1 00:00:00,500 --> 00:00:02,830 The following content is provided under a Creative 2 00:00:02,830 --> 00:00:04,370 Commons license. 3 00:00:04,370 --> 00:00:06,670 Your support will help MIT OpenCourseWare 4 00:00:06,670 --> 00:00:11,030 continue to offer high quality educational resources for free. 5 00:00:11,030 --> 00:00:13,660 To make a donation or view additional materials 6 00:00:13,660 --> 00:00:17,610 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,610 --> 00:00:18,540 at ocw.mit.edu. 8 00:00:25,462 --> 00:00:27,170 JOANNE STUBBE: So what I want to do today 9 00:00:27,170 --> 00:00:30,620 is continue where we left off to try 10 00:00:30,620 --> 00:00:37,570 to get further in into this module on regulation of iron 11 00:00:37,570 --> 00:00:39,670 right now in terms of humans. 12 00:00:39,670 --> 00:00:43,450 And we're talking about the fact that regulation occurs 13 00:00:43,450 --> 00:00:45,520 at the translational level. 14 00:00:45,520 --> 00:00:47,740 And so I'm going to introduce to you the model. 15 00:00:47,740 --> 00:00:51,250 And I introduced you, last time, to two key players 16 00:00:51,250 --> 00:00:53,590 that we'll look at in a little more detail-- 17 00:00:53,590 --> 00:00:57,220 proteins and little pieces of RNA. 18 00:00:57,220 --> 00:00:59,750 And what happens is the proteins bind 19 00:00:59,750 --> 00:01:04,870 to the little pieces of RNA and prevent the translation 20 00:01:04,870 --> 00:01:07,540 of the messenger RNA into protein, 21 00:01:07,540 --> 00:01:11,770 or prevent degradation of the messenger RNA, 22 00:01:11,770 --> 00:01:14,000 allowing the translation to proceed. 23 00:01:14,000 --> 00:01:16,150 So that's really the take-home message. 24 00:01:16,150 --> 00:01:18,220 And I'll show you what the model is. 25 00:01:18,220 --> 00:01:22,390 So the last time, we were talking about one 26 00:01:22,390 --> 00:01:29,560 of the protein players, IRP1 and IRP2. 27 00:01:29,560 --> 00:01:38,020 And I told you that IRP1 was a cytosolic aconitase 28 00:01:38,020 --> 00:01:42,130 and that you had seen the aconitase reaction, which 29 00:01:42,130 --> 00:01:45,070 I drew in the board last time, which is conversion of citrate 30 00:01:45,070 --> 00:01:46,340 into isocitrate. 31 00:01:46,340 --> 00:01:50,350 If you look at the model up there, citrate to isocitrate, 32 00:01:50,350 --> 00:01:55,000 you're simply losing a molecule of water. 33 00:01:55,000 --> 00:01:57,730 And then over here, you're generating isocitrate. 34 00:01:57,730 --> 00:02:02,740 And the chemistry is facilitated by the presence 35 00:02:02,740 --> 00:02:06,880 of a single unique iron in the form of iron 4 sulfur cluster, 36 00:02:06,880 --> 00:02:10,930 which was the first example of these kinds of clusters doing 37 00:02:10,930 --> 00:02:15,760 chemistry in addition to electron transfer 38 00:02:15,760 --> 00:02:18,190 reactions that you've been exposed to before. 39 00:02:18,190 --> 00:02:19,000 OK. 40 00:02:19,000 --> 00:02:23,780 So the question then is, what is the signal? 41 00:02:23,780 --> 00:02:28,570 And so we're going to see that the signal is 42 00:02:28,570 --> 00:02:30,280 going to be related to-- 43 00:02:30,280 --> 00:02:32,570 let me just get myself organized here. 44 00:02:32,570 --> 00:02:36,070 So the signal-- the question is, what's 45 00:02:36,070 --> 00:02:42,380 recognized under low iron and high iron conditions? 46 00:02:42,380 --> 00:02:45,190 So that's what we'll be talking about, 47 00:02:45,190 --> 00:02:50,170 and how does this switch work to allow translation or not 48 00:02:50,170 --> 00:02:52,480 allow translation to occur. 49 00:02:52,480 --> 00:02:53,920 And the other player that we need 50 00:02:53,920 --> 00:02:58,380 to be introduced to before we look at how this signal works 51 00:02:58,380 --> 00:03:02,680 is the iron responsive element. 52 00:03:02,680 --> 00:03:05,686 And this is a piece of RNA-- 53 00:03:05,686 --> 00:03:09,220 and I'll show you that on the next slide-- 54 00:03:09,220 --> 00:03:12,190 within the messenger RNA. 55 00:03:12,190 --> 00:03:13,060 OK. 56 00:03:13,060 --> 00:03:19,930 So you have a structure like this, 57 00:03:19,930 --> 00:03:23,380 and there are so many base pairs, 58 00:03:23,380 --> 00:03:26,470 in this little stem loop, that's part of the messenger RNA. 59 00:03:29,260 --> 00:03:37,150 And you have a three nucleotide sequence, 60 00:03:37,150 --> 00:03:40,420 and this is a bulge sequence. 61 00:03:40,420 --> 00:03:42,190 And what we're going to see is that there 62 00:03:42,190 --> 00:03:43,630 are many of these structures. 63 00:03:43,630 --> 00:03:45,580 People have now done a much more extensive-- 64 00:03:45,580 --> 00:03:48,650 this was the model that came forth a long time ago 65 00:03:48,650 --> 00:03:49,690 when it was discovered. 66 00:03:49,690 --> 00:03:51,190 It was discovered a long time ago, 67 00:03:51,190 --> 00:03:54,790 but people have since done a lot of bioinformatics analysis 68 00:03:54,790 --> 00:03:57,130 to try to define really what do we 69 00:03:57,130 --> 00:03:59,260 know about this little sequence here. 70 00:03:59,260 --> 00:04:00,560 Is it three nucleotides? 71 00:04:00,560 --> 00:04:01,060 Is it more? 72 00:04:01,060 --> 00:04:03,810 People now think it's a little bit more, 73 00:04:03,810 --> 00:04:07,480 but it-- there's variability in that you need a bulge. 74 00:04:07,480 --> 00:04:10,600 And so what's going to happen is our iron responsive 75 00:04:10,600 --> 00:04:13,630 protein is going to interact with this bulge, 76 00:04:13,630 --> 00:04:15,400 and that's going to be what's related 77 00:04:15,400 --> 00:04:19,630 to depending on the location of this bulge within the message. 78 00:04:19,630 --> 00:04:27,040 So we'll see this bulge can be at the 3 prime end or the 5 79 00:04:27,040 --> 00:04:29,070 prime end. 80 00:04:29,070 --> 00:04:33,370 And this location and its interaction with this protein 81 00:04:33,370 --> 00:04:36,370 is going to regulate the translational process. 82 00:04:36,370 --> 00:04:39,380 So that's what I'm going to be presenting to you. 83 00:04:39,380 --> 00:04:41,470 So what do I want to say? 84 00:04:45,470 --> 00:04:52,280 What I want to say then is for the iron response protein 1, 85 00:04:52,280 --> 00:04:59,940 it has, as we saw with the mitochondrial aconitase of 4 86 00:04:59,940 --> 00:05:01,460 iron 4 sulfur clusters. 87 00:05:01,460 --> 00:05:03,110 So that could be a switch. 88 00:05:03,110 --> 00:05:04,810 We're doing iron sensing. 89 00:05:04,810 --> 00:05:06,920 What's going to cause us-- 90 00:05:06,920 --> 00:05:10,520 what is the sensor of iron that allows us to translate or not 91 00:05:10,520 --> 00:05:13,090 translate all of the proteins-- transfer 92 00:05:13,090 --> 00:05:17,600 and receptor, transfer and ferroportin, all of the things 93 00:05:17,600 --> 00:05:21,150 we were introduced to in the previous lecture. 94 00:05:21,150 --> 00:05:29,460 So this protein has a 4 iron 4 sulfur cluster. 95 00:05:29,460 --> 00:05:31,760 And when it loads the cluster, as 96 00:05:31,760 --> 00:05:35,450 with mitochondrial aconitase, the protein is active. 97 00:05:35,450 --> 00:05:41,510 So it's found in the cytosol as opposed to the mitochondria. 98 00:05:41,510 --> 00:05:48,050 And it can convert citrate to isocitrate. 99 00:05:48,050 --> 00:05:48,890 OK. 100 00:05:48,890 --> 00:05:55,700 So the question is, what is the switch that allows this IRP1 101 00:05:55,700 --> 00:05:58,700 to interact with this little piece of RNA-- 102 00:05:58,700 --> 00:06:01,070 the stem loop piece of RNA? 103 00:06:01,070 --> 00:06:08,420 And so the switch is that you have to lose this cluster. 104 00:06:08,420 --> 00:06:11,920 And what you generate then is apoIRP1. 105 00:06:16,880 --> 00:06:22,190 And apoIRP1-- so somehow the cluster magically disappears. 106 00:06:22,190 --> 00:06:26,330 And when it disappears it can bind to the IRE. 107 00:06:26,330 --> 00:06:29,210 So in the apo form-- that means no metal-- 108 00:06:29,210 --> 00:06:32,040 it binds to IRE. 109 00:06:34,760 --> 00:06:45,350 Whereas in 4 iron 4 sulfur loaded form, it does not bind. 110 00:06:50,380 --> 00:06:52,210 So what that would imply-- 111 00:06:52,210 --> 00:06:54,910 if you think about it sort of superficially-- 112 00:06:54,910 --> 00:07:00,040 if you have low iron and there's no iron sulfur cluster, 113 00:07:00,040 --> 00:07:01,760 the apo form is going to bind. 114 00:07:01,760 --> 00:07:02,260 OK. 115 00:07:02,260 --> 00:07:04,093 I'm going to show you the model in a minute. 116 00:07:04,093 --> 00:07:04,660 OK. 117 00:07:04,660 --> 00:07:07,250 So the switch really is related to-- 118 00:07:07,250 --> 00:07:11,260 in this case, the sensor is related to-- the fact 119 00:07:11,260 --> 00:07:16,300 that we have a 4 iron 4 sulfur cluster. 120 00:07:16,300 --> 00:07:18,280 So we also have-- 121 00:07:18,280 --> 00:07:19,600 I told you before-- 122 00:07:19,600 --> 00:07:23,420 in addition to an IRP1, we have an IRP2. 123 00:07:23,420 --> 00:07:26,830 And IRP2 also looks, structurally, 124 00:07:26,830 --> 00:07:31,280 like a cytosolic aconitase, but it has no aconitase activity. 125 00:07:31,280 --> 00:07:31,780 OK. 126 00:07:31,780 --> 00:07:37,750 So we have the second protein, IRP2. 127 00:07:37,750 --> 00:07:45,970 It's also a cytosolic aconitase lookalike, 128 00:07:45,970 --> 00:07:49,290 but it has no activity. 129 00:07:49,290 --> 00:07:51,970 And why does it have no activity? 130 00:07:51,970 --> 00:07:54,910 You've seen, over here, the iron sulfur cluster 131 00:07:54,910 --> 00:07:57,220 is required to do the dehydration reaction. 132 00:07:57,220 --> 00:08:00,190 So it's required for activity in the mitochondrial enzyme. 133 00:08:00,190 --> 00:08:02,980 This has no iron sulfur cluster. 134 00:08:02,980 --> 00:08:08,635 So it has no iron sulfur cluster. 135 00:08:11,460 --> 00:08:13,590 And what it has in addition, even though 136 00:08:13,590 --> 00:08:15,740 it looks like IRP2-- 137 00:08:15,740 --> 00:08:21,570 the structurally homologous-- it has a 73 amino acid insert. 138 00:08:21,570 --> 00:08:25,080 So this is a distinction between the two. 139 00:08:25,080 --> 00:08:25,860 OK. 140 00:08:25,860 --> 00:08:29,070 But now, this raises the question here-- 141 00:08:29,070 --> 00:08:31,590 at least superficially, you can understand 142 00:08:31,590 --> 00:08:35,370 that you might be able to sense iron, 143 00:08:35,370 --> 00:08:38,360 because you have a cluster, and you can go to no cluster. 144 00:08:38,360 --> 00:08:40,020 OK. 145 00:08:40,020 --> 00:08:42,330 And you can go back and forth. 146 00:08:42,330 --> 00:08:44,720 And so, remember, in a couple of lectures 147 00:08:44,720 --> 00:08:46,770 ago I told you about biosynthetic pathways, 148 00:08:46,770 --> 00:08:50,370 and I showed you a picture of iron sulfur cluster 149 00:08:50,370 --> 00:08:52,560 assembly-- very complicated. 150 00:08:52,560 --> 00:08:55,530 At the end of the notes in this part of the lecture, 151 00:08:55,530 --> 00:08:57,180 you'll see what the model is. 152 00:08:57,180 --> 00:08:58,830 I'm not going to go through that. 153 00:08:58,830 --> 00:09:02,250 But how you assemble and disassemble, 154 00:09:02,250 --> 00:09:04,890 even though this model has been around for a long time, 155 00:09:04,890 --> 00:09:08,202 is only recently beginning to be understood. 156 00:09:08,202 --> 00:09:09,660 It's not trivial, because there are 157 00:09:09,660 --> 00:09:12,670 10 steps to assemble a 4 iron 4 sulfur cluster. 158 00:09:12,670 --> 00:09:13,350 OK. 159 00:09:13,350 --> 00:09:16,620 But here, we don't even have any iron. 160 00:09:16,620 --> 00:09:22,990 So how is the IRP2, which binds to the same IREs-- 161 00:09:22,990 --> 00:09:25,710 and again, in vivo we don't really know all of this. 162 00:09:25,710 --> 00:09:27,210 People are trying to sort that out 163 00:09:27,210 --> 00:09:32,130 as what the what the functions of the different proteins 164 00:09:32,130 --> 00:09:33,930 actually are. 165 00:09:33,930 --> 00:09:36,570 But how does it sense? 166 00:09:36,570 --> 00:09:43,650 And so I just told you that the apo form of the IRP1 binds. 167 00:09:43,650 --> 00:09:47,040 That's also true of the IRP2. 168 00:09:47,040 --> 00:09:48,900 And in fact, it can only be apo, because it 169 00:09:48,900 --> 00:09:50,560 can't bind a cluster. 170 00:09:50,560 --> 00:09:53,490 So the active form, the binding form, 171 00:09:53,490 --> 00:10:05,520 the apoIRP2 binds the IRE. 172 00:10:05,520 --> 00:10:07,440 And then the question is, what is the switch? 173 00:10:11,110 --> 00:10:16,000 And so what we'll see is that the switch relates to the fact 174 00:10:16,000 --> 00:10:21,100 that IRP2 gets degraded. 175 00:10:27,990 --> 00:10:31,890 So when IRP2 is degraded, it can't bind. 176 00:10:31,890 --> 00:10:34,290 And that's how you turn the thing off. 177 00:10:34,290 --> 00:10:36,790 So then that takes you back a step further-- 178 00:10:36,790 --> 00:10:39,960 how do you target IRP2 for degradation? 179 00:10:39,960 --> 00:10:41,700 And this goes back to one of the reasons 180 00:10:41,700 --> 00:10:45,360 that I'm going to spend some time talking about degradation 181 00:10:45,360 --> 00:10:47,580 in mammalian systems. 182 00:10:47,580 --> 00:10:49,260 And so it turns out-- 183 00:10:49,260 --> 00:10:52,815 how does this relate then to iron sensing? 184 00:10:57,320 --> 00:11:02,270 And what I'm going to show you is that you have an E3 ligase. 185 00:11:02,270 --> 00:11:04,380 I'll show you this in cartoon form. 186 00:11:04,380 --> 00:11:06,110 And I'll just say, see PowerPoint. 187 00:11:06,110 --> 00:11:07,040 We're not going to-- 188 00:11:07,040 --> 00:11:08,720 it's not really completely understood, 189 00:11:08,720 --> 00:11:11,280 so I'm not going to talk about it in detail. 190 00:11:11,280 --> 00:11:24,610 But what it has attached to it is an FXBL5 domain 191 00:11:24,610 --> 00:11:28,600 that looks like a protein we've seen before earlier 192 00:11:28,600 --> 00:11:30,280 that has an iron in it. 193 00:11:30,280 --> 00:11:32,770 So many of you probably don't remember hemerythrin, 194 00:11:32,770 --> 00:11:36,320 but that's the little enzyme in worms that reversibly binds 195 00:11:36,320 --> 00:11:38,800 oxygen. So that was incredible. 196 00:11:38,800 --> 00:11:41,950 It's structurally homologous to that little protein. 197 00:11:41,950 --> 00:11:49,820 So this is-- again, the details are not known. 198 00:11:49,820 --> 00:11:56,560 But it can bind iron and it can sense oxygen. 199 00:11:56,560 --> 00:12:01,690 So if you're at low iron, there's 200 00:12:01,690 --> 00:12:04,780 no iron bound to this little domain. 201 00:12:04,780 --> 00:12:06,160 And so there's a consequence. 202 00:12:06,160 --> 00:12:07,285 I'll show you what that is. 203 00:12:07,285 --> 00:12:09,980 But if it's high iron, it has a different consequence. 204 00:12:09,980 --> 00:12:12,790 So the sensing is back a step. 205 00:12:12,790 --> 00:12:15,550 Its back a step into-- 206 00:12:15,550 --> 00:12:18,700 remember, I told you E3 ubiquitin ligases are 207 00:12:18,700 --> 00:12:20,050 multienzyme complexes. 208 00:12:20,050 --> 00:12:22,430 So this is part-- you'll see in a minute-- 209 00:12:22,430 --> 00:12:25,930 of the multienzyme complex. 210 00:12:29,140 --> 00:12:32,530 And so under certain sets of conditions 211 00:12:32,530 --> 00:12:36,070 when you have high iron, what happens 212 00:12:36,070 --> 00:12:37,960 is this is targeted for degradation. 213 00:12:37,960 --> 00:12:41,060 I'll show you what the model is for how this works. 214 00:12:41,060 --> 00:12:42,670 So the models are the same. 215 00:12:42,670 --> 00:12:45,370 That is, apo in both cases bind. 216 00:12:45,370 --> 00:12:50,050 In one case, the iron sensor is directly related 217 00:12:50,050 --> 00:12:54,690 to the IRP itself, because it has an iron sulfur cluster. 218 00:12:54,690 --> 00:12:57,640 And in the second case, it's indirectly 219 00:12:57,640 --> 00:13:01,780 related to an iron cluster that's associated 220 00:13:01,780 --> 00:13:04,340 with the E3 ubiquitin ligase. 221 00:13:04,340 --> 00:13:06,760 So another point I think I want to raise-- 222 00:13:06,760 --> 00:13:09,020 and this will get us into the next module, 223 00:13:09,020 --> 00:13:11,440 which I'm not going to spend very much time on-- 224 00:13:11,440 --> 00:13:14,530 the reason that we have iron module juxtaposed 225 00:13:14,530 --> 00:13:17,140 to the reactive oxygen species is they're really 226 00:13:17,140 --> 00:13:18,790 intimately linked. 227 00:13:18,790 --> 00:13:20,500 We've talked about how iron 2 can 228 00:13:20,500 --> 00:13:25,180 generate hydroxide radical or hydrogen peroxide. 229 00:13:25,180 --> 00:13:29,560 These are iron sulfur clusters are also oxygen-sensitive. 230 00:13:29,560 --> 00:13:32,320 This is oxygen-dependent. 231 00:13:32,320 --> 00:13:34,600 So again, what you're seeing is not only 232 00:13:34,600 --> 00:13:38,470 do we have iron sensors, but we will 233 00:13:38,470 --> 00:13:48,850 see that iron sensing and oxygen sensing are linked. 234 00:13:48,850 --> 00:13:50,770 And I would say-- 235 00:13:50,770 --> 00:13:52,510 I was trying to make up your exam, 236 00:13:52,510 --> 00:13:54,070 and I was trying to put in a linkage 237 00:13:54,070 --> 00:13:57,680 so you would all of a sudden see this, and the more I read, 238 00:13:57,680 --> 00:13:59,170 the more confused I got. 239 00:13:59,170 --> 00:14:01,900 So the fact is, there are many, many papers published 240 00:14:01,900 --> 00:14:06,250 on this now, and the linkages-- the proteins involved-- 241 00:14:06,250 --> 00:14:07,730 do many things. 242 00:14:07,730 --> 00:14:10,230 And so sorting this out into a very simple model 243 00:14:10,230 --> 00:14:12,220 is really still tough. 244 00:14:12,220 --> 00:14:14,530 But what I what I believe right now 245 00:14:14,530 --> 00:14:17,950 is both iron and oxygen sensing are linked 246 00:14:17,950 --> 00:14:20,800 through this type of a model. 247 00:14:20,800 --> 00:14:25,510 So let me now just show you a little bit about IRP1. 248 00:14:25,510 --> 00:14:29,080 We know a lot about IRP1, because we have structures. 249 00:14:29,080 --> 00:14:33,130 So this is the structure actually 250 00:14:33,130 --> 00:14:37,840 of the cytosolic aconitase, and this is with the 4 iron 4 251 00:14:37,840 --> 00:14:40,060 sulfur cluster bound. 252 00:14:40,060 --> 00:14:43,150 So what happens when you get to the apo form? 253 00:14:43,150 --> 00:14:46,390 What happens in the apo form, you now 254 00:14:46,390 --> 00:14:50,730 have a little piece of RNA bound. 255 00:14:50,730 --> 00:14:53,470 And this little piece of RNA always 256 00:14:53,470 --> 00:14:55,720 has a bulge with a cysteine in it. 257 00:14:55,720 --> 00:14:57,360 And it always has some kind of a loop. 258 00:14:57,360 --> 00:15:00,850 And we'll see in a second that the sequence of that loop 259 00:15:00,850 --> 00:15:02,977 can be variable. 260 00:15:02,977 --> 00:15:04,060 But here you can see that. 261 00:15:04,060 --> 00:15:07,780 So these little balls here that are iron sulfur clusters 262 00:15:07,780 --> 00:15:12,460 are the cytidine bulge in this loop. 263 00:15:12,460 --> 00:15:14,740 So you see the thing changes confirmation 264 00:15:14,740 --> 00:15:16,360 and as binding to an IRE. 265 00:15:16,360 --> 00:15:19,295 So that is the switch. 266 00:15:19,295 --> 00:15:20,920 And then the question is, how does that 267 00:15:20,920 --> 00:15:23,970 work at level of controlling translation, which I'm 268 00:15:23,970 --> 00:15:25,220 going to show you in a second. 269 00:15:25,220 --> 00:15:27,600 OK. 270 00:15:27,600 --> 00:15:33,790 So where do we see these iron-responsive elements 271 00:15:33,790 --> 00:15:35,140 in our messenger RNA? 272 00:15:35,140 --> 00:15:38,980 So messenger RNA-- go back and look at all 273 00:15:38,980 --> 00:15:42,640 of the players I introduced you to the last time. 274 00:15:42,640 --> 00:15:47,150 We have a transferrin receptor that's involved in uptake. 275 00:15:47,150 --> 00:15:48,400 We have DMT1. 276 00:15:48,400 --> 00:15:52,510 That's a dimetal transporter involved in iron uptake. 277 00:15:52,510 --> 00:15:55,480 So intuitively, you should ask the question, 278 00:15:55,480 --> 00:16:03,560 if you are at low iron, do you want to take up more iron? 279 00:16:03,560 --> 00:16:06,210 So you want to turn on the transferrin receptor. 280 00:16:06,210 --> 00:16:10,720 You want to turn on the DMT1 protein. 281 00:16:10,720 --> 00:16:13,390 So I think most of it makes intuitive sense. 282 00:16:13,390 --> 00:16:17,590 The linkage to oxygen, I think, is less intuitive. 283 00:16:17,590 --> 00:16:20,770 If you have a lot of iron, what do you want to do? 284 00:16:20,770 --> 00:16:22,790 You want to store the iron. 285 00:16:22,790 --> 00:16:27,460 So you in some way want to make more of the ferritin. 286 00:16:27,460 --> 00:16:32,110 And then the other thing, this HIF 2 alpha 287 00:16:32,110 --> 00:16:35,860 is a transcription factor hypoxia-- 288 00:16:35,860 --> 00:16:39,305 inducible transcription factor-- that's linked to many, 289 00:16:39,305 --> 00:16:41,680 many things-- a huge number of people are working on this 290 00:16:41,680 --> 00:16:42,400 now-- 291 00:16:42,400 --> 00:16:46,210 one of which is this linkage to iron. 292 00:16:46,210 --> 00:16:50,110 But it senses anaerobiasis. 293 00:16:50,110 --> 00:16:54,100 And so you can see, it's also linked by one 294 00:16:54,100 --> 00:16:55,690 of these little elements. 295 00:16:55,690 --> 00:16:59,050 And the next slide just shows you a more recent one 296 00:16:59,050 --> 00:17:01,660 where people started doing a lot of bioinformatics on this. 297 00:17:01,660 --> 00:17:05,470 The previous slide was from a few years ago. 298 00:17:05,470 --> 00:17:07,359 And again, the details, what you need 299 00:17:07,359 --> 00:17:09,050 to see is you, in all cases, have 300 00:17:09,050 --> 00:17:12,490 stem loops, a little bulge of a cytosine, 301 00:17:12,490 --> 00:17:14,170 and then you have some kind of a loop 302 00:17:14,170 --> 00:17:17,060 at the top of the stem loop. 303 00:17:17,060 --> 00:17:22,390 And if you look down here and you go through-- 304 00:17:22,390 --> 00:17:24,339 so these little stem loops are going 305 00:17:24,339 --> 00:17:27,500 to be either at the 3 prime or the 5 prime end 306 00:17:27,500 --> 00:17:29,980 of your messenger RNA. 307 00:17:29,980 --> 00:17:33,160 And so, for example, one of the things you see 308 00:17:33,160 --> 00:17:36,700 is aminolevulinic acid. 309 00:17:36,700 --> 00:17:41,748 Does anybody know what pathway that's involved in? 310 00:17:41,748 --> 00:17:42,790 AUDIENCE: Heme synthesis. 311 00:17:42,790 --> 00:17:43,110 JOANNE STUBBE: Yeah. 312 00:17:43,110 --> 00:17:45,100 So it's a rate-limiting step in heme synthesis. 313 00:17:45,100 --> 00:17:47,200 So there would be a place that would make sense. 314 00:17:47,200 --> 00:17:51,340 Remember, I told you all the iron is in heme and hemoglobin. 315 00:17:51,340 --> 00:17:51,850 OK. 316 00:17:51,850 --> 00:17:54,182 So almost all of these stem loops that you'll see, 317 00:17:54,182 --> 00:17:56,140 if you go back and you look through your notes, 318 00:17:56,140 --> 00:18:00,070 will make sense in terms of the big picture of how 319 00:18:00,070 --> 00:18:03,460 you want to control the levels of these proteins 320 00:18:03,460 --> 00:18:06,820 to deal with high iron or to deal with low iron. 321 00:18:06,820 --> 00:18:07,690 OK. 322 00:18:07,690 --> 00:18:10,510 So that's iron 2. 323 00:18:10,510 --> 00:18:13,570 And so here's the picture of IRP2. 324 00:18:13,570 --> 00:18:19,150 And this is the model for how IRP2 works. 325 00:18:19,150 --> 00:18:24,010 And so here's the case when you have high iron. 326 00:18:24,010 --> 00:18:26,050 And when you have iron, this part here, 327 00:18:26,050 --> 00:18:35,380 the Fbox, Skp1, and Cul are all part of the SCF E3 ubiquitin 328 00:18:35,380 --> 00:18:36,050 ligase. 329 00:18:36,050 --> 00:18:38,090 And I don't expect you to remember the names, 330 00:18:38,090 --> 00:18:41,300 but remember I told you the E3 ubiquitin ligase is 331 00:18:41,300 --> 00:18:43,580 the one that does what? 332 00:18:43,580 --> 00:18:46,730 It attaches ubiquitin on to the proteins, 333 00:18:46,730 --> 00:18:49,770 targeting it for degradation. 334 00:18:49,770 --> 00:18:53,300 So this little part is the ubiquitin ligase. 335 00:18:53,300 --> 00:18:54,380 Here's your E2. 336 00:18:54,380 --> 00:18:58,400 Remember, you always need an E2 and an E3. 337 00:18:58,400 --> 00:19:02,610 And somehow the E2 is attaching this 338 00:19:02,610 --> 00:19:06,710 onto the IRP2, which is targeting it for degradation 339 00:19:06,710 --> 00:19:07,880 by the proteasome. 340 00:19:07,880 --> 00:19:08,510 OK. 341 00:19:08,510 --> 00:19:10,340 So this is exactly like the model 342 00:19:10,340 --> 00:19:14,250 we put forth a couple of lectures ago. 343 00:19:14,250 --> 00:19:18,560 So again, this is the part that's most interesting. 344 00:19:18,560 --> 00:19:21,650 If you go back and you look at hemerythrin, which irreversibly 345 00:19:21,650 --> 00:19:24,950 binds oxygen with two irons, you have a diirons site. 346 00:19:24,950 --> 00:19:26,600 And earlier in your notes, I showed you 347 00:19:26,600 --> 00:19:28,760 what that site looks like. 348 00:19:28,760 --> 00:19:33,440 This site is intact because the protein is folded. 349 00:19:33,440 --> 00:19:37,670 Under conditions of very low iron, what happens is 350 00:19:37,670 --> 00:19:40,310 this becomes unfolded. 351 00:19:40,310 --> 00:19:42,560 And then this part of the protein gets 352 00:19:42,560 --> 00:19:46,380 targeted for degradation by another E3 ligase-- 353 00:19:46,380 --> 00:19:48,020 not this one. 354 00:19:48,020 --> 00:19:50,890 And then you've lost your sensor. 355 00:19:50,890 --> 00:19:55,280 So the IRP2 remains stable. 356 00:19:55,280 --> 00:19:57,410 So you might think this is complicated-- 357 00:19:57,410 --> 00:20:00,050 and maybe I didn't spend enough time going through this-- 358 00:20:00,050 --> 00:20:02,850 but you should go back, and you should look at the explanation 359 00:20:02,850 --> 00:20:03,350 again. 360 00:20:03,350 --> 00:20:06,800 So the two key switches are here and here. 361 00:20:06,800 --> 00:20:10,108 This one is the more complicated switch. 362 00:20:10,108 --> 00:20:11,900 Everybody thought everything was understood 363 00:20:11,900 --> 00:20:16,310 once they found this little iron binding protein that models 364 00:20:16,310 --> 00:20:19,640 hemerythrin, but nothing could be farther from the truth. 365 00:20:19,640 --> 00:20:22,670 We still really don't understand, overall, 366 00:20:22,670 --> 00:20:24,230 how this fits into the big picture. 367 00:20:24,230 --> 00:20:27,050 But it's not an accident that iron and oxygen 368 00:20:27,050 --> 00:20:30,860 are required to fold this into this little bundle that 369 00:20:30,860 --> 00:20:33,920 looks like hemerythrin. 370 00:20:33,920 --> 00:20:35,150 So those are the switches. 371 00:20:35,150 --> 00:20:37,950 And so now, what I want to do is put forth a model. 372 00:20:37,950 --> 00:20:39,800 So let me see. 373 00:20:42,620 --> 00:20:45,620 So what is the model for how you want to turn these things off 374 00:20:45,620 --> 00:20:46,470 and on? 375 00:20:46,470 --> 00:20:46,970 OK. 376 00:20:50,260 --> 00:20:52,880 So we have two things-- we have IREs-- 377 00:20:52,880 --> 00:20:54,590 Iron-Responsive Elements-- that can 378 00:20:54,590 --> 00:21:00,800 bind in front of the message to be transcribed or at the end. 379 00:21:00,800 --> 00:21:05,120 So what we're going to look at two sets of conditions-- 380 00:21:05,120 --> 00:21:09,200 one is under conditions we have low iron, 381 00:21:09,200 --> 00:21:12,560 and one is under conditions where you have high iron. 382 00:21:12,560 --> 00:21:14,155 How do you sense those conditions? 383 00:21:14,155 --> 00:21:15,210 So that's the question. 384 00:21:15,210 --> 00:21:28,440 So again, we're sensing low versus high iron. 385 00:21:28,440 --> 00:21:31,680 So let's look at low iron first. 386 00:21:34,440 --> 00:21:38,100 So what we're going to see is we're 387 00:21:38,100 --> 00:21:39,480 going to have a stem loop. 388 00:21:39,480 --> 00:21:44,280 So here's my messenger RNA, and here's the 3 prime end, 389 00:21:44,280 --> 00:21:47,460 and here's the 5 prime end. 390 00:21:47,460 --> 00:21:54,060 And here, you initiate translation. 391 00:21:54,060 --> 00:21:57,150 And so if it binds-- 392 00:21:57,150 --> 00:22:03,750 if this protein IRP2 or IRP1, both in the apo form-- 393 00:22:03,750 --> 00:22:07,800 IRE-- and they do both bind. 394 00:22:07,800 --> 00:22:10,390 People are trying to sort all of this out. 395 00:22:10,390 --> 00:22:21,610 So this is IRP1 or IRP2. 396 00:22:21,610 --> 00:22:24,010 What happens to the translation? 397 00:22:24,010 --> 00:22:27,730 What happens to the translation is it's inhibited. 398 00:22:27,730 --> 00:22:30,790 So if you have this little stem loop 399 00:22:30,790 --> 00:22:35,080 in the front of your message, you inhibit translation. 400 00:22:41,260 --> 00:22:43,700 And so what I'm showing you now-- 401 00:22:43,700 --> 00:22:45,840 and then I will give you some examples-- 402 00:22:45,840 --> 00:22:47,430 is the key to thinking about this. 403 00:22:47,430 --> 00:22:49,750 And most of it actually is intuitive 404 00:22:49,750 --> 00:22:51,840 once you remember what all the factors are 405 00:22:51,840 --> 00:22:54,720 that are involved in iron homeostasis 406 00:22:54,720 --> 00:22:56,810 that we've already gone over. 407 00:22:56,810 --> 00:23:01,560 So binding inhibits translation. 408 00:23:06,920 --> 00:23:07,420 OK. 409 00:23:07,420 --> 00:23:11,610 So then we have the second case at low iron. 410 00:23:11,610 --> 00:23:15,960 And again, we have a 5 prime end, 411 00:23:15,960 --> 00:23:26,740 and then we have a 3 prime end, again, of our messenger RNA. 412 00:23:31,590 --> 00:23:35,370 And here is the initiation of translation. 413 00:23:38,040 --> 00:23:41,670 And in this case as well, you have the same sort 414 00:23:41,670 --> 00:23:44,010 of structures of stem loops. 415 00:23:44,010 --> 00:23:46,920 They are similar but distinct. 416 00:23:46,920 --> 00:23:51,070 And what can happen with the apo form of IRP1 or IRP2? 417 00:23:51,070 --> 00:23:53,790 Again, it can bind to these stem loops. 418 00:23:53,790 --> 00:23:56,570 So you can have-- 419 00:23:56,570 --> 00:23:57,915 this chalk is not working-- 420 00:24:01,410 --> 00:24:03,105 your proteins, they're all bound-- 421 00:24:06,020 --> 00:24:07,770 they may or may not be all bound. 422 00:24:07,770 --> 00:24:10,830 I don't think we know that much. 423 00:24:10,830 --> 00:24:14,250 But what you see is the number of stem loops at the 3 424 00:24:14,250 --> 00:24:15,990 prime end is variable. 425 00:24:15,990 --> 00:24:17,670 It depends on the message. 426 00:24:17,670 --> 00:24:26,655 So number of stem loops is variable. 427 00:24:29,520 --> 00:24:31,530 So what does this binding do? 428 00:24:31,530 --> 00:24:36,900 What this binding does is it prevents the messenger RNA 429 00:24:36,900 --> 00:24:37,980 from being degraded. 430 00:24:37,980 --> 00:24:42,670 So it basically stabilizes the messenger RNA. 431 00:24:42,670 --> 00:24:58,950 So this model binding prevents messenger RNA degradation. 432 00:24:58,950 --> 00:25:09,450 So it stabilizes the messenger RNA. 433 00:25:09,450 --> 00:25:11,210 So that's the model. 434 00:25:11,210 --> 00:25:11,710 OK. 435 00:25:11,710 --> 00:25:13,990 So now, let's just look at a couple of examples. 436 00:25:13,990 --> 00:25:17,770 And then what you can do later on 437 00:25:17,770 --> 00:25:21,910 is go back and think about this more of what's going on. 438 00:25:21,910 --> 00:25:25,120 So again, this is the model. 439 00:25:25,120 --> 00:25:28,930 And let me just make sure I go through the ones I want to do. 440 00:25:28,930 --> 00:25:31,180 So we're still at low iron. 441 00:25:31,180 --> 00:25:33,335 And we'll do two at low iron. 442 00:25:33,335 --> 00:25:34,960 And then we'll look at the consequences 443 00:25:34,960 --> 00:25:37,330 of what happens at high iron, and does 444 00:25:37,330 --> 00:25:39,730 it make intuitive sense based on what we think 445 00:25:39,730 --> 00:25:43,390 the function of these proteins are that are 446 00:25:43,390 --> 00:25:46,180 going to be translated? 447 00:25:46,180 --> 00:25:48,040 So let's look at low iron. 448 00:25:48,040 --> 00:25:50,680 So we're at low iron. 449 00:25:50,680 --> 00:25:54,270 And let's look at ferritin. 450 00:25:54,270 --> 00:25:54,770 OK. 451 00:25:54,770 --> 00:25:56,730 So what is ferritin? 452 00:25:56,730 --> 00:25:59,670 It's the iron storage protein. 453 00:25:59,670 --> 00:26:06,790 So under low iron, do we want to store iron? 454 00:26:06,790 --> 00:26:08,150 No. 455 00:26:08,150 --> 00:26:12,690 So if you have the choice of these two modes of regulation, 456 00:26:12,690 --> 00:26:15,150 what would you choose? 457 00:26:15,150 --> 00:26:18,810 Where would you put your iron-responsive element? 458 00:26:18,810 --> 00:26:22,640 At the 5 prime end the 3 prime end? 459 00:26:22,640 --> 00:26:26,030 So we're at low iron. 460 00:26:26,030 --> 00:26:29,120 We don't want to store iron. 461 00:26:29,120 --> 00:26:35,360 So we don't want storage. 462 00:26:39,270 --> 00:26:40,530 So what would we do? 463 00:26:40,530 --> 00:26:41,830 If these are the two choices-- 464 00:26:41,830 --> 00:26:44,190 and these are the two choices from experimental data. 465 00:26:44,190 --> 00:26:46,380 There are many other variations on this 466 00:26:46,380 --> 00:26:49,620 you could have imagined, but this is the model 467 00:26:49,620 --> 00:26:52,200 that everybody agrees on at this stage. 468 00:26:52,200 --> 00:26:55,360 So where would you put your stem loop? 469 00:26:55,360 --> 00:26:55,860 Yeah. 470 00:26:55,860 --> 00:26:57,420 You'd put it at the 5 prime end. 471 00:26:57,420 --> 00:26:59,970 And why would you put it at the 5 prime end? 472 00:26:59,970 --> 00:27:03,750 Because it prevents conversion of your message from ferritin 473 00:27:03,750 --> 00:27:05,400 into the protein. 474 00:27:05,400 --> 00:27:07,890 So you have less of the ferritin. 475 00:27:07,890 --> 00:27:10,200 So what you see now is you have-- 476 00:27:10,200 --> 00:27:15,390 again, so this is a stem loop at the 5 prime end 477 00:27:15,390 --> 00:27:28,120 prevents translation and have lower concentration 478 00:27:28,120 --> 00:27:28,620 of ferritin. 479 00:27:31,880 --> 00:27:34,670 So that's exactly what you would expect. 480 00:27:34,670 --> 00:27:39,260 Some of the others are less intuitive, 481 00:27:39,260 --> 00:27:41,330 but we've seen ferroportin. 482 00:27:41,330 --> 00:27:45,050 Remember, ferroportin is the iron 2 transporter 483 00:27:45,050 --> 00:27:48,230 in many cells which allows the iron to come from the inside 484 00:27:48,230 --> 00:27:50,840 to, get picked up by transferrin, 485 00:27:50,840 --> 00:27:52,850 and redistributed to the tissue. 486 00:27:52,850 --> 00:27:57,380 So it, in conjunction with the hepcidin peptide hormone 487 00:27:57,380 --> 00:28:02,870 we briefly talked about plays a really important role 488 00:28:02,870 --> 00:28:06,740 actually in controlling where the iron ends up going. 489 00:28:06,740 --> 00:28:10,160 And in fact, what you would like to be 490 00:28:10,160 --> 00:28:13,130 able to do-- say you had not very much iron, 491 00:28:13,130 --> 00:28:14,810 where would you want to put your iron? 492 00:28:14,810 --> 00:28:18,410 Would you want to put your iron in some metabolic pathway 493 00:28:18,410 --> 00:28:20,390 that's not so important, or would you 494 00:28:20,390 --> 00:28:23,450 want to put your iron in a metabolic pathway that's 495 00:28:23,450 --> 00:28:25,250 very important? 496 00:28:25,250 --> 00:28:26,990 You would want to put it into the pathway 497 00:28:26,990 --> 00:28:29,630 where you really need it to survive. 498 00:28:29,630 --> 00:28:33,570 And so this is a subtle tuning on all of this. 499 00:28:33,570 --> 00:28:36,260 And so an example of how this can 500 00:28:36,260 --> 00:28:39,620 be tuned if you look at an iron-responsive element binding 501 00:28:39,620 --> 00:28:44,770 protein is succinate dehydrogenase. 502 00:28:44,770 --> 00:28:48,170 Any of you ever heard of succinate dehydrogenase? 503 00:28:48,170 --> 00:28:50,000 And where have you heard of it? 504 00:28:50,000 --> 00:28:52,525 You have heard of it, you just probably don't remember it. 505 00:28:52,525 --> 00:28:53,710 [INTERPOSING VOICES] 506 00:28:53,710 --> 00:28:54,543 JOANNE STUBBE: Yeah. 507 00:28:54,543 --> 00:28:56,360 So it's in the TCA cycle. 508 00:28:56,360 --> 00:28:59,860 So it converts succinate, which is a hydrocarbon, 509 00:28:59,860 --> 00:29:05,510 into an olefin, an alpha beta unsat-- into fumarate. 510 00:29:05,510 --> 00:29:10,010 So remember the TCA cycle, you can tune it down 511 00:29:10,010 --> 00:29:12,110 or you can tune it up. 512 00:29:12,110 --> 00:29:14,990 So if you really were desperate for iron, 513 00:29:14,990 --> 00:29:19,700 you would probably tune down the TCA cycle. 514 00:29:19,700 --> 00:29:23,180 So in fact, if you look, you'll see a stem loop 515 00:29:23,180 --> 00:29:25,670 in front of succinate dehydrogenase 516 00:29:25,670 --> 00:29:29,330 which prevents its translation and tunes down the pathway. 517 00:29:29,330 --> 00:29:33,440 So there's a subtle example of how nature has-- at least is 518 00:29:33,440 --> 00:29:36,530 the way we rationalize the experimental observations 519 00:29:36,530 --> 00:29:38,300 of what nature has done. 520 00:29:38,300 --> 00:29:43,370 Now ferroportin, which is the way I started on this, 521 00:29:43,370 --> 00:29:44,660 sets priorities. 522 00:29:48,360 --> 00:29:54,508 And it does this in conjunction with hepcidin, which we already 523 00:29:54,508 --> 00:29:55,050 talked about. 524 00:29:55,050 --> 00:30:00,330 Remember, hepcidin can target ferroportins for degradation. 525 00:30:00,330 --> 00:30:06,240 And this allows the iron to be distributed in defined ways 526 00:30:06,240 --> 00:30:08,190 within the cell. 527 00:30:08,190 --> 00:30:12,600 And in fact, what you want to do, in this case, 528 00:30:12,600 --> 00:30:18,000 is have the stem loop at the 5 prime end 529 00:30:18,000 --> 00:30:22,740 so that you don't export the iron inside the cell 530 00:30:22,740 --> 00:30:24,360 to the outside. 531 00:30:24,360 --> 00:30:26,255 So that's what it does. 532 00:30:26,255 --> 00:30:27,630 And some of these, as I'm saying, 533 00:30:27,630 --> 00:30:29,580 are easier to rationalize than others. 534 00:30:29,580 --> 00:30:33,720 The ferritin one is really easy to rationalize. 535 00:30:33,720 --> 00:30:37,320 The ferroportin is easy to rationalize 536 00:30:37,320 --> 00:30:39,720 based on what I just told you. 537 00:30:39,720 --> 00:30:44,560 But what you see also is that in some of these systems-- 538 00:30:44,560 --> 00:30:47,520 I don't know how much you guys thought about RNA, 539 00:30:47,520 --> 00:30:51,000 but you know messenger RNA can be spliced. 540 00:30:51,000 --> 00:30:54,870 In different cells it's spliced differently. 541 00:30:54,870 --> 00:30:58,530 You've also seen that cell types, in terms 542 00:30:58,530 --> 00:31:02,100 of iron homeostasis, the enterocyte, 543 00:31:02,100 --> 00:31:05,640 the macrophage system in the spleen, 544 00:31:05,640 --> 00:31:08,460 red blood cells are much more important, it might be, 545 00:31:08,460 --> 00:31:11,100 if you're in some other tissue, the splicing site is different, 546 00:31:11,100 --> 00:31:12,392 and you don't have a stem loop. 547 00:31:12,392 --> 00:31:21,720 So you can alter the regulation by alternate splicing systems. 548 00:31:21,720 --> 00:31:27,690 So these are these two are at the 5 prime end. 549 00:31:27,690 --> 00:31:31,500 What about the transferrin receptor? 550 00:31:31,500 --> 00:31:35,310 So let me put this down here. 551 00:31:35,310 --> 00:31:36,810 What about the transferrin receptor? 552 00:31:36,810 --> 00:31:39,240 What would you expect at low iron-- 553 00:31:39,240 --> 00:31:40,560 we're still at low iron-- 554 00:31:43,200 --> 00:31:45,420 the regulation to be from the transferrin receptor? 555 00:31:48,110 --> 00:31:51,230 What do you want to do at low iron? 556 00:31:51,230 --> 00:31:54,710 So this is another example, low iron. 557 00:31:57,510 --> 00:32:00,660 Let's look at the transferrin receptor. 558 00:32:00,660 --> 00:32:02,400 What does the transferrin receptor do? 559 00:32:10,290 --> 00:32:11,420 Hopefully you know this. 560 00:32:11,420 --> 00:32:12,193 Yeah. 561 00:32:12,193 --> 00:32:14,803 AUDIENCE: [INAUDIBLE] 562 00:32:14,803 --> 00:32:16,470 JOANNE STUBBE: You need to speak louder, 563 00:32:16,470 --> 00:32:17,610 I can't hear anything you said. 564 00:32:17,610 --> 00:32:18,680 You just went like this. 565 00:32:18,680 --> 00:32:21,833 That didn't mean anything to me. 566 00:32:21,833 --> 00:32:23,250 AUDIENCE: It helps to intake iron. 567 00:32:23,250 --> 00:32:23,490 JOANNE STUBBE: Yeah. 568 00:32:23,490 --> 00:32:25,150 So it helps to intake iron. 569 00:32:25,150 --> 00:32:28,820 So if you have low iron, what do you want to do? 570 00:32:28,820 --> 00:32:30,153 AUDIENCE: You want to increase-- 571 00:32:30,153 --> 00:32:30,987 JOANNE STUBBE: Yeah. 572 00:32:30,987 --> 00:32:32,220 So you want to increase that. 573 00:32:32,220 --> 00:32:34,595 So where would you put the stem loop? 574 00:32:34,595 --> 00:32:35,870 AUDIENCE: 3 prime end. 575 00:32:35,870 --> 00:32:36,170 JOANNE STUBBE: Yeah. 576 00:32:36,170 --> 00:32:37,670 So you put it at the 3 prime end, 577 00:32:37,670 --> 00:32:40,070 because that stabilizes the messenger 578 00:32:40,070 --> 00:32:42,380 RNA of the transferrin. 579 00:32:42,380 --> 00:32:48,695 So here, at low iron, you want to increase iron uptake. 580 00:32:52,070 --> 00:33:01,832 And that means that if you have the 3 prime end, 581 00:33:01,832 --> 00:33:03,415 you're going to stabilize the message. 582 00:33:08,100 --> 00:33:12,600 So you can go through each one of the proteins 583 00:33:12,600 --> 00:33:16,500 that we discussed in the last lecture. 584 00:33:16,500 --> 00:33:19,500 And before you look at it, try to rationalize 585 00:33:19,500 --> 00:33:21,000 under different sets of conditions. 586 00:33:21,000 --> 00:33:23,190 This is low iron. 587 00:33:23,190 --> 00:33:28,010 What would you expect to happen at high iron? 588 00:33:28,010 --> 00:33:29,700 Here, let's just look at this one 589 00:33:29,700 --> 00:33:31,230 so I don't have to draw this again. 590 00:33:31,230 --> 00:33:36,600 But what would happen to the transfer receptor at high iron? 591 00:33:36,600 --> 00:33:39,880 Do you want to take more iron into the cell? 592 00:33:39,880 --> 00:33:41,100 No, you don't. 593 00:33:41,100 --> 00:33:42,690 So what you want to do is get rid 594 00:33:42,690 --> 00:33:44,520 of the transferrin receptor. 595 00:33:44,520 --> 00:33:46,890 So now what do you do? 596 00:33:46,890 --> 00:33:50,070 At high iron, if you're IRP1, you 597 00:33:50,070 --> 00:33:53,130 switch to pick up the iron sulfur cluster. 598 00:33:53,130 --> 00:33:55,350 It no longer binds. 599 00:33:55,350 --> 00:33:56,860 And so now what happens? 600 00:33:56,860 --> 00:33:59,670 So this is all bound. 601 00:33:59,670 --> 00:34:02,250 So it's stabilized and bound. 602 00:34:02,250 --> 00:34:05,450 In this case now, messenger RNA is degraded. 603 00:34:11,350 --> 00:34:15,370 So the big players in iron homeostasis, I think, 604 00:34:15,370 --> 00:34:16,820 are easy to rationalize. 605 00:34:16,820 --> 00:34:18,639 If once you know-- this might not 606 00:34:18,639 --> 00:34:21,489 be so rational why you would stabilize messenger RNA 607 00:34:21,489 --> 00:34:25,389 or whatever, but this is the way nature designed this. 608 00:34:25,389 --> 00:34:27,239 Once you remember this-- 609 00:34:27,239 --> 00:34:30,969 and remember, the switches are just apo binding, 610 00:34:30,969 --> 00:34:34,580 and somehow they sense iron and they no longer bind, 611 00:34:34,580 --> 00:34:36,699 whatever the details are-- 612 00:34:36,699 --> 00:34:39,310 you should be able to understand in different kinds of cell 613 00:34:39,310 --> 00:34:43,170 types how you might regulate the iron 614 00:34:43,170 --> 00:34:46,120 at the translational level. 615 00:34:46,120 --> 00:34:49,150 So I think that is all I wanted to say. 616 00:34:49,150 --> 00:34:54,190 This is just a summary of what we've done in the human part. 617 00:34:54,190 --> 00:34:56,969 And we're thinking about-- 618 00:34:56,969 --> 00:34:59,570 I gave you a big picture of what happens in humans. 619 00:34:59,570 --> 00:35:03,550 This is the summary of that big picture with all these factors 620 00:35:03,550 --> 00:35:06,850 that are regulated at the translational level 621 00:35:06,850 --> 00:35:09,950 by the iron-responsive binding proteins. 622 00:35:09,950 --> 00:35:12,670 And so you can go back and look at this cartoon. 623 00:35:12,670 --> 00:35:16,300 Whether you want to store it, whether you want to distribute 624 00:35:16,300 --> 00:35:19,660 it, whether you want to put some in the mitochondria-- 625 00:35:19,660 --> 00:35:22,600 all of that kind of stuff is regulated 626 00:35:22,600 --> 00:35:25,060 at the translational level. 627 00:35:25,060 --> 00:35:29,780 So in this module, the second lecture, 628 00:35:29,780 --> 00:35:35,560 which was longer than I wanted it to be-- but that's life-- 629 00:35:39,150 --> 00:35:42,720 was focused on the big picture for human 630 00:35:42,720 --> 00:35:44,220 and how iron is transported. 631 00:35:44,220 --> 00:35:50,070 And uptake, which we talked about by divalent metal irons, 632 00:35:50,070 --> 00:35:54,150 transporters, and by transferrin in the plus 3 633 00:35:54,150 --> 00:35:57,780 state, and this question of regulation 634 00:35:57,780 --> 00:35:59,010 at the translational level. 635 00:35:59,010 --> 00:36:02,460 Now everything-- the hepcidin, we didn't touch on very much. 636 00:36:02,460 --> 00:36:07,290 Very complicated, but it plays a major role systemically. 637 00:36:07,290 --> 00:36:10,740 Whereas these others-- what we were just talking about 638 00:36:10,740 --> 00:36:13,110 is more specific for each cell type. 639 00:36:13,110 --> 00:36:14,910 And different cell types want to have 640 00:36:14,910 --> 00:36:16,700 regulation in different ways. 641 00:36:16,700 --> 00:36:19,620 So the bigger picture is the hepcidin. 642 00:36:19,620 --> 00:36:22,320 And it was discovered a while back, 643 00:36:22,320 --> 00:36:25,290 but I still would say we don't understand 644 00:36:25,290 --> 00:36:27,240 a lot about what's going on in terms 645 00:36:27,240 --> 00:36:29,370 of that hormonal regulation. 646 00:36:29,370 --> 00:36:32,250 So now what I want to do, as advertised, 647 00:36:32,250 --> 00:36:37,035 is move into bacteria, and how do bacteria do the same thing. 648 00:36:37,035 --> 00:36:38,160 They have the same problem. 649 00:36:38,160 --> 00:36:39,900 We talked about metal homeostasis-- 650 00:36:39,900 --> 00:36:42,270 exact same problem in human and bacteria. 651 00:36:42,270 --> 00:36:45,240 But in the end, the bacteria want to survive 652 00:36:45,240 --> 00:36:47,070 and we want to survive, so we have 653 00:36:47,070 --> 00:36:53,550 the battle between the bacteria and us for iron. 654 00:36:53,550 --> 00:36:57,720 So what I want to do is introduce you to the bacteria-- 655 00:36:57,720 --> 00:37:02,200 generically, how they take up metal 656 00:37:02,200 --> 00:37:07,050 to use for the same things that we use it for-- 657 00:37:07,050 --> 00:37:10,350 a little less complicated, maybe, than humans. 658 00:37:10,350 --> 00:37:12,840 And then what we're going to do is 659 00:37:12,840 --> 00:37:18,100 I'll introduce you to this war between bacteria and humans. 660 00:37:18,100 --> 00:37:20,520 And then we're going to focus on one bacteria that's 661 00:37:20,520 --> 00:37:23,580 a major issue nowadays-- 662 00:37:23,580 --> 00:37:27,330 Staphylococcus aureus-- because of resistance problems. 663 00:37:27,330 --> 00:37:30,565 This is a problem that Liz's lab has worked on. 664 00:37:30,565 --> 00:37:32,190 And specifically, I'm going to give you 665 00:37:32,190 --> 00:37:39,030 one example of how Staphylococcus aureus gets iron 666 00:37:39,030 --> 00:37:40,110 out of our hemoglobin. 667 00:37:40,110 --> 00:37:42,450 That's going to be the example. 668 00:37:42,450 --> 00:37:46,020 And the system that you'll see, it's amazingly cool. 669 00:37:46,020 --> 00:37:48,450 But you'll see, there's still many things we don't really 670 00:37:48,450 --> 00:37:51,330 understand in a lot of detail. 671 00:37:51,330 --> 00:37:56,260 So what I want to do now is introduce you-- 672 00:37:56,260 --> 00:37:57,750 how am I doing timewise? 673 00:37:57,750 --> 00:37:58,880 OK. 674 00:37:58,880 --> 00:38:02,017 So what I really would like to do 675 00:38:02,017 --> 00:38:04,350 is draw this out on the board, because it's complicated. 676 00:38:04,350 --> 00:38:06,510 And I know what happens if you use PowerPoint, 677 00:38:06,510 --> 00:38:09,210 you go through it at 100 miles an hour. 678 00:38:09,210 --> 00:38:11,860 But I'm going to be using more PowerPoint 679 00:38:11,860 --> 00:38:13,530 to get through something. 680 00:38:13,530 --> 00:38:15,180 So anyhow, this is an overview of where 681 00:38:15,180 --> 00:38:18,930 we're going if you forget. 682 00:38:18,930 --> 00:38:22,380 So what I wanted to do, at least a little bit, 683 00:38:22,380 --> 00:38:26,460 we're going to be focusing on gram-positive and gram-negative 684 00:38:26,460 --> 00:38:27,090 systems. 685 00:38:27,090 --> 00:38:29,007 And I want to tell you what is the difference. 686 00:38:29,007 --> 00:38:31,860 You all know or have heard about gram-positive and gram-negative 687 00:38:31,860 --> 00:38:32,370 bacteria. 688 00:38:32,370 --> 00:38:33,637 They use different strategies. 689 00:38:33,637 --> 00:38:35,220 They use the same strategies, but they 690 00:38:35,220 --> 00:38:40,710 use distinct strategies because of their structures. 691 00:38:40,710 --> 00:38:47,320 And so what I want to do is give you an overview, 692 00:38:47,320 --> 00:38:52,740 and then we'll focus specifically on Staph aureus. 693 00:38:52,740 --> 00:38:58,350 So in gram-positive, here we have our plasma membrane. 694 00:39:02,830 --> 00:39:06,735 So this is the plasma membrane. 695 00:39:09,570 --> 00:39:15,000 And this big guy here is PG-- 696 00:39:15,000 --> 00:39:16,040 the peptidoglycan. 697 00:39:20,120 --> 00:39:23,980 And we'll see, in Staph aureus, the peptidoglycan 698 00:39:23,980 --> 00:39:25,460 is going to play a key role. 699 00:39:25,460 --> 00:39:28,080 So you need to understand the structure of the peptidoglycan. 700 00:39:28,080 --> 00:39:30,780 So I am going to spend a little bit of time 701 00:39:30,780 --> 00:39:32,730 describing to you the structure. 702 00:39:32,730 --> 00:39:37,170 It's also the major target of many antibacterial agents 703 00:39:37,170 --> 00:39:38,370 that are currently used. 704 00:39:38,370 --> 00:39:38,870 Why? 705 00:39:38,870 --> 00:39:41,080 Because it's unique to bacteria. 706 00:39:41,080 --> 00:39:45,270 So you have only this plasma membrane. 707 00:39:45,270 --> 00:39:46,620 There's no outer membrane. 708 00:39:46,620 --> 00:39:50,520 That's going to be distinct from gram-negative. 709 00:39:50,520 --> 00:39:54,750 And so the question is, how do you get iron 710 00:39:54,750 --> 00:39:58,110 from the outside to the inside? 711 00:39:58,110 --> 00:40:03,113 And so one of the ways you can take in iron is-- 712 00:40:03,113 --> 00:40:05,280 you've already seen this, and you've talked about it 713 00:40:05,280 --> 00:40:09,060 in detail in the first half of the course-- 714 00:40:09,060 --> 00:40:11,100 siderophores. 715 00:40:11,100 --> 00:40:12,840 And we've already talked about the fact 716 00:40:12,840 --> 00:40:16,380 that we have many, many different kinds 717 00:40:16,380 --> 00:40:18,210 of siderophores. 718 00:40:18,210 --> 00:40:20,310 And somehow these siderophores-- and we'll 719 00:40:20,310 --> 00:40:23,820 look at a few structures-- can get to the outside of a cell. 720 00:40:23,820 --> 00:40:27,390 They pick up iron in the plus 3 state, 721 00:40:27,390 --> 00:40:33,730 and then they need to bring it back to the plasma membrane. 722 00:40:33,730 --> 00:40:40,560 And then somehow it needs to get transported across the plasma 723 00:40:40,560 --> 00:40:41,230 membrane. 724 00:40:41,230 --> 00:40:44,460 This is a transporter. 725 00:40:44,460 --> 00:40:47,100 Most of them are called ABC transporters 726 00:40:47,100 --> 00:40:48,495 and they require ATP. 727 00:40:53,800 --> 00:40:57,640 And when they get across, they take the siderophore 728 00:40:57,640 --> 00:40:59,620 with the iron into the cell. 729 00:41:04,050 --> 00:41:06,720 So that looks simple enough. 730 00:41:06,720 --> 00:41:10,650 We'll see that the strategy of gram-negative bacteria 731 00:41:10,650 --> 00:41:14,820 is going to be distinct, because it has an outer membrane. 732 00:41:14,820 --> 00:41:20,190 So how would you get the iron out of the siderophore? 733 00:41:20,190 --> 00:41:24,000 And so I'm going to push this up, 734 00:41:24,000 --> 00:41:25,620 and I'll come back down again. 735 00:41:25,620 --> 00:41:30,030 So how do you get the iron out of the siderophore? 736 00:41:30,030 --> 00:41:33,450 And of course, what that depends on 737 00:41:33,450 --> 00:41:37,440 is the reduction potential of the iron. 738 00:41:37,440 --> 00:41:40,140 So we will see with enterobactin, in which you 739 00:41:40,140 --> 00:41:42,360 already looked at, the reduction potential 740 00:41:42,360 --> 00:41:48,420 under neutral conditions is minus 750 millivolts. 741 00:41:48,420 --> 00:41:51,030 Minus means that it's really hard to reduce. 742 00:41:51,030 --> 00:41:52,800 It wants to be oxidized. 743 00:41:52,800 --> 00:41:56,190 It's outside the realm of what you can do inside the cell. 744 00:41:56,190 --> 00:41:58,930 So And we want to reduce iron 3 to iron 2. 745 00:41:58,930 --> 00:41:59,910 Why? 746 00:41:59,910 --> 00:42:05,190 Because we increase the exchangeability of our ligands. 747 00:42:05,190 --> 00:42:07,440 That's why that was introduced before. 748 00:42:07,440 --> 00:42:12,160 So you could reduce this, potentially. 749 00:42:12,160 --> 00:42:14,400 And so I'll just put a question mark there. 750 00:42:14,400 --> 00:42:18,750 And so then what you have is a siderophore. 751 00:42:18,750 --> 00:42:21,180 So let me just write this down so you don't forget. 752 00:42:21,180 --> 00:42:22,740 So this is the siderophore. 753 00:42:27,770 --> 00:42:29,600 And then you have your iron. 754 00:42:29,600 --> 00:42:33,710 So what happens to the siderophore with no iron? 755 00:42:33,710 --> 00:42:38,480 It can now get recycled back to pick up more iron. 756 00:42:38,480 --> 00:42:40,700 So let me just put this here. 757 00:42:40,700 --> 00:42:41,435 This is recycled. 758 00:42:44,300 --> 00:42:49,210 And we're going to be focusing on here taking up iron 759 00:42:49,210 --> 00:42:51,650 from siderophores, but we'll see that you can take up iron 760 00:42:51,650 --> 00:42:52,523 from hemes. 761 00:42:52,523 --> 00:42:53,690 And you have the same issue. 762 00:42:53,690 --> 00:42:55,273 You're going to use the same strategy. 763 00:42:55,273 --> 00:42:56,710 You'll bring it into the cell. 764 00:42:56,710 --> 00:42:59,360 You've got to get the iron out of the heme, 765 00:42:59,360 --> 00:43:01,100 and you have to recycle it. 766 00:43:01,100 --> 00:43:03,950 So if you can't reduce it, what do you do? 767 00:43:03,950 --> 00:43:06,550 Does anybody remember what you do with enterobactin? 768 00:43:09,970 --> 00:43:11,210 Anybody remember the KD? 769 00:43:11,210 --> 00:43:13,130 We talked about this last time, but it bonds 770 00:43:13,130 --> 00:43:14,420 like a son of a gun. 771 00:43:14,420 --> 00:43:17,780 It's hard to reduce. 772 00:43:17,780 --> 00:43:20,480 You have ester linkages in enterobactin. 773 00:43:20,480 --> 00:43:22,430 If you go back and look at the structure, 774 00:43:22,430 --> 00:43:25,550 there are proteins that can hydrolize the ester linkages. 775 00:43:25,550 --> 00:43:28,250 So ring opens-- makes it bind less tightly, 776 00:43:28,250 --> 00:43:29,840 and so it can be released. 777 00:43:29,840 --> 00:43:39,467 So in the case of enterobactin, you have an esterase. 778 00:43:39,467 --> 00:43:41,050 So let me just show you that, and then 779 00:43:41,050 --> 00:43:43,970 we'll come back again to the gram-negative. 780 00:43:43,970 --> 00:43:45,790 But if you look at the siderophores, 781 00:43:45,790 --> 00:43:47,890 there are 500 siderophores. 782 00:43:47,890 --> 00:43:51,003 Here is enterobactin. here are the esters. 783 00:43:51,003 --> 00:43:52,420 You can hydrolyze them to release. 784 00:43:52,420 --> 00:43:55,180 You can't reduce, because, again, 785 00:43:55,180 --> 00:43:57,680 the more negative, the more it wants to be oxidized. 786 00:43:57,680 --> 00:44:00,710 And the range of reduction inside the cell 787 00:44:00,710 --> 00:44:02,920 is maybe minus 500. 788 00:44:02,920 --> 00:44:06,197 You can't get that much above that. 789 00:44:06,197 --> 00:44:07,780 But if you look down here at citrate-- 790 00:44:07,780 --> 00:44:10,510 remember, we were talking about citrate-- 791 00:44:10,510 --> 00:44:15,190 unusual in that citrate is part of this aconitase IRP1 and IRP2 792 00:44:15,190 --> 00:44:15,980 system. 793 00:44:15,980 --> 00:44:17,980 But what's the reduction potentially 794 00:44:17,980 --> 00:44:19,360 are completely different. 795 00:44:19,360 --> 00:44:22,210 So if you had iron citrate, you could easily reduce it 796 00:44:22,210 --> 00:44:23,680 under physiological conditions. 797 00:44:23,680 --> 00:44:28,240 So the strategies you need to be able to release the iron 798 00:44:28,240 --> 00:44:30,520 to then use the iron for what you want to do 799 00:44:30,520 --> 00:44:34,660 is distinct depending on the siderophore. 800 00:44:34,660 --> 00:44:38,770 So if we go back, now let's just look over here 801 00:44:38,770 --> 00:44:42,350 and I'll draw that in parallel. 802 00:44:42,350 --> 00:44:46,250 So what's the difference between gram-positive 803 00:44:46,250 --> 00:44:49,582 and gram-negative? 804 00:44:49,582 --> 00:44:50,540 So let's draw that out. 805 00:44:50,540 --> 00:44:53,690 And then what I'm going to show you, rapidly, is, 806 00:44:53,690 --> 00:44:57,210 again, the strategies with heme are subtly different, but very, 807 00:44:57,210 --> 00:44:58,940 very similar. 808 00:44:58,940 --> 00:45:00,860 We have different sets of proteins. 809 00:45:00,860 --> 00:45:07,290 So with gram-negative we have an outer membrane. 810 00:45:07,290 --> 00:45:08,540 So this is gram-negative. 811 00:45:11,850 --> 00:45:19,768 And what we have in the outer membrane are proteins. 812 00:45:19,768 --> 00:45:20,810 It has a lot of proteins. 813 00:45:23,770 --> 00:45:28,270 And it has a big protein with a ball in it. 814 00:45:28,270 --> 00:45:33,010 And these proteins-- it has 27 beta strands, and these 815 00:45:33,010 --> 00:45:34,720 are beta barrels. 816 00:45:34,720 --> 00:45:36,940 So there are many, many of these proteins. 817 00:45:36,940 --> 00:45:40,660 In fact if any of you heard Dan Cohn's talk this past semester, 818 00:45:40,660 --> 00:45:43,930 he's figured out how do these things get made down here 819 00:45:43,930 --> 00:45:45,580 and get inserted in the outer membrane. 820 00:45:45,580 --> 00:45:47,420 It's an interesting problem. 821 00:45:47,420 --> 00:45:53,350 So these are beta barrel proteins, 822 00:45:53,350 --> 00:45:55,575 and they have 27 strands. 823 00:46:00,010 --> 00:46:05,980 We then have a peptidoglycan. 824 00:46:05,980 --> 00:46:10,660 But the peptidoglycan is distinct. 825 00:46:10,660 --> 00:46:13,000 It's much smaller. 826 00:46:13,000 --> 00:46:15,010 It doesn't take up anywhere near as much space. 827 00:46:15,010 --> 00:46:17,895 And then you have your plasma membrane. 828 00:46:23,530 --> 00:46:25,690 And then in the plasma membrane-- 829 00:46:25,690 --> 00:46:27,739 so this is a plasma membrane-- 830 00:46:33,120 --> 00:46:35,070 you still need to do the same thing. 831 00:46:35,070 --> 00:46:39,180 You need to get your siderophore into the cell. 832 00:46:39,180 --> 00:46:40,380 So what do you have here? 833 00:46:40,380 --> 00:46:42,000 You still have transporters. 834 00:46:44,700 --> 00:46:50,310 And those transporters are going to allow your siderophore 835 00:46:50,310 --> 00:46:52,890 to go into the cell, just like we saw with the gram-positive. 836 00:46:52,890 --> 00:46:56,610 So over here then, we have a siderophore-- 837 00:46:56,610 --> 00:47:00,360 again, the same types of siderophores. 838 00:47:00,360 --> 00:47:04,910 So somehow it needs to get inside the cell. 839 00:47:04,910 --> 00:47:07,590 And we have many of these beta barrels, and a lot of them 840 00:47:07,590 --> 00:47:09,858 are specific for a given siderophores. 841 00:47:09,858 --> 00:47:11,400 There are many, many of these things. 842 00:47:11,400 --> 00:47:12,317 We'll look at E. coli. 843 00:47:12,317 --> 00:47:17,297 There are 10 different ways to get iron from the environment 844 00:47:17,297 --> 00:47:17,880 into the cell. 845 00:47:17,880 --> 00:47:21,300 That tells you how important all of this is. 846 00:47:21,300 --> 00:47:24,840 And it turns out that you also have, 847 00:47:24,840 --> 00:47:29,010 inside the cell, a periplasmic binding protein 848 00:47:29,010 --> 00:47:30,930 that can pick up the iron when it 849 00:47:30,930 --> 00:47:32,550 gets transferred across here. 850 00:47:32,550 --> 00:47:42,265 So you have a periplasmic binding protein. 851 00:47:46,380 --> 00:47:50,460 And one of the questions is, how does the siderophore 852 00:47:50,460 --> 00:47:51,780 get transferred? 853 00:47:51,780 --> 00:47:56,100 And to do that in gram-negative bacteria, you need a machine. 854 00:47:56,100 --> 00:47:58,440 And that machine is composed of three proteins. 855 00:47:58,440 --> 00:48:00,390 It's called the tan protein. 856 00:48:00,390 --> 00:48:03,870 If you look over there in pink, you have tanB. 857 00:48:03,870 --> 00:48:04,590 It's exbB. 858 00:48:08,010 --> 00:48:12,390 And this should be not C, but exbD. 859 00:48:12,390 --> 00:48:14,280 So there are three proteins required. 860 00:48:14,280 --> 00:48:18,060 And they somehow can use the proton motor force 861 00:48:18,060 --> 00:48:22,020 from the inner plasma membrane to allow transport 862 00:48:22,020 --> 00:48:24,160 across the outer membrane. 863 00:48:24,160 --> 00:48:35,090 So in all of these, one has tanB. 864 00:48:35,090 --> 00:48:38,890 So this is tanB. 865 00:48:38,890 --> 00:48:43,820 And tanB can recognize part of the beta barrel. 866 00:48:43,820 --> 00:48:47,660 So it interacts with the beta barrel protein. 867 00:48:47,660 --> 00:48:53,390 And this is exbD and exbB. 868 00:48:56,960 --> 00:49:00,230 And again, you generate a proton motor force 869 00:49:00,230 --> 00:49:04,400 which allows the siderophore to get into the cell. 870 00:49:04,400 --> 00:49:09,080 It then gets transferred to a periplasmic binding protein. 871 00:49:09,080 --> 00:49:10,670 And then what does it have to do? 872 00:49:10,670 --> 00:49:17,770 So from here, it has to go through our transporter. 873 00:49:17,770 --> 00:49:21,025 So let me put this up here, just like we just did before. 874 00:49:24,610 --> 00:49:28,030 So your siderophore-- so you can't see the bottom 875 00:49:28,030 --> 00:49:30,550 of my transporter-- 876 00:49:33,400 --> 00:49:37,810 comes through So this is the plasma membrane. 877 00:49:40,325 --> 00:49:41,200 And what do you have? 878 00:49:41,200 --> 00:49:42,340 You have the same problem. 879 00:49:42,340 --> 00:49:45,130 You have to get the iron out of the siderophore. 880 00:49:45,130 --> 00:49:47,760 And so the problem is exactly the same and gram-negative 881 00:49:47,760 --> 00:49:49,120 and gram-positive. 882 00:49:49,120 --> 00:49:51,160 So you somehow have to get it in. 883 00:49:51,160 --> 00:49:54,580 It's more complicated to get it in with gram-negative because 884 00:49:54,580 --> 00:49:57,730 of the different constructions of the peptidoglycans 885 00:49:57,730 --> 00:49:59,110 and the outer membrane. 886 00:49:59,110 --> 00:50:03,190 The other thing I wanted to say about the other outer membrane 887 00:50:03,190 --> 00:50:04,810 is-- 888 00:50:04,810 --> 00:50:06,478 which I don't know if you guys know, 889 00:50:06,478 --> 00:50:08,020 but I think it's incredibly important 890 00:50:08,020 --> 00:50:12,040 and is a major issue in a human disease-- 891 00:50:12,040 --> 00:50:15,940 the fact that, in addition to this outer membrane 892 00:50:15,940 --> 00:50:19,090 in these beta barrels, the whole outer surface 893 00:50:19,090 --> 00:50:21,670 is covered with sort of amazing molecules 894 00:50:21,670 --> 00:50:23,080 called lipopolysaccharides. 895 00:50:23,080 --> 00:50:31,293 So the whole outer surface is covered with LPS-- 896 00:50:31,293 --> 00:50:33,460 I'm not going to write it out-- lipopolysaccharides. 897 00:50:33,460 --> 00:50:36,970 Which, actually, one of my best friends 898 00:50:36,970 --> 00:50:39,540 elucidated the whole pathway for how that works. 899 00:50:39,540 --> 00:50:43,000 It's a beautiful, beautiful set of biochemical studies 900 00:50:43,000 --> 00:50:44,800 to figure out how this thing is made. 901 00:50:44,800 --> 00:50:45,640 It's got lipids. 902 00:50:45,640 --> 00:50:46,780 It's got all these sugars. 903 00:50:46,780 --> 00:50:49,330 It's got all this stuff hanging off of it. 904 00:50:49,330 --> 00:50:52,890 And this thing is really important in human health. 905 00:50:52,890 --> 00:50:54,710 If you read about infections, they're 906 00:50:54,710 --> 00:50:57,520 always talking about lipopolysaccharides. 907 00:50:57,520 --> 00:51:01,450 So what I'm going to do next time, just by way of showing 908 00:51:01,450 --> 00:51:04,240 you to introduce you to this-- 909 00:51:04,240 --> 00:51:06,190 you can see here in the next cartoon 910 00:51:06,190 --> 00:51:09,130 we have the same problem when we want to take up hemes 911 00:51:09,130 --> 00:51:10,750 as opposed to siderophores. 912 00:51:10,750 --> 00:51:12,880 And we're going to focus on hemes. 913 00:51:12,880 --> 00:51:15,670 So this is a cartoon, very similar to the one you just 914 00:51:15,670 --> 00:51:16,670 saw. 915 00:51:16,670 --> 00:51:18,880 And there are a couple of proteins on the outside 916 00:51:18,880 --> 00:51:20,260 that you need to think about. 917 00:51:20,260 --> 00:51:24,460 How are you going to get the heme across the peptidoglycan 918 00:51:24,460 --> 00:51:25,650 or into the cells? 919 00:51:25,650 --> 00:51:27,750 So the model is very similar. 920 00:51:27,750 --> 00:51:28,750 You should look at that. 921 00:51:28,750 --> 00:51:31,690 And then we'll see this is what your problem 922 00:51:31,690 --> 00:51:33,310 set is going to be on. 923 00:51:33,310 --> 00:51:35,410 This is for Staph aureus. 924 00:51:35,410 --> 00:51:38,830 And we'll see that if you get a heme, 925 00:51:38,830 --> 00:51:42,280 there's going to be bucket brigade that can transfer 926 00:51:42,280 --> 00:51:45,580 the heme through proteins covalently bound 927 00:51:45,580 --> 00:51:48,280 to the peptidoglycan into the cell. 928 00:51:48,280 --> 00:51:51,760 It's sort an amazing system, and that's 929 00:51:51,760 --> 00:51:54,820 what we're going to talk about for probably the first half 930 00:51:54,820 --> 00:51:56,240 of the next lecture. 931 00:51:56,240 --> 00:51:58,365 So you're going to have to read on that on your own 932 00:51:58,365 --> 00:52:00,060 to solve the. problem.