective. 121 00:06:29,950 --> 00:06:34,150 So loop 6 plays a key role, as does, 122 00:06:34,150 --> 00:06:37,990 you can see from your problem set, loop 1. 123 00:06:37,990 --> 00:06:40,930 So what does loop 6 do in the model? 124 00:06:40,930 --> 00:06:43,780 The cartoon is shown over here. 125 00:06:43,780 --> 00:06:47,200 Under these conditions where you have low sterol, 126 00:06:47,200 --> 00:06:50,770 is below whatever the membrane concentration 127 00:06:50,770 --> 00:06:53,950 is that you looked at for the recitation 128 00:06:53,950 --> 00:06:57,400 this week, we're down 3% or less or something 129 00:06:57,400 --> 00:07:02,830 like that, what happens is this little zip code is exposed. 130 00:07:02,830 --> 00:07:05,920 And it in some way recruits proteins 131 00:07:05,920 --> 00:07:08,140 that are involved in another complex process 132 00:07:08,140 --> 00:07:11,140 that we aren't going to talk about where you can bud off 133 00:07:11,140 --> 00:07:15,880 little vesicles where proteins of interest and also lipids 134 00:07:15,880 --> 00:07:21,040 can be moved from one membrane to another, the ER in this case 135 00:07:21,040 --> 00:07:22,780 to the Golgi. 136 00:07:22,780 --> 00:07:26,530 And so this interaction is a GTPase here. 137 00:07:26,530 --> 00:07:28,930 There are a couple of additional proteins 138 00:07:28,930 --> 00:07:30,100 that have been identified. 139 00:07:30,100 --> 00:07:32,380 We're not going to talk about the details. 140 00:07:32,380 --> 00:07:37,120 But this is the key to allow movement into the Golgi, which 141 00:07:37,120 --> 00:07:40,630 then you have the defined proteases 142 00:07:40,630 --> 00:07:43,270 that we've talked about before which allow cutting 143 00:07:43,270 --> 00:07:46,210 and allow the little piece with the helix loop helix 144 00:07:46,210 --> 00:07:48,070 at the N-terminus to become soluble 145 00:07:48,070 --> 00:07:51,460 and then move to the cytosol. 146 00:07:51,460 --> 00:07:54,880 So we want to ask the question now and spend 147 00:07:54,880 --> 00:07:57,040 a little bit of time, what happens 148 00:07:57,040 --> 00:07:59,300 with high sterol concentrations? 149 00:07:59,300 --> 00:08:03,520 So everything we've looked at has 150 00:08:03,520 --> 00:08:08,090 been-- this is at low sterol concentrations. 151 00:08:08,090 --> 00:08:12,760 And under these conditions, this zip code 152 00:08:12,760 --> 00:08:25,010 targets SCAP and SRE-BP to the Golgi. 153 00:08:27,760 --> 00:08:30,840 So now we want to go to the second set of conditions. 154 00:08:30,840 --> 00:08:33,409 And again, in this week's recitation, 155 00:08:33,409 --> 00:08:37,580 we're focused on high sterol, low sterol concentrations. 156 00:08:37,580 --> 00:08:44,330 High sterol, what is it that allows this to prevent movement 157 00:08:44,330 --> 00:08:47,030 into the Golgi so you can get this processing so you 158 00:08:47,030 --> 00:08:53,660 can turn on HMG reductase and LDL receptor biosynthesis. 159 00:08:53,660 --> 00:08:56,780 And so the proposal has been-- 160 00:08:56,780 --> 00:08:59,870 so in some way this is connected to sterol, 161 00:08:59,870 --> 00:09:05,480 so we're at 5% or 6% sterol in the ER membrane. 162 00:09:05,480 --> 00:09:09,587 That's what we're going to discuss today, 163 00:09:09,587 --> 00:09:11,420 for those of you who haven't had recitation. 164 00:09:11,420 --> 00:09:14,300 How do you know what the turn on versus the turn off 165 00:09:14,300 --> 00:09:17,490 is for sterol levels? 166 00:09:17,490 --> 00:09:19,900 Where does it bind? 167 00:09:19,900 --> 00:09:21,280 How does it bind? 168 00:09:21,280 --> 00:09:24,730 All of that becomes pretty interesting. 169 00:09:24,730 --> 00:09:29,080 And in high sterol we now introduce yet another player. 170 00:09:29,080 --> 00:09:39,295 So in addition to SCAP we now have to pay attention to INSIG. 171 00:09:39,295 --> 00:09:40,420 So that's the other player. 172 00:09:40,420 --> 00:09:42,850 We'll see that INSIG is this protein that's 173 00:09:42,850 --> 00:09:46,430 the linchpin for all the regulatory mechanisms. 174 00:09:46,430 --> 00:09:51,180 So if we go back here, what do we see about INSIG? 175 00:09:51,180 --> 00:09:52,000 It's small. 176 00:09:52,000 --> 00:09:53,440 It's much smaller than SCAP. 177 00:09:53,440 --> 00:09:57,240 SCAP is this huge protein, 1,200 amino acids. 178 00:09:57,240 --> 00:10:01,300 Here's 200 amino acids, all transmembrane. 179 00:10:01,300 --> 00:10:03,400 Recently, actually, there was a structure 180 00:10:03,400 --> 00:10:07,020 not of the human system, but of a bacterial system. 181 00:10:07,020 --> 00:10:09,880 It's not found in the vast majority of bacteria, 182 00:10:09,880 --> 00:10:14,110 but they found one and so they've gotten some-- 183 00:10:14,110 --> 00:10:16,340 they have proposed some model for how 184 00:10:16,340 --> 00:10:18,800 INSIG could be in the membrane. 185 00:10:18,800 --> 00:10:20,560 Now, one of the things that I find 186 00:10:20,560 --> 00:10:23,603 confusing to think about the molecular basis for what's 187 00:10:23,603 --> 00:10:25,270 going on-- which we don't know anything. 188 00:10:25,270 --> 00:10:27,400 You're looking at a cartoon level-- 189 00:10:27,400 --> 00:10:30,970 is we have so many transmembrane helices, 190 00:10:30,970 --> 00:10:32,560 but is this thing a monomer? 191 00:10:32,560 --> 00:10:33,580 Is it a dimer? 192 00:10:33,580 --> 00:10:34,630 Is it a tetramer? 193 00:10:34,630 --> 00:10:36,640 Is it a hexamer? 194 00:10:36,640 --> 00:10:38,470 And how do you look at that? 195 00:10:38,470 --> 00:10:40,120 Because when you solubilize it, you 196 00:10:40,120 --> 00:10:42,910 have to put it in detergent, et cetera. 197 00:10:42,910 --> 00:10:46,360 And is what you see in the test tube, how do you relate it 198 00:10:46,360 --> 00:10:48,260 back to what's going on inside the cell? 199 00:10:48,260 --> 00:10:50,320 And I think we really don't know. 200 00:10:50,320 --> 00:10:54,040 Most of these things-- both SCAP, which 201 00:10:54,040 --> 00:10:56,320 is thought to be a tetramer, and now 202 00:10:56,320 --> 00:10:58,090 in this new paper they're claiming it's 203 00:10:58,090 --> 00:11:00,190 a trimer of dimers. 204 00:11:00,190 --> 00:11:03,040 Just add to the complexity of trying to figure out 205 00:11:03,040 --> 00:11:04,930 how all these things interact. 206 00:11:04,930 --> 00:11:07,420 So that's the issue with doing these kinds of experiments. 207 00:11:07,420 --> 00:11:08,920 We don't have very good experiments. 208 00:11:08,920 --> 00:11:11,110 We need people to invent new ways 209 00:11:11,110 --> 00:11:15,280 of trying to ferret out how these things interact 210 00:11:15,280 --> 00:11:17,610 within the membrane. 211 00:11:17,610 --> 00:11:22,180 So here INSIG is going to be a key player 212 00:11:22,180 --> 00:11:24,820 and SCAP is also a key player. 213 00:11:24,820 --> 00:11:27,880 And so somehow, in the presence of sterol, 214 00:11:27,880 --> 00:11:31,540 so we're at high sterol, in the presence of INSIG, 215 00:11:31,540 --> 00:11:33,940 we need to get rid of the zip code. 216 00:11:33,940 --> 00:11:35,150 That's the bottom line. 217 00:11:35,150 --> 00:11:44,050 So the model is in the presence of sterol, we remove-- 218 00:11:44,050 --> 00:11:49,450 we don't really remove it, but we hide the zip code. 219 00:11:53,770 --> 00:11:57,190 And when you hide the zip code, so this is shown-- 220 00:11:57,190 --> 00:11:59,800 I'm not going to draw this out on the board 221 00:11:59,800 --> 00:12:02,540 because I think they draw out better than I can do it, 222 00:12:02,540 --> 00:12:05,590 and we really don't know what that's going on anyhow. 223 00:12:05,590 --> 00:12:07,810 But again, this comes from a region 224 00:12:07,810 --> 00:12:11,410 where it's accessible to another region 225 00:12:11,410 --> 00:12:14,650 where these proteins can no longer bind 226 00:12:14,650 --> 00:12:19,420 to do the transport of these proteins into the Golgi. 227 00:12:19,420 --> 00:12:24,100 So that means you never get processing of SRE-BP 228 00:12:24,100 --> 00:12:26,140 to become active. 229 00:12:26,140 --> 00:12:28,210 Does everybody understand that? 230 00:12:28,210 --> 00:12:30,760 So one of the questions is, if I asked 231 00:12:30,760 --> 00:12:33,340 you to design an experiment, hopefully you're 232 00:12:33,340 --> 00:12:37,270 now starting to be able to think about designing experiments. 233 00:12:37,270 --> 00:12:38,770 Is there any kind of an experiment 234 00:12:38,770 --> 00:12:43,180 you might think you could do to-- 235 00:12:43,180 --> 00:12:45,400 a simple experiment that you might 236 00:12:45,400 --> 00:12:50,650 be able to do or try to do that would allow you to show 237 00:12:50,650 --> 00:12:52,900 that you you'd undergone a conformational change 238 00:12:52,900 --> 00:12:55,990 in a loop 6. 239 00:12:55,990 --> 00:12:57,340 Anybody think of something? 240 00:12:57,340 --> 00:13:00,390 So loop 6 this is big, huge-- 241 00:13:00,390 --> 00:13:04,690 it's proposed to be a big, huge piece of polypeptide 242 00:13:04,690 --> 00:13:08,110 and it's proposed to undergo-- 243 00:13:08,110 --> 00:13:10,470 this is a cartoon, but a tremendous conformational 244 00:13:10,470 --> 00:13:11,325 change. 245 00:13:11,325 --> 00:13:12,700 And so what you need is some kind 246 00:13:12,700 --> 00:13:16,360 of a simple probe that might tell you that it's undergone 247 00:13:16,360 --> 00:13:17,680 a conformational change. 248 00:13:17,680 --> 00:13:21,610 And what might some kind of a probe like that be? 249 00:13:21,610 --> 00:13:24,450 How could you design something like that? 250 00:13:24,450 --> 00:13:24,950 Yeah? 251 00:13:24,950 --> 00:13:26,492 AUDIENCE: A FRET experiment? 252 00:13:26,492 --> 00:13:28,950 JOANNE STUBBE: OK, so that would be one thing you could do. 253 00:13:28,950 --> 00:13:31,140 You couldn't just do a FRET experiment to do it. 254 00:13:31,140 --> 00:13:32,340 What would you have to do? 255 00:13:32,340 --> 00:13:33,250 AUDIENCE: Incorporate fluorophores. 256 00:13:33,250 --> 00:13:34,350 JOANNE STUBBE: Yeah, so you'd have 257 00:13:34,350 --> 00:13:35,690 to incorporate fluorophores. 258 00:13:35,690 --> 00:13:38,370 So the issue with FRET is not only now 259 00:13:38,370 --> 00:13:40,890 do you have one problem, so you can put it one place, 260 00:13:40,890 --> 00:13:46,117 but now you've got to figure out where to put the second FRET. 261 00:13:46,117 --> 00:13:47,700 So that would be a lot of experiments, 262 00:13:47,700 --> 00:13:49,950 but now you can do mutagenesis, so you could probably 263 00:13:49,950 --> 00:13:52,200 do an experiment like that. 264 00:13:52,200 --> 00:13:55,920 Is there anything else you can think about? 265 00:13:55,920 --> 00:13:56,973 Yeah? 266 00:13:56,973 --> 00:13:58,390 AUDIENCE: So I don't know how much 267 00:13:58,390 --> 00:14:01,230 is known about the conformational rigidity of loop 268 00:14:01,230 --> 00:14:02,380 6 and also I'm not sure-- 269 00:14:02,380 --> 00:14:04,160 I've ever seen this in a membrane protein, 270 00:14:04,160 --> 00:14:07,680 but you can maybe look at proton exchange, 271 00:14:07,680 --> 00:14:09,640 like [INAUDIBLE] backbone exchange. 272 00:14:09,640 --> 00:14:11,765 JOANNE STUBBE: So that's a sophisticated experiment 273 00:14:11,765 --> 00:14:15,040 and I would say there probably wouldn't be my first choice. 274 00:14:15,040 --> 00:14:17,770 But the idea that it's accessible, 275 00:14:17,770 --> 00:14:20,050 is there any kind of an enzyme that you might 276 00:14:20,050 --> 00:14:22,157 be able to use that could-- 277 00:14:22,157 --> 00:14:23,740 again, you'd have to be lucky, but you 278 00:14:23,740 --> 00:14:25,740 could look at the sequence and think about this. 279 00:14:25,740 --> 00:14:27,880 Is there any kind of an enzyme you 280 00:14:27,880 --> 00:14:31,000 might be able to use that could sense 281 00:14:31,000 --> 00:14:32,270 a change in the conformation? 282 00:14:32,270 --> 00:14:37,600 And if the model is right, it's on the cytoplasmic face. 283 00:14:37,600 --> 00:14:40,780 And so the answer is people use trypsin. 284 00:14:40,780 --> 00:14:42,625 So if you go back and look at the sequence, 285 00:14:42,625 --> 00:14:43,458 there's an arginine. 286 00:14:43,458 --> 00:14:47,200 And actually, I wouldn't have expected you to see this, 287 00:14:47,200 --> 00:14:49,780 although I think it is mentioned in one of the papers 288 00:14:49,780 --> 00:14:52,360 where it becomes much more accessible in one 289 00:14:52,360 --> 00:14:54,992 state than the other, so you get proteolytic clipping. 290 00:14:54,992 --> 00:14:56,450 But those are the kinds of things-- 291 00:14:56,450 --> 00:14:59,140 what other kind of experiment could you do? 292 00:14:59,140 --> 00:15:02,020 So you think you're undergoing a conformational change, 293 00:15:02,020 --> 00:15:04,840 what kinds of probes did Liz talk 294 00:15:04,840 --> 00:15:09,110 about that might allow you to see some kind of change 295 00:15:09,110 --> 00:15:09,970 in conformation? 296 00:15:09,970 --> 00:15:12,370 So we have fluorescence probes, which we haven't really 297 00:15:12,370 --> 00:15:15,360 talked about yet. 298 00:15:15,360 --> 00:15:18,510 Can you think of what other kinds of probes? 299 00:15:18,510 --> 00:15:21,310 She spent a whole recitation on it. 300 00:15:21,310 --> 00:15:22,270 AUDIENCE: [INAUDIBLE]. 301 00:15:22,270 --> 00:15:23,570 JOANNE STUBBE: The what? 302 00:15:23,570 --> 00:15:25,990 AUDIENCE: As far as binding [INAUDIBLE],, 303 00:15:25,990 --> 00:15:27,740 you would do it in cross-linking. 304 00:15:27,740 --> 00:15:29,480 JOANNE STUBBE: Yeah, so some kind of cross-linking. 305 00:15:29,480 --> 00:15:31,063 You might get information out of that. 306 00:15:31,063 --> 00:15:34,640 To do that again, you've got to put in the cysteine. 307 00:15:34,640 --> 00:15:37,490 So here you might have issues because you have 308 00:15:37,490 --> 00:15:38,850 all these cysteines down there. 309 00:15:38,850 --> 00:15:40,440 So the question is, could you do that? 310 00:15:40,440 --> 00:15:42,780 Cysteine is the most easy-- 311 00:15:42,780 --> 00:15:44,900 that's the easiest side chain to modify. 312 00:15:44,900 --> 00:15:48,013 On the other hand, these cysteine's really 313 00:15:48,013 --> 00:15:49,430 playing a functional role, there's 314 00:15:49,430 --> 00:15:50,730 no way you could modify it. 315 00:15:50,730 --> 00:15:52,220 But those are the kinds of things. 316 00:15:52,220 --> 00:15:55,400 Hopefully you're starting to get a battery of tools 317 00:15:55,400 --> 00:15:57,800 that you're learning about with different systems 318 00:15:57,800 --> 00:16:00,020 that you could further probe this, 319 00:16:00,020 --> 00:16:02,640 but you can just see that this is, I think, 320 00:16:02,640 --> 00:16:05,000 a really challenging problem. 321 00:16:05,000 --> 00:16:08,930 So the idea is it does undergo a conformational change 322 00:16:08,930 --> 00:16:12,620 and you can no longer see the zip code. 323 00:16:12,620 --> 00:16:14,420 It disappears. 324 00:16:14,420 --> 00:16:19,490 And it resides, and so the model, then, becomes here 325 00:16:19,490 --> 00:16:27,340 that the sterol binds to the sterol sensitive binding domain 326 00:16:27,340 --> 00:16:29,390 and recruits INSIG. 327 00:16:29,390 --> 00:16:30,520 Is that true? 328 00:16:30,520 --> 00:16:33,690 Could INSIG bind first and that the two of them together? 329 00:16:33,690 --> 00:16:36,260 And in the paper that you were looking at in recitation 330 00:16:36,260 --> 00:16:40,790 this week, the concentrations of INSIG were elevated 331 00:16:40,790 --> 00:16:43,640 and they got a result that you may or may not 332 00:16:43,640 --> 00:16:46,910 have been able to predict what the outcome would be. 333 00:16:46,910 --> 00:16:51,950 So again, the order of binding and interaction, 334 00:16:51,950 --> 00:16:54,440 I think, really still remains something 335 00:16:54,440 --> 00:16:57,000 that needs to be studied. 336 00:16:57,000 --> 00:16:59,420 So we just form a complex in this case. 337 00:16:59,420 --> 00:17:05,170 So we form a complex. 338 00:17:05,170 --> 00:17:06,430 So in the membrane-- 339 00:17:08,950 --> 00:17:10,599 we have our ER membrane. 340 00:17:10,599 --> 00:17:11,829 We have INSIG. 341 00:17:11,829 --> 00:17:16,380 I'm not going to draw the-- we have INSIG. 342 00:17:16,380 --> 00:17:19,099 It overlaps somehow with SCAP. 343 00:17:24,069 --> 00:17:27,010 And then SCAP somehow binds the sterol, 344 00:17:27,010 --> 00:17:31,880 so you need to have a sterol here. 345 00:17:31,880 --> 00:17:35,540 And this is a sterol-sensitive domain. 346 00:17:35,540 --> 00:17:43,852 And then up here you have a loop and the zip code hides. 347 00:17:43,852 --> 00:17:44,685 So that's the model. 348 00:17:48,960 --> 00:17:51,220 And so that's as sophisticated as we 349 00:17:51,220 --> 00:17:54,040 can get at this stage, which is, for a chemist, 350 00:17:54,040 --> 00:17:56,560 not particularly sophisticated. 351 00:17:56,560 --> 00:17:57,830 But that's still the model. 352 00:17:57,830 --> 00:18:00,460 The model has been in the literature for a long time 353 00:18:00,460 --> 00:18:02,060 and we don't know that much about it. 354 00:18:02,060 --> 00:18:05,160 Here is just another cartoon from another paper 355 00:18:05,160 --> 00:18:09,490 where, again, the little gold thing here is the INSIG. 356 00:18:09,490 --> 00:18:11,420 This is the SCAP. 357 00:18:11,420 --> 00:18:15,010 Here is the sterol responsive binding element. 358 00:18:15,010 --> 00:18:20,260 And here they don't show the hiding of the little zip code. 359 00:18:20,260 --> 00:18:28,270 So this is the model that people have put forward 360 00:18:28,270 --> 00:18:33,160 for how low cholesterol levels allow 361 00:18:33,160 --> 00:18:35,110 you to turn on the genes required 362 00:18:35,110 --> 00:18:38,620 to make more cholesterol or to take it from the diet. 363 00:18:38,620 --> 00:18:41,530 So that's a transcription model. 364 00:18:41,530 --> 00:18:45,550 So in the last lecture now what I want to focus on 365 00:18:45,550 --> 00:18:49,060 is post-transcriptional regulation. 366 00:18:49,060 --> 00:18:51,580 So this is lecture 5. 367 00:18:51,580 --> 00:18:53,740 We're talking about post-transcriptional. 368 00:18:59,662 --> 00:19:01,120 And what you're going to see, we're 369 00:19:01,120 --> 00:19:10,450 going to focus on again is our common player in both 370 00:19:10,450 --> 00:19:12,730 of these regulatory mechanisms. 371 00:19:12,730 --> 00:19:17,310 INSIG played a key role in keeping 372 00:19:17,310 --> 00:19:20,240 SRE-BP in the ER membrane. 373 00:19:20,240 --> 00:19:25,840 We're going to say INSIG plays a key role also in destroying-- 374 00:19:25,840 --> 00:19:28,570 so at high cholesterol concentrations, 375 00:19:28,570 --> 00:19:31,747 you don't want to make any more HMG-CoA reductase. 376 00:19:31,747 --> 00:19:33,580 And if you have a lot of it in the membrane, 377 00:19:33,580 --> 00:19:35,170 you want to get rid of it. 378 00:19:35,170 --> 00:19:42,760 So INSIG and HMG-R are going to interact with each other based 379 00:19:42,760 --> 00:19:43,690 on-- 380 00:19:43,690 --> 00:19:44,995 so this is a high sterol. 381 00:19:49,050 --> 00:19:53,110 And what's going to happen is, in a sophisticated way, 382 00:19:53,110 --> 00:20:04,700 HMG-R, which makes cholesterol, is targeted for degradation. 383 00:20:04,700 --> 00:20:05,872 So this is the model. 384 00:20:05,872 --> 00:20:08,330 We're going to come back to this model over and over again. 385 00:20:08,330 --> 00:20:08,830 Sorry. 386 00:20:08,830 --> 00:20:11,070 My handwriting is getting worse and worse. 387 00:20:11,070 --> 00:20:12,050 So we have INSIG. 388 00:20:12,050 --> 00:20:14,767 We have HMG-R. We want to get rid of it 389 00:20:14,767 --> 00:20:16,850 because we don't want to make anymore cholesterol. 390 00:20:16,850 --> 00:20:17,808 That's the bottom line. 391 00:20:17,808 --> 00:20:22,940 So INSIG is a player in both of these mechanisms. 392 00:20:22,940 --> 00:20:26,120 So what I want to do now in lecture 5-- 393 00:20:26,120 --> 00:20:27,170 that's what this is here. 394 00:20:27,170 --> 00:20:29,870 What I want to do in lecture 5 is really 395 00:20:29,870 --> 00:20:32,000 talk about how you do degradation 396 00:20:32,000 --> 00:20:35,510 in eukaryotic cells in general. 397 00:20:35,510 --> 00:20:38,000 And then what I'm going to do is come back 398 00:20:38,000 --> 00:20:42,110 and ask the question, how is HMG-R, 399 00:20:42,110 --> 00:20:45,170 HMG-CoA reductase targeted for degradation. 400 00:20:45,170 --> 00:20:47,060 So that's the overview of where we're going. 401 00:20:47,060 --> 00:20:52,310 It's pretty simple and we'll see that mechanism 402 00:20:52,310 --> 00:20:55,700 of the proteosome, it's much more complicated, 403 00:20:55,700 --> 00:21:01,730 but there are many similarities between this chamber of doom 404 00:21:01,730 --> 00:21:06,200 and the one you saw with ClpX and ClpP 405 00:21:06,200 --> 00:21:09,020 where you've spent a lot of time discussing what 406 00:21:09,020 --> 00:21:11,510 we know about it from a more chemical 407 00:21:11,510 --> 00:21:14,240 and biochemical perspective. 408 00:21:14,240 --> 00:21:17,750 And what I'm going to show you at the very end is 409 00:21:17,750 --> 00:21:23,490 not only does HMG-CoA reductase get targeted, but now 410 00:21:23,490 --> 00:21:25,370 in the last couple of years, they 411 00:21:25,370 --> 00:21:29,420 found that all of the proteins involved in cholesterol 412 00:21:29,420 --> 00:21:34,760 homeostasis get targeted by different mechanisms 413 00:21:34,760 --> 00:21:35,960 to get degraded. 414 00:21:35,960 --> 00:21:38,210 So protein degradation inside the cell 415 00:21:38,210 --> 00:21:40,080 is extremely complicated. 416 00:21:40,080 --> 00:21:42,110 So what I'm going to do is give you 417 00:21:42,110 --> 00:21:46,980 an outline of a generic picture of how it gets degraded. 418 00:21:46,980 --> 00:21:51,530 And the caveat is this is a very active area of research 419 00:21:51,530 --> 00:21:54,260 and I think you'll see why it's so complicated. 420 00:21:54,260 --> 00:21:57,560 And then the next problem set, problem set 8, 421 00:21:57,560 --> 00:21:59,240 there's been a model in the literature 422 00:21:59,240 --> 00:22:02,060 that this is the equipment that targets 423 00:22:02,060 --> 00:22:03,983 this protein for degradation. 424 00:22:03,983 --> 00:22:05,900 And I'm going to give you a bunch of data that 425 00:22:05,900 --> 00:22:08,900 says that may not be correct. 426 00:22:08,900 --> 00:22:10,550 So this is what you're dealing with. 427 00:22:10,550 --> 00:22:14,060 Every time you pick up a journal, there's another model 428 00:22:14,060 --> 00:22:17,507 and perhaps there are five or six different ways-- 429 00:22:17,507 --> 00:22:19,340 not that many-- three or four different ways 430 00:22:19,340 --> 00:22:21,220 that you can mediate the degradation. 431 00:22:21,220 --> 00:22:24,650 And we're in the process of trying to unravel this. 432 00:22:24,650 --> 00:22:27,180 So this is where we're going. 433 00:22:27,180 --> 00:22:32,270 And so what I want to do is start out by looking at-- 434 00:22:32,270 --> 00:22:34,190 and then if we have time at the end, 435 00:22:34,190 --> 00:22:35,900 I'll come back to both recitations. 436 00:22:35,900 --> 00:22:37,608 But I probably won't have time at the end 437 00:22:37,608 --> 00:22:39,575 because I want to move on to the next modules. 438 00:22:42,210 --> 00:22:44,400 But you'll see that the recitations really 439 00:22:44,400 --> 00:22:47,410 are pretty much linked to what we're talking about in class. 440 00:22:47,410 --> 00:22:50,640 So it's unfortunate that they weren't timed a little better, 441 00:22:50,640 --> 00:22:52,680 but that's the way life is when you're trying 442 00:22:52,680 --> 00:22:55,590 to balance all of these acts. 443 00:22:55,590 --> 00:22:56,590 So this is the overview. 444 00:22:56,590 --> 00:22:58,257 I'm not going to draw this on the board, 445 00:22:58,257 --> 00:23:00,360 but I'm going to walk through it step by step. 446 00:23:00,360 --> 00:23:02,790 So this is a cartoon overview. 447 00:23:02,790 --> 00:23:06,300 You can see it's pretty old. 448 00:23:06,300 --> 00:23:09,960 We've learned some stuff, but there's a lot of stuff 449 00:23:09,960 --> 00:23:11,320 that remains unknown. 450 00:23:11,320 --> 00:23:12,930 So let's just work through the cartoon 451 00:23:12,930 --> 00:23:16,380 and then we'll walk through who the players are, 452 00:23:16,380 --> 00:23:18,490 what the model is and then in the end, 453 00:23:18,490 --> 00:23:21,670 we're going to return to HMG-CoA reductase. 454 00:23:21,670 --> 00:23:27,630 So we have a protein and we need to target it for degradation. 455 00:23:27,630 --> 00:23:30,780 How does anything know that this is targeted for degradation? 456 00:23:30,780 --> 00:23:32,600 The protein's the same in the beginning. 457 00:23:32,600 --> 00:23:38,250 In the end, how do we know why this protein 458 00:23:38,250 --> 00:23:41,580 has a different kind of half life than some other protein? 459 00:23:41,580 --> 00:23:45,300 We haven't discussed that, but we asked the same question 460 00:23:45,300 --> 00:23:48,300 in bacterial systems and I'm going to spend 461 00:23:48,300 --> 00:23:49,750 not very much time on it. 462 00:23:49,750 --> 00:23:53,160 But the N-terminus of the protein 463 00:23:53,160 --> 00:23:56,100 can be modified in many ways. 464 00:23:56,100 --> 00:24:01,020 This is called the N-end rule, totally mind boggling. 465 00:24:01,020 --> 00:24:05,520 I might give you a few examples of this on a problem set. 466 00:24:05,520 --> 00:24:09,240 But you can add on amino acids or take off amino acids. 467 00:24:09,240 --> 00:24:14,190 It changes the lifetime of the protein from minutes to hours. 468 00:24:14,190 --> 00:24:16,230 So this is like-- when this first came out, I, 469 00:24:16,230 --> 00:24:18,030 said there's no way that can be true. 470 00:24:18,030 --> 00:24:20,190 So we're talking about a few amino acids, 471 00:24:20,190 --> 00:24:22,770 just like we're talking about these zip codes over here. 472 00:24:22,770 --> 00:24:24,240 It's true. 473 00:24:24,240 --> 00:24:28,620 And the way the rules work or have evolved, 474 00:24:28,620 --> 00:24:30,660 they're different in all organisms. 475 00:24:30,660 --> 00:24:34,680 They're in all organisms, but they're all distinct. 476 00:24:34,680 --> 00:24:37,230 So another way that I think is key, 477 00:24:37,230 --> 00:24:39,090 and we're still trying to figure this out 478 00:24:39,090 --> 00:24:42,480 is that many proteins, are post translationally modified 479 00:24:42,480 --> 00:24:46,800 by phosphorylation or hydroxylation or whatever. 480 00:24:46,800 --> 00:24:48,870 I think that's also a key thing that's going 481 00:24:48,870 --> 00:24:51,720 to target them for degradation. 482 00:24:51,720 --> 00:24:52,830 So we have a protein. 483 00:24:52,830 --> 00:24:55,320 Somehow it's going to get targeted for degradation. 484 00:24:55,320 --> 00:24:56,190 What does that? 485 00:24:56,190 --> 00:25:00,330 So it turns out we're going to be introduced to a molecule you 486 00:25:00,330 --> 00:25:03,060 saw in your first recitation, ubiquitin, 487 00:25:03,060 --> 00:25:06,210 small little protein, like a rock, 76 amino acids. 488 00:25:06,210 --> 00:25:07,800 What is it doing? 489 00:25:07,800 --> 00:25:11,280 It's like the SSRA tag except more complicated, 490 00:25:11,280 --> 00:25:13,210 that you saw before. 491 00:25:13,210 --> 00:25:16,100 And then we're going to be introduced to three proteins-- 492 00:25:16,100 --> 00:25:23,390 E1, E2, and E3, an activating enzyme, a conjugating enzyme, 493 00:25:23,390 --> 00:25:24,510 and a ligase. 494 00:25:24,510 --> 00:25:26,340 And I'll sort of define for you what 495 00:25:26,340 --> 00:25:28,930 the function of these proteins are. 496 00:25:28,930 --> 00:25:30,750 And you'll see that they require energy. 497 00:25:30,750 --> 00:25:32,970 Maybe not surprising, the Nobel Prize 498 00:25:32,970 --> 00:25:36,930 was given for the work on discovery 499 00:25:36,930 --> 00:25:39,990 of how this little system works a number of years ago, 500 00:25:39,990 --> 00:25:43,740 where a major player in that was a mechanistic entomologist 501 00:25:43,740 --> 00:25:46,000 named Ernie Rose, who nobody ever heard of. 502 00:25:46,000 --> 00:25:48,870 I remember when the Nobel Prize came out, 503 00:25:48,870 --> 00:25:52,180 the chemist was saying, who the hell is this guy? 504 00:25:52,180 --> 00:25:54,060 Well, so that's because, again, they don't 505 00:25:54,060 --> 00:25:55,530 care about how enzymes work. 506 00:25:55,530 --> 00:25:57,350 But what's amazing is this guy is one 507 00:25:57,350 --> 00:25:58,650 of the most brilliant people. 508 00:25:58,650 --> 00:26:01,080 He's dead now, but he's one of the most brilliant people 509 00:26:01,080 --> 00:26:01,995 I've ever met. 510 00:26:01,995 --> 00:26:04,620 And he was [INAUDIBLE]---- he did thousands of things that were 511 00:26:04,620 --> 00:26:07,350 really creative and important, but this is the one, 512 00:26:07,350 --> 00:26:09,600 because he was hooked in with the guys that were doing 513 00:26:09,600 --> 00:26:14,140 the biology, that allowed him to elucidate what was going on. 514 00:26:14,140 --> 00:26:17,970 So we're going to take this a little ubiquitin 515 00:26:17,970 --> 00:26:19,740 and somehow this equipment is going 516 00:26:19,740 --> 00:26:24,300 to attach the ubiquitin onto the protein that's 517 00:26:24,300 --> 00:26:26,880 targeted for degradation. 518 00:26:26,880 --> 00:26:29,760 And we'll see that you have to have multiple ubiquitins 519 00:26:29,760 --> 00:26:32,580 attached to get degraded. 520 00:26:32,580 --> 00:26:36,990 That being said, we now know that almost all proteins 521 00:26:36,990 --> 00:26:39,540 can be ubiquitinated. 522 00:26:39,540 --> 00:26:43,140 We know ubiquitin has something like 20 homologues, 523 00:26:43,140 --> 00:26:47,100 look alikes, and they all do different biology. 524 00:26:47,100 --> 00:26:49,980 So this is another example of post-translational 525 00:26:49,980 --> 00:26:51,230 modification. 526 00:26:51,230 --> 00:26:54,425 We're only going to focus on targeting for degradation. 527 00:26:54,425 --> 00:26:55,800 That's what I'm going to show you 528 00:26:55,800 --> 00:27:00,750 but the ubiquitinome is quite complicated. 529 00:27:00,750 --> 00:27:04,260 So once it gets the ubiquitins attached, what do you see here? 530 00:27:04,260 --> 00:27:06,630 You see the proteosome. 531 00:27:06,630 --> 00:27:08,880 This is the chamber of doom. 532 00:27:08,880 --> 00:27:12,270 We'll come back and look at that, just like clip x 533 00:27:12,270 --> 00:27:13,430 and clip p. 534 00:27:13,430 --> 00:27:15,990 So you have clip p here, the chamber of doom. 535 00:27:15,990 --> 00:27:19,350 And then you have little pieces on the top 536 00:27:19,350 --> 00:27:22,950 and the bottom of that, which would be sort of like clip x. 537 00:27:22,950 --> 00:27:26,640 Hexameric ATPase we'll see is much more complicated 538 00:27:26,640 --> 00:27:28,640 in human cells. 539 00:27:28,640 --> 00:27:29,890 And so what do you have to do? 540 00:27:29,890 --> 00:27:31,860 You have to unfold the protein. 541 00:27:31,860 --> 00:27:34,230 You have to thread it into the chamber of doom. 542 00:27:34,230 --> 00:27:37,200 You have to break it down into pieces 543 00:27:37,200 --> 00:27:39,570 and you spit out the pieces. 544 00:27:39,570 --> 00:27:42,960 This process requires ATP like you studied 545 00:27:42,960 --> 00:27:45,420 in the bacterial proteosome. 546 00:27:45,420 --> 00:27:51,360 And then there are actually many different proteosomes 547 00:27:51,360 --> 00:27:53,000 in human cells and I'm just going 548 00:27:53,000 --> 00:27:56,940 to talk about the generic proteosome. 549 00:27:56,940 --> 00:28:00,930 So that's the cartoon overview. 550 00:28:00,930 --> 00:28:07,080 So I want to say a few things about-- 551 00:28:07,080 --> 00:28:11,990 so let's start by looking at the proteosome. 552 00:28:15,930 --> 00:28:19,570 And again, this is the human proteosome. 553 00:28:19,570 --> 00:28:22,320 And if you look at these big machines, 554 00:28:22,320 --> 00:28:24,570 you've already learned one way you characterize 555 00:28:24,570 --> 00:28:29,640 them is by their sedimentation in some kind 556 00:28:29,640 --> 00:28:31,950 of a centrifugal field. 557 00:28:31,950 --> 00:28:35,010 And so these things migrate. 558 00:28:35,010 --> 00:28:40,120 Like, a 26S particle only has a sedimentation value of 26. 559 00:28:40,120 --> 00:28:47,130 So it's huge, and it's 2.5 megadaltons. 560 00:28:47,130 --> 00:28:51,390 So this is a huge machine just like the ones 561 00:28:51,390 --> 00:28:54,300 you've been studying in the first part of the course. 562 00:28:54,300 --> 00:28:58,080 So it turns out this can be divided into two parts 563 00:28:58,080 --> 00:29:01,380 as you've already seen and you can see over there. 564 00:29:01,380 --> 00:29:09,810 You have the 20S, which is the core proteosome. 565 00:29:09,810 --> 00:29:15,150 And then you have a 19S lid. 566 00:29:15,150 --> 00:29:20,640 Actually, you can have multiple lids and in these lids 567 00:29:20,640 --> 00:29:23,680 there can be 20 proteins, 15 to 20 proteins. 568 00:29:23,680 --> 00:29:27,930 So the lid contain 15 to 20 proteins. 569 00:29:27,930 --> 00:29:32,010 We'll come back and look at this a little bit. 570 00:29:32,010 --> 00:29:34,100 And so this is going to be in-- 571 00:29:34,100 --> 00:29:39,600 and among these things are the AAA plus ATPases, 572 00:29:39,600 --> 00:29:42,180 which are actually quite distinct from what 573 00:29:42,180 --> 00:29:45,400 you're going to see, what you have seen 574 00:29:45,400 --> 00:29:48,090 in the bacterial proteosome. 575 00:29:48,090 --> 00:29:53,190 So here again is going to be the 20S core. 576 00:29:53,190 --> 00:29:56,280 Here are the proteins, so this 20S core. 577 00:29:56,280 --> 00:29:59,340 Here are the proteins involved in the lid. 578 00:29:59,340 --> 00:30:02,250 Some are tightly bound, some are not tightly bound. 579 00:30:02,250 --> 00:30:07,520 Remember, we had a hexameric ATPase, so RP-- 580 00:30:07,520 --> 00:30:09,690 I can't remember the acronyms-- 581 00:30:09,690 --> 00:30:15,690 RPT, and there are six different ATPases, not one, six. 582 00:30:15,690 --> 00:30:18,240 But they form a hexameric structure. 583 00:30:18,240 --> 00:30:20,460 And then you have a lot of additional proteins 584 00:30:20,460 --> 00:30:23,400 that we're going to come back and look at, but one of you 585 00:30:23,400 --> 00:30:27,930 might expect would be something that could recognize ubiquitin, 586 00:30:27,930 --> 00:30:32,730 just like you had something that recognized the SSRA tag. 587 00:30:32,730 --> 00:30:37,410 It turns out ubiquitin is recycled inside the cell. 588 00:30:37,410 --> 00:30:42,480 So the equipment that allows you to cut off the ubiquitin so it 589 00:30:42,480 --> 00:30:45,560 can be used again, de-ubiquinating enzymes, 590 00:30:45,560 --> 00:30:50,220 is also located in the lid. 591 00:30:50,220 --> 00:30:53,490 And you can also imagine that could be many kinds of adapter 592 00:30:53,490 --> 00:30:55,560 proteins because we're going to be 593 00:30:55,560 --> 00:30:59,490 able to degrade many, many, many proteins under different sets 594 00:30:59,490 --> 00:31:00,130 of conditions. 595 00:31:00,130 --> 00:31:08,700 So this changes in composition, as opposed to the chamber 596 00:31:08,700 --> 00:31:12,810 of doom, the 20S proteasome. 597 00:31:12,810 --> 00:31:19,885 So let's look again at the core particle, so the core protease. 598 00:31:19,885 --> 00:31:22,620 Let's abbreviate it CP. 599 00:31:22,620 --> 00:31:24,180 And what do we know about this? 600 00:31:24,180 --> 00:31:25,650 What we know is the following-- 601 00:31:28,290 --> 00:31:36,885 that it forms four heptameric rings. 602 00:31:45,190 --> 00:31:50,450 And the rings, so each one of these is a 7-mer. 603 00:31:50,450 --> 00:31:53,070 And it turns out we have two kinds. 604 00:31:53,070 --> 00:31:55,020 They're actually pretty similar to each other, 605 00:31:55,020 --> 00:31:59,930 just like the proteosome from bacteria. 606 00:31:59,930 --> 00:32:04,820 But we have alpha, we have beta, we have beta, 607 00:32:04,820 --> 00:32:07,010 and we have alpha. 608 00:32:07,010 --> 00:32:10,370 And we call them-- we put i's next to them 609 00:32:10,370 --> 00:32:13,640 because, again, they're not the same. 610 00:32:13,640 --> 00:32:15,398 So they're all different. 611 00:32:15,398 --> 00:32:16,940 So they're all structurally the same, 612 00:32:16,940 --> 00:32:18,910 but they're all different. 613 00:32:18,910 --> 00:32:24,680 So i can be 1 through 7. 614 00:32:24,680 --> 00:32:28,640 So what do we know from studies that people have done? 615 00:32:28,640 --> 00:32:31,920 The key thing is alpha. 616 00:32:31,920 --> 00:32:36,610 So these alphas at the top and the bottom 617 00:32:36,610 --> 00:32:41,260 are inactive in terms of chemistry of peptide bond 618 00:32:41,260 --> 00:32:43,300 hydrolysis. 619 00:32:43,300 --> 00:32:46,000 So all of the chemistry-- 620 00:32:46,000 --> 00:32:52,630 so these are each the beta heptamer is also-- 621 00:32:52,630 --> 00:32:53,630 these are in the center. 622 00:32:53,630 --> 00:32:55,600 These are active. 623 00:32:55,600 --> 00:32:58,630 So the activity is here and it's flanked 624 00:32:58,630 --> 00:33:03,660 by to heptameric rings that are inactive. 625 00:33:03,660 --> 00:33:06,340 And so what do we know about beta? 626 00:33:06,340 --> 00:33:11,530 So even though we have beta i, where this is 1 through 7, 627 00:33:11,530 --> 00:33:17,620 it turns out that four out of the seven betas are inactive. 628 00:33:17,620 --> 00:33:21,970 So again, you saw the complexity with Saunders' talk 629 00:33:21,970 --> 00:33:27,410 on single molecule stuff on ClpP, right? 630 00:33:27,410 --> 00:33:36,070 So four of the seven betas are inactive. 631 00:33:38,770 --> 00:33:42,330 So that might not be so different. 632 00:33:42,330 --> 00:33:45,100 But I think every proteosome, even though the architecture is 633 00:33:45,100 --> 00:33:48,760 sort of similar, has evolved slightly different strategies 634 00:33:48,760 --> 00:33:50,950 to deal with the same problem. 635 00:33:50,950 --> 00:33:54,130 But what's interesting here is, it 636 00:33:54,130 --> 00:33:55,970 doesn't matter which one is which, 637 00:33:55,970 --> 00:33:58,210 but the three betas that are active 638 00:33:58,210 --> 00:34:02,590 all have different specificities of peptide bond hydrolysis. 639 00:34:02,590 --> 00:34:08,080 So B1 has D,E specificity. 640 00:34:08,080 --> 00:34:10,920 Hopefully you all know what that means. 641 00:34:10,920 --> 00:34:18,179 That means simply, for example, if you had an aspartate 642 00:34:18,179 --> 00:34:21,300 and this is where the peptide bond cleavages, 643 00:34:21,300 --> 00:34:25,870 they recognize aspartate in the P1 binding site. 644 00:34:25,870 --> 00:34:32,300 So if they recognize aspartate and a glutamate, B2-- 645 00:34:32,300 --> 00:34:34,830 or I might have the numbers mixed up-- 646 00:34:34,830 --> 00:34:37,489 recognize lysine and arginine. 647 00:34:37,489 --> 00:34:38,670 What does that look like? 648 00:34:38,670 --> 00:34:40,889 We've seen this now a hundred times. 649 00:34:40,889 --> 00:34:43,230 That should remind you of trypsin. 650 00:34:43,230 --> 00:34:47,489 So we have yet another lysine-arginine-dependent 651 00:34:47,489 --> 00:34:51,360 protease, and these are all over the place in the body. 652 00:34:51,360 --> 00:34:54,150 So it's not just this one little site. 653 00:34:54,150 --> 00:34:55,870 It is, maybe, in the proteosome. 654 00:34:55,870 --> 00:34:57,990 But if you look at blood coagulation, 655 00:34:57,990 --> 00:35:03,198 there was something like 15 lysine-dependent proteases, 656 00:35:03,198 --> 00:35:04,740 and they've got to all be controlled, 657 00:35:04,740 --> 00:35:07,410 otherwise we would clot all the time. 658 00:35:07,410 --> 00:35:08,524 Yeah? 659 00:35:08,524 --> 00:35:12,050 AUDIENCE: When you say four of the seven [INAUDIBLE] 660 00:35:12,050 --> 00:35:12,900 are inactive. 661 00:35:12,900 --> 00:35:13,740 JOANNE STUBBE: Yeah. 662 00:35:13,740 --> 00:35:15,183 AUDIENCE: Do you mean that-- 663 00:35:15,183 --> 00:35:17,350 JOANNE STUBBE: They can't catalyze any peptide bond. 664 00:35:17,350 --> 00:35:19,725 AUDIENCE: But is it, like, in some of the other proteases 665 00:35:19,725 --> 00:35:21,240 we saw where it changes? 666 00:35:21,240 --> 00:35:24,090 Or is it for a given or a specific molecule, a specific 667 00:35:24,090 --> 00:35:26,910 protease it's always the same four units that are inactive? 668 00:35:26,910 --> 00:35:29,368 JOANNE STUBBE: It's always the same four that are inactive, 669 00:35:29,368 --> 00:35:32,070 but whether they're locate-- how do you call 1, 2, 3, 4, 670 00:35:32,070 --> 00:35:34,300 and how they assemble? 671 00:35:34,300 --> 00:35:36,870 An interesting question that actually people 672 00:35:36,870 --> 00:35:38,700 are studying in thermophilic bacteria. 673 00:35:38,700 --> 00:35:41,220 But you can imagine if they had be-- 674 00:35:41,220 --> 00:35:42,990 I don't think they have to be predisposed. 675 00:35:42,990 --> 00:35:44,430 That's why I'm saying the numbers don't 676 00:35:44,430 --> 00:35:45,513 make that much difference. 677 00:35:45,513 --> 00:35:46,590 AUDIENCE: [INAUDIBLE]. 678 00:35:46,590 --> 00:35:49,650 JOANNE STUBBE: So it doesn't have to be B1, B2, B3, B4, B5, 679 00:35:49,650 --> 00:35:51,150 and you always see the same. 680 00:35:51,150 --> 00:35:55,760 I don't think that's true, but I don't really know. 681 00:35:55,760 --> 00:35:58,040 So you have a different specificity there. 682 00:35:58,040 --> 00:36:01,932 And the third one, which they call-- 683 00:36:01,932 --> 00:36:03,390 I don't remember what they call it. 684 00:36:03,390 --> 00:36:05,520 They call it B4 in the paper, so maybe they 685 00:36:05,520 --> 00:36:15,330 more know more about this than I do, but you have hydrophobics 686 00:36:15,330 --> 00:36:17,350 and you have aromatics. 687 00:36:17,350 --> 00:36:21,082 Where have you seen that kind of a protease before? 688 00:36:21,082 --> 00:36:23,080 Yeah, kind of with trypsin. 689 00:36:23,080 --> 00:36:25,600 So you're seeing this-- these are the common proteases you 690 00:36:25,600 --> 00:36:26,990 find all over the place. 691 00:36:26,990 --> 00:36:30,310 I mean, we use them as tools all the time as biochemists, 692 00:36:30,310 --> 00:36:31,780 these three. 693 00:36:31,780 --> 00:36:35,150 There are many variations on this theme. 694 00:36:35,150 --> 00:36:41,890 So anyhow, so what you have then is basically heptameric units 695 00:36:41,890 --> 00:36:44,230 where the activity is here. 696 00:36:44,230 --> 00:36:45,160 These are inactive. 697 00:36:45,160 --> 00:36:47,530 And somehow you have to get the protein that's 698 00:36:47,530 --> 00:36:52,990 going to be degraded just like you did in the clip p protein, 699 00:36:52,990 --> 00:36:56,860 get it into the chamber of doom. 700 00:36:56,860 --> 00:37:00,790 So what do we know about the mechanism of cleavage? 701 00:37:00,790 --> 00:37:04,360 So I'm not going to go through the mechanism in detail, 702 00:37:04,360 --> 00:37:07,540 but the mechanism I'm going to say a few things. 703 00:37:07,540 --> 00:37:18,550 The mechanism of peptide bond cleavage 704 00:37:18,550 --> 00:37:22,060 is distinct in that what did you see in clip p? 705 00:37:22,060 --> 00:37:25,000 You had a serine-type protease that 706 00:37:25,000 --> 00:37:27,950 involved covalent catalysis. 707 00:37:27,950 --> 00:37:33,850 Here, what you have in the human system is a threonine. 708 00:37:33,850 --> 00:37:35,530 So that's sort of unusual. 709 00:37:35,530 --> 00:37:37,180 There were been a number of these 710 00:37:37,180 --> 00:37:41,680 since this was discovered a while back. 711 00:37:41,680 --> 00:37:47,620 There have been a number of threonine proteases, 712 00:37:47,620 --> 00:37:51,100 so this is the rest of the proteosome. 713 00:37:54,420 --> 00:37:57,840 So it turns out that threonine is at the N-terminus 714 00:37:57,840 --> 00:37:59,730 of the proteosome. 715 00:37:59,730 --> 00:38:02,550 So that becomes important in terms of its chemistry. 716 00:38:02,550 --> 00:38:04,785 So this is the N-terminus. 717 00:38:11,170 --> 00:38:13,570 And the two things that you could 718 00:38:13,570 --> 00:38:17,200 picture that might be involved in catalysis, 719 00:38:17,200 --> 00:38:20,770 based on what you've learned about the bacterial protease, 720 00:38:20,770 --> 00:38:22,780 is that you have a serine, you have a threonine. 721 00:38:22,780 --> 00:38:29,140 They both have OHs that could be involved in covalent catalysis. 722 00:38:29,140 --> 00:38:35,130 So this OH is thought to be involved in covalent catalysis. 723 00:38:38,670 --> 00:38:40,650 And remember, what do you have and what do you 724 00:38:40,650 --> 00:38:47,930 have in the case of the clip p protein? 725 00:38:47,930 --> 00:38:51,510 What also is required besides a serine? 726 00:38:51,510 --> 00:38:53,190 You need some kind of general acid, 727 00:38:53,190 --> 00:38:56,130 some kind of general base catalyst in all 728 00:38:56,130 --> 00:38:57,930 these proteases. 729 00:38:57,930 --> 00:39:02,880 In the case of serine proteases, it's usually histadine. 730 00:39:02,880 --> 00:39:05,730 It's not a histadine in this case. 731 00:39:05,730 --> 00:39:10,770 It is the N-terminal amino group of the protein 732 00:39:10,770 --> 00:39:12,810 that's proposed to be the N-terminal amino group 733 00:39:12,810 --> 00:39:13,460 of the protein. 734 00:39:13,460 --> 00:39:17,520 Now, if you look through the PKAs amino groups 735 00:39:17,520 --> 00:39:22,500 at the N-terminus versus lysine, they're always lower. 736 00:39:22,500 --> 00:39:36,460 So this amino group, so the N-terminus amino group 737 00:39:36,460 --> 00:39:38,200 is thought to be the general base 738 00:39:38,200 --> 00:39:44,230 catalyst and the general acid catalyst in the mechanism. 739 00:39:44,230 --> 00:39:45,760 So that's the proposal. 740 00:39:45,760 --> 00:39:48,760 And I must say, I don't think we know a whole heck of a lot. 741 00:39:48,760 --> 00:39:50,620 I haven't read the literature [INAUDIBLE].. 742 00:39:50,620 --> 00:39:52,210 There's not that many people working 743 00:39:52,210 --> 00:39:55,840 on the mechanism at this stage. 744 00:39:55,840 --> 00:39:58,030 So this is the proposed mechanism, 745 00:39:58,030 --> 00:40:00,310 so just put it in quotes, "proposed". 746 00:40:00,310 --> 00:40:03,010 And it's completely sort of analogous 747 00:40:03,010 --> 00:40:08,230 to what you went through in the first part of the first part 748 00:40:08,230 --> 00:40:09,970 of the semester. 749 00:40:09,970 --> 00:40:12,490 So the amino group is deprotonated. 750 00:40:12,490 --> 00:40:16,390 It's got to be deprotonated to function as a general base 751 00:40:16,390 --> 00:40:21,040 catalyst, proposed to deprotonate 752 00:40:21,040 --> 00:40:24,190 the hydroxyl group of a threonine, 753 00:40:24,190 --> 00:40:27,900 activated for nucleophilic attack somehow. 754 00:40:27,900 --> 00:40:29,860 Do you form an oxyanion hole? 755 00:40:29,860 --> 00:40:32,520 Whenever you see brackets, that means we don't see it 756 00:40:32,520 --> 00:40:34,420 and its a proposed intermediate. 757 00:40:34,420 --> 00:40:37,720 From chemical studies, we know tetrahedral intermediates 758 00:40:37,720 --> 00:40:39,610 exist. 759 00:40:39,610 --> 00:40:42,040 In proteases, no one has ever seen 760 00:40:42,040 --> 00:40:43,940 a tetrahedral intermediate. 761 00:40:43,940 --> 00:40:46,930 So all these things you see in all these mechanisms 762 00:40:46,930 --> 00:40:50,680 are a figment of people's imaginations based 763 00:40:50,680 --> 00:40:53,620 on really sort of a thorough understanding of the chemistry 764 00:40:53,620 --> 00:40:54,620 of what's going on. 765 00:40:54,620 --> 00:40:56,600 So when whenever you see brackets, 766 00:40:56,600 --> 00:40:58,390 that means there's no direct evidence 767 00:40:58,390 --> 00:41:02,080 or it's reasonable mechanism based on the chemistry. 768 00:41:02,080 --> 00:41:04,840 And then what we need to do is we want to break down 769 00:41:04,840 --> 00:41:07,060 this tetrahedral intermediate. 770 00:41:07,060 --> 00:41:08,920 And we want to cleave the amide bond, which 771 00:41:08,920 --> 00:41:12,640 is the goal of this proteosome. 772 00:41:12,640 --> 00:41:16,540 And to do that, we can now use this amino group which we've 773 00:41:16,540 --> 00:41:19,840 initially used as a general base catalyst, 774 00:41:19,840 --> 00:41:21,250 as the general acid catalyst. 775 00:41:21,250 --> 00:41:25,150 So that's where the general acid catalysis comes in, 776 00:41:25,150 --> 00:41:28,150 and you see this over and over again in biology. 777 00:41:28,150 --> 00:41:32,110 You have one group and it can function as an acid and base 778 00:41:32,110 --> 00:41:32,982 catalyst. 779 00:41:32,982 --> 00:41:34,690 And it gives you-- what does it give you? 780 00:41:34,690 --> 00:41:36,430 It gives you an acyl-enzyme. 781 00:41:36,430 --> 00:41:38,560 So you've seen that before. 782 00:41:38,560 --> 00:41:42,250 And now you just do the reverse of this reaction 783 00:41:42,250 --> 00:41:44,650 where this forms as a general base catalyst 784 00:41:44,650 --> 00:41:49,390 to activate water, forming a tetrahedral intermediate, 785 00:41:49,390 --> 00:41:51,672 and then loss of water to regenerate 786 00:41:51,672 --> 00:41:52,630 your starting material. 787 00:41:52,630 --> 00:41:54,760 So that's a working hypothesis and I don't really 788 00:41:54,760 --> 00:41:58,150 want to spend any more time on that. 789 00:41:58,150 --> 00:42:03,950 So that's the chemistry of the core particle. 790 00:42:03,950 --> 00:42:05,980 And really sort of what we want to do now 791 00:42:05,980 --> 00:42:09,340 is focus on the real chemistry that's going to go on, 792 00:42:09,340 --> 00:42:11,770 that's going to allow us to mediate degradation 793 00:42:11,770 --> 00:42:15,820 of proteins of interest as a regulatory mechanism. 794 00:42:15,820 --> 00:42:19,690 So the second thing that I want to talk about, 795 00:42:19,690 --> 00:42:21,430 the second player I want to talk about 796 00:42:21,430 --> 00:42:29,060 is ubiquitin, which you have all seen. 797 00:42:29,060 --> 00:42:37,130 And this is the key tag that targets, 798 00:42:37,130 --> 00:42:39,710 although it's a major tag. 799 00:42:39,710 --> 00:42:43,490 But I think the tagging is much more complicated, as I tried 800 00:42:43,490 --> 00:42:45,205 to indicate in the first slide. 801 00:42:45,205 --> 00:42:46,080 We just went over it. 802 00:42:46,080 --> 00:42:48,500 You need something else to target your protein 803 00:42:48,500 --> 00:42:51,890 for degradation because you need to target the ubiquitination 804 00:42:51,890 --> 00:42:53,310 in the first place. 805 00:42:53,310 --> 00:42:56,005 So something has got to be special about protein A 806 00:42:56,005 --> 00:43:00,260 and protein D that controls lifetime inside the cell. 807 00:43:00,260 --> 00:43:02,230 And this is a lot of people working on that. 808 00:43:02,230 --> 00:43:05,100 So tag the targets. 809 00:43:05,100 --> 00:43:06,980 Let's call it a protein of the interest. 810 00:43:06,980 --> 00:43:18,420 So this would be HMG-CoA reductase for degradation. 811 00:43:21,515 --> 00:43:22,890 So this is a key player and we're 812 00:43:22,890 --> 00:43:25,890 going to spend a little bit of time talking about that. 813 00:43:25,890 --> 00:43:29,650 But furthermore, one ubiquitin is not enough. 814 00:43:29,650 --> 00:43:31,710 You need to have polyubiquitins. 815 00:43:31,710 --> 00:43:34,500 So what we're going to see is-- 816 00:43:34,500 --> 00:43:36,150 and I think this is still the rule, 817 00:43:36,150 --> 00:43:42,360 it keeps changing-- but you need polyubiquitins 818 00:43:42,360 --> 00:43:47,730 where n is greater than or equal to 4 for this targeting 819 00:43:47,730 --> 00:43:49,920 to work, so one's not enough. 820 00:43:49,920 --> 00:43:51,640 So now we're faced with the problem, 821 00:43:51,640 --> 00:43:53,910 how do we stick this ubiquitin onto the protein 822 00:43:53,910 --> 00:43:55,290 that's going to get degraded? 823 00:43:55,290 --> 00:43:57,330 So that's what we're focusing on. 824 00:43:57,330 --> 00:44:01,860 So let's look a little bit at the structure of ubiquitin. 825 00:44:01,860 --> 00:44:03,780 So here's the structure of ubiquitin, 826 00:44:03,780 --> 00:44:09,037 and ubiquitin is 76 amino acids. 827 00:44:09,037 --> 00:44:10,620 It's a compact-- you've already looked 828 00:44:10,620 --> 00:44:15,900 at the structure of this guy and it's compact a little protein 829 00:44:15,900 --> 00:44:21,540 that has a C-terminal glycine. 830 00:44:21,540 --> 00:44:24,980 Let me write this out. 831 00:44:24,980 --> 00:44:28,840 So this is a C-terminal glycine, which is a key player. 832 00:44:28,840 --> 00:44:30,880 So in some of the pictures that you're 833 00:44:30,880 --> 00:44:34,630 going to see that I draw on the board, 834 00:44:34,630 --> 00:44:36,730 this is going to be a key player so I might write 835 00:44:36,730 --> 00:44:40,600 G76 or something like that, so glycine 76 836 00:44:40,600 --> 00:44:41,833 is in all of these things. 837 00:44:41,833 --> 00:44:43,750 And it's going to be making the linkages we're 838 00:44:43,750 --> 00:44:44,708 going to be looking at. 839 00:44:44,708 --> 00:44:46,723 So you just need to remember that. 840 00:44:46,723 --> 00:44:48,640 And if you look at where it is, if you go back 841 00:44:48,640 --> 00:44:50,680 and you look at the structure, it's 842 00:44:50,680 --> 00:44:54,610 on a flexible loop at the end. 843 00:44:54,610 --> 00:44:58,450 So the other thing that you need to know about ubiquitin when 844 00:44:58,450 --> 00:45:09,100 we look at the structure is that it has seven lysines. 845 00:45:09,100 --> 00:45:10,865 And the lysines, because this is so small, 846 00:45:10,865 --> 00:45:13,120 are located on the surface. 847 00:45:13,120 --> 00:45:16,930 For targeting ubiquitin, targeting proteins 848 00:45:16,930 --> 00:45:19,360 for degradation, we're really going to be focusing on, 849 00:45:19,360 --> 00:45:21,640 like, lysine 48. 850 00:45:21,640 --> 00:45:23,740 But all of the lysines can get modified. 851 00:45:23,740 --> 00:45:25,240 That's just the complexity of it. 852 00:45:25,240 --> 00:45:30,270 So we're going to focus on lysine 48. 853 00:45:35,690 --> 00:45:38,890 So the key thing is what happens. 854 00:45:38,890 --> 00:45:41,210 What I'm going to show you is what the structure is. 855 00:45:41,210 --> 00:45:45,080 So we have a protein of interest that's 856 00:45:45,080 --> 00:45:46,620 targeted for degradation, and we're 857 00:45:46,620 --> 00:45:49,310 going to talk about how it's targeted. 858 00:45:49,310 --> 00:45:57,750 It has a lysine on its surface, a lysine on its surface, which 859 00:45:57,750 --> 00:46:06,810 is somehow then going to be attached to glycine 76 860 00:46:06,810 --> 00:46:09,450 on the C-terminus of ubiquitin. 861 00:46:09,450 --> 00:46:15,178 So this is going to be part of ubiquitin. 862 00:46:15,178 --> 00:46:16,220 So let me just write it-- 863 00:46:16,220 --> 00:46:17,887 I'm not going to write this out anymore, 864 00:46:17,887 --> 00:46:21,170 but this is a C-terminus. 865 00:46:21,170 --> 00:46:24,253 So what's unusual about this bond? 866 00:46:27,360 --> 00:46:32,200 I mean, do you normally see that bond in proteins? 867 00:46:32,200 --> 00:46:32,700 No. 868 00:46:32,700 --> 00:46:34,890 Normally, you see it with alpha amino groups, 869 00:46:34,890 --> 00:46:38,100 now you're seeing it with the epsilon amino group of lysine. 870 00:46:38,100 --> 00:46:40,920 So this is an isopeptide and everything 871 00:46:40,920 --> 00:46:44,820 with ubiquitin in chemistry is through isopeptide linkages. 872 00:46:44,820 --> 00:46:48,540 So this is, again, this is a lysine here. 873 00:46:48,540 --> 00:46:50,720 And we will see that all the proteins targeted 874 00:46:50,720 --> 00:46:54,310 have lysines on the surface that can do covalent chemistry. 875 00:46:54,310 --> 00:46:56,640 And we'll see how with ubiquitin. 876 00:46:56,640 --> 00:46:58,230 So this is an isopeptide. 877 00:47:02,520 --> 00:47:05,940 Again, this is the epsilon amino group, not 878 00:47:05,940 --> 00:47:07,870 the alpha amino group. 879 00:47:07,870 --> 00:47:12,720 And this is the C-terminal flexible chain of ubiquitin. 880 00:47:12,720 --> 00:47:16,810 And then, ultimately, what we need 881 00:47:16,810 --> 00:47:20,110 to get to when we're looking at the biosynthetic pathway 882 00:47:20,110 --> 00:47:24,410 is how do we attach all of these things? 883 00:47:24,410 --> 00:47:27,360 So the question is, what are the linkages 884 00:47:27,360 --> 00:47:29,310 between the ubiquitins? 885 00:47:29,310 --> 00:47:32,190 They're going to be isopeptide linkages, 886 00:47:32,190 --> 00:47:34,710 and they're isopeptide linkages between the lysine 887 00:47:34,710 --> 00:47:37,410 on the ubiquitin and the C-terminal glycine 888 00:47:37,410 --> 00:47:38,940 of another ubiquitin. 889 00:47:38,940 --> 00:47:42,870 So what you have here again is an isopeptide. 890 00:47:45,950 --> 00:47:50,470 And I think once you get this down in your head 891 00:47:50,470 --> 00:47:52,710 as to what's going on, the chemistry is going 892 00:47:52,710 --> 00:47:54,520 to be really straightforward. 893 00:47:54,520 --> 00:48:00,940 So again, what you have in the case of ubiquitin, 894 00:48:00,940 --> 00:48:02,500 you have lysine. 895 00:48:02,500 --> 00:48:06,940 Remember, I told you we were going to focus on lysine 48, 896 00:48:06,940 --> 00:48:08,260 so lysine 48. 897 00:48:08,260 --> 00:48:18,770 Again, it is surface exposed and if forms a covalent linkage 898 00:48:18,770 --> 00:48:25,280 with this is glycine 76 at the C-terminus. 899 00:48:25,280 --> 00:48:28,220 So again, we have this isopeptide linkage. 900 00:48:28,220 --> 00:48:31,120 So that you're going to see over and over and over again. 901 00:48:31,120 --> 00:48:32,713 Does everybody get that? 902 00:48:32,713 --> 00:48:34,130 I don't think it's so hard to see, 903 00:48:34,130 --> 00:48:37,700 but it's just different from what you've seen before. 904 00:48:37,700 --> 00:48:38,780 So now I want to do-- 905 00:48:38,780 --> 00:48:40,460 I told you at the very beginning, 906 00:48:40,460 --> 00:48:41,790 how do we do all of that? 907 00:48:41,790 --> 00:48:45,110 Well, the time is over. 908 00:48:45,110 --> 00:48:45,688 I'm sorry. 909 00:48:45,688 --> 00:48:46,730 The time is over already. 910 00:48:46,730 --> 00:48:48,750 It just goes by so fast. 911 00:48:48,750 --> 00:48:50,270 I can't stand this. 912 00:48:50,270 --> 00:48:52,400 I didn't even get to the exciting part. 913 00:48:52,400 --> 00:48:55,370 So next time, next time we'll have to come back 914 00:48:55,370 --> 00:48:59,120 and we will talk about E1, E2, E3. 915 00:48:59,120 --> 00:49:01,970 The chemistry is straightforward and it's analogous to chemistry 916 00:49:01,970 --> 00:49:03,600 you've already seen before. 917 00:49:03,600 --> 00:49:05,270 And then we're going to briefly look 918 00:49:05,270 --> 00:49:09,470 at how this relates to HMG-CoA reductase, degradation, 919 00:49:09,470 --> 00:49:11,540 which I'll show you what the factors are, 920 00:49:11,540 --> 00:49:15,070 but it's still pretty much a black box in my opinion.