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 00:00:29,620 --> 00:00:32,500 JOANNE STUBBE: --iron homeostasis in module 6. 9 00:00:32,500 --> 00:00:36,820 And then, we're going to move on to module 7. 10 00:00:36,820 --> 00:00:38,920 And the readings have been posted. 11 00:00:38,920 --> 00:00:42,970 And the PowerPoint has been posted for today's lecture 12 00:00:42,970 --> 00:00:44,240 as well. 13 00:00:44,240 --> 00:00:44,740 OK. 14 00:00:44,740 --> 00:00:48,670 So we were talking about last time peptidoglycans 15 00:00:48,670 --> 00:00:52,550 in gram-positive bacteria. 16 00:00:52,550 --> 00:00:54,280 And they're actually quite thick, 17 00:00:54,280 --> 00:00:56,000 depending on the organism. 18 00:00:56,000 --> 00:00:58,540 And so, what is the strategy nature 19 00:00:58,540 --> 00:01:03,820 uses to be able to pick up iron where 20 00:01:03,820 --> 00:01:07,870 you have a bacteria surrounded by this huge peptidoglycan? 21 00:01:07,870 --> 00:01:12,130 And the strategy in Staph aureus that has evolved 22 00:01:12,130 --> 00:01:15,160 is they attach everything, almost everything, 23 00:01:15,160 --> 00:01:18,040 covalently to the peptidoglycan itself. 24 00:01:18,040 --> 00:01:21,730 So at the end of the last lecture, we were looking at-- 25 00:01:21,730 --> 00:01:24,640 we introduced you to the operon and Staph aureus. 26 00:01:24,640 --> 00:01:27,400 And there were proteins called sortases, 27 00:01:27,400 --> 00:01:30,850 which are transpeptidases, really, basically. 28 00:01:30,850 --> 00:01:34,570 And they have different sequence specificities. 29 00:01:34,570 --> 00:01:36,550 And I'll just go back one. 30 00:01:36,550 --> 00:01:40,270 So where we were last time, we have this operon. 31 00:01:40,270 --> 00:01:42,520 And all of the proteins that are going 32 00:01:42,520 --> 00:01:47,150 to be involved in uptake of heme into the cell 33 00:01:47,150 --> 00:01:50,530 are called the Isd proteins. 34 00:01:50,530 --> 00:01:53,690 And so, here is the operon. 35 00:01:53,690 --> 00:01:57,100 And you can see that each part of this operon 36 00:01:57,100 --> 00:01:58,990 is activated by a Fur box. 37 00:01:58,990 --> 00:02:02,320 A Fur box is the transcriptional factor 38 00:02:02,320 --> 00:02:06,370 that turns off or on regulation at the transcriptional level 39 00:02:06,370 --> 00:02:08,830 by the presence or absence of iron, which we're not 40 00:02:08,830 --> 00:02:11,230 going to talk about further. 41 00:02:11,230 --> 00:02:14,560 And what I wanted to point out here before we move on 42 00:02:14,560 --> 00:02:18,130 is that all of the yellow anchors 43 00:02:18,130 --> 00:02:22,390 are proteins that are attached covalently by Sortase A, which 44 00:02:22,390 --> 00:02:24,700 has a defined zip code. 45 00:02:24,700 --> 00:02:30,235 The blue in IsdC has its own sortase, 46 00:02:30,235 --> 00:02:35,590 Sortase B. And IsdE is not attached covalently 47 00:02:35,590 --> 00:02:38,630 to the peptidoglycan, but actually has 48 00:02:38,630 --> 00:02:41,110 a membrane that's attached covalently 49 00:02:41,110 --> 00:02:43,630 to a lipid, which is then inserted 50 00:02:43,630 --> 00:02:47,110 into the membrane, which is the other strategy that's 51 00:02:47,110 --> 00:02:48,590 used in these systems. 52 00:02:48,590 --> 00:02:50,383 And let me just point out-- you saw this. 53 00:02:50,383 --> 00:02:52,300 I'm not going to spend much time on this today 54 00:02:52,300 --> 00:02:54,980 because you've already had a problem in your last problem 55 00:02:54,980 --> 00:02:55,660 set on this. 56 00:02:55,660 --> 00:03:00,220 But you have these little N domains, or the NEAT domains, 57 00:03:00,220 --> 00:03:05,250 which are 120 amino acid domains that allow you to pick up heme, 58 00:03:05,250 --> 00:03:09,700 and are involved in transfer of heme down a bucket brigade. 59 00:03:09,700 --> 00:03:13,930 So what we're trying to do is attach these Isd proteins 60 00:03:13,930 --> 00:03:16,000 to the peptidoglycan. 61 00:03:16,000 --> 00:03:18,430 How it does that, how they end up being organized, 62 00:03:18,430 --> 00:03:22,330 how many there are, is all an active area of research. 63 00:03:22,330 --> 00:03:23,860 We don't really know that. 64 00:03:23,860 --> 00:03:25,810 Somebody might know it, but it's not 65 00:03:25,810 --> 00:03:27,520 in the published literature. 66 00:03:27,520 --> 00:03:29,290 And so, the idea is, again, you can 67 00:03:29,290 --> 00:03:32,430 see all of these proteins in some way 68 00:03:32,430 --> 00:03:37,450 are tethered via usually a single transmembrane spanning 69 00:03:37,450 --> 00:03:39,070 a region in the protein. 70 00:03:39,070 --> 00:03:43,480 And here is one Isd protein here with a little zip code. 71 00:03:43,480 --> 00:03:46,900 And it involves the Sortase A, which you've now 72 00:03:46,900 --> 00:03:48,460 seen over and over again, happens 73 00:03:48,460 --> 00:03:51,430 to have a cysteine in its active site 74 00:03:51,430 --> 00:03:54,220 to go through a covalent bond formation. 75 00:03:54,220 --> 00:03:58,030 And you're going to be cleaving one peptide bond 76 00:03:58,030 --> 00:04:00,040 and forming another peptide bond. 77 00:04:00,040 --> 00:04:02,030 Where have you seen that before? 78 00:04:02,030 --> 00:04:03,820 That's how you did cross-linking, 79 00:04:03,820 --> 00:04:07,600 the transpeptidation reaction we talked 80 00:04:07,600 --> 00:04:11,950 about last time that allows you to form the bag. 81 00:04:11,950 --> 00:04:16,089 And the bag of peptidoglycans becomes cross-linked. 82 00:04:16,089 --> 00:04:20,329 And that's the key to survival of the organism. 83 00:04:20,329 --> 00:04:23,950 And so, you cleave this bond between T and G. 84 00:04:23,950 --> 00:04:26,920 The specificity is understood. 85 00:04:26,920 --> 00:04:31,390 And people in the Pentelutes Lab use sortase quite a bit. 86 00:04:31,390 --> 00:04:34,930 But it has been engineered by the Liu lab 87 00:04:34,930 --> 00:04:37,910 to be more efficient as a catalyst. 88 00:04:37,910 --> 00:04:39,980 So this is a natural catalyst. 89 00:04:39,980 --> 00:04:42,790 You do a transpeptidation reaction. 90 00:04:42,790 --> 00:04:44,890 And then you have-- 91 00:04:44,890 --> 00:04:46,570 this is to me, what's amazing. 92 00:04:46,570 --> 00:04:51,670 You have this lipid II, which is that C55 isoprene 93 00:04:51,670 --> 00:04:54,160 system with the pentaglycine hanging off 94 00:04:54,160 --> 00:04:56,890 the pentapeptide, all of which is generated 95 00:04:56,890 --> 00:04:58,420 on the inside of the cell, and gets 96 00:04:58,420 --> 00:05:01,110 transferred to the outside of the cell. 97 00:05:01,110 --> 00:05:06,130 One, then, is set up to do peptide bond formation, 98 00:05:06,130 --> 00:05:11,140 just like we saw with the transpeptidase reaction. 99 00:05:11,140 --> 00:05:18,160 And so, you end up with then this lipid, lipid II attached 100 00:05:18,160 --> 00:05:20,590 to the Isd protein. 101 00:05:20,590 --> 00:05:23,380 And then, that's taken-- 102 00:05:23,380 --> 00:05:25,450 so this is the detailed chemistry 103 00:05:25,450 --> 00:05:26,590 that we just discussed. 104 00:05:26,590 --> 00:05:28,965 And you can go through it again if you don't remember it. 105 00:05:28,965 --> 00:05:31,450 But it's very similar to things we've now 106 00:05:31,450 --> 00:05:34,480 seen many, many times in this course. 107 00:05:34,480 --> 00:05:40,840 So once you attach this, what happens is it gets transferred. 108 00:05:40,840 --> 00:05:43,840 The whole thing now gets transferred by the mechanism 109 00:05:43,840 --> 00:05:45,970 similar to forming the polymer. 110 00:05:45,970 --> 00:05:49,150 But now you have the pentaglycine with an Isd 111 00:05:49,150 --> 00:05:51,760 protein attached as well. 112 00:05:51,760 --> 00:05:58,060 And so you can see, then, this is the growing peptidoglycan 113 00:05:58,060 --> 00:06:01,060 with alternating units of N-Acetylglucosamine and 114 00:06:01,060 --> 00:06:04,270 N-Acetylmuramic acid we talked about last time. 115 00:06:04,270 --> 00:06:07,920 And now we have the Isd protein covalently attached. 116 00:06:07,920 --> 00:06:10,720 So this is a pretty amazing strategy. 117 00:06:10,720 --> 00:06:13,330 And you can imagine trying to study this 118 00:06:13,330 --> 00:06:15,490 under natural systems. 119 00:06:15,490 --> 00:06:17,653 With everything attached, it's quite challenging. 120 00:06:17,653 --> 00:06:19,570 And so the problem you had in the problem set, 121 00:06:19,570 --> 00:06:20,530 what did they do? 122 00:06:20,530 --> 00:06:22,270 They cut everything off. 123 00:06:22,270 --> 00:06:25,660 And all you're looking at is a little piece 124 00:06:25,660 --> 00:06:28,240 of the protein, not the whole protein anchored 125 00:06:28,240 --> 00:06:32,560 in this way with the right topology in the active site. 126 00:06:32,560 --> 00:06:37,420 So, to me, even thinking about how you make a peptidoglycan 127 00:06:37,420 --> 00:06:41,760 that's 50-50 nanometers thick-- 128 00:06:41,760 --> 00:06:43,060 so you add things. 129 00:06:43,060 --> 00:06:47,590 How does it construct itself, a sacculus around this system 130 00:06:47,590 --> 00:06:49,180 and build up? 131 00:06:49,180 --> 00:06:50,510 I think nobody knows that. 132 00:06:50,510 --> 00:06:53,920 And recent studies have been able to the first time 133 00:06:53,920 --> 00:06:59,020 to, in Staph aureus, reconstitute transpeptidase 134 00:06:59,020 --> 00:07:01,310 and a glycosyltransferase reaction. 135 00:07:01,310 --> 00:07:04,630 So maybe that, with a sort of an imaging technology 136 00:07:04,630 --> 00:07:07,660 we have, maybe in the next five or six years somebody 137 00:07:07,660 --> 00:07:11,240 will figure out how this amazing thing is organized. 138 00:07:11,240 --> 00:07:16,270 So anyhow, we were able to attach these things covalently. 139 00:07:16,270 --> 00:07:17,530 And so, here we are. 140 00:07:17,530 --> 00:07:20,410 We've got all of these things anchored. 141 00:07:20,410 --> 00:07:24,100 And then, in your problem set-- 142 00:07:24,100 --> 00:07:25,870 and in the last part of this, I just 143 00:07:25,870 --> 00:07:27,940 wanted to very briefly go through how 144 00:07:27,940 --> 00:07:33,850 are people trying to study how the heme that's up here 145 00:07:33,850 --> 00:07:36,410 gets transferred. 146 00:07:36,410 --> 00:07:39,100 And so, this is a cartoon model. 147 00:07:39,100 --> 00:07:40,810 And so, we have hemoglobin. 148 00:07:40,810 --> 00:07:42,640 We've been able to get-- 149 00:07:42,640 --> 00:07:45,610 we've been able to lyse red blood cells, 150 00:07:45,610 --> 00:07:49,780 get hemoglobin to come out, or haptoglobin hemoglobin. 151 00:07:49,780 --> 00:07:53,680 And now somehow you have to extract the heme. 152 00:07:53,680 --> 00:07:59,770 And that's done by two proteins, ISB and H. 153 00:07:59,770 --> 00:08:06,080 And then we somehow have to get it to the plasma membrane. 154 00:08:06,080 --> 00:08:13,270 And so, the model is that this is transferred from IsdB 155 00:08:13,270 --> 00:08:15,130 through a NEAT domain. 156 00:08:15,130 --> 00:08:17,590 So all of these transfers up to here 157 00:08:17,590 --> 00:08:20,270 are through these little domains that can bind heme. 158 00:08:20,270 --> 00:08:21,520 And we have a structure of it. 159 00:08:21,520 --> 00:08:23,960 I'll briefly show you that. 160 00:08:23,960 --> 00:08:27,460 And then the question, is there an order? 161 00:08:27,460 --> 00:08:35,919 If you get it out of IsdB versus IsdH, is it transferred? 162 00:08:35,919 --> 00:08:43,179 Here we have A closer to this than B. But is C closer than A? 163 00:08:43,179 --> 00:08:45,080 What is the organization? 164 00:08:45,080 --> 00:08:46,990 How many of these guys are there? 165 00:08:46,990 --> 00:08:50,650 Can they be transferred back and forth between the IsdAs? 166 00:08:50,650 --> 00:08:52,510 I mean, there's 100 questions you can ask. 167 00:08:52,510 --> 00:08:56,200 And I think we don't know the answer to any of them. 168 00:08:56,200 --> 00:08:59,470 And the methodology, the problems 169 00:08:59,470 --> 00:09:00,970 that you looked at, again, they were 170 00:09:00,970 --> 00:09:05,470 looking at something that wasn't the intact system. 171 00:09:05,470 --> 00:09:08,470 And the key to all of this, of course, is the kinetics. 172 00:09:08,470 --> 00:09:11,295 And I think when you start taking apart the pieces-- 173 00:09:11,295 --> 00:09:13,090 so you have this piece and this piece, 174 00:09:13,090 --> 00:09:16,000 and half the spinach is missing, then 175 00:09:16,000 --> 00:09:18,460 you have this issue of how do you design the experiment 176 00:09:18,460 --> 00:09:20,320 so you have the right concentrations, 177 00:09:20,320 --> 00:09:24,640 so they meet in an efficient fashion? 178 00:09:24,640 --> 00:09:27,940 And so the kinetics of what you were looking at in your problem 179 00:09:27,940 --> 00:09:32,020 set are not realistic. 180 00:09:32,020 --> 00:09:34,690 Because things aren't covalently attached. 181 00:09:34,690 --> 00:09:36,310 And so the question is, does it really 182 00:09:36,310 --> 00:09:38,680 give you a good representation of what's 183 00:09:38,680 --> 00:09:40,630 going on in the intact system. 184 00:09:40,630 --> 00:09:44,080 So the model has been from studies like the ones 185 00:09:44,080 --> 00:09:49,180 that you saw in your problem set, that there is a pathway. 186 00:09:49,180 --> 00:09:52,690 That this protein can extract-- 187 00:09:52,690 --> 00:09:56,020 the B and the H proteins can extract the heme. 188 00:09:56,020 --> 00:09:58,470 It's transferred to the A protein. 189 00:09:58,470 --> 00:10:02,290 And so, here we have, can it be transferred directly 190 00:10:02,290 --> 00:10:04,200 to the C protein? 191 00:10:04,200 --> 00:10:06,100 Or does it need to go through the A protein? 192 00:10:06,100 --> 00:10:09,350 So then you can start doing experiments like that. 193 00:10:09,350 --> 00:10:11,890 And you can take this protein and look for transfer 194 00:10:11,890 --> 00:10:13,720 to this protein. 195 00:10:13,720 --> 00:10:16,030 Or you can add in this protein and see 196 00:10:16,030 --> 00:10:19,900 if the rate, if you set the experiment up correctly, 197 00:10:19,900 --> 00:10:20,750 increases. 198 00:10:20,750 --> 00:10:22,270 You can also ask the question, are 199 00:10:22,270 --> 00:10:25,510 these reactions stoichiometric or catalytic, 200 00:10:25,510 --> 00:10:27,850 which was also asked in the problems 201 00:10:27,850 --> 00:10:30,130 that you were looking at. 202 00:10:30,130 --> 00:10:33,850 If there is an order, how fast is 203 00:10:33,850 --> 00:10:37,540 the transfer from here to here versus here to here? 204 00:10:37,540 --> 00:10:40,270 And if it's really fast, what can happen 205 00:10:40,270 --> 00:10:43,060 is you don't need stoichiometric amounts of this. 206 00:10:43,060 --> 00:10:45,160 You can use small amounts of this 207 00:10:45,160 --> 00:10:46,810 to get the transfer to work. 208 00:10:46,810 --> 00:10:50,140 And those are the kinds of questions that people 209 00:10:50,140 --> 00:10:51,520 are actually focused on. 210 00:10:51,520 --> 00:10:56,830 So this is one model that came out of the types of studies 211 00:10:56,830 --> 00:10:59,020 that you guys had on your problem set. 212 00:10:59,020 --> 00:11:04,720 And eventually, you need to get down here to transfer this 213 00:11:04,720 --> 00:11:08,980 to the ATPase, which allows the heme to be transferred 214 00:11:08,980 --> 00:11:10,090 into the cytosol. 215 00:11:10,090 --> 00:11:12,280 So these proteins are structurally 216 00:11:12,280 --> 00:11:15,280 distinct from this protein, which is structurally 217 00:11:15,280 --> 00:11:16,630 distinct from that protein. 218 00:11:16,630 --> 00:11:19,240 We have structures of all these things. 219 00:11:19,240 --> 00:11:21,310 And they all do heme transfer. 220 00:11:21,310 --> 00:11:24,100 That's what their function actually is. 221 00:11:24,100 --> 00:11:27,640 And so the details, the molecular details 222 00:11:27,640 --> 00:11:30,490 of how the heme is transferred is a major focus 223 00:11:30,490 --> 00:11:33,170 of a lot of energy right now. 224 00:11:33,170 --> 00:11:36,410 And so, if you actually look at this, 225 00:11:36,410 --> 00:11:39,880 I want to just say a few things about how this happens. 226 00:11:39,880 --> 00:11:42,250 And so, one of the things that you 227 00:11:42,250 --> 00:11:49,850 need to think about, so what are the methods 228 00:11:49,850 --> 00:12:02,580 to examine Isd-heme transfer? 229 00:12:02,580 --> 00:12:08,480 And this is through these NEAT domains, 230 00:12:08,480 --> 00:12:11,330 which I defined before. 231 00:12:11,330 --> 00:12:13,730 And so, one of the first things that people did 232 00:12:13,730 --> 00:12:25,620 was number one, you need to clone, express, purify, 233 00:12:25,620 --> 00:12:28,910 the Isd proteins. 234 00:12:28,910 --> 00:12:31,940 And most of these studies have been done 235 00:12:31,940 --> 00:12:38,205 in pieces versus full length. 236 00:12:38,205 --> 00:12:40,580 And when you do-- so you could try to do the whole thing. 237 00:12:40,580 --> 00:12:42,650 Some of these things have three NEAT domains. 238 00:12:42,650 --> 00:12:44,120 Some have one NEAT domain. 239 00:12:44,120 --> 00:12:46,400 If you look in those cartoons I gave you, 240 00:12:46,400 --> 00:12:52,140 the number of NEAT domains are defined by sequences. 241 00:12:52,140 --> 00:12:55,828 So you can study the full-length thing. 242 00:12:55,828 --> 00:12:58,370 Or the other one-- and, in fact, I think in your problem set, 243 00:12:58,370 --> 00:13:00,037 I can't remember, but in the problem set 244 00:13:00,037 --> 00:13:02,750 I think you actually did an expe-- was 245 00:13:02,750 --> 00:13:06,890 an experiment that you had one NEAT domain versus two. 246 00:13:06,890 --> 00:13:10,070 And what was the differences in the transfer? 247 00:13:10,070 --> 00:13:12,000 So you can make these things. 248 00:13:12,000 --> 00:13:14,785 But when you make them, heme is quite often-- because there 249 00:13:14,785 --> 00:13:17,390 is biosynthetic machinery like there 250 00:13:17,390 --> 00:13:22,350 is to insert iron clusters, biosynthetic iron clusters, 251 00:13:22,350 --> 00:13:26,120 there's also biosynthetic machinery to insert hemes. 252 00:13:26,120 --> 00:13:28,320 So it comes out-- these come out to be apo. 253 00:13:31,280 --> 00:13:33,290 So then, what you need to do is you 254 00:13:33,290 --> 00:13:39,240 need to add apo plus protoporphyrin 255 00:13:39,240 --> 00:13:42,630 IX to get presumably holo-- 256 00:13:45,630 --> 00:13:49,350 holo, whatever Isd is. 257 00:13:49,350 --> 00:13:51,420 And then, you need to purify that so you 258 00:13:51,420 --> 00:13:52,502 can get rid of any heme. 259 00:13:52,502 --> 00:13:54,460 You don't have a bunch of heme floating around. 260 00:13:54,460 --> 00:13:58,120 So, it's, again, yet another purification 261 00:13:58,120 --> 00:14:03,760 to look at all of this stuff that may or may not be easy. 262 00:14:03,760 --> 00:14:07,500 And so, then after you do that, what you want to do 263 00:14:07,500 --> 00:14:16,440 is you need to characterize the spectra of the Isd 264 00:14:16,440 --> 00:14:20,040 proteins loaded with heme. 265 00:14:20,040 --> 00:14:22,620 And so this is the key to the solution 266 00:14:22,620 --> 00:14:24,660 of the problem, which is the same thing you 267 00:14:24,660 --> 00:14:27,340 saw in your problem set. 268 00:14:27,340 --> 00:14:29,730 But if you look at hemes-- 269 00:14:29,730 --> 00:14:33,240 and I'll show you one set of data, a different one from what 270 00:14:33,240 --> 00:14:34,800 you had in your problem set. 271 00:14:34,800 --> 00:14:38,790 But hemes, you all know this from looking at hemoglobin, 272 00:14:38,790 --> 00:14:41,190 when you prick yourself and you bleed, if it's red 273 00:14:41,190 --> 00:14:43,860 or it's blue, depending on the oxygenation state, 274 00:14:43,860 --> 00:14:47,860 they have a very strong band called the Soret band 275 00:14:47,860 --> 00:14:50,880 at 400 nanometers-- 276 00:14:50,880 --> 00:14:57,900 about 400. 277 00:14:57,900 --> 00:14:59,220 And that's a key thing. 278 00:14:59,220 --> 00:15:01,320 Why do they like the Soret band? 279 00:15:01,320 --> 00:15:04,290 Because it has extremely high extinction coefficient. 280 00:15:04,290 --> 00:15:05,680 I don't know what the number is. 281 00:15:05,680 --> 00:15:10,888 But its extinction coefficient at 400 is approximately 10 282 00:15:10,888 --> 00:15:11,430 to the fifth. 283 00:15:14,430 --> 00:15:16,410 So it's easy to see that's why people have 284 00:15:16,410 --> 00:15:18,780 studied hemes over the years. 285 00:15:18,780 --> 00:15:23,370 Because heme is so much easier to see than any non-heme iron 286 00:15:23,370 --> 00:15:24,510 system. 287 00:15:24,510 --> 00:15:27,270 And what you can see here, which also turn out 288 00:15:27,270 --> 00:15:30,600 to be quite useful, is these much weaker bands 289 00:15:30,600 --> 00:15:34,170 between 500 and 650. 290 00:15:34,170 --> 00:15:37,830 And those bands can also be used if you have enough sample 291 00:15:37,830 --> 00:15:38,700 to look at this. 292 00:15:38,700 --> 00:15:40,110 And they're more distinct. 293 00:15:40,110 --> 00:15:42,990 They're indicative of the coordination environment 294 00:15:42,990 --> 00:15:45,677 in heme, whether it's hexacoordinate 295 00:15:45,677 --> 00:15:47,010 or whether it's pentacoordinate. 296 00:15:47,010 --> 00:15:50,250 So all of this, I'm not going to talk about the spectroscopy. 297 00:15:50,250 --> 00:15:53,010 The spectroscopy of hemes has been extremely well-studied 298 00:15:53,010 --> 00:15:55,800 and is extremely rich. 299 00:15:55,800 --> 00:15:57,180 And so, you need to do that. 300 00:15:57,180 --> 00:16:01,560 You need to have your little proteins characterized 301 00:16:01,560 --> 00:16:03,510 and loaded with heme. 302 00:16:03,510 --> 00:16:06,180 You need to quantitate the amount of heme balance. 303 00:16:06,180 --> 00:16:09,240 They want to make sure you don't have any free heme around which 304 00:16:09,240 --> 00:16:11,940 can interfere with all of your experiments. 305 00:16:11,940 --> 00:16:14,580 And then you can ask the question-- 306 00:16:14,580 --> 00:16:16,320 you can start asking your questions 307 00:16:16,320 --> 00:16:19,050 once you've got that information. 308 00:16:19,050 --> 00:16:23,940 If you start out with IsdA loaded with heme, 309 00:16:23,940 --> 00:16:29,760 does it get transferred to IsdC in the apo form? 310 00:16:29,760 --> 00:16:32,880 And so, how do you monitor this reaction? 311 00:16:32,880 --> 00:16:34,600 Anybody got any suggestions? 312 00:16:34,600 --> 00:16:36,420 One way would be-- so you want to-- 313 00:16:36,420 --> 00:16:38,580 the question that they're focused on here 314 00:16:38,580 --> 00:16:50,820 is, does IsdA with a heme plus apo-IsdC, 315 00:16:50,820 --> 00:16:54,090 does it transfer the heme to give you 316 00:16:54,090 --> 00:17:02,670 apo-IsdA plus heme-IsdC? 317 00:17:05,609 --> 00:17:09,990 And the question is, what's the mechanism of this transfer? 318 00:17:09,990 --> 00:17:13,890 So I just told you we have spectroscopy. 319 00:17:13,890 --> 00:17:16,550 So I'm going to show you you could potentially do that. 320 00:17:16,550 --> 00:17:17,609 That's not so easy to do. 321 00:17:17,609 --> 00:17:19,067 But is there another method you can 322 00:17:19,067 --> 00:17:21,839 think of so we could monitor the reaction? 323 00:17:21,839 --> 00:17:22,604 You need an assay. 324 00:17:31,057 --> 00:17:32,890 And that's what you saw in your problem set. 325 00:17:32,890 --> 00:17:38,890 So we can take advantage of the region of the Soret band, 326 00:17:38,890 --> 00:17:41,920 or perhaps in the longer wavelength. 327 00:17:41,920 --> 00:17:44,650 What other method might you be able to use 328 00:17:44,650 --> 00:17:47,740 to monitor this reaction that we've 329 00:17:47,740 --> 00:17:54,330 learned about recently in recitations, which hasn't been 330 00:17:54,330 --> 00:17:56,220 used in the papers that you've read, 331 00:17:56,220 --> 00:17:58,710 but actually has been used to study these systems? 332 00:18:01,854 --> 00:18:02,727 AUDIENCE: Mass spec? 333 00:18:02,727 --> 00:18:03,560 JoANNE STUBBE: Yeah. 334 00:18:03,560 --> 00:18:04,730 Mass spec. 335 00:18:04,730 --> 00:18:07,890 And so, what people have done-- so you have to carry-- 336 00:18:07,890 --> 00:18:10,420 what you will see is that if you look 337 00:18:10,420 --> 00:18:13,910 at the sizes of these things, some have two NEAT domains, 338 00:18:13,910 --> 00:18:15,920 some have one. 339 00:18:15,920 --> 00:18:19,940 The apo also is distinct in size from the non-apo. 340 00:18:19,940 --> 00:18:23,270 So theoretically, if you do all of your homework, 341 00:18:23,270 --> 00:18:25,700 you can use mass spec quantitatively 342 00:18:25,700 --> 00:18:26,960 to measure these reactions. 343 00:18:26,960 --> 00:18:30,560 And a lot of people have recently gone to that method, 344 00:18:30,560 --> 00:18:32,990 because this method is challenging. 345 00:18:32,990 --> 00:18:36,410 Now, again, the caveat is, so you always 346 00:18:36,410 --> 00:18:45,500 need to remember this, is that all the Isd proteins are not 347 00:18:45,500 --> 00:18:48,290 covalently attached to the peptidoglycan. 348 00:18:52,550 --> 00:18:54,980 So to me, this immediately raises 349 00:18:54,980 --> 00:18:58,770 this issue of how do you decide how to do your experiments? 350 00:18:58,770 --> 00:19:01,520 So how much-- do you use micromolar? 351 00:19:01,520 --> 00:19:04,700 Do you use millimolar? 352 00:19:04,700 --> 00:19:07,400 Is it widely different, depending on whether it's 353 00:19:07,400 --> 00:19:09,790 attached or not attached? 354 00:19:09,790 --> 00:19:12,110 And all of that is going to affect 355 00:19:12,110 --> 00:19:14,060 the kinetics of this transfer. 356 00:19:14,060 --> 00:19:15,310 So you can see transfer. 357 00:19:15,310 --> 00:19:18,920 But what you really want is the rate constants for transfer. 358 00:19:18,920 --> 00:19:23,075 So you have two questions, is do you see transfer? 359 00:19:25,800 --> 00:19:29,130 So that's the first question. 360 00:19:29,130 --> 00:19:32,760 And so, one could tell that by either of these two assays. 361 00:19:32,760 --> 00:19:35,970 So you could use one and two. 362 00:19:35,970 --> 00:19:39,060 And what you really want are the rate constants for transfer. 363 00:19:44,570 --> 00:19:46,360 And the rate constants for transfer 364 00:19:46,360 --> 00:19:49,410 are dependent on the concentrations. 365 00:19:49,410 --> 00:19:52,200 So how you set this up is something 366 00:19:52,200 --> 00:19:55,380 you've got to do a lot of messing around with. 367 00:19:55,380 --> 00:19:58,950 And so, if you look at this model, 368 00:19:58,950 --> 00:20:02,220 you can ask the question, how is this transfer occurred? 369 00:20:02,220 --> 00:20:05,190 Do you go through a ternary complex? 370 00:20:05,190 --> 00:20:08,700 So does this form a complex with this? 371 00:20:08,700 --> 00:20:12,120 And then the transfer occurs through the complex? 372 00:20:12,120 --> 00:20:14,700 Or you can ask a question, does the heme 373 00:20:14,700 --> 00:20:18,260 dissociate, and then the heme get picked up? 374 00:20:18,260 --> 00:20:21,250 So you can ask the question is, what is the order of addition? 375 00:20:26,890 --> 00:20:34,950 So you can look at the mechanism of transfer, 376 00:20:34,950 --> 00:20:41,010 and specifically, the order of addition. 377 00:20:41,010 --> 00:20:44,180 Do you need the second protein there to see transfer? 378 00:20:44,180 --> 00:20:47,400 So they've done a whole bunch of experiments like this. 379 00:20:47,400 --> 00:20:54,110 I'll show you using the Soret band what they actually monitor 380 00:20:54,110 --> 00:20:56,370 in this particular reaction. 381 00:20:56,370 --> 00:20:59,280 But they did an experiment where they just took 382 00:20:59,280 --> 00:21:03,960 IsdA, IsdA loaded with heme, and asked the question, 383 00:21:03,960 --> 00:21:06,570 does heme go into solution? 384 00:21:06,570 --> 00:21:08,810 So that's not a trivial experiment either 385 00:21:08,810 --> 00:21:09,840 because it can rebind. 386 00:21:09,840 --> 00:21:12,490 It depends on the on-rate and the off-rate. 387 00:21:12,490 --> 00:21:15,060 So you have to have a way of making sure 388 00:21:15,060 --> 00:21:16,830 that if it comes off, you pull it 389 00:21:16,830 --> 00:21:20,050 to the right to be able to measure the rate constant. 390 00:21:20,050 --> 00:21:23,880 So this whole problem is really associated 391 00:21:23,880 --> 00:21:26,640 with thinking about detailed kinetic models, which 392 00:21:26,640 --> 00:21:28,180 I'm not going to go into. 393 00:21:28,180 --> 00:21:30,270 But if you look at the data, that's 394 00:21:30,270 --> 00:21:32,490 what you need to think about to believe the data, 395 00:21:32,490 --> 00:21:35,370 whether the data has been interpreted correctly, that I'm 396 00:21:35,370 --> 00:21:36,810 very briefly going to show you. 397 00:21:36,810 --> 00:21:38,665 You need to derive the equations, 398 00:21:38,665 --> 00:21:40,290 and look at what your expectations are. 399 00:21:40,290 --> 00:21:44,220 Are they consistent with the kinetic analysis 400 00:21:44,220 --> 00:21:45,540 of what's going on? 401 00:21:45,540 --> 00:21:47,890 So here, they see a rate constant. 402 00:21:47,890 --> 00:21:52,050 So here, they're just looking simply at this reaction. 403 00:21:52,050 --> 00:22:00,950 Does this go to IsdA apo plus heme? 404 00:22:03,960 --> 00:22:05,590 And if they look at that rate constant, 405 00:22:05,590 --> 00:22:10,210 it's .0007 per second. 406 00:22:10,210 --> 00:22:13,590 So it's really slow. 407 00:22:13,590 --> 00:22:16,752 So then the question is, is this transfer-- 408 00:22:16,752 --> 00:22:18,210 does anybody remember with the rate 409 00:22:18,210 --> 00:22:21,570 constants were for transfer in the problem you worked at? 410 00:22:21,570 --> 00:22:22,440 Was it seconds? 411 00:22:22,440 --> 00:22:23,970 Was it minutes? 412 00:22:23,970 --> 00:22:26,325 Did you think about it? 413 00:22:26,325 --> 00:22:27,700 I don't remember what the numbers 414 00:22:27,700 --> 00:22:28,670 are off the top of my head. 415 00:22:28,670 --> 00:22:30,180 AUDIENCE: Like, tenths per second. 416 00:22:30,180 --> 00:22:31,013 JoANNE STUBBE: Yeah. 417 00:22:31,013 --> 00:22:33,740 So that's much faster than this. 418 00:22:33,740 --> 00:22:36,040 And so, this is a really low number. 419 00:22:36,040 --> 00:22:37,780 But the question is, how low-- remember, 420 00:22:37,780 --> 00:22:39,853 we're missing part of a whole system. 421 00:22:39,853 --> 00:22:41,770 And so it could be really low, just because we 422 00:22:41,770 --> 00:22:45,760 don't have the system set up to mimic what 423 00:22:45,760 --> 00:22:47,510 we see in the native organism. 424 00:22:47,510 --> 00:22:50,170 So that's the problem you always face. 425 00:22:50,170 --> 00:22:54,190 And so, if you look at the data, here's what they're monitoring. 426 00:22:54,190 --> 00:22:57,790 So here's the Soret band at 400. 427 00:22:57,790 --> 00:23:00,970 And so you need to get a good spectrum and convince yourself 428 00:23:00,970 --> 00:23:04,360 you're looking at the stoichiometric loading. 429 00:23:04,360 --> 00:23:06,010 So you need to know how much heme. 430 00:23:06,010 --> 00:23:09,070 Because that's going to affect your absorption spectrum. 431 00:23:09,070 --> 00:23:12,110 So you need to know how much is loaded. 432 00:23:12,110 --> 00:23:15,220 And then you can do the experiment outlined over there. 433 00:23:15,220 --> 00:23:20,050 And what they're monitoring is these small changes. 434 00:23:20,050 --> 00:23:23,968 And so, when they do that, they come up with, 435 00:23:23,968 --> 00:23:25,510 if you look at the analysis, and I'll 436 00:23:25,510 --> 00:23:28,360 show you a few pieces of data, they come up with-- 437 00:23:28,360 --> 00:23:32,860 they favor the model associated with the kinetic analysis 438 00:23:32,860 --> 00:23:37,450 of all of their data, that you form a tertiary complex-- 439 00:23:37,450 --> 00:23:40,300 sorry, a binary complex of the two proteins, 440 00:23:40,300 --> 00:23:43,240 and that the heme is transferred from one to the other. 441 00:23:43,240 --> 00:23:49,900 And then the apo IsdA dissociates. 442 00:23:49,900 --> 00:23:51,790 So this is analogous to what you've seen. 443 00:23:51,790 --> 00:23:53,630 And here's some data. 444 00:23:53,630 --> 00:23:55,500 So they've done it with every single pair 445 00:23:55,500 --> 00:23:57,250 in this particular paper. 446 00:23:57,250 --> 00:23:59,500 And what you can do-- and they've 447 00:23:59,500 --> 00:24:01,800 done all the experiments the same way, 448 00:24:01,800 --> 00:24:04,060 been able to see differences in Soret bands. 449 00:24:04,060 --> 00:24:07,120 These were all done with changes in the spectra, 450 00:24:07,120 --> 00:24:09,400 the visible spectra. 451 00:24:09,400 --> 00:24:11,920 But if you look at this, for example, 452 00:24:11,920 --> 00:24:18,370 from the transfer of the heme from methemoglobin. 453 00:24:18,370 --> 00:24:20,890 So that's the first step up here. 454 00:24:20,890 --> 00:24:24,640 And you look at all the rate constants they measured. 455 00:24:24,640 --> 00:24:29,090 The fastest number is 0.31 per second. 456 00:24:29,090 --> 00:24:30,740 So that's slow. 457 00:24:30,740 --> 00:24:31,920 But you might expect it. 458 00:24:31,920 --> 00:24:34,145 It might not be so easy to extract the heme out 459 00:24:34,145 --> 00:24:35,330 of hemoglobin. 460 00:24:35,330 --> 00:24:39,200 So you might expect this to be slow. 461 00:24:39,200 --> 00:24:41,870 But, again, if you look here, and this is a thing 462 00:24:41,870 --> 00:24:44,510 that I think hopefully some of you thought about, 463 00:24:44,510 --> 00:24:46,580 if you look at the rate constants, 464 00:24:46,580 --> 00:24:49,830 these reactions are all bimolecular. 465 00:24:49,830 --> 00:24:51,170 But what do they have up there? 466 00:24:51,170 --> 00:24:53,780 They have first order processes. 467 00:24:53,780 --> 00:24:57,050 So that has got to be telling you something about 468 00:24:57,050 --> 00:24:59,780 the interaction-- let's go down-- 469 00:24:59,780 --> 00:25:05,260 whether this interaction is rapid and reversible. 470 00:25:05,260 --> 00:25:08,585 And in the paper describing this work in detail, 471 00:25:08,585 --> 00:25:11,210 and I give you the reference in the PowerPoint for those of you 472 00:25:11,210 --> 00:25:12,752 want to look at it, you need to think 473 00:25:12,752 --> 00:25:14,352 about the kinetic analysis. 474 00:25:14,352 --> 00:25:16,060 So when you're looking at rate constants, 475 00:25:16,060 --> 00:25:18,530 you need to think about whether it's first or second order. 476 00:25:18,530 --> 00:25:20,300 Somehow they have to get together. 477 00:25:20,300 --> 00:25:22,860 If this is doing half and rapid and reversible, 478 00:25:22,860 --> 00:25:25,400 you still have a term for that equilibrium step. 479 00:25:25,400 --> 00:25:28,370 But then you're looking at a first-order process. 480 00:25:31,520 --> 00:25:32,507 So, if you look-- 481 00:25:32,507 --> 00:25:34,340 I'm not going to go through the whole thing. 482 00:25:34,340 --> 00:25:39,920 But if you look at the transfer from holo IsdB, 483 00:25:39,920 --> 00:25:41,750 so that's the second step. 484 00:25:41,750 --> 00:25:45,848 It got the B, got the heme out of the hemoglobin. 485 00:25:45,848 --> 00:25:47,390 And then you can look at the transfer 486 00:25:47,390 --> 00:25:49,010 to all the other proteins. 487 00:25:49,010 --> 00:25:53,010 You see that in this case one is 114 per second. 488 00:25:53,010 --> 00:25:55,095 So that's a fast transfer. 489 00:25:55,095 --> 00:25:55,970 So you can look down. 490 00:25:55,970 --> 00:25:58,190 And they've done every single one of these steps. 491 00:25:58,190 --> 00:26:01,220 And they've also then also asked the question, 492 00:26:01,220 --> 00:26:04,130 can these proteins act catalytically? 493 00:26:04,130 --> 00:26:06,110 So then they put it in a small amount of one, 494 00:26:06,110 --> 00:26:08,060 and look at the rate constants in the presence 495 00:26:08,060 --> 00:26:10,220 or the absence of one. 496 00:26:10,220 --> 00:26:14,240 And they conclude that these proteins can act catalytically 497 00:26:14,240 --> 00:26:15,160 as well. 498 00:26:15,160 --> 00:26:16,160 So these are the kinds-- 499 00:26:16,160 --> 00:26:17,720 I don't want to spend a lot of time 500 00:26:17,720 --> 00:26:20,960 discussing this detailed setup. 501 00:26:20,960 --> 00:26:26,540 Because I think you still have to worry about being covalently 502 00:26:26,540 --> 00:26:28,650 attached to the peptidoglycan. 503 00:26:28,650 --> 00:26:31,970 But these rate constants are pretty darn fast. 504 00:26:31,970 --> 00:26:34,280 And so then the other thing that's 505 00:26:34,280 --> 00:26:37,610 interesting is, why does it have an order? 506 00:26:37,610 --> 00:26:39,090 Does it have an order? 507 00:26:39,090 --> 00:26:42,980 So if you were going to take B, can I transfer it to C? 508 00:26:42,980 --> 00:26:46,410 And what are the differences in the rate constants? 509 00:26:46,410 --> 00:26:50,240 And here, it's 114 versus 15. 510 00:26:50,240 --> 00:26:52,760 So now the question is, did they set up the experiment 511 00:26:52,760 --> 00:26:54,200 correctly? 512 00:26:54,200 --> 00:26:56,420 They probably used all the same concentrations 513 00:26:56,420 --> 00:26:57,360 in the experiment. 514 00:26:57,360 --> 00:26:59,880 But one might have a higher affinity than the other. 515 00:26:59,880 --> 00:27:02,920 And so, you need to think about all that stuff. 516 00:27:02,920 --> 00:27:04,640 And if they thought about that correctly, 517 00:27:04,640 --> 00:27:06,470 they really have learned something 518 00:27:06,470 --> 00:27:11,270 about the order of addition and the ability of these proteins 519 00:27:11,270 --> 00:27:12,830 to act catalytically. 520 00:27:12,830 --> 00:27:15,560 So this is state of the art right now, 521 00:27:15,560 --> 00:27:18,280 the way people are studying this. 522 00:27:18,280 --> 00:27:21,680 And this kinetic data has allowed 523 00:27:21,680 --> 00:27:23,210 us to come up with that model. 524 00:27:23,210 --> 00:27:26,420 I just showed you that there is an ordered way 525 00:27:26,420 --> 00:27:30,230 to transfer these systems. 526 00:27:30,230 --> 00:27:32,330 And then I just want to say very briefly-- 527 00:27:32,330 --> 00:27:36,290 I just want to show you very briefly the structures 528 00:27:36,290 --> 00:27:39,840 of these, and just show you again 529 00:27:39,840 --> 00:27:42,680 where the state of the art is in this area. 530 00:27:42,680 --> 00:27:44,270 All of these NEAT domains-- 531 00:27:44,270 --> 00:27:47,450 so NEAT domains are the heme-binding domains, 532 00:27:47,450 --> 00:27:49,110 120 amino acids. 533 00:27:49,110 --> 00:27:51,250 They are found in A, B, C, and H. 534 00:27:51,250 --> 00:27:53,000 They are found in four different proteins. 535 00:27:56,030 --> 00:27:59,210 This is super position of all the NEAT domains. 536 00:27:59,210 --> 00:28:01,530 You can see they all look alike. 537 00:28:01,530 --> 00:28:04,580 Furthermore, if you go down here, and you look at binding, 538 00:28:04,580 --> 00:28:08,900 they all have a pentacoordinate heme 539 00:28:08,900 --> 00:28:12,920 with one axial ligand being a tyrosine. 540 00:28:12,920 --> 00:28:17,690 The other one, the top face, is apo in this version of it. 541 00:28:17,690 --> 00:28:20,690 So they also have a structure of two 542 00:28:20,690 --> 00:28:22,680 of these things bound together. 543 00:28:22,680 --> 00:28:25,250 Again, these are little domains. 544 00:28:25,250 --> 00:28:27,560 And so the question, then, you have to ask yourself, 545 00:28:27,560 --> 00:28:32,600 which is this question of rates of exchange of ligands. 546 00:28:32,600 --> 00:28:34,790 How is this transferred? 547 00:28:34,790 --> 00:28:39,020 Do you have-- how does this interface help this guy 548 00:28:39,020 --> 00:28:42,090 move from this protein to that protein? 549 00:28:42,090 --> 00:28:45,170 And that's what people are focusing their energies on, 550 00:28:45,170 --> 00:28:48,230 trying to think about the detailed structures 551 00:28:48,230 --> 00:28:52,370 to come up with a model for how this transfer occurs. 552 00:28:52,370 --> 00:28:55,340 And I'll just show you, this is a-- 553 00:28:55,340 --> 00:28:59,000 if you go all the way down, you're 554 00:28:59,000 --> 00:29:02,510 going to go from C to E. E has a different structure. 555 00:29:02,510 --> 00:29:05,180 So the mechanism of heme transfer is different. 556 00:29:05,180 --> 00:29:06,440 People have a model for that. 557 00:29:06,440 --> 00:29:08,450 You need to think about the details. 558 00:29:08,450 --> 00:29:13,970 And now, furthermore, you can go from E to F, 559 00:29:13,970 --> 00:29:18,140 all the way through the plasma membrane to the ATPase, 560 00:29:18,140 --> 00:29:20,610 which then helps you get the heme, 561 00:29:20,610 --> 00:29:24,480 provides the driving force getting heme into the cytosol. 562 00:29:24,480 --> 00:29:26,490 So we have a lot of structural data. 563 00:29:26,490 --> 00:29:28,460 But what's disappointing, I think, 564 00:29:28,460 --> 00:29:31,130 from reading those papers, which I have read, 565 00:29:31,130 --> 00:29:33,680 is we still really don't have a good model for how 566 00:29:33,680 --> 00:29:35,930 these transfers actually work. 567 00:29:35,930 --> 00:29:38,490 So this is an active area of research, 568 00:29:38,490 --> 00:29:43,520 and the people interested in the bioinorganic chemistry 569 00:29:43,520 --> 00:29:46,560 and how you get heme into cells. 570 00:29:46,560 --> 00:29:53,730 So that is the end of module 6. 571 00:29:53,730 --> 00:29:56,340 I think we've learned a lot in the last few years 572 00:29:56,340 --> 00:29:58,320 about these proteins. 573 00:29:58,320 --> 00:30:01,350 But, as you can see, we still have a long way 574 00:30:01,350 --> 00:30:06,280 to go in terms of molecular understanding. 575 00:30:06,280 --> 00:30:09,760 And so the next module, module 7, 576 00:30:09,760 --> 00:30:12,640 is going to be the shortest module. 577 00:30:12,640 --> 00:30:14,980 And I'll give you an outline of what 578 00:30:14,980 --> 00:30:19,516 I'm going to be talking about. 579 00:30:19,516 --> 00:30:25,040 And then, today's-- the first lecture is much longer than 580 00:30:25,040 --> 00:30:25,890 the second lecture. 581 00:30:25,890 --> 00:30:27,860 And we'll see the second lecture is 582 00:30:27,860 --> 00:30:29,870 going to be focused a lot on what we're 583 00:30:29,870 --> 00:30:31,400 doing in recitation this week. 584 00:30:31,400 --> 00:30:34,910 So if you notice, maybe you haven't, but we 585 00:30:34,910 --> 00:30:35,960 posted the readings. 586 00:30:35,960 --> 00:30:38,777 And one of the papers for the course, 587 00:30:38,777 --> 00:30:41,110 this part of the course, the lecture part of the course, 588 00:30:41,110 --> 00:30:42,560 is a Carroll paper you're supposed 589 00:30:42,560 --> 00:30:44,000 to read for recitation. 590 00:30:44,000 --> 00:30:45,770 So there's a lot of overlap. 591 00:30:45,770 --> 00:30:47,930 And so, the second lecture will be much shorter 592 00:30:47,930 --> 00:30:50,720 because we're going to draw on what we're doing in recitation, 593 00:30:50,720 --> 00:30:53,870 actually today. 594 00:30:53,870 --> 00:30:57,410 So let me give you the outline. 595 00:30:57,410 --> 00:31:04,250 So module 7 is the shortest. 596 00:31:04,250 --> 00:31:06,140 And this is the required reading. 597 00:31:06,140 --> 00:31:12,000 So we've posted a review article by Winterbourn, who, 598 00:31:12,000 --> 00:31:13,250 in my opinion-- 599 00:31:13,250 --> 00:31:17,360 this area of reactive oxygen species 600 00:31:17,360 --> 00:31:21,200 and how [INAUDIBLE] to how you control 601 00:31:21,200 --> 00:31:22,455 reactive oxygen species. 602 00:31:22,455 --> 00:31:24,080 I'm going to show you they can be good. 603 00:31:24,080 --> 00:31:25,430 They can be bad. 604 00:31:25,430 --> 00:31:29,690 Just like we saw with iron, it's all a question of homeostasis. 605 00:31:29,690 --> 00:31:33,110 The most thoughtful discussions have 606 00:31:33,110 --> 00:31:38,420 been described by Winterbourn, who 607 00:31:38,420 --> 00:31:41,030 is in New Zealand, who really thinks 608 00:31:41,030 --> 00:31:43,400 about the kinetics of what's going on. 609 00:31:43,400 --> 00:31:45,770 And I would argue, you can't do anything in this field 610 00:31:45,770 --> 00:31:47,720 without thinking about kinetics, which 611 00:31:47,720 --> 00:31:49,670 most people, most MDs in this field, 612 00:31:49,670 --> 00:31:51,590 don't think about at all. 613 00:31:51,590 --> 00:31:53,950 So the literature is a mess. 614 00:31:56,990 --> 00:32:00,800 But I think the last few years it's become-- 615 00:32:00,800 --> 00:32:04,100 it's starting to get unmuddy. 616 00:32:04,100 --> 00:32:06,890 And I think it's an incredibly important area. 617 00:32:06,890 --> 00:32:08,910 I guarantee you that that's true. 618 00:32:08,910 --> 00:32:12,920 So unmuddying an incredibly important area 619 00:32:12,920 --> 00:32:14,538 is going to be up to you guys. 620 00:32:14,538 --> 00:32:16,580 But I think it's going to happen in the next five 621 00:32:16,580 --> 00:32:17,390 years or something. 622 00:32:17,390 --> 00:32:19,430 I think we've already learned a lot 623 00:32:19,430 --> 00:32:21,110 in the last couple of years. 624 00:32:21,110 --> 00:32:22,790 So I'm going to have an outline. 625 00:32:22,790 --> 00:32:24,140 So we have that paper. 626 00:32:24,140 --> 00:32:25,700 And then we have the Carroll paper 627 00:32:25,700 --> 00:32:31,140 that you guys hopefully have already looked at in some form. 628 00:32:31,140 --> 00:32:31,640 All right. 629 00:32:31,640 --> 00:32:34,190 So here, let me just switch. 630 00:32:34,190 --> 00:32:35,630 So where are we going? 631 00:32:35,630 --> 00:32:40,970 And so, we're going to have a couple of lectures. 632 00:32:40,970 --> 00:32:42,880 First of all, what is ROS? 633 00:32:47,670 --> 00:32:51,795 So ROS-- a reactive oxygen species. 634 00:32:59,100 --> 00:33:02,610 So automatically, there are a bunch 635 00:33:02,610 --> 00:33:04,210 of molecules that are reactive. 636 00:33:04,210 --> 00:33:05,760 And so, what you need to think about 637 00:33:05,760 --> 00:33:07,860 is what does reactive mean? 638 00:33:07,860 --> 00:33:11,870 So the first thing is we're going to identify them. 639 00:33:11,870 --> 00:33:14,700 The second thing is we're going to look at the chemical 640 00:33:14,700 --> 00:33:15,540 reactivity. 641 00:33:19,280 --> 00:33:23,540 And, again, the question of chemical reactivity 642 00:33:23,540 --> 00:33:24,870 can be quite complex. 643 00:33:24,870 --> 00:33:28,500 But I'm going to give you my view of the chemical reactivity 644 00:33:28,500 --> 00:33:31,400 and what that view is based on. 645 00:33:31,400 --> 00:33:35,100 And then we'll very briefly look over-- 646 00:33:35,100 --> 00:33:39,740 since we move from an anaerobic world, whatever, 647 00:33:39,740 --> 00:33:42,290 a billion years ago into an aerobic world, 648 00:33:42,290 --> 00:33:44,750 like we learned from the last module, 649 00:33:44,750 --> 00:33:47,570 the question is, how do we defend ourself 650 00:33:47,570 --> 00:33:51,140 against the presence of oxygen with reduced metals? 651 00:33:51,140 --> 00:33:53,630 And that's the issue we raised last time. 652 00:33:53,630 --> 00:33:56,090 So what are our defense mechanisms? 653 00:33:59,670 --> 00:34:02,880 Because we saw we had copper. 654 00:34:02,880 --> 00:34:05,490 We had iron. 655 00:34:05,490 --> 00:34:07,440 And now we have oxygen. And we'll 656 00:34:07,440 --> 00:34:11,330 see that that can be a recipe for disaster 657 00:34:11,330 --> 00:34:14,190 unless you can figure out how to control all of that. 658 00:34:14,190 --> 00:34:17,850 So, again, it's all homeostasis. 659 00:34:17,850 --> 00:34:19,560 So that's the second. 660 00:34:19,560 --> 00:34:23,020 That will be the first part of today's lecture. 661 00:34:23,020 --> 00:34:26,940 Then we're going to move into the question 662 00:34:26,940 --> 00:34:39,929 of the battle between bacteria or viruses 663 00:34:39,929 --> 00:34:41,560 or parasites in humans. 664 00:34:45,040 --> 00:34:48,940 And what I'm going to talk about specifically 665 00:34:48,940 --> 00:34:52,989 is destruction of bacteria by neutrophils. 666 00:34:57,170 --> 00:35:00,080 And we'll see that neutrophils are white blood cells. 667 00:35:05,900 --> 00:35:08,810 And we will see that they are the first responders. 668 00:35:08,810 --> 00:35:11,722 So if you have a bacteria in our bloodstream, 669 00:35:11,722 --> 00:35:13,430 the first guys there are the neutrophils. 670 00:35:13,430 --> 00:35:16,130 And that's what we're going to focus on. 671 00:35:16,130 --> 00:35:17,310 So let me see. 672 00:35:17,310 --> 00:35:17,810 All right. 673 00:35:17,810 --> 00:35:18,650 I'll go over here. 674 00:35:23,600 --> 00:35:26,000 So neutrophils are the first responders. 675 00:35:26,000 --> 00:35:32,420 Now, we know quite a bit about this. 676 00:35:32,420 --> 00:35:35,780 And really, what we're going to be focusing on 677 00:35:35,780 --> 00:35:38,840 in both today's lecture and in the next lecture 678 00:35:38,840 --> 00:35:43,340 is the group of isozymes called Nox proteins. 679 00:35:46,170 --> 00:35:48,970 N-O-X-- NADPH oxidases. 680 00:36:01,280 --> 00:36:03,950 And we're going to talk about that particular protein. 681 00:36:03,950 --> 00:36:06,770 And we're going to be specifically focused on Nox2. 682 00:36:09,440 --> 00:36:13,280 And we'll see that Nox2's professional job, 683 00:36:13,280 --> 00:36:16,700 we'll talk about that, is to generate a reactive oxygen 684 00:36:16,700 --> 00:36:20,540 species, superoxide, which is then going to be used 685 00:36:20,540 --> 00:36:23,630 in some way to kill bacteria. 686 00:36:23,630 --> 00:36:26,180 So we're going to be talking about neutrophils. 687 00:36:26,180 --> 00:36:28,670 We're going to be talking about the Nox protein. 688 00:36:28,670 --> 00:36:32,150 Also, if you've read the recitation paper for today, 689 00:36:32,150 --> 00:36:34,180 what are we talking about in signaling 690 00:36:34,180 --> 00:36:35,660 that's oxygen-dependent? 691 00:36:35,660 --> 00:36:37,320 The Nox proteins. 692 00:36:37,320 --> 00:36:38,450 So here we have bad. 693 00:36:38,450 --> 00:36:39,890 We're killing the bacteria. 694 00:36:39,890 --> 00:36:41,400 Here we have good. 695 00:36:41,400 --> 00:36:43,670 We're using the Nox proteins for signaling. 696 00:36:43,670 --> 00:36:47,630 So that's sort of the take-home message is homeostasis. 697 00:36:47,630 --> 00:36:50,510 How do you control it for bad versus for good? 698 00:36:50,510 --> 00:36:54,620 We've already seen that in the case of the iron system. 699 00:36:54,620 --> 00:36:57,740 So the other protein we're going to talk 700 00:36:57,740 --> 00:37:00,410 about today, or probably won't get that far today-- 701 00:37:00,410 --> 00:37:01,850 how bad am I? 702 00:37:01,850 --> 00:37:05,256 So we have another protein called myeloperoxidase. 703 00:37:10,890 --> 00:37:12,780 And we're going to see that this-- 704 00:37:12,780 --> 00:37:17,130 so this guy is going to be involved with superoxide. 705 00:37:17,130 --> 00:37:22,950 This guy is involved in the neutrophils with generating 706 00:37:22,950 --> 00:37:24,030 hypochlorous acid. 707 00:37:24,030 --> 00:37:25,260 So these are the proteins. 708 00:37:25,260 --> 00:37:28,548 And these proteins together-- 709 00:37:28,548 --> 00:37:31,170 I'll give you the model, the current model. 710 00:37:31,170 --> 00:37:33,420 But the current model I'm going to give you 711 00:37:33,420 --> 00:37:35,500 is much simpler than reality. 712 00:37:35,500 --> 00:37:37,770 But those are the proteins we're going to focus on. 713 00:37:37,770 --> 00:37:39,810 And those are two of the reactive species 714 00:37:39,810 --> 00:37:41,910 we're going to be focusing on. 715 00:37:41,910 --> 00:37:47,580 And then the second lecture goes back to the Nox2 proteins, 716 00:37:47,580 --> 00:37:52,490 and the question now of not killing, but signaling. 717 00:37:52,490 --> 00:37:57,570 And as we already saw in the last recitation, 718 00:37:57,570 --> 00:37:58,650 how were we signaling? 719 00:37:58,650 --> 00:38:04,170 We were signaling by a reaction of sulfide groups 720 00:38:04,170 --> 00:38:05,610 with hydrogen peroxide, which can 721 00:38:05,610 --> 00:38:07,710 be generated from superoxide. 722 00:38:07,710 --> 00:38:09,760 I'll show you how that happens. 723 00:38:09,760 --> 00:38:10,740 So, signaling. 724 00:38:10,740 --> 00:38:15,030 And we're focusing on signaling by sulfenylation which, again, 725 00:38:15,030 --> 00:38:21,850 is the topic of today's recitation 726 00:38:21,850 --> 00:38:26,140 of the epidermal growth factor receptor. 727 00:38:26,140 --> 00:38:27,670 So that's where we're going. 728 00:38:27,670 --> 00:38:29,702 And I'm going to give you that-- we're 729 00:38:29,702 --> 00:38:30,910 going to follow this outline. 730 00:38:30,910 --> 00:38:32,770 And I think you'll get a pretty good feeling 731 00:38:32,770 --> 00:38:35,860 for it, an overview of reactive oxygen species, 732 00:38:35,860 --> 00:38:39,610 even though we're not going through it in a lot of details. 733 00:38:39,610 --> 00:38:42,490 It's really complicated. 734 00:38:42,490 --> 00:38:44,980 So what I want to do before I get to this slide 735 00:38:44,980 --> 00:38:46,290 is give you the big picture. 736 00:38:50,540 --> 00:38:52,520 So this is the take-home message. 737 00:38:52,520 --> 00:38:53,630 So we have a big picture. 738 00:38:58,370 --> 00:39:04,760 And so here we have cell. 739 00:39:04,760 --> 00:39:11,300 And in this cell we have reactive oxygen species. 740 00:39:11,300 --> 00:39:15,860 And there were also things called reactive nitrogen 741 00:39:15,860 --> 00:39:17,280 species. 742 00:39:17,280 --> 00:39:19,940 And you'll see that in some of the slides in the PowerPoint 743 00:39:19,940 --> 00:39:20,730 presentation. 744 00:39:20,730 --> 00:39:22,940 We're not going to talk about that chemistry. 745 00:39:22,940 --> 00:39:23,870 It's interesting. 746 00:39:23,870 --> 00:39:27,230 If I had an extra three or four lectures I would also 747 00:39:27,230 --> 00:39:29,540 be talking about that. 748 00:39:29,540 --> 00:39:30,470 It's central. 749 00:39:30,470 --> 00:39:34,460 It's as important as reactive oxygen species. 750 00:39:34,460 --> 00:39:37,520 But I've decided to focus on-- 751 00:39:37,520 --> 00:39:39,140 that's what I've decided to focus on. 752 00:39:39,140 --> 00:39:41,780 So we have hydroxide radical. 753 00:39:41,780 --> 00:39:46,160 We have hydrogen peroxide. 754 00:39:46,160 --> 00:39:49,250 We have superoxide. 755 00:39:49,250 --> 00:39:53,420 And we have hypochlorous acid. 756 00:39:53,420 --> 00:39:55,220 So these are the four species that we're 757 00:39:55,220 --> 00:39:57,050 going to be focused on. 758 00:39:57,050 --> 00:40:00,080 And you can already see that some of them are radical. 759 00:40:00,080 --> 00:40:02,180 And some of them are not. 760 00:40:02,180 --> 00:40:04,610 So reactive oxygen species doesn't 761 00:40:04,610 --> 00:40:06,620 mean they have a free radical. 762 00:40:06,620 --> 00:40:09,350 They can do one-electron or two-electron chemistry. 763 00:40:09,350 --> 00:40:12,630 And we'll talk briefly about that. 764 00:40:12,630 --> 00:40:16,880 And so, the question is, where do these come from? 765 00:40:16,880 --> 00:40:19,880 So remember, we made a transition from an anaerobic 766 00:40:19,880 --> 00:40:23,210 to an aerobic world a billion years ago. 767 00:40:23,210 --> 00:40:28,205 And during that process, we have a respiratory chain. 768 00:40:34,880 --> 00:40:38,420 In humans, the respiratory chain is found in the mitochondria. 769 00:40:38,420 --> 00:40:40,750 Otherwise, it's found in the plasma membrane. 770 00:40:40,750 --> 00:40:50,920 So we have complexes I, II, and III. 771 00:40:50,920 --> 00:40:55,400 And these guys, to chemistry, their ultimate goal, 772 00:40:55,400 --> 00:40:57,740 if you're in an oxygen-dependent world, 773 00:40:57,740 --> 00:41:00,200 is to reduce oxygen to water. 774 00:41:00,200 --> 00:41:03,950 So this is a goal, is oxygen to water. 775 00:41:03,950 --> 00:41:07,130 Although, you all know in bacteria if there's no oxygen, 776 00:41:07,130 --> 00:41:12,140 you have to have some other terminal electron accepter. 777 00:41:12,140 --> 00:41:18,410 And so what you get from these complexes is uncoupling. 778 00:41:18,410 --> 00:41:22,310 So 100% of the time it doesn't do what you want it to do. 779 00:41:22,310 --> 00:41:23,720 And so, you have to-- 780 00:41:23,720 --> 00:41:26,870 you get side reactions that you have to deal with. 781 00:41:26,870 --> 00:41:27,370 All right. 782 00:41:27,370 --> 00:41:28,745 Now, what did I do with my chalk? 783 00:41:28,745 --> 00:41:29,300 Anyhow. 784 00:41:29,300 --> 00:41:30,440 So you get uncoupling. 785 00:41:35,250 --> 00:41:37,590 So that's one way they're generated. 786 00:41:37,590 --> 00:41:41,010 A second way they're generated we've already been through. 787 00:41:41,010 --> 00:41:44,930 We went from an anaerobic world to an aerobic world. 788 00:41:44,930 --> 00:41:49,140 What do we have under those conditions? 789 00:41:49,140 --> 00:41:52,170 We have iron, and we have copper. 790 00:41:52,170 --> 00:41:54,690 And, again, we have oxygen. And so, we 791 00:41:54,690 --> 00:41:57,732 can generate reactive oxygen species. 792 00:41:57,732 --> 00:41:59,190 We've already talked about the fact 793 00:41:59,190 --> 00:42:01,820 that iron just isn't freely floating around 794 00:42:01,820 --> 00:42:03,000 inside the cell. 795 00:42:03,000 --> 00:42:04,550 But if something happens and you have 796 00:42:04,550 --> 00:42:06,810 an imbalance in iron homeostasis, 797 00:42:06,810 --> 00:42:10,950 it leads to imbalance in oxygen homeostasis. 798 00:42:10,950 --> 00:42:14,370 And hopefully, you remember that one of the proteins regulated 799 00:42:14,370 --> 00:42:17,190 by the iron-responsive element, iron-responsive binding 800 00:42:17,190 --> 00:42:21,030 proteins, was the oxygen transcription, 801 00:42:21,030 --> 00:42:25,350 oxygen-sensing transcription factor. 802 00:42:25,350 --> 00:42:27,270 Another way that we get these things 803 00:42:27,270 --> 00:42:34,470 are from xenobiotics or environmental pollutants. 804 00:42:40,340 --> 00:42:43,790 If you smoke, which I guess people don't do. 805 00:42:43,790 --> 00:42:46,070 But when I was in graduate school everybody smoked. 806 00:42:46,070 --> 00:42:47,540 And they smoked in the lab. 807 00:42:47,540 --> 00:42:49,460 Anyhow-- you get-- 808 00:42:49,460 --> 00:42:53,970 so pollutants can generate reactive oxygen species. 809 00:42:53,970 --> 00:42:56,540 So that's how we get them. 810 00:42:56,540 --> 00:42:57,890 What do we do with them? 811 00:42:57,890 --> 00:43:00,710 So there's some important things that we can do with them. 812 00:43:00,710 --> 00:43:01,400 Here it is. 813 00:43:01,400 --> 00:43:02,300 This is what I want. 814 00:43:02,300 --> 00:43:04,140 So what are we going to do with them? 815 00:43:04,140 --> 00:43:13,360 So one of the things we can do is we have white blood cells 816 00:43:13,360 --> 00:43:14,770 and neutrophils. 817 00:43:18,130 --> 00:43:27,100 And we kill bacteria or viruses or parasites. 818 00:43:27,100 --> 00:43:29,688 So that's one of the good things we can do with them. 819 00:43:29,688 --> 00:43:31,480 A second thing we're going to do with them, 820 00:43:31,480 --> 00:43:36,580 which is what we're focusing on, is signaling. 821 00:43:36,580 --> 00:43:39,280 And we'll see that while we're looking 822 00:43:39,280 --> 00:43:45,610 at signaling of growth factors or hormones or cytokines, which 823 00:43:45,610 --> 00:43:49,420 is good signaling, we'll see that you can 824 00:43:49,420 --> 00:43:51,490 have all kinds of signaling. 825 00:43:51,490 --> 00:43:53,900 So it's not limited to the one system 826 00:43:53,900 --> 00:43:56,110 we're going to be studying. 827 00:43:56,110 --> 00:43:58,390 So it's very broadly defined. 828 00:43:58,390 --> 00:44:00,760 This is a huge area of research right now, 829 00:44:00,760 --> 00:44:02,260 people looking at this. 830 00:44:02,260 --> 00:44:05,170 And the third thing that happens when 831 00:44:05,170 --> 00:44:07,270 this is completely out of control 832 00:44:07,270 --> 00:44:10,580 is you modify, you damage all the macromolecules, 833 00:44:10,580 --> 00:44:12,820 the small molecules inside the cell. 834 00:44:12,820 --> 00:44:15,490 So you have extensive damage. 835 00:44:15,490 --> 00:44:26,196 You can have extensive damage of DNA-- 836 00:44:26,196 --> 00:44:28,450 this thing is not writing very well-- proteins. 837 00:44:31,930 --> 00:44:33,220 But it's not limited to that. 838 00:44:33,220 --> 00:44:34,540 You have lipids. 839 00:44:34,540 --> 00:44:39,340 Lipids are modified by hydrogen peroxide. 840 00:44:39,340 --> 00:44:40,750 And so, that's the big picture. 841 00:44:40,750 --> 00:44:42,970 That's where we're going. 842 00:44:42,970 --> 00:44:44,613 We're focusing on these guys. 843 00:44:44,613 --> 00:44:46,030 And we're focusing on good things. 844 00:44:46,030 --> 00:44:47,405 And we're focusing on bad things. 845 00:44:47,405 --> 00:44:49,240 And how do we control all of that. 846 00:44:52,660 --> 00:44:55,290 So in the next-- 847 00:44:55,290 --> 00:45:00,550 what I want to do is show you why this is-- 848 00:45:00,550 --> 00:45:02,260 why I decided to talk about this. 849 00:45:02,260 --> 00:45:05,458 I've always found this area fascinating. 850 00:45:05,458 --> 00:45:07,750 And, I must say, I've been going to meetings off and on 851 00:45:07,750 --> 00:45:08,530 for decades. 852 00:45:08,530 --> 00:45:11,350 And I sort of quit going because I lost information 853 00:45:11,350 --> 00:45:14,620 every time I went to a meeting because it was so confusing. 854 00:45:14,620 --> 00:45:16,480 Because everybody used different cell types. 855 00:45:16,480 --> 00:45:17,920 And they have different kinds of assays. 856 00:45:17,920 --> 00:45:20,590 And they didn't pay attention to what the assays were really 857 00:45:20,590 --> 00:45:21,580 telling them. 858 00:45:21,580 --> 00:45:25,060 But now, I think we're at the time when people really 859 00:45:25,060 --> 00:45:27,070 need-- people are doing good experiments. 860 00:45:27,070 --> 00:45:29,740 I think we've turned a corner. 861 00:45:29,740 --> 00:45:32,440 And so, one of the things that has 862 00:45:32,440 --> 00:45:37,960 been in the front pages of all the newspapers since 2007, 863 00:45:37,960 --> 00:45:41,080 there was-- we actually-- there was Jim Collins, who 864 00:45:41,080 --> 00:45:46,810 was at Boston University, received a huge amount of press 865 00:45:46,810 --> 00:45:49,780 on a paper he published in 2007. 866 00:45:49,780 --> 00:45:53,630 And since that time it's been extremely controversial. 867 00:45:53,630 --> 00:45:57,170 And so, I think it brings up a lot of issues about, 868 00:45:57,170 --> 00:46:00,290 again, how you do controlled experiments. 869 00:46:00,290 --> 00:46:02,440 So his observation and his conclusions 870 00:46:02,440 --> 00:46:04,420 were, from the experiments he published, 871 00:46:04,420 --> 00:46:05,960 were very interesting. 872 00:46:05,960 --> 00:46:08,557 So we just talked about antibiotics. 873 00:46:08,557 --> 00:46:09,390 What do they target? 874 00:46:09,390 --> 00:46:10,750 They target cell walls. 875 00:46:10,750 --> 00:46:13,270 We have penicillin, vancomycin. 876 00:46:13,270 --> 00:46:14,860 They can target the ribosome. 877 00:46:14,860 --> 00:46:17,050 You saw that in the first part of the course. 878 00:46:17,050 --> 00:46:18,730 You have aminoglycosides. 879 00:46:18,730 --> 00:46:20,810 They can target DNA replication. 880 00:46:20,810 --> 00:46:22,960 So those are the three sort of major targets. 881 00:46:22,960 --> 00:46:25,240 You have the quinolones that do that. 882 00:46:25,240 --> 00:46:28,990 His conclusion from the paper is that all 883 00:46:28,990 --> 00:46:32,260 of these things, the mechanism of cell kill 884 00:46:32,260 --> 00:46:36,010 is not involved with the primary targets at all. 885 00:46:36,010 --> 00:46:39,940 But it's involved with a downstream target, that 886 00:46:39,940 --> 00:46:46,750 somehow these guys undo oxygen homeostasis, 887 00:46:46,750 --> 00:46:52,000 resulting in bad radicals, reactive oxygen species, that 888 00:46:52,000 --> 00:46:56,260 end up damaging macromolecules, damaging the cell, 889 00:46:56,260 --> 00:46:59,160 and resulting in bacterial cell death. 890 00:46:59,160 --> 00:47:01,240 So that's the model. 891 00:47:01,240 --> 00:47:02,950 This has been quite controversial. 892 00:47:02,950 --> 00:47:04,900 I've given you some papers to read. 893 00:47:04,900 --> 00:47:08,170 The latest paper was just published online 894 00:47:08,170 --> 00:47:10,540 saying reactive oxygen species play 895 00:47:10,540 --> 00:47:14,530 an important role in bactericidal activity 896 00:47:14,530 --> 00:47:17,050 of the quinolones that targets topoisomerase 897 00:47:17,050 --> 00:47:19,960 in DNA replication. 898 00:47:19,960 --> 00:47:21,880 But we've had two articles published 899 00:47:21,880 --> 00:47:25,720 in Science saying killing by bacterial antibiotics does not 900 00:47:25,720 --> 00:47:28,510 depend on reactive oxygen species. 901 00:47:28,510 --> 00:47:31,390 So we've had quite inflammatory responses 902 00:47:31,390 --> 00:47:34,870 to this paper, which I at one stage 903 00:47:34,870 --> 00:47:37,300 didn't believe anything that he did. 904 00:47:37,300 --> 00:47:39,880 Because I think if you look at a lot 905 00:47:39,880 --> 00:47:41,740 of the original experiments, they 906 00:47:41,740 --> 00:47:45,490 use reagents that were completely nonspecific for what 907 00:47:45,490 --> 00:47:46,990 they thought they were. 908 00:47:46,990 --> 00:47:48,700 But I think I am now-- 909 00:47:48,700 --> 00:47:51,010 I think his observations, in fact, were correct. 910 00:47:51,010 --> 00:47:54,520 But not-- some of his observations were correct. 911 00:47:54,520 --> 00:47:59,140 But the reasoning behind the observations has changed. 912 00:47:59,140 --> 00:48:01,580 And I think there is something about this. 913 00:48:01,580 --> 00:48:02,950 You do signaling up here. 914 00:48:02,950 --> 00:48:04,540 And then where do you see something? 915 00:48:04,540 --> 00:48:06,550 Way down here, because you trigger off 916 00:48:06,550 --> 00:48:07,930 a signaling cascade. 917 00:48:07,930 --> 00:48:11,290 And that's the way the world works inside the cell. 918 00:48:11,290 --> 00:48:13,780 So a second example of this which 919 00:48:13,780 --> 00:48:17,200 also received a lot of press, this guy-- everybody 920 00:48:17,200 --> 00:48:18,760 know who this guy is? 921 00:48:18,760 --> 00:48:19,943 The DNA guy. 922 00:48:19,943 --> 00:48:21,610 The famous-- he's a male chauvinist pig, 923 00:48:21,610 --> 00:48:25,150 but the DNA guy. 924 00:48:25,150 --> 00:48:27,360 Anyhow, what he-- there's been a big fight. 925 00:48:27,360 --> 00:48:31,450 Are reactive oxygen species good or bad in fighting cancer? 926 00:48:31,450 --> 00:48:34,630 So some people say, again, it's the reactive oxygen 927 00:48:34,630 --> 00:48:37,600 species that eventually kill the cancer cells. 928 00:48:37,600 --> 00:48:39,280 Again, you do something up here. 929 00:48:39,280 --> 00:48:40,840 You trigger off a set of events. 930 00:48:40,840 --> 00:48:45,250 You generate a pathway that generates reactive oxygen 931 00:48:45,250 --> 00:48:48,250 species that helps kill the tumor cells. 932 00:48:48,250 --> 00:48:51,070 Or do they stimulate growth-- 933 00:48:51,070 --> 00:48:53,980 reactive oxygen species stimulate growth? 934 00:48:53,980 --> 00:48:56,560 Or do they trigger apoptosis? 935 00:48:56,560 --> 00:48:59,170 So people are still debating that. 936 00:48:59,170 --> 00:49:01,690 And there's probably some truth in both of these statements, 937 00:49:01,690 --> 00:49:03,023 depending on how you look at it. 938 00:49:03,023 --> 00:49:06,820 Anyhow, that's just to get you thinking about the fact 939 00:49:06,820 --> 00:49:11,560 that this is an important area that a lot of people 940 00:49:11,560 --> 00:49:14,500 are actually focusing energy on. 941 00:49:14,500 --> 00:49:18,550 So what I want to do now is we've given a big overview. 942 00:49:18,550 --> 00:49:21,100 What I want to do now is look at what 943 00:49:21,100 --> 00:49:24,070 are the identification of the species. 944 00:49:29,470 --> 00:49:34,830 And the species we're going to be focusing on, again, 945 00:49:34,830 --> 00:49:37,405 are superoxide-- 946 00:49:37,405 --> 00:49:41,110 I'll write this down later-- hydrogen peroxide, 947 00:49:41,110 --> 00:49:49,070 H202, hypochlorous acid, and hydroxide radical. 948 00:49:49,070 --> 00:49:55,150 So this is a scheme that was taken from a Winterbourn 949 00:49:55,150 --> 00:49:55,750 article. 950 00:49:55,750 --> 00:49:57,000 So it's in all of her papers. 951 00:49:57,000 --> 00:50:00,730 I don't know if it's the exact same scheme in the reading 952 00:50:00,730 --> 00:50:03,070 assignment, but you'll see something similar. 953 00:50:03,070 --> 00:50:03,820 And we're not-- 954 00:50:03,820 --> 00:50:06,670 I told you we're not going to talk about reactive oxygen 955 00:50:06,670 --> 00:50:07,180 species. 956 00:50:07,180 --> 00:50:10,090 We're only focusing on reactive-- 957 00:50:10,090 --> 00:50:11,890 sorry-- reactive nitrogen species. 958 00:50:11,890 --> 00:50:14,560 We're really focusing on reactive oxygen species. 959 00:50:14,560 --> 00:50:17,110 But this gives you the big picture with both. 960 00:50:17,110 --> 00:50:19,210 So where are we going to be focusing? 961 00:50:19,210 --> 00:50:23,590 Oxygen picks up an electron and goes to superoxide. 962 00:50:23,590 --> 00:50:27,610 Superoxide, in the presence of another electron and protons, 963 00:50:27,610 --> 00:50:30,170 goes to hydrogen peroxide. 964 00:50:30,170 --> 00:50:32,530 So we're going to be focusing here. 965 00:50:32,530 --> 00:50:35,520 Hydrogen peroxide in the presence of Iron(II)-- 966 00:50:35,520 --> 00:50:37,570 if we've somehow screwed up our iron 967 00:50:37,570 --> 00:50:42,260 and where in the reduced state generates hydroxide radical. 968 00:50:42,260 --> 00:50:45,730 Hydrogen peroxide with myeloperoxidase 969 00:50:45,730 --> 00:50:51,130 in the neutrophils forms hypochlorous acid. 970 00:50:51,130 --> 00:50:54,040 And so-- at which eventually can chlorinate everything. 971 00:50:54,040 --> 00:50:55,570 They chlorinate amino acids. 972 00:50:55,570 --> 00:50:58,450 It chlorinates lipids, and can result 973 00:50:58,450 --> 00:51:04,130 in extensive damage to whatever hypochlorous acid is 974 00:51:04,130 --> 00:51:05,360 adjacent to. 975 00:51:05,360 --> 00:51:08,540 So this is where we're going to be focusing, 976 00:51:08,540 --> 00:51:09,910 this part of the scheme. 977 00:51:09,910 --> 00:51:12,940 Those of you who have seen reactive nitrogen 978 00:51:12,940 --> 00:51:16,150 in the species can look at how that gets integrated 979 00:51:16,150 --> 00:51:18,490 into this big picture. 980 00:51:18,490 --> 00:51:21,675 So I just want to write down one of these things 981 00:51:21,675 --> 00:51:24,280 that I think it's important to think about. 982 00:51:24,280 --> 00:51:26,680 Ultimately, we're doing chemistry. 983 00:51:26,680 --> 00:51:28,840 This-- whoops. 984 00:51:28,840 --> 00:51:30,450 I mean, this is like, it's terrible. 985 00:51:30,450 --> 00:51:31,930 It's all of a sudden, I look up. 986 00:51:31,930 --> 00:51:32,860 It's a good thing I looked up. 987 00:51:32,860 --> 00:51:34,193 Because I would have kept going. 988 00:51:34,193 --> 00:51:34,860 Anyhow. 989 00:51:34,860 --> 00:51:35,410 Sorry. 990 00:51:35,410 --> 00:51:36,310 The time is over. 991 00:51:36,310 --> 00:51:39,430 But next time we will come back, and we 992 00:51:39,430 --> 00:51:43,600 will talk about what I outlined on the board. 993 00:51:43,600 --> 00:51:45,440 And I didn't even digress today. 994 00:51:45,440 --> 00:51:46,940 I'm just-- anyhow. 995 00:51:46,940 --> 00:51:48,910 All right.