1 00:00:00,500 --> 00:00:02,820 The following content is provided under a Creative 2 00:00:02,820 --> 00:00:04,360 Commons license. 3 00:00:04,360 --> 00:00:06,660 Your support will help MIT OpenCourseWare 4 00:00:06,660 --> 00:00:11,020 continue to offer high quality educational resources for free. 5 00:00:11,020 --> 00:00:13,650 To make a donation, or view additional materials 6 00:00:13,650 --> 00:00:17,600 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,600 --> 00:00:18,550 at ocw.mit.edu. 8 00:00:25,682 --> 00:00:27,140 JOANNE STUBBE: In the last lecture, 9 00:00:27,140 --> 00:00:29,780 we were focused on the proteasome. 10 00:00:29,780 --> 00:00:33,350 And we were focusing on how you targeted 11 00:00:33,350 --> 00:00:37,040 the protein of interest for degradation 12 00:00:37,040 --> 00:00:38,780 by attachment of ubiquitin. 13 00:00:38,780 --> 00:00:41,180 So here's the model that I presented 14 00:00:41,180 --> 00:00:42,290 at the very beginning. 15 00:00:42,290 --> 00:00:47,600 And right now, we're focused on how do you attach ubiquitins 16 00:00:47,600 --> 00:00:50,970 to a protein of interest that's going to be degraded. 17 00:00:50,970 --> 00:00:54,290 OK, and so here's the protein of interest 18 00:00:54,290 --> 00:00:57,920 the last time we talked about all the linkages. 19 00:00:57,920 --> 00:01:01,190 We're going to be isopeptides where 20 00:01:01,190 --> 00:01:04,780 you have lysine on the surface, or a lysine that's accessible, 21 00:01:04,780 --> 00:01:08,120 that can then get attached to ubiquitin, which at a C 22 00:01:08,120 --> 00:01:10,970 terminal end has glycine. 23 00:01:16,400 --> 00:01:17,980 So that's glycine 76-- 24 00:01:17,980 --> 00:01:20,830 that's the linkage common to everything. 25 00:01:20,830 --> 00:01:24,220 And then this ubiquitin, and you can see from the structure, 26 00:01:24,220 --> 00:01:27,280 has a number of lysines attached depending 27 00:01:27,280 --> 00:01:30,910 on what the function is of the ubiquitin. 28 00:01:30,910 --> 00:01:32,470 It can be attached almost anywhere. 29 00:01:32,470 --> 00:01:35,750 For the proteasome, we're focused on lysine 48. 30 00:01:35,750 --> 00:01:39,880 OK, which again, makes an isopeptide linkage here. 31 00:01:39,880 --> 00:01:42,790 And you need multiple ubiquitins to be 32 00:01:42,790 --> 00:01:46,360 able to get your protein of interest degraded. 33 00:01:46,360 --> 00:01:52,840 OK, so what I want to do now is talk about the equipment that's 34 00:01:52,840 --> 00:01:55,150 required to attach this ubiquitin 35 00:01:55,150 --> 00:01:59,540 molecule through the glycine to the protein of interest. 36 00:01:59,540 --> 00:02:04,720 And so what we're focused on is the three enzymes-- 37 00:02:04,720 --> 00:02:06,640 E1, E2, E3. 38 00:02:06,640 --> 00:02:09,179 So we want to look at the attachment. 39 00:02:13,500 --> 00:02:19,090 And we're going to look at E1, E2, E3, 40 00:02:19,090 --> 00:02:22,210 and what their function is in general. 41 00:02:22,210 --> 00:02:27,880 And so E1-- and it turns out in human systems there are only 42 00:02:27,880 --> 00:02:29,260 two of these proteins-- 43 00:02:32,440 --> 00:02:35,260 by, again, by homology. 44 00:02:35,260 --> 00:02:38,350 And this is going to be what they 45 00:02:38,350 --> 00:02:42,520 call the activating enzyme. 46 00:02:46,560 --> 00:02:51,653 OK, and so E1 is going to be-- so 47 00:02:51,653 --> 00:02:53,320 since we have thousands of proteins that 48 00:02:53,320 --> 00:02:56,140 are going to be degraded and only one of these E1s, 49 00:02:56,140 --> 00:02:59,710 it's going to play a role in many, many reactions. 50 00:02:59,710 --> 00:03:01,780 It's sort of the lynchpin. 51 00:03:01,780 --> 00:03:04,120 Over there there's a cartoon of what I'm 52 00:03:04,120 --> 00:03:06,610 going to write on the board. 53 00:03:06,610 --> 00:03:11,320 But E1-- and when we look at the chemistry, 54 00:03:11,320 --> 00:03:15,640 a key player in the chemistry are cysteines and covalent 55 00:03:15,640 --> 00:03:18,620 catalysis by forming thioesters. 56 00:03:18,620 --> 00:03:21,250 OK, you've now seen this many times 57 00:03:21,250 --> 00:03:22,850 in the polyketide synthases. 58 00:03:22,850 --> 00:03:24,790 You've seen it in fatty acids synthases. 59 00:03:24,790 --> 00:03:27,100 You've seen it in cysteine proteases. 60 00:03:27,100 --> 00:03:32,080 This is the motif that nature uses over and over again. 61 00:03:32,080 --> 00:03:38,770 And what she does is takes ATP and then she takes ubiquitin. 62 00:03:38,770 --> 00:03:43,990 And I'm just going to put G 76 at the C terminal end. 63 00:03:43,990 --> 00:03:46,910 So this is the C terminal carboxylate. 64 00:03:49,420 --> 00:03:54,760 And what she does then is uses ATP to activate the carboxylate 65 00:03:54,760 --> 00:03:58,690 so that it can be attached to the cysteine, which 66 00:03:58,690 --> 00:04:00,460 then forms the thioester. 67 00:04:00,460 --> 00:04:03,010 OK, and sort of the strategy is the same. 68 00:04:03,010 --> 00:04:06,130 I'm not going to write out the details of this strategy. 69 00:04:06,130 --> 00:04:10,270 But there are two ways ATP can be used. 70 00:04:10,270 --> 00:04:12,670 What does ATP use to activate this 71 00:04:12,670 --> 00:04:14,730 into a good dehydrating agent? 72 00:04:14,730 --> 00:04:17,519 What does nature do? 73 00:04:17,519 --> 00:04:20,546 What are the two options? 74 00:04:20,546 --> 00:04:21,779 AUDIENCE: [INAUDIBLE] 75 00:04:21,779 --> 00:04:25,450 JOANNE STUBBE: So adenylate or phosphorylate, alpha or gamma, 76 00:04:25,450 --> 00:04:27,740 you see this over, and over, and over again. 77 00:04:27,740 --> 00:04:35,290 You have seen this used many, many times in the first half 78 00:04:35,290 --> 00:04:36,150 of this course. 79 00:04:36,150 --> 00:04:41,440 We have talked about the tRNA synthetases, the activating 80 00:04:41,440 --> 00:04:45,070 domains of the non-ribosomal peptide synthetases. 81 00:04:45,070 --> 00:04:49,240 And so what you have done in this reaction 82 00:04:49,240 --> 00:04:54,410 is activated the carboxylate. 83 00:04:54,410 --> 00:04:56,410 And just remember this so I don't 84 00:04:56,410 --> 00:04:57,890 have to keep writing this down. 85 00:04:57,890 --> 00:04:58,690 The G is there. 86 00:04:58,690 --> 00:05:02,440 It's always attached to the C-terminus of ubiquitin. 87 00:05:02,440 --> 00:05:03,620 And now what happens-- 88 00:05:03,620 --> 00:05:05,320 so this is activated. 89 00:05:05,320 --> 00:05:07,300 And so you have a base. 90 00:05:07,300 --> 00:05:10,830 You never do any chemistry with a thiol. 91 00:05:10,830 --> 00:05:15,430 And so one then forms that a thioester by nucleophilic 92 00:05:15,430 --> 00:05:18,070 attack on the carbonyl. 93 00:05:18,070 --> 00:05:22,120 So what one ends up with then in the first step 94 00:05:22,120 --> 00:05:26,660 is ubiquitin attached covalently to E1. 95 00:05:26,660 --> 00:05:30,910 OK, so let me put that up. 96 00:05:30,910 --> 00:05:36,550 Again, all of this is written down in your hand outs. 97 00:05:36,550 --> 00:05:41,290 And so what we have now is E1, where we 98 00:05:41,290 --> 00:05:46,310 have S attached to ubiquitin. 99 00:05:46,310 --> 00:05:49,880 OK, so that's the activating step. 100 00:05:49,880 --> 00:05:51,864 The second step is-- 101 00:05:51,864 --> 00:05:54,100 involves an E2. 102 00:05:54,100 --> 00:06:00,200 And that's called the ubiquitin conjugating enzyme. 103 00:06:00,200 --> 00:06:12,010 So this is going to react with E2, the ubiquitin conjugating 104 00:06:12,010 --> 00:06:13,720 enzyme. 105 00:06:13,720 --> 00:06:19,540 What we see in humans again, that there are about 40 106 00:06:19,540 --> 00:06:21,050 of these proteins. 107 00:06:21,050 --> 00:06:23,080 And we still have thousands of proteins 108 00:06:23,080 --> 00:06:25,520 that are going to be targeted for degradation. 109 00:06:25,520 --> 00:06:28,570 So that would imply that these E2s 110 00:06:28,570 --> 00:06:31,260 can be used in multiple processes 111 00:06:31,260 --> 00:06:34,520 and during the targeting process for degradation. 112 00:06:34,520 --> 00:06:36,480 So we're going to see that E2-- 113 00:06:36,480 --> 00:06:44,650 and so there are 40 of these. 114 00:06:44,650 --> 00:06:53,960 And we'll see that E2 also has a sulfhydryl group that 115 00:06:53,960 --> 00:06:57,870 is going to play a key role in this reaction. 116 00:06:57,870 --> 00:07:00,200 So what we're going to do now is a simple 117 00:07:00,200 --> 00:07:03,800 thiotransesterification. 118 00:07:03,800 --> 00:07:09,860 So we're going to transfer the ubiquitin to E2 119 00:07:09,860 --> 00:07:11,750 by thiotransesterification. 120 00:07:11,750 --> 00:07:14,125 So again, you have base. 121 00:07:17,460 --> 00:07:20,510 And you liberate E1. 122 00:07:20,510 --> 00:07:33,140 And now what you have is E2 with the attached ubiquitin. 123 00:07:33,140 --> 00:07:36,498 OK, so there were 40 of these things. 124 00:07:36,498 --> 00:07:38,040 And what does that have to tell you-- 125 00:07:38,040 --> 00:07:40,780 what does that tell you about E1 and E2? 126 00:07:40,780 --> 00:07:42,950 I haven't given you any information about structure, 127 00:07:42,950 --> 00:07:45,110 but there are 40 E2's, so they obviously all 128 00:07:45,110 --> 00:07:46,400 have different structures. 129 00:07:46,400 --> 00:07:49,400 But these things have got to be really flexible. 130 00:07:49,400 --> 00:07:51,560 And so we have a few structures, but I 131 00:07:51,560 --> 00:07:54,740 don't think it even comes close to allowing 132 00:07:54,740 --> 00:07:57,620 us to understand really sort of the specificity 133 00:07:57,620 --> 00:08:00,560 of these processes. 134 00:08:00,560 --> 00:08:03,363 And this is a major focus of many people now. 135 00:08:03,363 --> 00:08:05,030 They've been discovered for a long time, 136 00:08:05,030 --> 00:08:06,553 but people are still messing around. 137 00:08:06,553 --> 00:08:07,970 And I think part of the complexity 138 00:08:07,970 --> 00:08:10,580 relates to the flexibility, which makes 139 00:08:10,580 --> 00:08:13,460 them harder to crystallize. 140 00:08:13,460 --> 00:08:20,060 OK, and so E3 are the ubiquitin ligases. 141 00:08:24,950 --> 00:08:26,920 And the latest paper I read-- 142 00:08:26,920 --> 00:08:32,120 they keep finding new ones-- but there are greater than 600 143 00:08:32,120 --> 00:08:34,090 of these guys. 144 00:08:34,090 --> 00:08:36,950 OK, so even there, since more proteins 145 00:08:36,950 --> 00:08:39,809 are targeted for degradation than 600, 146 00:08:39,809 --> 00:08:43,490 E3s are going to be used multiple times. 147 00:08:43,490 --> 00:08:49,310 And E3s are able to form protein complexes. 148 00:08:53,380 --> 00:08:56,770 And again, I'm giving you sort of a generic overview. 149 00:08:56,770 --> 00:08:59,410 But you'll see in the next module, 150 00:08:59,410 --> 00:09:02,410 one of the key proteins involved in iron homeostasis 151 00:09:02,410 --> 00:09:05,040 gets targeted for degradation by a ubiquitin 152 00:09:05,040 --> 00:09:10,330 ligase that exists in a complicated protein complex. 153 00:09:10,330 --> 00:09:12,310 So that and then adds to the complexity 154 00:09:12,310 --> 00:09:14,950 you need to target all of your proteins 155 00:09:14,950 --> 00:09:19,180 specifically for degradation in some fashion. 156 00:09:19,180 --> 00:09:22,240 OK, so what does E3 do? 157 00:09:22,240 --> 00:09:26,440 So we have an E2. 158 00:09:26,440 --> 00:09:32,370 And we have an E2 with ubiquitin attached. 159 00:09:32,370 --> 00:09:38,250 And E2 needs to interact with E3. 160 00:09:38,250 --> 00:09:40,830 And this, again, is a complex. 161 00:09:40,830 --> 00:09:46,160 It is not a single, necessarily a single polypeptide. 162 00:09:46,160 --> 00:09:51,610 And E3 interacts-- ultimately what we want to do 163 00:09:51,610 --> 00:09:54,580 is attach ubiquitin into our protein of interest. 164 00:09:54,580 --> 00:10:00,680 So E3 interacts with the protein of interest. 165 00:10:00,680 --> 00:10:02,800 And as I alluded to before, but I'm not 166 00:10:02,800 --> 00:10:06,070 going to talk about in any detail, what 167 00:10:06,070 --> 00:10:10,090 is the basis of the interaction of a protein? 168 00:10:10,090 --> 00:10:10,885 It's going around. 169 00:10:10,885 --> 00:10:12,590 It's doing its function. 170 00:10:12,590 --> 00:10:14,950 How do you target it for degradation? 171 00:10:14,950 --> 00:10:18,010 In general, you target for degradation 172 00:10:18,010 --> 00:10:24,010 by the N-terminal modification or 173 00:10:24,010 --> 00:10:27,340 by post-translational modification. 174 00:10:27,340 --> 00:10:28,920 And every protein is different. 175 00:10:28,920 --> 00:10:30,220 So you might phosphorylate it. 176 00:10:30,220 --> 00:10:32,680 You might hydroxylate it. 177 00:10:32,680 --> 00:10:38,100 In fact, I told you and Lizzie told you in the section 178 00:10:38,100 --> 00:10:41,050 on ClipX and ClipP, that there was this thing called 179 00:10:41,050 --> 00:10:42,670 the N-end rule. 180 00:10:42,670 --> 00:10:45,130 OK, so one of the things you can do 181 00:10:45,130 --> 00:10:50,140 is you can attach different amino acids to the N-terminus. 182 00:10:50,140 --> 00:10:54,520 And in fact, tRNAs are actually used to do that. 183 00:10:54,520 --> 00:10:56,872 And we're not going to talk about the details of that. 184 00:10:56,872 --> 00:10:59,080 But these things, you know, you have so many proteins 185 00:10:59,080 --> 00:11:00,370 that are floating around. 186 00:11:00,370 --> 00:11:02,432 How you're going to subtly control 187 00:11:02,432 --> 00:11:04,390 when you're going to degrade it is not trivial. 188 00:11:04,390 --> 00:11:08,570 And that's a focus of many people's attention. 189 00:11:08,570 --> 00:11:11,560 So the model here is that-- remember, everything we're 190 00:11:11,560 --> 00:11:14,020 doing is isopeptide linkages. 191 00:11:14,020 --> 00:11:16,510 OK so, this needs to be set up. 192 00:11:16,510 --> 00:11:21,250 This complex needs to be set up so the lysine to which 193 00:11:21,250 --> 00:11:24,070 the ubiquitin is going to be attached 194 00:11:24,070 --> 00:11:27,100 needs to be adjacent to the ubiquitin. 195 00:11:27,100 --> 00:11:32,920 So you have-- a lysine needs to be activated 196 00:11:32,920 --> 00:11:35,710 for nucleophilic attack. 197 00:11:35,710 --> 00:11:40,750 And now you've attached your ubiquitin. 198 00:11:40,750 --> 00:11:43,540 So this is a direct transfer. 199 00:11:43,540 --> 00:11:46,420 Again, you're forming the isopeptides. 200 00:11:46,420 --> 00:11:50,110 And remember that we said in the very beginning, 201 00:11:50,110 --> 00:11:51,790 you don't just have one ubiquitin. 202 00:11:51,790 --> 00:11:52,930 You have many ubiquitins. 203 00:11:52,930 --> 00:11:54,490 Somebody asked me after class, how 204 00:11:54,490 --> 00:11:58,000 do you get the many ubiquitins attached? 205 00:11:58,000 --> 00:12:00,370 It turns out that people have started 206 00:12:00,370 --> 00:12:02,560 to study this in some detail. 207 00:12:02,560 --> 00:12:06,160 And in many cases, the E2s and E3s 208 00:12:06,160 --> 00:12:12,100 interact in a processive way to attach multiple ubiquitins. 209 00:12:12,100 --> 00:12:15,640 I gave you a reference if anybody 210 00:12:15,640 --> 00:12:16,720 wants to read about this. 211 00:12:16,720 --> 00:12:19,180 It was published a couple of years 212 00:12:19,180 --> 00:12:22,390 ago about how you control polyubiquitination. 213 00:12:22,390 --> 00:12:25,030 And one of the handouts, they had an E4. 214 00:12:25,030 --> 00:12:31,330 There could be E4s that also act process to attach ubiquitin. 215 00:12:31,330 --> 00:12:38,620 So you can attach more than one to give you, basically, 216 00:12:38,620 --> 00:12:47,220 the protein of interest with the ubiquitins actually attached. 217 00:12:47,220 --> 00:12:50,530 OK, so that is the machinery. 218 00:12:50,530 --> 00:12:52,330 We know what a bit about this machinery, 219 00:12:52,330 --> 00:12:56,920 but that's all I'm going to actually say in this class. 220 00:12:56,920 --> 00:13:01,220 But every system-- there are a lot of people studying this. 221 00:13:01,220 --> 00:13:02,980 And there are very few of these that 222 00:13:02,980 --> 00:13:06,440 are understood in really sort of molecular detail. 223 00:13:06,440 --> 00:13:11,200 So here again is, again, that you have these kinds 224 00:13:11,200 --> 00:13:14,710 of isopeptide linkages. 225 00:13:14,710 --> 00:13:16,990 Here is that ubiquitin with the carboxylate. 226 00:13:16,990 --> 00:13:20,950 And here is the ubiquitin with a lysine 48 227 00:13:20,950 --> 00:13:24,640 which you can attach additional ubiquitins to. 228 00:13:24,640 --> 00:13:27,590 And here is another cartoon of this overall process. 229 00:13:27,590 --> 00:13:30,310 So what I didn't tell you in general 230 00:13:30,310 --> 00:13:34,090 is that E3s come in flavors. 231 00:13:36,850 --> 00:13:41,470 So they have little domains in them. 232 00:13:41,470 --> 00:13:44,257 They have HECT-- is that what it's called? 233 00:13:44,257 --> 00:13:46,090 I can't remember the names of these domains. 234 00:13:46,090 --> 00:13:51,640 I haven't-- yeah, HECT, H-E-C-T domains, HECT domains, 235 00:13:51,640 --> 00:13:59,500 RING domains, U domains, all of which are distinct and play 236 00:13:59,500 --> 00:14:04,010 a role in the details of how this process actually works. 237 00:14:04,010 --> 00:14:05,770 So again, this is something I don't expect 238 00:14:05,770 --> 00:14:08,140 you to remember the details from, 239 00:14:08,140 --> 00:14:10,660 but this is what you can imagine happening. 240 00:14:10,660 --> 00:14:12,820 So in the cartoon over here, this 241 00:14:12,820 --> 00:14:15,130 is what I just described, that we 242 00:14:15,130 --> 00:14:19,810 have an E2 that has-- this little ball is ubiquitin. 243 00:14:19,810 --> 00:14:23,680 Here is our protein of interest, S. And so what happens is, 244 00:14:23,680 --> 00:14:28,030 E2 is attaching ubiquitin to the protein of interest. 245 00:14:28,030 --> 00:14:30,195 OK, so that's one possibility. 246 00:14:30,195 --> 00:14:34,660 And in this case, the transfer is directly from E2. 247 00:14:34,660 --> 00:14:37,360 And that's what the ones that have been studied, 248 00:14:37,360 --> 00:14:41,230 the RING-finger-containing domains, do. 249 00:14:41,230 --> 00:14:44,530 Alternatively, you can imagine that E2 250 00:14:44,530 --> 00:14:48,430 could transfer ubiquitin to E3. 251 00:14:48,430 --> 00:14:51,340 And once it attaches it to E3, E3 252 00:14:51,340 --> 00:14:54,190 could attach it to the protein of interest. 253 00:14:54,190 --> 00:14:56,010 So all of those are possible. 254 00:14:56,010 --> 00:14:59,680 And there has been one case where that's been studied, 255 00:14:59,680 --> 00:15:02,123 where E3 attaches to ubiquitin. 256 00:15:02,123 --> 00:15:03,790 So I think you're going to find actually 257 00:15:03,790 --> 00:15:05,710 many variations of this theme. 258 00:15:05,710 --> 00:15:08,950 This is an old paper, I think. 259 00:15:08,950 --> 00:15:12,460 And I think the more people study it, 260 00:15:12,460 --> 00:15:15,880 probably the more complex it will end up getting. 261 00:15:15,880 --> 00:15:18,880 And so that's basically sort of the machinery-- 262 00:15:18,880 --> 00:15:21,760 with the major consideration, which 263 00:15:21,760 --> 00:15:25,540 I think is actually quite interesting from a biochemical 264 00:15:25,540 --> 00:15:28,390 point of view, is the N-end rule, 265 00:15:28,390 --> 00:15:31,750 how do you modify the N to target it for degradation. 266 00:15:31,750 --> 00:15:33,490 Does it have a half-life of two minutes? 267 00:15:33,490 --> 00:15:36,400 Does it have a half-life of two hours? 268 00:15:36,400 --> 00:15:38,110 And what governs all of that? 269 00:15:38,110 --> 00:15:41,260 And you can imagine that post-translational modification 270 00:15:41,260 --> 00:15:45,250 also governs the half-life-- so both of those possible. 271 00:15:45,250 --> 00:15:51,490 And those of you interested in polyubiquitination 272 00:15:51,490 --> 00:15:52,900 can look at that reference. 273 00:15:52,900 --> 00:15:55,800 And in fact, that paper uses methodologies 274 00:15:55,800 --> 00:15:58,090 we've talked about in class and recitation too. 275 00:15:58,090 --> 00:16:02,050 They use rapid chemical quench technology 276 00:16:02,050 --> 00:16:04,000 to measure the rate constants for putting 277 00:16:04,000 --> 00:16:05,860 on multiple ubiquitins. 278 00:16:05,860 --> 00:16:09,730 So this rapid chemical quenched technology continues 279 00:16:09,730 --> 00:16:11,680 to appear over and over again when 280 00:16:11,680 --> 00:16:14,050 you want to look at more details about how 281 00:16:14,050 --> 00:16:16,870 these systems actually work. 282 00:16:16,870 --> 00:16:22,780 OK, so that is allowing us to get to the stage 283 00:16:22,780 --> 00:16:26,920 where the ubiquitin is attached to the protein of interest. 284 00:16:26,920 --> 00:16:35,200 So and that is via the chamber of doom, the 20s proteosome. 285 00:16:35,200 --> 00:16:38,270 But now what we also would like to look at a little bit-- 286 00:16:38,270 --> 00:16:41,680 and this is a very active area of research-- 287 00:16:41,680 --> 00:16:42,940 is the lid. 288 00:16:42,940 --> 00:16:47,530 OK, and you saw ClipX in addition to ClipP before. 289 00:16:47,530 --> 00:16:50,650 And so I just want to spend a minute 290 00:16:50,650 --> 00:16:57,310 on the 19s lid of the proteosome. 291 00:17:01,360 --> 00:17:05,398 And this lid has proteins coming and going. 292 00:17:05,398 --> 00:17:06,940 And when you isolate it, you probably 293 00:17:06,940 --> 00:17:09,609 lose proteins that are loosely bound. 294 00:17:09,609 --> 00:17:11,380 So this is, again, a complex-- 295 00:17:11,380 --> 00:17:14,670 you can tell that from this cartoon over here-- 296 00:17:14,670 --> 00:17:18,640 a machine of 15 to 20 proteins. 297 00:17:22,670 --> 00:17:24,930 OK, and if you look at this machine, 298 00:17:24,930 --> 00:17:26,680 there has been a lot of people-- actually, 299 00:17:26,680 --> 00:17:29,080 one of Bob Sauer's students at Berkeley 300 00:17:29,080 --> 00:17:33,190 has spent a lot of time studying this human counterpart 301 00:17:33,190 --> 00:17:36,490 and has done a lot of really beautiful cryoEM on this. 302 00:17:36,490 --> 00:17:38,980 So again, this methodology we've been talking about 303 00:17:38,980 --> 00:17:39,910 has been used. 304 00:17:39,910 --> 00:17:41,440 Why are they using cryoEM? 305 00:17:41,440 --> 00:17:43,390 Because you can imagine, this is really hard 306 00:17:43,390 --> 00:17:47,290 to get a picture of because it's moving around a lot. 307 00:17:47,290 --> 00:17:49,420 So if you look over here, what you 308 00:17:49,420 --> 00:17:56,515 will see is that you have a species called Rp2-- 309 00:17:56,515 --> 00:17:58,300 Rpt. 310 00:17:58,300 --> 00:18:00,070 And there are six of these. 311 00:18:00,070 --> 00:18:01,780 So they're all slightly different. 312 00:18:01,780 --> 00:18:05,970 And this is part of an AAA ATPase system. 313 00:18:05,970 --> 00:18:12,610 So you have the Rpt equivalent 1 through 6. 314 00:18:12,610 --> 00:18:13,620 And this is an ATPase. 315 00:18:16,150 --> 00:18:25,180 And it sits on top of the proteosome. 316 00:18:25,180 --> 00:18:29,290 OK, so its function is exactly like what you guys learned 317 00:18:29,290 --> 00:18:31,270 about with ClipX and ClipP. 318 00:18:31,270 --> 00:18:32,560 What does it do? 319 00:18:32,560 --> 00:18:36,310 It's going to pull to try to unfold the protein. 320 00:18:36,310 --> 00:18:39,550 And it's going to use ATP hydrolysis to then try 321 00:18:39,550 --> 00:18:42,760 to thread the protein into the chamber of doom. 322 00:18:42,760 --> 00:18:46,360 So the model, which hasn't been anywhere near as well-studied-- 323 00:18:46,360 --> 00:18:49,150 the best-studied system is the one that Liz talked about. 324 00:18:49,150 --> 00:18:51,123 That's why we chose to look at this. 325 00:18:51,123 --> 00:18:52,540 It's sort of doing the same thing. 326 00:18:52,540 --> 00:18:56,260 It's just there is orders of level more complexity 327 00:18:56,260 --> 00:18:57,340 associated with this. 328 00:18:57,340 --> 00:18:59,380 You can imagine how complicated this 329 00:18:59,380 --> 00:19:01,570 is in terms of thinking about-- 330 00:19:01,570 --> 00:19:04,390 from the Saunders single-molecule talk, 331 00:19:04,390 --> 00:19:07,400 you can imagine this is even more complicated. 332 00:19:07,400 --> 00:19:12,630 So what do you have here that's also similar to the ClipX-ClipP 333 00:19:12,630 --> 00:19:13,360 system? 334 00:19:13,360 --> 00:19:15,130 Here is a hexamer. 335 00:19:15,130 --> 00:19:18,160 And remember that we looked at beta and alpha, 336 00:19:18,160 --> 00:19:20,000 they were sevenmers. 337 00:19:20,000 --> 00:19:23,650 So again, just like with ClipX and ClipP, you have a mismatch. 338 00:19:23,650 --> 00:19:35,370 So we have a hexamer-heptamer mismatch 339 00:19:35,370 --> 00:19:36,890 just like you did before. 340 00:19:36,890 --> 00:19:41,400 And why nature has chosen to do this, I don't know. 341 00:19:41,400 --> 00:19:44,070 But remember, even the beta-- 342 00:19:44,070 --> 00:19:45,720 the alpha subunits are inactive. 343 00:19:45,720 --> 00:19:48,420 The beta subunits are-- only three out of the seven 344 00:19:48,420 --> 00:19:49,020 are active. 345 00:19:49,020 --> 00:19:52,290 So it's just really quite complex. 346 00:19:52,290 --> 00:19:56,050 Now, what are the other things that could be involved-- 347 00:19:56,050 --> 00:19:58,020 these other proteins could be involved in? 348 00:19:58,020 --> 00:20:00,620 Well now, what's distinct from the two 349 00:20:00,620 --> 00:20:03,990 you had in the ClipX-P system, you 350 00:20:03,990 --> 00:20:08,040 had something that recognized the ssRNA tag. 351 00:20:08,040 --> 00:20:10,300 So you had adapter proteins. 352 00:20:10,300 --> 00:20:12,000 So here what we need is something 353 00:20:12,000 --> 00:20:16,530 that recognizes the ubiquitins. 354 00:20:16,530 --> 00:20:19,550 And these could be-- and in the handouts that I've given you, 355 00:20:19,550 --> 00:20:21,480 they tell you which one of these is which. 356 00:20:21,480 --> 00:20:23,910 I'm not going to talk about that detail. 357 00:20:23,910 --> 00:20:33,350 But you have Rpn proteins that recognize ubiquitins. 358 00:20:33,350 --> 00:20:34,800 OK, and I have another-- 359 00:20:34,800 --> 00:20:38,370 people are starting to get cryoEM pictures of all of this. 360 00:20:38,370 --> 00:20:41,430 This is a paper in 2012. 361 00:20:41,430 --> 00:20:43,800 Here is the protein of interest. 362 00:20:43,800 --> 00:20:48,330 Here is the AAA-type ATPase system 363 00:20:48,330 --> 00:20:51,480 that needs to unfold the protein of interest 364 00:20:51,480 --> 00:20:55,470 and thread it into the chamber of doom. 365 00:20:55,470 --> 00:20:59,840 And you have binding sites for the polyubiquitin tail. 366 00:20:59,840 --> 00:21:06,940 OK, so that's one thing you need to do with your lid proteins. 367 00:21:06,940 --> 00:21:09,870 A second thing you need to do is that nature 368 00:21:09,870 --> 00:21:13,000 recycles the ubiquitins. 369 00:21:13,000 --> 00:21:19,470 So what you have is enzymes that are called deubiquitin enzymes. 370 00:21:19,470 --> 00:21:22,850 I think that's whatever they label down here. 371 00:21:22,850 --> 00:21:27,250 R11 in this molecule, Up6 are deubiquitin-- 372 00:21:27,250 --> 00:21:27,750 sorry. 373 00:21:30,460 --> 00:21:34,200 [INAUDIBLE] DUBs-- OK, yeah, so they are. 374 00:21:34,200 --> 00:21:37,620 Both of these guys that are involved in clipping off 375 00:21:37,620 --> 00:21:39,210 the ubiquitins and recycling. 376 00:21:39,210 --> 00:21:48,325 So you have another set of proteins, deubiquitinating 377 00:21:48,325 --> 00:21:48,825 enzymes. 378 00:21:52,160 --> 00:21:56,510 And you have an isopeptide linkage, remember. 379 00:21:56,510 --> 00:21:58,820 And what kind of an enzyme might you 380 00:21:58,820 --> 00:22:04,210 expect a deubiquitinating enzyme to be? 381 00:22:04,210 --> 00:22:05,710 What kind of activity would it have? 382 00:22:08,640 --> 00:22:10,130 You want to cut these things off. 383 00:22:10,130 --> 00:22:10,370 What are you-- 384 00:22:10,370 --> 00:22:10,400 AUDIENCE: Protease. 385 00:22:10,400 --> 00:22:11,608 JOANNE STUBBE: --going to do? 386 00:22:11,608 --> 00:22:12,620 Protease, OK. 387 00:22:12,620 --> 00:22:15,370 And it turns out almost all of them, there-- 388 00:22:15,370 --> 00:22:17,560 again, we're identifying them continually. 389 00:22:17,560 --> 00:22:20,090 They're not so sequence-identifiable 390 00:22:20,090 --> 00:22:22,610 by looking at bioinformatics. 391 00:22:22,610 --> 00:22:26,900 The ones that have been looked at or all cysteine proteases. 392 00:22:26,900 --> 00:22:31,300 So the ones that have been studied in detail 393 00:22:31,300 --> 00:22:33,140 are cysteine proteases. 394 00:22:33,140 --> 00:22:37,760 But remember, they're recognizing 395 00:22:37,760 --> 00:22:42,620 isopeptide linkages, not peptide linkages. 396 00:22:42,620 --> 00:22:45,770 And as in the case of cysteine proteases, 397 00:22:45,770 --> 00:22:46,910 what do they involve? 398 00:22:46,910 --> 00:22:48,740 They involve covalent catalysis. 399 00:22:48,740 --> 00:22:50,490 So again, here is another example of stuff 400 00:22:50,490 --> 00:22:52,282 you learned in the first part of the course 401 00:22:52,282 --> 00:22:53,810 that you're going to-- you see over, 402 00:22:53,810 --> 00:22:56,570 and over, and over again in nature. 403 00:22:56,570 --> 00:23:00,140 And hopefully, this is now becoming second nature 404 00:23:00,140 --> 00:23:04,100 to you guys, that these kinds of processes actually happen. 405 00:23:04,100 --> 00:23:08,480 OK, so this is the lid. 406 00:23:08,480 --> 00:23:11,450 I'm not going to say anymore about that. 407 00:23:11,450 --> 00:23:12,650 You see the equipment. 408 00:23:12,650 --> 00:23:14,390 You see how complicated it is. 409 00:23:14,390 --> 00:23:17,930 And every system you study in biology, 410 00:23:17,930 --> 00:23:19,820 and if you care about the regulation, 411 00:23:19,820 --> 00:23:22,400 you're probably kind of have to think about degradation. 412 00:23:22,400 --> 00:23:24,830 And you're going to have to individually look 413 00:23:24,830 --> 00:23:27,590 at the proteins of interest and figure out 414 00:23:27,590 --> 00:23:31,490 what the E2s and the E3s are and what 415 00:23:31,490 --> 00:23:35,040 the signals are that control this overall process. 416 00:23:35,040 --> 00:23:38,210 So this was just taken out of some recent review, 417 00:23:38,210 --> 00:23:41,750 but it just gives you an idea of where-- 418 00:23:41,750 --> 00:23:44,690 you see this is a couple of years old now-- but, 419 00:23:44,690 --> 00:23:46,970 where you see this kind of machinery. 420 00:23:46,970 --> 00:23:49,620 We're going to see it in the next section. 421 00:23:49,620 --> 00:23:53,060 We're going to see a key player in sensing iron 422 00:23:53,060 --> 00:23:57,140 is degraded by ubiquitination. 423 00:23:57,140 --> 00:23:59,840 In addition, you can imagine progression through the cell 424 00:23:59,840 --> 00:24:03,980 cycle, apoptosis, immune surveillance, 425 00:24:03,980 --> 00:24:08,220 they're all regulated by protein-mediated degradation. 426 00:24:08,220 --> 00:24:11,900 So this is a fundamental mechanism of regulation. 427 00:24:11,900 --> 00:24:14,870 And so having, I think, a cartoon overview that I've 428 00:24:14,870 --> 00:24:16,610 given you in class is really important 429 00:24:16,610 --> 00:24:18,152 to have in the back of your mind when 430 00:24:18,152 --> 00:24:22,670 you're thinking about the system that you might be working on. 431 00:24:22,670 --> 00:24:26,550 And this was a paper that was very recently published. 432 00:24:26,550 --> 00:24:29,690 And so we've been focusing on cholesterol homeostasis. 433 00:24:29,690 --> 00:24:33,440 And remember, when I introduced this topic, 434 00:24:33,440 --> 00:24:38,000 we were talking about Insig and HMG-CoA Reductase. 435 00:24:38,000 --> 00:24:42,230 And HMG-CoA Reductase is targeted for degradation 436 00:24:42,230 --> 00:24:42,980 by Insig. 437 00:24:42,980 --> 00:24:45,770 That's why we made this digression. 438 00:24:45,770 --> 00:24:50,360 And if you go back now and look at what people have pulled out 439 00:24:50,360 --> 00:24:52,280 of the literature-- we're going to look today 440 00:24:52,280 --> 00:24:57,470 very briefly at gp78 in your problem set due this week. 441 00:24:57,470 --> 00:25:00,680 We'll see that gp78, which people thought 442 00:25:00,680 --> 00:25:03,050 was the whole story, is not the whole story, 443 00:25:03,050 --> 00:25:06,358 that there is another E3. 444 00:25:06,358 --> 00:25:08,150 Hopefully you will get that out of the data 445 00:25:08,150 --> 00:25:11,380 that I've given you in problem set three. 446 00:25:11,380 --> 00:25:17,050 And there is yet another system involved 447 00:25:17,050 --> 00:25:21,820 in cholesterol degradation of HMG-CoA Reductase, 448 00:25:21,820 --> 00:25:27,190 but it's not limited to HMG-CoA Reductase. 449 00:25:27,190 --> 00:25:31,990 One also has degradation of the transcription factors SREBP. 450 00:25:31,990 --> 00:25:33,880 We've talked about those. 451 00:25:33,880 --> 00:25:37,420 They use different targeting enzymes. 452 00:25:37,420 --> 00:25:39,310 And furthermore, a lot of people have 453 00:25:39,310 --> 00:25:42,940 been studying the enzymes involved in cholesterol efflux. 454 00:25:42,940 --> 00:25:47,440 And again, these enzymes here are also 455 00:25:47,440 --> 00:25:49,160 targeted for degradation. 456 00:25:49,160 --> 00:25:53,200 So the timing of all this and what's recognized 457 00:25:53,200 --> 00:25:57,100 is central to think-- people thinking about regulation, 458 00:25:57,100 --> 00:26:03,340 not only in systems in general, but cholesterol, specifically. 459 00:26:03,340 --> 00:26:07,330 OK, so that's a summary of everything we've said. 460 00:26:07,330 --> 00:26:08,800 And finally, what I want to do now 461 00:26:08,800 --> 00:26:14,260 is just come back to where we started in this section 462 00:26:14,260 --> 00:26:16,090 to finish up. 463 00:26:16,090 --> 00:26:18,400 And where we started was, we were 464 00:26:18,400 --> 00:26:24,310 looking at the second mechanism of regulation 465 00:26:24,310 --> 00:26:27,370 and the key role of Insig, that you've already seen, 466 00:26:27,370 --> 00:26:32,740 plays a key role in SREBP control, 467 00:26:32,740 --> 00:26:35,740 keeping it in the endoplasmic reticulation. 468 00:26:35,740 --> 00:26:39,970 So now we're coming back and looking 469 00:26:39,970 --> 00:26:47,200 at HMG Reductase and the role of Insig 470 00:26:47,200 --> 00:26:53,880 in targeting its degradation. 471 00:26:58,120 --> 00:27:00,970 And so we've seen these players now 472 00:27:00,970 --> 00:27:02,420 over, and over, and over again. 473 00:27:02,420 --> 00:27:04,960 So I'm not going to keep drawing the structures out 474 00:27:04,960 --> 00:27:06,850 on the board. 475 00:27:06,850 --> 00:27:12,730 But remember, if you have high cholesterol, what 476 00:27:12,730 --> 00:27:15,130 do you want to do with HMG-CoA Reductase, 477 00:27:15,130 --> 00:27:16,510 if you have high cholesterol? 478 00:27:19,030 --> 00:27:20,000 Do what? 479 00:27:20,000 --> 00:27:20,880 AUDIENCE: Inhibit it. 480 00:27:20,880 --> 00:27:22,713 JOANNE STUBBE: Yeah, you want to inhibit it. 481 00:27:22,713 --> 00:27:27,180 And so the way you inhibit it is you 482 00:27:27,180 --> 00:27:30,420 target it to remain in the ER. 483 00:27:30,420 --> 00:27:32,460 And so the question then is then, 484 00:27:32,460 --> 00:27:37,840 how does Insig and HMGR in the presence of cholesterol-- 485 00:27:37,840 --> 00:27:41,250 and it turns out, the signal is not cholesterol itself, 486 00:27:41,250 --> 00:27:43,410 but the signal is lanosterol. 487 00:27:47,600 --> 00:27:50,930 And we talked about that very briefly a couple of times. 488 00:27:50,930 --> 00:27:53,660 Where do you see lenosterol? 489 00:27:53,660 --> 00:27:56,435 If you go back to the biosynthetic pathway, 490 00:27:56,435 --> 00:27:57,560 it's sort of in the middle. 491 00:28:00,530 --> 00:28:02,740 So you have acetyl-CoA. 492 00:28:06,080 --> 00:28:09,200 You have lanosterol. 493 00:28:09,200 --> 00:28:12,800 And you have another 19 steps before you get to cholesterol. 494 00:28:12,800 --> 00:28:16,970 And somehow, this senses lanosterol. 495 00:28:16,970 --> 00:28:19,652 And people are trying to understand the details of that. 496 00:28:19,652 --> 00:28:21,110 How do you really know that's true? 497 00:28:21,110 --> 00:28:22,735 That's not such an easy thing, as we've 498 00:28:22,735 --> 00:28:25,190 talked about in recitation. 499 00:28:25,190 --> 00:28:29,870 So what we want to do then is retain 500 00:28:29,870 --> 00:28:33,080 HMG-CoA Reductase in the ER. 501 00:28:33,080 --> 00:28:35,470 So these are both ER-bound. 502 00:28:38,210 --> 00:28:41,330 And in the presence of lanosterol, 503 00:28:41,330 --> 00:28:45,110 we want to target HMG-CoA Reductase for degradation. 504 00:28:45,110 --> 00:28:47,780 That's the goal. 505 00:28:47,780 --> 00:28:51,650 The question is, is how did people go about studying that? 506 00:28:51,650 --> 00:28:55,550 OK, and so it turns out that they 507 00:28:55,550 --> 00:29:03,740 have discovered three proteins, at least 508 00:29:03,740 --> 00:29:06,140 in one of these systems. 509 00:29:06,140 --> 00:29:09,650 And the protein that I'm going to talk about for a very 510 00:29:09,650 --> 00:29:14,750 brief period of time is gp78. 511 00:29:14,750 --> 00:29:17,510 That was glycoprotein78, tells you 512 00:29:17,510 --> 00:29:19,250 something about its molecular weight. 513 00:29:19,250 --> 00:29:23,520 Again, I don't expect you to remember the details. 514 00:29:23,520 --> 00:29:28,160 But gp78 interacts with Insig. 515 00:29:28,160 --> 00:29:29,750 OK and if you go back and you look 516 00:29:29,750 --> 00:29:32,130 at the little cartoons I've given you, 517 00:29:32,130 --> 00:29:35,960 Insig, again, has lots of transmembrane helices 518 00:29:35,960 --> 00:29:39,770 and is stuck in the ER. 519 00:29:39,770 --> 00:29:42,110 So what do we know about gp78? 520 00:29:42,110 --> 00:29:46,140 And again, you see these cartoons that Liz has used 521 00:29:46,140 --> 00:29:48,950 and I've been using, since we really 522 00:29:48,950 --> 00:29:53,510 know nothing about the detailed structures of these systems. 523 00:29:53,510 --> 00:29:58,250 What we know is, at the N-terminus, 524 00:29:58,250 --> 00:30:02,270 we have an Insig binding site. 525 00:30:08,930 --> 00:30:10,573 And so people had to study that. 526 00:30:10,573 --> 00:30:11,740 And how did they study that? 527 00:30:11,740 --> 00:30:15,160 Probably by mechanisms similar to what you had to-- 528 00:30:15,160 --> 00:30:19,990 what you thought about looking at problem set seven. 529 00:30:19,990 --> 00:30:26,620 It turns out that gp78 is a ubiquitin ligase, 530 00:30:26,620 --> 00:30:28,450 so it's an E3. 531 00:30:28,450 --> 00:30:36,370 So this is an E3 ubiquitin ligase. 532 00:30:36,370 --> 00:30:40,870 So this is-- gp78 is an E3 ubiquitin ligase. 533 00:30:40,870 --> 00:30:44,800 It has a RING domain. 534 00:30:44,800 --> 00:30:46,960 Remember we said there were little domains that 535 00:30:46,960 --> 00:30:49,870 alter the way you stick the ubiquitin on. 536 00:30:49,870 --> 00:30:52,720 Again, we don't know the details about this. 537 00:30:52,720 --> 00:30:59,170 It has another little domain called Ubc7. 538 00:30:59,170 --> 00:31:00,880 We're really into acronym worlds. 539 00:31:00,880 --> 00:31:04,960 But what you need to know is that this is an E2-conjugating 540 00:31:04,960 --> 00:31:06,200 enzyme. 541 00:31:06,200 --> 00:31:09,160 So what you have now is an E3, they can bind an E2. 542 00:31:09,160 --> 00:31:14,380 That's the cartoons we just went through over here, E3 binding 543 00:31:14,380 --> 00:31:15,070 to E2. 544 00:31:15,070 --> 00:31:16,835 E3 is the gp78. 545 00:31:16,835 --> 00:31:21,430 E2 is this little protein domain. 546 00:31:21,430 --> 00:31:24,190 And I think what's really interesting 547 00:31:24,190 --> 00:31:29,530 about this protein is it has another little domain called 548 00:31:29,530 --> 00:31:31,180 VPC. 549 00:31:31,180 --> 00:31:32,275 And this is an ATPase. 550 00:31:38,420 --> 00:31:41,930 And if you think about this, if you want to target something 551 00:31:41,930 --> 00:31:48,260 for degradation, where is the proteosome located 552 00:31:48,260 --> 00:31:49,900 that we've been talking about? 553 00:31:49,900 --> 00:31:52,130 Where is it located in the cell? 554 00:31:52,130 --> 00:31:53,970 Actually, there are multiple proteosomes, 555 00:31:53,970 --> 00:31:57,698 but the ones we've been focused on, where is it located? 556 00:31:57,698 --> 00:31:58,650 AUDIENCE: Cytosol. 557 00:31:58,650 --> 00:32:03,030 JOANNE STUBBE: Yeah, cytosol, so this is a membrane protein. 558 00:32:03,030 --> 00:32:08,520 So how do you get this membrane protein into the proteosome? 559 00:32:08,520 --> 00:32:10,220 OK, that's not trivial. 560 00:32:10,220 --> 00:32:13,280 And this protein, this VPC domain, 561 00:32:13,280 --> 00:32:17,180 uses energy somehow to pull this out of the membrane 562 00:32:17,180 --> 00:32:20,340 so it can get degraded in the proteosome. 563 00:32:30,700 --> 00:32:43,310 So the VPC domain, well, pulls HMGR out of membrane. 564 00:32:47,680 --> 00:33:01,750 And so it gets degraded in the cytosol by the proteosome, 565 00:33:01,750 --> 00:33:04,590 complicated, actually quite interesting-- yeah? 566 00:33:04,590 --> 00:33:07,200 AUDIENCE: Is it at all understood how that pulling out 567 00:33:07,200 --> 00:33:07,910 happens? 568 00:33:07,910 --> 00:33:10,400 JOANNE STUBBE: I don't-- you know, maybe, I don't know how. 569 00:33:10,400 --> 00:33:12,025 I haven't found anything, but I haven't 570 00:33:12,025 --> 00:33:15,350 looked through the literature of any of this, the details. 571 00:33:15,350 --> 00:33:20,190 My guess is the answer is no, but you can go look it up. 572 00:33:20,190 --> 00:33:21,740 And one of the questions you can ask 573 00:33:21,740 --> 00:33:25,010 is how frequent does that happen? 574 00:33:25,010 --> 00:33:26,540 How often do you want to degrade-- 575 00:33:26,540 --> 00:33:30,110 do you have this domain, and how often is that domain used? 576 00:33:30,110 --> 00:33:32,420 And what are the characteristics of that domain? 577 00:33:32,420 --> 00:33:33,710 Probably a lot more is known. 578 00:33:33,710 --> 00:33:35,502 I don't really know off the top of my head. 579 00:33:37,880 --> 00:33:40,130 So this is a cartoon model. 580 00:33:40,130 --> 00:33:42,970 And so I'm not going to draw the model out. 581 00:33:42,970 --> 00:33:49,150 So I'll say the model, you can just see your PowerPoint. 582 00:33:49,150 --> 00:33:52,288 OK, and so this is the same kind of cartoon 583 00:33:52,288 --> 00:33:53,830 we've been using over and over again. 584 00:33:53,830 --> 00:33:56,050 So Insig is the center guy. 585 00:33:56,050 --> 00:33:59,470 Insig interrupts with SCAP and cholesterol 586 00:33:59,470 --> 00:34:05,230 to keep SREBP in the ER membrane so you don't activate 587 00:34:05,230 --> 00:34:08,440 transcription of cholesterol biosynthesis or the LDL 588 00:34:08,440 --> 00:34:08,949 receptor. 589 00:34:08,949 --> 00:34:11,179 We spent a lot of time on this. 590 00:34:11,179 --> 00:34:14,830 So here, Insig is here again. 591 00:34:14,830 --> 00:34:19,120 And it interacts with gp78, which 592 00:34:19,120 --> 00:34:24,400 interacts with these other two proteins, the E2 593 00:34:24,400 --> 00:34:28,960 and whatever this protein is that helps extract it 594 00:34:28,960 --> 00:34:30,550 from the membrane. 595 00:34:30,550 --> 00:34:34,630 A key player in all of this is lanosterol. 596 00:34:34,630 --> 00:34:38,380 You have lanosterol in the membranes. 597 00:34:38,380 --> 00:34:41,320 So you could do, potentially, a similar study that we talked 598 00:34:41,320 --> 00:34:45,870 about in recitation this past week to look at 599 00:34:45,870 --> 00:34:47,860 do you see a switch with lanosterol, what are 600 00:34:47,860 --> 00:34:49,540 the lanosterol concentrations? 601 00:34:49,540 --> 00:34:53,260 What are the concentrations of lanosterol? 602 00:34:53,260 --> 00:34:55,870 And this is a cartoon showing this. 603 00:34:55,870 --> 00:34:58,390 I have no idea about the details of this cartoon, 604 00:34:58,390 --> 00:35:01,390 but what you're going to do then is 605 00:35:01,390 --> 00:35:08,240 attach the ubiquitin using this E2-E3 machinery 606 00:35:08,240 --> 00:35:11,740 onto HMG-CoA Reductase. 607 00:35:11,740 --> 00:35:13,460 And remember, that protein-- 608 00:35:13,460 --> 00:35:16,090 we've looked at that now a number of times-- 609 00:35:16,090 --> 00:35:22,270 has a steroid-- sterol-sensor domain, which is lanosterol. 610 00:35:22,270 --> 00:35:24,430 And it also has a cytoplasmic face. 611 00:35:24,430 --> 00:35:27,100 That's the HMG-CoA Reductase. 612 00:35:27,100 --> 00:35:28,670 You can cut this off. 613 00:35:28,670 --> 00:35:29,770 It's also active. 614 00:35:29,770 --> 00:35:32,410 And we've talked about that a number of times. 615 00:35:32,410 --> 00:35:35,590 And so what they have here is just a cartoon of attaching 616 00:35:35,590 --> 00:35:39,250 ubiquitin, which then, in the end, magic, 617 00:35:39,250 --> 00:35:44,450 you end up with degradation of your membrane-bound system. 618 00:35:44,450 --> 00:35:49,960 So this is a major mechanism of regulation involving 619 00:35:49,960 --> 00:35:52,150 cholesterol homeostasis. 620 00:35:52,150 --> 00:35:55,120 But what you see when you look at the problem set 621 00:35:55,120 --> 00:35:58,340 that I've given you is that it's more complicated than that. 622 00:35:58,340 --> 00:36:01,660 So you can knock out genes and you still get it degraded. 623 00:36:01,660 --> 00:36:02,830 What is the timescale? 624 00:36:02,830 --> 00:36:04,360 How do you do the experiments? 625 00:36:04,360 --> 00:36:07,090 And I think that's what people are seeing 626 00:36:07,090 --> 00:36:09,220 with all of these things. 627 00:36:09,220 --> 00:36:12,340 And in part, it becomes complicated 628 00:36:12,340 --> 00:36:15,070 because, if these proteins need to be modified in some way, 629 00:36:15,070 --> 00:36:18,010 it's not so easy to tell whether they've been modified, 630 00:36:18,010 --> 00:36:23,660 and what it is that is recognized by the E3 ligase. 631 00:36:23,660 --> 00:36:27,330 OK, so I think this is sort of an exciting and interesting 632 00:36:27,330 --> 00:36:27,830 area. 633 00:36:27,830 --> 00:36:30,400 And we need some new breakthroughs 634 00:36:30,400 --> 00:36:35,560 so that we can better understand how these degradation 635 00:36:35,560 --> 00:36:40,760 systems are integrated into regulation in general. 636 00:36:40,760 --> 00:36:46,160 So that's just a summary of the role of Insig, 637 00:36:46,160 --> 00:36:48,070 in the presence of cholesterol-- 638 00:36:48,070 --> 00:36:53,590 or lanosterol, in keeping the levels of cholesterol low. 639 00:36:53,590 --> 00:36:59,200 OK, so we finished the section on cholesterol. 640 00:36:59,200 --> 00:37:01,330 I think I've introduced you to a lot 641 00:37:01,330 --> 00:37:03,460 of different kinds of concepts. 642 00:37:03,460 --> 00:37:08,950 I've told you how important it is in terms of therapeutics. 643 00:37:08,950 --> 00:37:12,610 People are continually studying this, 644 00:37:12,610 --> 00:37:15,940 as you saw by the news article that Liz 645 00:37:15,940 --> 00:37:18,010 had given me last time. 646 00:37:18,010 --> 00:37:22,210 We have this PCSK9 that's in clinical trials, 647 00:37:22,210 --> 00:37:23,710 in addition to the statins. 648 00:37:23,710 --> 00:37:27,610 And I think it's going to be on people's radar screens 649 00:37:27,610 --> 00:37:29,530 for some time to come. 650 00:37:29,530 --> 00:37:34,180 So I think cholesterol is cool because 651 00:37:34,180 --> 00:37:37,740 of the spectacular discoveries of receptor-mediated 652 00:37:37,740 --> 00:37:41,620 endocytosis of transcription factors 653 00:37:41,620 --> 00:37:44,020 that are found in the ER as opposed 654 00:37:44,020 --> 00:37:45,940 to being found in the nucleus. 655 00:37:45,940 --> 00:37:50,440 And we've also introduced you to another generic mechanism 656 00:37:50,440 --> 00:37:55,570 of control, that by protein-mediated degradation. 657 00:37:55,570 --> 00:38:03,200 So that's the end of Module 5. 658 00:38:03,200 --> 00:38:05,000 And what I'm going to do now-- 659 00:38:05,000 --> 00:38:09,110 and we've posted this information. 660 00:38:09,110 --> 00:38:11,230 Again, the information will always 661 00:38:11,230 --> 00:38:15,850 be posted ahead of class so that you can actually 662 00:38:15,850 --> 00:38:18,370 have the PowerPoints out there. 663 00:38:18,370 --> 00:38:21,490 Some things, I'm not going to write down. 664 00:38:21,490 --> 00:38:24,840 In this section, there is a lot more phenomenology. 665 00:38:24,840 --> 00:38:27,490 What I'll try to do is give you an overview 666 00:38:27,490 --> 00:38:29,710 of why I've picked this phenomenology, 667 00:38:29,710 --> 00:38:32,170 but I'm not going to write down-- it takes a long time 668 00:38:32,170 --> 00:38:34,360 to write down all of the phenomenology 669 00:38:34,360 --> 00:38:35,110 on the blackboard. 670 00:38:35,110 --> 00:38:36,318 And I'm not going to do that. 671 00:38:36,318 --> 00:38:39,520 So integrating your notes of the things 672 00:38:39,520 --> 00:38:42,730 I'm going to write down with your PowerPoint, I think, 673 00:38:42,730 --> 00:38:44,960 is really important for you to do. 674 00:38:44,960 --> 00:38:47,455 And I would suggest that you bring the PowerPoint 675 00:38:47,455 --> 00:38:49,060 so you can see what's written down 676 00:38:49,060 --> 00:38:52,090 and where you might want to stick in a piece of paper 677 00:38:52,090 --> 00:38:54,580 where I expand on something or really tell you 678 00:38:54,580 --> 00:38:57,460 something in much more detail than what's 679 00:38:57,460 --> 00:38:59,620 written in the PowerPoint. 680 00:38:59,620 --> 00:39:05,650 So Module 6, so as I just told you at the very beginning, 681 00:39:05,650 --> 00:39:08,590 these modules are not really linked together 682 00:39:08,590 --> 00:39:10,900 except through thinking about homeostasis. 683 00:39:10,900 --> 00:39:13,270 Everything in the cell is homeostasis. 684 00:39:15,595 --> 00:39:16,970 In the first lecture, we're going 685 00:39:16,970 --> 00:39:21,320 to be talking about metals and metal homeostasis in general 686 00:39:21,320 --> 00:39:23,140 using the periodic table. 687 00:39:23,140 --> 00:39:26,920 OK, but then what I'm going to do is focus on a single metal. 688 00:39:26,920 --> 00:39:31,240 And the single metal I'm going to be focused on is iron. 689 00:39:31,240 --> 00:39:36,190 And so the reading is also posted. 690 00:39:36,190 --> 00:39:38,440 And there are three things for you to read. 691 00:39:38,440 --> 00:39:44,330 One is to think about iron in the geochemical world. 692 00:39:44,330 --> 00:39:46,300 You know, why is iron so important? 693 00:39:46,300 --> 00:39:48,160 If you look at the periodic table, 694 00:39:48,160 --> 00:39:50,020 why aren't we using aluminum? 695 00:39:50,020 --> 00:39:53,100 It's the most abundant in the earth's crust. 696 00:39:53,100 --> 00:39:56,630 OK well using iron and not aluminum? 697 00:39:56,630 --> 00:39:59,980 Well, as chemists, we ought to be able to think about that. 698 00:39:59,980 --> 00:40:01,450 Silicon is the other thing that's 699 00:40:01,450 --> 00:40:03,910 one of the most abundant things in the earth's crust. 700 00:40:03,910 --> 00:40:08,320 Why aren't we using silica and aluminum as life-- 701 00:40:08,320 --> 00:40:09,820 as the basis for life? 702 00:40:09,820 --> 00:40:11,860 And this article, I think it's very 703 00:40:11,860 --> 00:40:13,750 interesting from a chemical perspective 704 00:40:13,750 --> 00:40:16,670 telling you about how to think about these kinds of things. 705 00:40:16,670 --> 00:40:17,870 Why is it true? 706 00:40:17,870 --> 00:40:20,260 And I'll give you a little bit of background on that. 707 00:40:20,260 --> 00:40:22,120 And then you can do as much or as little 708 00:40:22,120 --> 00:40:26,620 thinking about it as you choose. 709 00:40:26,620 --> 00:40:29,500 So the first one, I'm just going to give you 710 00:40:29,500 --> 00:40:34,930 an overview of why metals are so darn important 711 00:40:34,930 --> 00:40:37,300 and try to convince you that you should all 712 00:40:37,300 --> 00:40:40,400 know a lot more about metals than probably most of you 713 00:40:40,400 --> 00:40:44,320 have thought about from an introductory course. 714 00:40:44,320 --> 00:40:48,670 Then in Lecture 2, we're going to talk about metal homeostasis 715 00:40:48,670 --> 00:40:50,290 in general. 716 00:40:50,290 --> 00:40:51,700 And that's going to be-- 717 00:40:51,700 --> 00:40:53,797 that could be applied to any of the metals 718 00:40:53,797 --> 00:40:55,630 I'm going to show you in the periodic table, 719 00:40:55,630 --> 00:40:58,528 but I'm going to focus on iron. 720 00:40:58,528 --> 00:41:00,070 And then in the second lecture, we're 721 00:41:00,070 --> 00:41:06,580 going to focus on iron homeostasis in humans. 722 00:41:06,580 --> 00:41:09,010 And we're going to look at iron transport 723 00:41:09,010 --> 00:41:11,190 from the diet, where we heard this from. 724 00:41:11,190 --> 00:41:13,530 How does it get taken into the cell? 725 00:41:13,530 --> 00:41:16,030 It can get taken into the cell-- we'll see a number of ways. 726 00:41:16,030 --> 00:41:17,447 But receptor-mediated endocytosis, 727 00:41:17,447 --> 00:41:19,390 and they told us where have we seen that? 728 00:41:22,620 --> 00:41:26,350 There is a protein that allows iron to be transferred around. 729 00:41:26,350 --> 00:41:27,920 Just like with cholesterol, you had 730 00:41:27,920 --> 00:41:31,905 to figure out how to keep this insoluble thing soluble 731 00:41:31,905 --> 00:41:34,530 with-- we're going to see there is a lot of problems with iron, 732 00:41:34,530 --> 00:41:37,780 so we need to figure out how to control iron's chemical 733 00:41:37,780 --> 00:41:38,630 reactivity. 734 00:41:38,630 --> 00:41:41,380 So we use a protein to do that. 735 00:41:41,380 --> 00:41:42,460 There is a transferrin. 736 00:41:42,460 --> 00:41:44,380 It's a little protein called transferrin. 737 00:41:44,380 --> 00:41:46,780 There is a transferrin receptor. 738 00:41:46,780 --> 00:41:47,780 We'll talk about that. 739 00:41:47,780 --> 00:41:55,360 And then there are many levels at which iron is regulated. 740 00:41:55,360 --> 00:41:57,220 Probably the most important regulation 741 00:41:57,220 --> 00:42:00,840 is a peptide hormone that I'll briefly mention, 742 00:42:00,840 --> 00:42:02,590 but that's not what I'm going to focus on. 743 00:42:02,590 --> 00:42:04,480 What I'm going to focus on is a new kind 744 00:42:04,480 --> 00:42:09,130 of regulation based on regulation 745 00:42:09,130 --> 00:42:14,500 of the translational process and proteins binding to RNA. 746 00:42:14,500 --> 00:42:18,110 And right now, that's a very active area of research here. 747 00:42:18,110 --> 00:42:20,230 It doesn't have to be a protein binding to RNA, 748 00:42:20,230 --> 00:42:22,240 but small molecules binding to RNA. 749 00:42:22,240 --> 00:42:25,610 Riboswitches are being found all over the place. 750 00:42:25,610 --> 00:42:31,510 And so I'm going to introduce you to translational control 751 00:42:31,510 --> 00:42:33,190 by proteins binding to RNA. 752 00:42:33,190 --> 00:42:37,120 And then the third and fourth lectures 753 00:42:37,120 --> 00:42:40,300 are going to be focused on more on bacteria. 754 00:42:40,300 --> 00:42:43,030 We know a lot about bacterial systems. 755 00:42:47,440 --> 00:42:51,670 Almost all bacteria require iron to survive. 756 00:42:51,670 --> 00:42:54,010 And Liz is the expert, so she can 757 00:42:54,010 --> 00:42:56,170 correct anything I say incorrectly 758 00:42:56,170 --> 00:42:57,100 during this lecture. 759 00:43:00,460 --> 00:43:02,980 Where did bacteria get their ion from? 760 00:43:02,980 --> 00:43:06,860 Some bacteria get their iron from rocks. 761 00:43:06,860 --> 00:43:09,680 How the heck do you get iron out of a rock? 762 00:43:09,680 --> 00:43:12,200 OK, well, bacteria have figured that out. 763 00:43:12,200 --> 00:43:14,420 We on the other hand are way up here. 764 00:43:14,420 --> 00:43:15,720 We can eat bacteria. 765 00:43:15,720 --> 00:43:16,740 We can eat plants. 766 00:43:16,740 --> 00:43:19,010 They've already figured out how to get the iron out 767 00:43:19,010 --> 00:43:20,100 of the rocks. 768 00:43:20,100 --> 00:43:22,730 And so our problem is much easier. 769 00:43:22,730 --> 00:43:25,400 But so bacteria are amazingly creative. 770 00:43:25,400 --> 00:43:28,610 And I've just chosen one of the creative ways 771 00:43:28,610 --> 00:43:32,852 to look at how iron is obtained. 772 00:43:32,852 --> 00:43:34,310 So we're going to talk a little bit 773 00:43:34,310 --> 00:43:36,620 about the host-pathogen battle. 774 00:43:36,620 --> 00:43:38,390 And I'm going to use specifically 775 00:43:38,390 --> 00:43:41,570 Staphylococcus aureus as an example because 776 00:43:41,570 --> 00:43:46,550 of the resistance problems we currently have in the clinic. 777 00:43:46,550 --> 00:43:48,110 You can get an iron in many forms. 778 00:43:48,110 --> 00:43:49,910 We're going to focus on getting iron 779 00:43:49,910 --> 00:43:53,630 in the form of heme, which is a major source of iron 780 00:43:53,630 --> 00:43:54,590 for this organism. 781 00:43:54,590 --> 00:43:56,427 OK, so that's where we're going. 782 00:43:56,427 --> 00:43:58,010 Will we get finished in four lectures? 783 00:43:58,010 --> 00:44:00,390 Probably not. 784 00:44:00,390 --> 00:44:03,110 Anyhow, so what I'm going to do today 785 00:44:03,110 --> 00:44:05,900 is the first five or six slides of PowerPoint. 786 00:44:05,900 --> 00:44:08,330 And it's more phenomenology. 787 00:44:08,330 --> 00:44:11,750 And then we'll get into it, the more details, 788 00:44:11,750 --> 00:44:13,130 in the next lecture. 789 00:44:13,130 --> 00:44:16,460 So here is the bottom bottle that-- do any of you 790 00:44:16,460 --> 00:44:18,190 take Flintstone vitamins? 791 00:44:18,190 --> 00:44:20,100 Anyway, I'm not supposed to digress. 792 00:44:20,100 --> 00:44:22,400 I can't swallow vitamins, though I like them 793 00:44:22,400 --> 00:44:23,750 because they taste good. 794 00:44:23,750 --> 00:44:25,376 AUDIENCE: [INAUDIBLE] 795 00:44:25,376 --> 00:44:26,185 JOANNE STUBBE: Huh? 796 00:44:26,185 --> 00:44:27,250 AUDIENCE: When we were little. 797 00:44:27,250 --> 00:44:29,125 JOANNE STUBBE: Do you remember-- does anybody 798 00:44:29,125 --> 00:44:31,130 remember who this guy is? 799 00:44:31,130 --> 00:44:31,720 No, OK-- 800 00:44:31,720 --> 00:44:32,270 AUDIENCE: [INAUDIBLE] 801 00:44:32,270 --> 00:44:33,950 JOANNE STUBBE: Oh yeah, all right, so [INAUDIBLE] Fred. 802 00:44:33,950 --> 00:44:36,530 OK, well you know, I always have this generation-- 803 00:44:36,530 --> 00:44:39,350 I'm much older than you. 804 00:44:39,350 --> 00:44:41,290 So anyhow, I mean, what you learned about 805 00:44:41,290 --> 00:44:44,660 in the introductory course 5.07 is, 806 00:44:44,660 --> 00:44:46,880 you learned a lot about the vitamin bottle, 807 00:44:46,880 --> 00:44:50,000 really, how the vitamins that you have, vitamin A, vitamin 808 00:44:50,000 --> 00:44:53,540 C, vitamin, all the vitamin Bs, et cetera, what they do 809 00:44:53,540 --> 00:44:56,240 is greatly expand the repertoire of reactions 810 00:44:56,240 --> 00:45:00,620 that enzymes can catalyze in all your metabolic pathways. 811 00:45:00,620 --> 00:45:04,220 What you don't learn about in most introductory courses 812 00:45:04,220 --> 00:45:05,940 is the minerals. 813 00:45:05,940 --> 00:45:07,770 OK, so they're on the bottle too, 814 00:45:07,770 --> 00:45:10,880 but you sort of ignore all of that stuff. 815 00:45:10,880 --> 00:45:12,730 And you know, you need iron. 816 00:45:12,730 --> 00:45:13,550 You need copper. 817 00:45:13,550 --> 00:45:15,470 You need calcium. 818 00:45:15,470 --> 00:45:17,570 You need zinc, et cetera. 819 00:45:17,570 --> 00:45:19,730 And so what I want to do is to try 820 00:45:19,730 --> 00:45:23,180 to give you very briefly an overview of why 821 00:45:23,180 --> 00:45:25,730 these metals are so important. 822 00:45:25,730 --> 00:45:29,120 And again, the focus is going to be on iron. 823 00:45:32,070 --> 00:45:35,140 OK so here is our periodic table. 824 00:45:35,140 --> 00:45:42,090 And these are the metals that are found inside of us-- 825 00:45:42,090 --> 00:45:44,820 yeah, I guess maybe. 826 00:45:44,820 --> 00:45:45,900 We don't have tungsten. 827 00:45:45,900 --> 00:45:47,070 Liz, do we have tungsten? 828 00:45:47,070 --> 00:45:49,110 We don't have tungsten. 829 00:45:49,110 --> 00:45:50,740 I don't think we have tungsten in us. 830 00:45:50,740 --> 00:45:55,050 So these are found in bacteria and us. 831 00:45:55,050 --> 00:45:58,290 And so if you look at this, all of these guys over here, 832 00:45:58,290 --> 00:45:59,790 where have we seen magnesium before? 833 00:45:59,790 --> 00:46:01,832 I've been talking about that over and over again. 834 00:46:01,832 --> 00:46:03,510 Magnesium is bound to all nucleotides. 835 00:46:03,510 --> 00:46:04,960 We're going to see this again and again. 836 00:46:04,960 --> 00:46:07,502 We're going to talk about-- a little bit about the proper use 837 00:46:07,502 --> 00:46:11,940 of magnesium which makes it function in that capacity 838 00:46:11,940 --> 00:46:15,330 to neutralize the charge on nucleotides. 839 00:46:15,330 --> 00:46:20,030 Sodium, and potassium, and iron, conduction-- calcium 840 00:46:20,030 --> 00:46:22,080 is involved in signaling. 841 00:46:22,080 --> 00:46:24,390 But what we're going to be focusing on 842 00:46:24,390 --> 00:46:26,390 are the transition metals. 843 00:46:26,390 --> 00:46:28,650 OK, and specifically within the transition 844 00:46:28,650 --> 00:46:33,960 metals, what we're going to be focusing on is iron. 845 00:46:33,960 --> 00:46:36,900 And this is-- it's hard to measure 846 00:46:36,900 --> 00:46:41,760 the concentrations in their localizations within the cell. 847 00:46:41,760 --> 00:46:44,430 But you can measure the total concentration 848 00:46:44,430 --> 00:46:46,890 by just taking your cell and then 849 00:46:46,890 --> 00:46:49,860 submitting it to some kind of mass spec analysis. 850 00:46:49,860 --> 00:46:53,580 We can see iron versus manganese. 851 00:46:53,580 --> 00:46:56,700 And we're going to, again, be focused on iron, 852 00:46:56,700 --> 00:46:59,670 which accounts for about 8%. 853 00:46:59,670 --> 00:47:04,500 And it's been estimated in this article that approximately 50% 854 00:47:04,500 --> 00:47:07,930 of all the proteins have some kind of metal bound. 855 00:47:07,930 --> 00:47:09,770 OK, it might involved in catalysis. 856 00:47:09,770 --> 00:47:10,380 It might not. 857 00:47:10,380 --> 00:47:15,300 In fact, the metals more likely are not involved in catalysis. 858 00:47:15,300 --> 00:47:17,340 And we'll look at that distribution. 859 00:47:17,340 --> 00:47:20,100 So we'll come back to this a couple of times, 860 00:47:20,100 --> 00:47:23,250 but we're going to be focusing over here. 861 00:47:23,250 --> 00:47:25,860 And what are the properties of metals 862 00:47:25,860 --> 00:47:28,860 that make them so special to increase 863 00:47:28,860 --> 00:47:32,970 the repertoire of reactions that can 864 00:47:32,970 --> 00:47:34,800 be catalyzed inside our bodies? 865 00:47:34,800 --> 00:47:37,050 OK, so these guys are unique from a lot 866 00:47:37,050 --> 00:47:39,750 of the reactions you've already studied 867 00:47:39,750 --> 00:47:42,300 in your introductory biochemistry course. 868 00:47:42,300 --> 00:47:44,940 And so what I want to do is sort of just give you 869 00:47:44,940 --> 00:47:47,580 a general overview of where you see 870 00:47:47,580 --> 00:47:49,740 metals involved in catalysis. 871 00:47:49,740 --> 00:47:51,620 And then we're going to focus on iron only. 872 00:47:54,690 --> 00:47:59,070 OK, so where do we see catalysis? 873 00:47:59,070 --> 00:48:00,450 We see iron transport. 874 00:48:00,450 --> 00:48:05,410 We need to get iron, potassium, and sodium in the right places, 875 00:48:05,410 --> 00:48:06,930 or we're in trouble. 876 00:48:06,930 --> 00:48:10,330 Signaling-- signal transduction, we use calcium all the time. 877 00:48:10,330 --> 00:48:14,220 There is huge numbers of people studying calcium signaling. 878 00:48:14,220 --> 00:48:16,380 Where have you seen oxygen transport? 879 00:48:16,380 --> 00:48:20,340 In us-- we're in serious trouble if oxygen 880 00:48:20,340 --> 00:48:24,260 can't be carried by our hemoglobin to our tissues. 881 00:48:24,260 --> 00:48:26,010 And I'll show you a little bit about that. 882 00:48:26,010 --> 00:48:29,190 So oxygen transport is really important. 883 00:48:29,190 --> 00:48:32,790 Of central importance is electron 884 00:48:32,790 --> 00:48:36,720 transfer and proton-coupled electron transfer. 885 00:48:36,720 --> 00:48:38,190 Where have you seen that? 886 00:48:38,190 --> 00:48:40,420 You've seen that in the respiratory chain. 887 00:48:40,420 --> 00:48:43,430 If you go back and you look at complex I, complex II, complex 888 00:48:43,430 --> 00:48:45,600 III, you see all these metals in there. 889 00:48:45,600 --> 00:48:47,360 What are they doing? 890 00:48:47,360 --> 00:48:49,500 They're doing electron transfer reactions. 891 00:48:49,500 --> 00:48:52,920 We'll talk a little bit, but not much, about that. 892 00:48:52,920 --> 00:48:58,860 So not only is electron transfer involved in respiration. 893 00:48:58,860 --> 00:49:01,590 Electron transfer plays a central role 894 00:49:01,590 --> 00:49:04,590 in all of the environmental chemistry. 895 00:49:04,590 --> 00:49:07,260 And so while, in many introductory classes, 896 00:49:07,260 --> 00:49:08,940 they don't talk about this-- 897 00:49:08,940 --> 00:49:12,120 we talk about humans, because most people are 898 00:49:12,120 --> 00:49:15,420 more interested in disease-- 899 00:49:15,420 --> 00:49:18,300 the coolest chemistry, without question, hands down, 900 00:49:18,300 --> 00:49:21,520 is absolutely associated with the bacteria and the Archae. 901 00:49:21,520 --> 00:49:23,880 OK, they do, like, amazing things. 902 00:49:23,880 --> 00:49:27,690 How do you take nitrogen and do an eight-electron reduction 903 00:49:27,690 --> 00:49:29,340 to ammonia? 904 00:49:29,340 --> 00:49:31,050 How do we do that as chemists? 905 00:49:31,050 --> 00:49:36,090 200 atmospheres pressure in a 400 degrees. 906 00:49:36,090 --> 00:49:38,010 This is an incredibly important reaction. 907 00:49:38,010 --> 00:49:41,240 Where does all the nitrogen from our amino acids come from? 908 00:49:41,240 --> 00:49:43,140 What about our nucleic acids? 909 00:49:43,140 --> 00:49:44,617 And we skip all this stuff. 910 00:49:44,617 --> 00:49:47,200 This is like-- I mean, this, to me, is sort of, like, amazing. 911 00:49:47,200 --> 00:49:49,620 Another thing we skip all the time 912 00:49:49,620 --> 00:49:52,710 is where does oxygen, how does oxygen-- 913 00:49:52,710 --> 00:49:56,880 how does light take water and make oxygen gas? 914 00:49:56,880 --> 00:49:59,430 Without that, we'd be in serious trouble. 915 00:49:59,430 --> 00:50:04,070 The bacteria would definitely be taking over the world. 916 00:50:04,070 --> 00:50:07,050 And this, I'll show you, is sort of an amazing reaction-- 917 00:50:07,050 --> 00:50:08,290 nucleotide reduction. 918 00:50:08,290 --> 00:50:11,130 We may never get there, but the last module 919 00:50:11,130 --> 00:50:15,960 is, I'm going to show you, you're making deoxynucleotides. 920 00:50:15,960 --> 00:50:21,180 The enzymes can use manganese, iron, cobalt, and iron sulfur. 921 00:50:21,180 --> 00:50:23,820 So they use a wide range of metals 922 00:50:23,820 --> 00:50:26,510 to make the building blocks required for DNA. 923 00:50:26,510 --> 00:50:28,260 OK, signaling, we've all-- 924 00:50:28,260 --> 00:50:31,590 I just talked about calcium as a signaling agent. 925 00:50:31,590 --> 00:50:34,680 But now it's becoming clear, because of studies 926 00:50:34,680 --> 00:50:37,170 from the lipid group, and studies 927 00:50:37,170 --> 00:50:39,480 from Chris Chang who is a former lipid group 928 00:50:39,480 --> 00:50:44,010 member, signaling of metals is much more common 929 00:50:44,010 --> 00:50:45,480 than we thought. 930 00:50:45,480 --> 00:50:47,850 And people are proposing that, not only 931 00:50:47,850 --> 00:50:52,020 is zinc a signaling agent, but also copper. 932 00:50:52,020 --> 00:50:53,700 And there is a lot of problems in nerve 933 00:50:53,700 --> 00:50:55,710 cells with oxidative damage which 934 00:50:55,710 --> 00:50:57,250 we're going to come back to. 935 00:50:57,250 --> 00:51:01,650 So thinking about the levels and sensing of these levels I think 936 00:51:01,650 --> 00:51:03,750 is going to be a future area that's going 937 00:51:03,750 --> 00:51:06,760 to be very exciting to study. 938 00:51:06,760 --> 00:51:09,000 You have to regulate these metals. 939 00:51:09,000 --> 00:51:10,950 Transcriptional, translational levels, 940 00:51:10,950 --> 00:51:12,750 we're going to talk about. 941 00:51:12,750 --> 00:51:16,780 And they're involved in many kinds of catalysis. 942 00:51:16,780 --> 00:51:19,380 So let me just close by showing you 943 00:51:19,380 --> 00:51:23,670 one last slide, oxygen carriers. 944 00:51:23,670 --> 00:51:25,500 You've seen this before. 945 00:51:25,500 --> 00:51:26,760 That's hemoglobin. 946 00:51:26,760 --> 00:51:28,770 You've all studied, hopefully, hemoglobin 947 00:51:28,770 --> 00:51:31,500 and the cooperative binding of oxygen, how it binds, 948 00:51:31,500 --> 00:51:35,670 how it's released-- sort of an amazing machine. 949 00:51:35,670 --> 00:51:38,490 That's not the only way that organisms 950 00:51:38,490 --> 00:51:43,080 reversibly bind oxygen. This guy, the horseshoe crab, 951 00:51:43,080 --> 00:51:46,000 it uses copper. 952 00:51:46,000 --> 00:51:48,220 This guy-- these are worms. 953 00:51:48,220 --> 00:51:49,600 These are found in-- 954 00:51:49,600 --> 00:51:51,575 they're found in the sea, right? 955 00:51:51,575 --> 00:51:53,620 So you go to Woods Hole and they'll 956 00:51:53,620 --> 00:51:55,030 extract these worms for you. 957 00:51:55,030 --> 00:51:56,950 Anyhow, what do they have? 958 00:51:56,950 --> 00:51:59,140 They have a diiron cluster. 959 00:51:59,140 --> 00:52:01,450 And the strategies of both-- they all 960 00:52:01,450 --> 00:52:04,360 have to reversibly bind oxygen. And they've all 961 00:52:04,360 --> 00:52:06,580 adapted to their environments to be 962 00:52:06,580 --> 00:52:09,500 able to do this in an efficient way. 963 00:52:09,500 --> 00:52:12,220 So what I'll do next time is come back and-- 964 00:52:12,220 --> 00:52:13,780 let me just do one more thing. 965 00:52:13,780 --> 00:52:15,730 Anyhow, this is-- think about this. 966 00:52:15,730 --> 00:52:16,840 Put it on under pillow. 967 00:52:16,840 --> 00:52:17,950 Think about how it works. 968 00:52:17,950 --> 00:52:18,550 Look at this. 969 00:52:18,550 --> 00:52:22,300 This is the cofactor of nitrogenase. 970 00:52:22,300 --> 00:52:24,400 Not only does it have iron and molybdenum, 971 00:52:24,400 --> 00:52:26,320 but look at that guy in the center-- 972 00:52:26,320 --> 00:52:30,640 carbon, carbon 4 minus. 973 00:52:30,640 --> 00:52:31,600 Think about that. 974 00:52:31,600 --> 00:52:34,080 We'll come back next time.