1 00:00:00,500 --> 00:00:03,270 The following content is provided under a Creative 2 00:00:03,270 --> 00:00:04,630 Commons license. 3 00:00:04,630 --> 00:00:07,140 Your support will help MIT OpenCourseWare 4 00:00:07,140 --> 00:00:11,470 continue to offer high-quality educational resources for free. 5 00:00:11,470 --> 00:00:14,100 To make a donation or view additional materials 6 00:00:14,100 --> 00:00:18,050 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:18,050 --> 00:00:19,000 at ocw.mit.edu. 8 00:00:24,261 --> 00:00:27,030 JOANNE STUBBE: OK, so I think what I'm going to do is I'll 9 00:00:27,030 --> 00:00:29,670 give you an overview of what the hypothesis is that we'll 10 00:00:29,670 --> 00:00:33,690 be talking about in class the next time, 11 00:00:33,690 --> 00:00:37,360 probably the next time and a little bit into Wednesday. 12 00:00:37,360 --> 00:00:39,250 And then this module will be over. 13 00:00:39,250 --> 00:00:44,760 So this is covering the second lecture, the good NADPH, 14 00:00:44,760 --> 00:00:47,680 the good function of NADPH oxidases. 15 00:00:47,680 --> 00:00:53,800 OK, so it's taken from this Kate Carroll's paper who-- 16 00:00:53,800 --> 00:00:55,902 and we'll continue where we left off last time. 17 00:00:55,902 --> 00:00:58,110 We'd gone through part of the paper you were supposed 18 00:00:58,110 --> 00:01:00,510 to read in Angewandte Chemie, but we hadn't 19 00:01:00,510 --> 00:01:02,970 gotten through the whole paper. 20 00:01:02,970 --> 00:01:05,640 And there were parts of it that I wanted 21 00:01:05,640 --> 00:01:07,480 to look at in more detail. 22 00:01:07,480 --> 00:01:10,026 But this is sort of a model. 23 00:01:10,026 --> 00:01:11,400 So this is really sort of-- and I 24 00:01:11,400 --> 00:01:14,290 think the model is easy to understand. 25 00:01:14,290 --> 00:01:18,420 And these kinds of models have been in the literature 26 00:01:18,420 --> 00:01:20,040 for 10 or 15 years. 27 00:01:20,040 --> 00:01:23,400 But really, there hasn't been experiments 28 00:01:23,400 --> 00:01:26,520 that sort of show how this all goes together 29 00:01:26,520 --> 00:01:27,990 using multiple methods. 30 00:01:27,990 --> 00:01:30,660 And the underlying theme-- 31 00:01:30,660 --> 00:01:36,155 and let me also say, in the handout I will give you, 32 00:01:36,155 --> 00:01:39,882 I've given you an Annual Reviews in Biochemistry paper 33 00:01:39,882 --> 00:01:41,340 by the [INAUDIBLE] lab, which shows 34 00:01:41,340 --> 00:01:44,890 how much more complicated life is than this simple cartoon. 35 00:01:44,890 --> 00:01:47,340 OK, so everything we look at, you start out 36 00:01:47,340 --> 00:01:48,792 with the simple cartoon. 37 00:01:48,792 --> 00:01:50,250 But when you study it, the more you 38 00:01:50,250 --> 00:01:53,250 study it, the more complicated it gets. 39 00:01:53,250 --> 00:01:58,560 So if you care about things only at the circle and square level, 40 00:01:58,560 --> 00:01:59,631 then you get a cartoon. 41 00:01:59,631 --> 00:02:01,380 But if you really care about how it works, 42 00:02:01,380 --> 00:02:04,140 you have to do a lot of work to figure it out. 43 00:02:04,140 --> 00:02:09,630 OK, so this sort of model is similar to all-- 44 00:02:09,630 --> 00:02:13,400 we're going to be talking about epidermal growth factor. 45 00:02:13,400 --> 00:02:18,705 And it's similar to how many growth factors work 46 00:02:18,705 --> 00:02:22,230 that I think wasn't outlined in this paper, 47 00:02:22,230 --> 00:02:25,020 but maybe it was the first paper, 48 00:02:25,020 --> 00:02:28,410 that this model is sort of a generic model for many 49 00:02:28,410 --> 00:02:30,240 of these systems. 50 00:02:30,240 --> 00:02:33,420 Although, the signaling pathways are distinct. 51 00:02:33,420 --> 00:02:39,130 So you have the epidermal growth factor receptor, 52 00:02:39,130 --> 00:02:41,320 which has a single transmembrane region. 53 00:02:41,320 --> 00:02:46,040 And this little-- what is this little domain in the cytosol? 54 00:02:46,040 --> 00:02:48,850 That's the tyrosine kinase activity. 55 00:02:48,850 --> 00:02:52,550 And it can react on itself. 56 00:02:52,550 --> 00:02:57,420 And this little ball is the growth factor. 57 00:02:57,420 --> 00:02:59,970 And so the model has been that, when 58 00:02:59,970 --> 00:03:05,080 the growth factor is around, that the proteins can dimerize. 59 00:03:05,080 --> 00:03:07,150 And so that's what's indicated here. 60 00:03:07,150 --> 00:03:10,540 So this is the model that was taken out of the paper. 61 00:03:10,540 --> 00:03:15,240 And in the presence of ATP, they can phosphorylate each other. 62 00:03:15,240 --> 00:03:19,380 OK, so that kinase domain has become active. 63 00:03:19,380 --> 00:03:22,290 And one of the questions is how do 64 00:03:22,290 --> 00:03:25,070 you know that phosphorylation makes it active. 65 00:03:25,070 --> 00:03:27,880 So you have to design experiments to test this. 66 00:03:27,880 --> 00:03:30,750 And if you go back, and you think about 67 00:03:30,750 --> 00:03:35,070 in introductory biochemistry course, you saw many cascades. 68 00:03:35,070 --> 00:03:37,380 And sometimes, phosphorylation activates. 69 00:03:37,380 --> 00:03:39,000 And sometimes, it inactivates. 70 00:03:39,000 --> 00:03:43,620 So all of that needs to be studied in some fashion. 71 00:03:43,620 --> 00:03:46,230 And so what we're going to be talking 72 00:03:46,230 --> 00:03:51,960 about in the module 7, which is the reactive oxygen species, 73 00:03:51,960 --> 00:03:57,960 is this protein, the NOX protein, NADPH oxidases. 74 00:03:57,960 --> 00:04:02,650 And what's the function of NADPH oxidases? 75 00:04:02,650 --> 00:04:04,170 What are the cofactors required? 76 00:04:04,170 --> 00:04:08,470 Anybody get that out of the paper? 77 00:04:08,470 --> 00:04:10,260 So you have an NADPH oxidase. 78 00:04:10,260 --> 00:04:12,726 So obviously, what's one of the cofactors? 79 00:04:12,726 --> 00:04:13,460 AUDIENCE: NADPH. 80 00:04:13,460 --> 00:04:14,390 JOANNE STUBBE: NADPH. 81 00:04:14,390 --> 00:04:19,120 OK, so what is another cofactor? 82 00:04:19,120 --> 00:04:21,000 Did any of you look at that? 83 00:04:21,000 --> 00:04:24,590 Or you haven't read the handout, first handout 84 00:04:24,590 --> 00:04:27,660 on reactive oxygen species, because it's also-- 85 00:04:27,660 --> 00:04:32,730 there are six isozymes of these NADPH oxidases. 86 00:04:32,730 --> 00:04:36,090 And they all form different complexes, 87 00:04:36,090 --> 00:04:40,530 but the basic chemistry is the same in all of them. 88 00:04:40,530 --> 00:04:43,510 So does anybody know? 89 00:04:43,510 --> 00:04:44,940 No. 90 00:04:44,940 --> 00:04:46,230 Any guesses? 91 00:04:46,230 --> 00:04:50,400 Ultimately, we have to convert the function of this protein-- 92 00:04:50,400 --> 00:04:54,750 hopefully, you know this is an unusual function. 93 00:04:54,750 --> 00:04:57,240 It converts oxygen into superoxide, 94 00:04:57,240 --> 00:05:00,120 which is one of those reactive oxygen 95 00:05:00,120 --> 00:05:02,490 species we saw this morning. 96 00:05:02,490 --> 00:05:08,610 OK, and this is the only known protein that does that, 97 00:05:08,610 --> 00:05:13,290 that's specific function is to generate superoxide. 98 00:05:13,290 --> 00:05:15,600 A lot of times, you do generate superoxide, 99 00:05:15,600 --> 00:05:18,420 but it's an artifact of uncoupling reactions 100 00:05:18,420 --> 00:05:22,120 or other consequences inside the cell. 101 00:05:22,120 --> 00:05:25,920 So we have NADPH, and that's on this side 102 00:05:25,920 --> 00:05:27,030 and the cytosolic side. 103 00:05:27,030 --> 00:05:30,765 This is the extracellular side. 104 00:05:30,765 --> 00:05:34,354 No guesses as to what the cofactor is? 105 00:05:34,354 --> 00:05:38,910 OK, so it has two kinds of cofactors-- 106 00:05:38,910 --> 00:05:48,300 FAD, OK, and the second cofactor are two heme b systems. 107 00:05:48,300 --> 00:05:49,910 So you have three cofactors-- 108 00:05:49,910 --> 00:05:59,890 FAD that are bound within the membrane and two hemes. 109 00:05:59,890 --> 00:06:04,740 And so that's true of all these systems. 110 00:06:04,740 --> 00:06:12,480 So NADPH is a two-electron donor. 111 00:06:12,480 --> 00:06:14,820 And what are we generating over here? 112 00:06:14,820 --> 00:06:19,350 We're generating superoxide from oxygen. So that's one electron. 113 00:06:19,350 --> 00:06:25,680 So the major function of NADPH is not to go from two electrons 114 00:06:25,680 --> 00:06:28,530 to one electron, but it transfers electrons 115 00:06:28,530 --> 00:06:29,520 to the flavin. 116 00:06:29,520 --> 00:06:32,520 They have similar redox potentials inside the cell. 117 00:06:32,520 --> 00:06:35,730 And then the flavin, because it's long and planar, 118 00:06:35,730 --> 00:06:39,230 can do both two-electron and one-electron chemistry. 119 00:06:39,230 --> 00:06:43,260 And if you have a heme, you can only do one-electron chemistry 120 00:06:43,260 --> 00:06:46,470 because you toggle between iron(III) and iron(II). 121 00:06:46,470 --> 00:06:50,010 So we'll talk about that in class next time. 122 00:06:50,010 --> 00:06:54,090 But somehow, you do chemistry over here. 123 00:06:54,090 --> 00:06:56,050 And this to me is the most amazing thing, 124 00:06:56,050 --> 00:06:59,280 and it should have upset you when you read this paper. 125 00:06:59,280 --> 00:07:00,687 OK, you should now start-- 126 00:07:00,687 --> 00:07:02,020 we're at the end of this course. 127 00:07:02,020 --> 00:07:03,900 You should now start, you know, you say 128 00:07:03,900 --> 00:07:05,514 how the heck can that happen. 129 00:07:05,514 --> 00:07:06,680 I don't believe any of this. 130 00:07:06,680 --> 00:07:08,110 This is the way I read all papers. 131 00:07:08,110 --> 00:07:09,190 I don't believe of this. 132 00:07:09,190 --> 00:07:09,420 Right? 133 00:07:09,420 --> 00:07:09,919 [LAUGHTER] 134 00:07:09,919 --> 00:07:10,870 So I come in. 135 00:07:10,870 --> 00:07:12,134 I'm at the one extreme. 136 00:07:12,134 --> 00:07:14,550 I don't believe anything I read because I think people are 137 00:07:14,550 --> 00:07:16,960 really sloppy experimentally. 138 00:07:16,960 --> 00:07:21,000 But you guys should start thinking like that. 139 00:07:21,000 --> 00:07:23,680 So we're going to generate superoxide. 140 00:07:23,680 --> 00:07:26,634 And it's on the outside of the cell. 141 00:07:26,634 --> 00:07:30,900 OK, This is a transmembrane system. 142 00:07:30,900 --> 00:07:34,260 So is there anything that links, if you read this paper 143 00:07:34,260 --> 00:07:36,390 carefully, and we'll look at data, but is there 144 00:07:36,390 --> 00:07:40,199 anything that links these two proteins in this paper 145 00:07:40,199 --> 00:07:41,490 that you were supposed to read? 146 00:07:50,762 --> 00:07:52,740 Which, even if you read it, there's 147 00:07:52,740 --> 00:07:54,150 not that much data there. 148 00:07:54,150 --> 00:07:57,810 You know, I guess there is a lot of data within each panel. 149 00:07:57,810 --> 00:08:00,900 But what the data is in each panel, they show you something, 150 00:08:00,900 --> 00:08:03,400 and then they quantitate it adjacent to it. 151 00:08:03,400 --> 00:08:06,000 So it's the same data just transferred 152 00:08:06,000 --> 00:08:07,860 into some kind of a bar graph because that's 153 00:08:07,860 --> 00:08:11,190 the way biologists do things, rather than actually 154 00:08:11,190 --> 00:08:14,910 drawing some kind of-- 155 00:08:14,910 --> 00:08:18,240 showing you some kind of a model with kinetics on it. 156 00:08:18,240 --> 00:08:20,310 So what do we know about these two proteins 157 00:08:20,310 --> 00:08:23,370 from what's in this paper? 158 00:08:23,370 --> 00:08:25,415 Any of you get anything out of that? 159 00:08:25,415 --> 00:08:26,690 AUDIENCE: They form a complex. 160 00:08:26,690 --> 00:08:28,398 JOANNE STUBBE: Yeah, they form a complex. 161 00:08:28,398 --> 00:08:30,100 So we'll come to that. 162 00:08:30,100 --> 00:08:32,880 But one of the experiments they did was they 163 00:08:32,880 --> 00:08:36,210 had antibodies to this, antibodies to this, 164 00:08:36,210 --> 00:08:38,460 and they showed you a picture where 165 00:08:38,460 --> 00:08:40,390 green and red went to yellow. 166 00:08:40,390 --> 00:08:45,110 And that was the evidence that these things might be linked. 167 00:08:45,110 --> 00:08:48,060 So what's unusual is that you're generating 168 00:08:48,060 --> 00:08:50,210 superoxide extracellularly. 169 00:08:50,210 --> 00:08:53,010 OK, and it's charged. 170 00:08:53,010 --> 00:08:56,130 In general, it doesn't go through membranes. 171 00:08:56,130 --> 00:09:00,510 But what you see, if you read the paper again, 172 00:09:00,510 --> 00:09:04,800 is the function of the NOX protein 173 00:09:04,800 --> 00:09:09,600 is to modulate the activity of the tyrosine kinase, which 174 00:09:09,600 --> 00:09:10,920 is intracellular. 175 00:09:10,920 --> 00:09:13,290 So the first thing that's bizarre 176 00:09:13,290 --> 00:09:19,158 is how do you get this superoxide inside the cell. 177 00:09:19,158 --> 00:09:23,340 OK, so that should have bothered-- it still bothers me. 178 00:09:23,340 --> 00:09:26,720 I mean, we have a model for how this works, 179 00:09:26,720 --> 00:09:29,970 but that's what the model is in all of these systems. 180 00:09:29,970 --> 00:09:35,810 And superoxide we'll see in the presence of protons. 181 00:09:35,810 --> 00:09:38,740 And protons, you don't have to have 182 00:09:38,740 --> 00:09:41,450 very acidic to have protons. 183 00:09:41,450 --> 00:09:44,180 It can cause disproportionation of superoxide 184 00:09:44,180 --> 00:09:46,940 to form oxygen and hydrogen peroxide. 185 00:09:46,940 --> 00:09:49,230 That happens actually quite fast. 186 00:09:49,230 --> 00:09:53,990 So the proposal is that somehow this disproportionates 187 00:09:53,990 --> 00:09:55,370 to form hydrogen peroxide. 188 00:09:55,370 --> 00:09:59,030 And it's the hydrogen peroxide that gets inside the cell. 189 00:09:59,030 --> 00:10:00,440 Hydrogen peroxide is neutral. 190 00:10:00,440 --> 00:10:02,620 Superoxide is charged. 191 00:10:02,620 --> 00:10:08,540 OK, and so the model has been that this molecule, AQP-- 192 00:10:08,540 --> 00:10:09,890 does anybody know what that was? 193 00:10:09,890 --> 00:10:10,160 Did-- 194 00:10:10,160 --> 00:10:11,170 AUDIENCE: It's an aquaporin. 195 00:10:11,170 --> 00:10:12,720 JOANNE STUBBE: Yeah, so aquaporin, 196 00:10:12,720 --> 00:10:13,640 so what does that do? 197 00:10:13,640 --> 00:10:16,940 They won the Nobel Prize for this two years ago 198 00:10:16,940 --> 00:10:18,557 for it supposedly is a water channel. 199 00:10:18,557 --> 00:10:19,640 AUDIENCE: For water, yeah. 200 00:10:19,640 --> 00:10:22,340 JOANNE STUBBE: Yeah, and we three-dimensional structures 201 00:10:22,340 --> 00:10:24,240 that people have gotten very excited about. 202 00:10:24,240 --> 00:10:26,157 So hydrogen peroxide sort of looks like water. 203 00:10:26,157 --> 00:10:28,490 AUDIENCE: And you can fit it through the aquaporin pore. 204 00:10:28,490 --> 00:10:30,640 JOANNE STUBBE: Yeah, so well, that's the model. 205 00:10:30,640 --> 00:10:33,932 Yeah, and there is a model from people 206 00:10:33,932 --> 00:10:35,390 that have looked at the structures, 207 00:10:35,390 --> 00:10:37,640 have modeled that this could go through. 208 00:10:37,640 --> 00:10:39,780 So now you generate hydrogen peroxide. 209 00:10:39,780 --> 00:10:43,430 OK, and so the hydrogen peroxide, 210 00:10:43,430 --> 00:10:48,020 what we've been talking about is post-translational modification 211 00:10:48,020 --> 00:10:49,590 by reacting-- 212 00:10:49,590 --> 00:10:50,510 what are we reacting? 213 00:10:50,510 --> 00:10:52,117 We're reacting a sulfhydrl group. 214 00:10:52,117 --> 00:10:53,450 Let's see if I brought my chalk. 215 00:10:57,410 --> 00:10:58,730 I don't have any good chalk. 216 00:10:58,730 --> 00:11:00,715 They don't make fat chalk anymore. 217 00:11:00,715 --> 00:11:06,440 All right, so they're converting-- 218 00:11:06,440 --> 00:11:08,900 we what did we talk about last time? 219 00:11:08,900 --> 00:11:11,350 We were talking about this reaction. 220 00:11:11,350 --> 00:11:12,725 This was the major focus. 221 00:11:15,480 --> 00:11:18,000 So we're forming the sulfenic acid. 222 00:11:18,000 --> 00:11:20,510 OK, and this whole paper is about, 223 00:11:20,510 --> 00:11:24,150 number one, do you form sulfenic acid inside the cell. 224 00:11:24,150 --> 00:11:26,330 And does it affect signaling? 225 00:11:26,330 --> 00:11:28,535 That's the focus. 226 00:11:28,535 --> 00:11:32,520 And her hypothesis was that it does. 227 00:11:32,520 --> 00:11:34,520 And so then you need to know something 228 00:11:34,520 --> 00:11:36,470 about the signaling pathway, which 229 00:11:36,470 --> 00:11:37,670 other people have studied. 230 00:11:37,670 --> 00:11:41,390 And so there are apparently two signaling pathways, 231 00:11:41,390 --> 00:11:43,670 which we're not going to pay that much attention to, 232 00:11:43,670 --> 00:11:47,240 but you need to pay attention to it enough because some 233 00:11:47,240 --> 00:11:49,595 of the figures in the paper-- 234 00:11:49,595 --> 00:11:53,100 what did some of the figures in the paper look at? 235 00:11:53,100 --> 00:11:54,077 Anybody remember that? 236 00:11:54,077 --> 00:11:55,160 Were you confused by that? 237 00:11:55,160 --> 00:11:59,357 It was in figure 1 if I remember correctly. 238 00:11:59,357 --> 00:12:00,940 AUDIENCE: Yeah, was it phosphorylation 239 00:12:00,940 --> 00:12:01,900 of these [INAUDIBLE]. 240 00:12:01,900 --> 00:12:04,540 JOANNE STUBBE: Right, so they looked at phosphorylation. 241 00:12:04,540 --> 00:12:05,639 And how can you do that? 242 00:12:05,639 --> 00:12:06,680 You can have an antibody. 243 00:12:06,680 --> 00:12:07,430 AUDIENCE: Western. 244 00:12:07,430 --> 00:12:09,304 JOANNE STUBBE: Yeah, by some kind of Western. 245 00:12:09,304 --> 00:12:12,595 So in the first figure, if you hadn't looked carefully 246 00:12:12,595 --> 00:12:13,970 at this figure, you wouldn't know 247 00:12:13,970 --> 00:12:16,050 why they were looking at phosphorylation at all. 248 00:12:16,050 --> 00:12:18,860 But what they're trying to look at, like in many 249 00:12:18,860 --> 00:12:21,860 of these systems, is a signaling pathway 250 00:12:21,860 --> 00:12:24,400 where something phosphorylates then dephosphorylates. 251 00:12:24,400 --> 00:12:27,860 OK, so these are the two signaling pathways, 252 00:12:27,860 --> 00:12:31,700 and they integrated that into their analysis. 253 00:12:31,700 --> 00:12:38,840 And so now the key questions, if you look at the tyrosine kinase 254 00:12:38,840 --> 00:12:42,620 activity, why would you think-- 255 00:12:42,620 --> 00:12:45,920 what did you learn in this paper sort of grossly 256 00:12:45,920 --> 00:12:51,560 about the potential for sulfenylation? 257 00:12:51,560 --> 00:12:52,790 Anybody read? 258 00:12:52,790 --> 00:12:55,887 Did anybody look at the supplementary information? 259 00:12:59,226 --> 00:13:01,300 I think that's always the best part of the paper. 260 00:13:01,300 --> 00:13:04,890 See, this is what the issue is nowadays. 261 00:13:04,890 --> 00:13:06,260 Now I'm digressing again. 262 00:13:06,260 --> 00:13:07,550 So I'm allowed to do this. 263 00:13:07,550 --> 00:13:09,860 I only have another five lectures to teach, 264 00:13:09,860 --> 00:13:11,450 and I'm finished anyhow. 265 00:13:11,450 --> 00:13:14,580 I mean, to me, it's sort of like that's where all the data is. 266 00:13:14,580 --> 00:13:17,960 And so nowadays, nobody puts data in papers. 267 00:13:17,960 --> 00:13:19,915 OK, it's impossible to evaluate a paper. 268 00:13:19,915 --> 00:13:21,665 It's all in the supplementary information. 269 00:13:21,665 --> 00:13:24,290 And what irritates the hell out of me 270 00:13:24,290 --> 00:13:27,120 is that most people just dump it. 271 00:13:27,120 --> 00:13:30,170 That is they don't present it in a way that's thoughtful 272 00:13:30,170 --> 00:13:31,220 so that someone like me-- 273 00:13:31,220 --> 00:13:31,910 I don't care. 274 00:13:31,910 --> 00:13:33,868 Most people don't want to read all the details. 275 00:13:33,868 --> 00:13:34,426 That's fine. 276 00:13:34,426 --> 00:13:36,050 But then if you're going to present it, 277 00:13:36,050 --> 00:13:39,860 it should be so that I can read it and go back and forth 278 00:13:39,860 --> 00:13:41,210 and figure out what's going on. 279 00:13:41,210 --> 00:13:44,840 Anyhow, here's a case where they talk-- 280 00:13:44,840 --> 00:13:47,390 do you remember that they talked about the kinase activity 281 00:13:47,390 --> 00:13:48,650 in this paper? 282 00:13:48,650 --> 00:13:50,240 Anybody remember that? 283 00:13:50,240 --> 00:13:55,094 We will get to that data if I get off this slide. 284 00:13:55,094 --> 00:13:57,302 AUDIENCE: I think in Blake they talked about the fact 285 00:13:57,302 --> 00:13:59,492 that it enhances the kinase activity if you 286 00:13:59,492 --> 00:14:01,200 have the post-translational modification. 287 00:14:01,200 --> 00:14:03,980 JOANNE STUBBE: Right, so what did they measure? 288 00:14:03,980 --> 00:14:05,070 Did you look at that? 289 00:14:05,070 --> 00:14:08,260 There's a figure where they focused on that. 290 00:14:08,260 --> 00:14:15,610 I can't remember what figure it was, probably figure 4, 5. 291 00:14:15,610 --> 00:14:17,045 So were you confused? 292 00:14:17,045 --> 00:14:18,420 It was one thing where you should 293 00:14:18,420 --> 00:14:21,050 have been confused because you wouldn't know 294 00:14:21,050 --> 00:14:23,934 what any of the words meant. 295 00:14:23,934 --> 00:14:25,450 So well, no. 296 00:14:25,450 --> 00:14:26,602 So what did you do? 297 00:14:26,602 --> 00:14:28,060 I mean, I had to do the same thing. 298 00:14:28,060 --> 00:14:29,560 I went and googled it and looked up, 299 00:14:29,560 --> 00:14:31,184 and I figured out what the words meant. 300 00:14:31,184 --> 00:14:32,142 And then I understood. 301 00:14:32,142 --> 00:14:34,600 And we're going to go through that because the reagents are 302 00:14:34,600 --> 00:14:35,200 a key thing. 303 00:14:35,200 --> 00:14:37,270 You need to understand what the reagents do 304 00:14:37,270 --> 00:14:40,240 to be able to think about what you're looking at. 305 00:14:40,240 --> 00:14:44,000 So anyhow, we'll come back to this, but, you know, we have-- 306 00:14:44,000 --> 00:14:46,750 this is a major target of drugs. 307 00:14:46,750 --> 00:14:49,630 There are drugs that are used clinically 308 00:14:49,630 --> 00:14:53,290 in treatment of cancer that target the epidermal growth 309 00:14:53,290 --> 00:14:55,030 factor receptor. 310 00:14:55,030 --> 00:14:57,850 And they inhibit the kinase domain, which 311 00:14:57,850 --> 00:15:02,030 were used to ask the question-- 312 00:15:02,030 --> 00:15:05,200 remember we talked about this last time, the importance 313 00:15:05,200 --> 00:15:07,630 of reversibility of this. 314 00:15:07,630 --> 00:15:11,170 And if you modify it, what is the effect? 315 00:15:11,170 --> 00:15:16,060 OK, so is the effect that you alter the downstream signaling, 316 00:15:16,060 --> 00:15:18,940 whatever is downstream if you know something about that? 317 00:15:18,940 --> 00:15:23,350 Or the other effect can be is do you modulate 318 00:15:23,350 --> 00:15:25,370 the catalytic activity. 319 00:15:25,370 --> 00:15:30,146 And does anybody get that out of one of the figures? 320 00:15:30,146 --> 00:15:32,850 Or was this paper so hard we didn't even get that? 321 00:15:35,730 --> 00:15:37,110 Look in figure-- 322 00:15:37,110 --> 00:15:39,750 I need a copy of the paper. 323 00:15:39,750 --> 00:15:44,480 Oh, I don't think this paper was that hard, 324 00:15:44,480 --> 00:15:47,210 but you had to work at it because the figures were small, 325 00:15:47,210 --> 00:15:48,810 and there was a lot of information. 326 00:15:48,810 --> 00:15:52,250 So if you look at figure 5, anybody look at figure 5? 327 00:15:52,250 --> 00:15:54,150 We'll come back to that in a minute. 328 00:15:59,265 --> 00:16:02,070 So to me, when I look at-- and you'll see the way 329 00:16:02,070 --> 00:16:02,900 I write this down. 330 00:16:02,900 --> 00:16:04,867 You have a panel of stuff. 331 00:16:04,867 --> 00:16:06,450 You have a panel of all these figures. 332 00:16:06,450 --> 00:16:09,180 Well, you know, usually, the title tells you 333 00:16:09,180 --> 00:16:10,824 what the whole figure is about. 334 00:16:10,824 --> 00:16:12,240 But then at the end of this, you'd 335 00:16:12,240 --> 00:16:13,698 like to be able to look at the data 336 00:16:13,698 --> 00:16:18,480 without any input from the person writing the paper 337 00:16:18,480 --> 00:16:20,050 and draw your own conclusions. 338 00:16:20,050 --> 00:16:22,770 So at the end of this, this is what I do in every paper. 339 00:16:22,770 --> 00:16:26,670 I look at the figures, and then I draw a conclusion 340 00:16:26,670 --> 00:16:27,807 from the figures. 341 00:16:27,807 --> 00:16:28,890 And if I can't draw this-- 342 00:16:28,890 --> 00:16:30,181 I do this with my students too. 343 00:16:30,181 --> 00:16:32,010 If I can't understand from looking 344 00:16:32,010 --> 00:16:34,440 at the figures what the key conclusions are, 345 00:16:34,440 --> 00:16:36,350 they didn't write a good paper. 346 00:16:36,350 --> 00:16:39,000 So this here, if you look at figure 5, 347 00:16:39,000 --> 00:16:41,070 you will see that there's activity. 348 00:16:41,070 --> 00:16:44,020 And so remember, if you're going to do this, 349 00:16:44,020 --> 00:16:45,780 why would you want to do it? 350 00:16:45,780 --> 00:16:47,280 It's got to have some effect. 351 00:16:47,280 --> 00:16:48,000 Right? 352 00:16:48,000 --> 00:16:49,740 Or it's not interesting. 353 00:16:49,740 --> 00:16:52,440 And so the key issue is that you could get chemistry 354 00:16:52,440 --> 00:16:56,280 like this that's happening, and it isn't interesting. 355 00:16:56,280 --> 00:16:58,290 And it's very challenging to look 356 00:16:58,290 --> 00:17:00,510 at this chemistry inside the cell 357 00:17:00,510 --> 00:17:04,900 because thiols get oxidized fairly easily. 358 00:17:04,900 --> 00:17:07,069 So what you want to do is not only-- 359 00:17:07,069 --> 00:17:10,170 and this is what we were focused on last time 360 00:17:10,170 --> 00:17:12,190 is seeing if this happens. 361 00:17:12,190 --> 00:17:15,010 OK, we haven't gotten to inside the cell yet. 362 00:17:15,010 --> 00:17:19,606 But then the question is is it connected to signaling. 363 00:17:19,606 --> 00:17:22,680 And that could be related to the activity 364 00:17:22,680 --> 00:17:27,630 of the tyrosine kinase, which triggers off 365 00:17:27,630 --> 00:17:29,610 the phosphorylation cascades. 366 00:17:29,610 --> 00:17:31,350 In a way you, you have to look up 367 00:17:31,350 --> 00:17:34,120 what other people have done to sort of understand that. 368 00:17:34,120 --> 00:17:40,060 OK, so now we have the issue is that we made hydrogen peroxide. 369 00:17:40,060 --> 00:17:44,770 OK, supposedly, by this model, it's gotten inside the cell. 370 00:17:44,770 --> 00:17:48,770 And so now the model is that it does this. 371 00:17:48,770 --> 00:17:53,040 OK, so what you see is the issue with this model, which 372 00:17:53,040 --> 00:17:55,740 is why people have been fighting over this, 373 00:17:55,740 --> 00:17:58,140 is the rate constant for this reaction. 374 00:17:58,140 --> 00:18:02,056 Even if you have a thiolate, it's not very fast. 375 00:18:02,056 --> 00:18:05,400 OK, so that's something we'll talk about in class. 376 00:18:05,400 --> 00:18:08,400 But the Winterbourn paper, when you read that, 377 00:18:08,400 --> 00:18:10,210 focuses on the rate constant. 378 00:18:10,210 --> 00:18:11,910 So this is a second order rate constant. 379 00:18:11,910 --> 00:18:15,900 It's like 1 per molar per second, really slow. 380 00:18:15,900 --> 00:18:18,700 So if this was happening over the period of hours, 381 00:18:18,700 --> 00:18:21,930 and your signaling is finished in 15 minutes, 382 00:18:21,930 --> 00:18:23,740 you've got a serious problem. 383 00:18:23,740 --> 00:18:26,520 So you have to deal with that problem. 384 00:18:26,520 --> 00:18:29,620 And people are finally dealing with that problem. 385 00:18:29,620 --> 00:18:33,030 And there are proteins you'll see in the next lecture 386 00:18:33,030 --> 00:18:36,540 that, you know, the pK of the sulfur 387 00:18:36,540 --> 00:18:38,220 isn't all that perturbed, but they're 388 00:18:38,220 --> 00:18:41,540 able to react with hydrogen peroxide much, much faster. 389 00:18:41,540 --> 00:18:43,870 And that that's going to be a key piece of information. 390 00:18:43,870 --> 00:18:46,470 So if you get modification, then the question 391 00:18:46,470 --> 00:18:48,990 is what is the consequences of the modification, which 392 00:18:48,990 --> 00:18:50,440 is what the paper is about. 393 00:18:50,440 --> 00:18:52,770 So the other key player in all of this, 394 00:18:52,770 --> 00:18:54,540 if you have something phosphorylated, 395 00:18:54,540 --> 00:18:56,910 so here we are something phosphorylated. 396 00:18:56,910 --> 00:18:57,800 So that's a kinase. 397 00:18:57,800 --> 00:18:59,550 It phosphorylates itself. 398 00:18:59,550 --> 00:19:01,509 But then whenever we have a kinase, 399 00:19:01,509 --> 00:19:04,050 we usually have something that clips off the phosphate, which 400 00:19:04,050 --> 00:19:05,020 is a phosphatase. 401 00:19:05,020 --> 00:19:07,660 So that's PTP. 402 00:19:07,660 --> 00:19:10,050 And we'll see that the phosphatases 403 00:19:10,050 --> 00:19:15,482 we're dealing with all have cysteines in the active site. 404 00:19:15,482 --> 00:19:17,720 OK, and the cysteines are all-- 405 00:19:17,720 --> 00:19:27,545 so the PTPs all have an active site cysteine. 406 00:19:27,545 --> 00:19:30,520 And this active site cysteine-- you've seen this over and over 407 00:19:30,520 --> 00:19:32,580 again-- is involved in covalent catalysis. 408 00:19:32,580 --> 00:19:35,940 OK, we've seen hundreds of examples of this now. 409 00:19:35,940 --> 00:19:38,050 So you might not know what's involved, 410 00:19:38,050 --> 00:19:41,110 but that would be a good guess based on everything we've seen. 411 00:19:41,110 --> 00:19:44,810 So it turns out the question is this is the active form. 412 00:19:44,810 --> 00:19:48,350 OK, so how could you shut it off? 413 00:19:48,350 --> 00:19:53,590 You might be able to shut it off by sulfenylation. 414 00:19:53,590 --> 00:19:58,270 So this would be active, and this would be inactive. 415 00:19:58,270 --> 00:20:01,060 So what you're looking at is, again, 416 00:20:01,060 --> 00:20:03,640 another method of post-translational 417 00:20:03,640 --> 00:20:06,250 modification-- this is the hypothesis-- 418 00:20:06,250 --> 00:20:10,060 that can affect the activity in these cascades. 419 00:20:10,060 --> 00:20:13,600 And does anybody remember what conclusion people 420 00:20:13,600 --> 00:20:16,670 drew about the phosphatase? 421 00:20:16,670 --> 00:20:19,840 That was another figure in the paper. 422 00:20:19,840 --> 00:20:21,160 Does anybody remember? 423 00:20:21,160 --> 00:20:22,630 There's a lot of information. 424 00:20:22,630 --> 00:20:25,780 But in the end, there aren't that many conclusions 425 00:20:25,780 --> 00:20:26,530 you could draw. 426 00:20:26,530 --> 00:20:29,740 But part of the problem is that I haven't gone 427 00:20:29,740 --> 00:20:32,290 over this cascade in lecture. 428 00:20:32,290 --> 00:20:33,370 AUDIENCE: They were dark. 429 00:20:33,370 --> 00:20:35,286 JOANNE STUBBE: So I realize that's putting you 430 00:20:35,286 --> 00:20:36,230 at a disadvantage. 431 00:20:36,230 --> 00:20:39,340 But you've had a couple of weeks to read this now, so yeah. 432 00:20:39,340 --> 00:20:40,810 AUDIENCE: They were differentially 433 00:20:40,810 --> 00:20:43,610 sulfenylated depending on whether or not 434 00:20:43,610 --> 00:20:44,610 you had EGF present. 435 00:20:44,610 --> 00:20:47,680 JOANNE STUBBE: Right, so they were differentially-- so nobody 436 00:20:47,680 --> 00:20:50,070 knew what the phosphatase was. 437 00:20:50,070 --> 00:20:52,360 OK, so they had a bunch of candidates 438 00:20:52,360 --> 00:20:54,377 because we have the whole genome sequence. 439 00:20:54,377 --> 00:20:55,960 We know what all the phosphatases are. 440 00:20:55,960 --> 00:20:58,010 Just like we know there are 500 kinases, 441 00:20:58,010 --> 00:20:59,800 there are 100 phosphatases. 442 00:20:59,800 --> 00:21:03,310 And they know which ones are connected to certain kinds 443 00:21:03,310 --> 00:21:04,750 of signaling pathways. 444 00:21:04,750 --> 00:21:08,290 So one of the key conclusions from this paper is that they 445 00:21:08,290 --> 00:21:11,350 identified, or they claimed to identify-- you may or may not-- 446 00:21:11,350 --> 00:21:13,270 when we get that far, you can look at the data 447 00:21:13,270 --> 00:21:14,710 and see whether you believe that. 448 00:21:14,710 --> 00:21:16,430 Based on what they reported here, 449 00:21:16,430 --> 00:21:19,360 they claim to know that it was SHP. 450 00:21:19,360 --> 00:21:20,230 I think it is. 451 00:21:20,230 --> 00:21:23,020 I don't remember the name of the phosphatase, 452 00:21:23,020 --> 00:21:27,040 but that's the one that was modified. 453 00:21:27,040 --> 00:21:30,220 And it turns out even the phosphatase 454 00:21:30,220 --> 00:21:33,850 can be modified further in a cascade 455 00:21:33,850 --> 00:21:36,220 by proteins called peroxiredoxins, 456 00:21:36,220 --> 00:21:38,460 which aren't in this paper. 457 00:21:38,460 --> 00:21:40,140 Anyhow, so that's the overview. 458 00:21:40,140 --> 00:21:42,790 And what I wanted to do was spend a little bit 459 00:21:42,790 --> 00:21:45,280 of time going back through what we 460 00:21:45,280 --> 00:21:47,895 had gone through last time, not this part. 461 00:21:47,895 --> 00:21:50,020 We're going to go through this very fast because we 462 00:21:50,020 --> 00:21:51,820 got this far. 463 00:21:51,820 --> 00:21:57,470 Again, the development of a specific reaction with either 464 00:21:57,470 --> 00:22:03,770 iodo-dimedone or dimedone to modify either the sulfenic acid 465 00:22:03,770 --> 00:22:05,660 or the sulfhydryl group. 466 00:22:05,660 --> 00:22:10,230 OK, and the issue is you can do this inside the cell. 467 00:22:10,230 --> 00:22:12,630 These are cell permeable. 468 00:22:12,630 --> 00:22:15,695 But then how do you ever find it? 469 00:22:15,695 --> 00:22:19,590 Right, so you've got 10,000 proteins. 470 00:22:19,590 --> 00:22:21,970 They can all get modified to some extent. 471 00:22:21,970 --> 00:22:23,940 We don't know how much of the protein is there. 472 00:22:23,940 --> 00:22:27,480 We don't know whether it's been partially modified. 473 00:22:27,480 --> 00:22:31,510 But there's no way to identify this currently. 474 00:22:31,510 --> 00:22:33,600 And of course, the first thing is 475 00:22:33,600 --> 00:22:35,730 you're assuming that the linkage is stable. 476 00:22:35,730 --> 00:22:37,090 That's important. 477 00:22:37,090 --> 00:22:39,330 But even so, there's no handle on this. 478 00:22:39,330 --> 00:22:43,800 So the focus of the Nature Chemical Biology paper 479 00:22:43,800 --> 00:22:45,490 was to figure out how to make this 480 00:22:45,490 --> 00:22:49,080 so you could find this inside the cell 481 00:22:49,080 --> 00:22:51,180 and use this in some way. 482 00:22:51,180 --> 00:22:55,080 And so the mass spec method we had focused on last time, which 483 00:22:55,080 --> 00:22:57,894 is also used again in this paper, 484 00:22:57,894 --> 00:23:00,060 but if you didn't read supplementary information you 485 00:23:00,060 --> 00:23:07,940 won't know that, is they use isotopically labeled materials. 486 00:23:07,940 --> 00:23:12,970 OK, so this is only if you have an extra mass of 6. 487 00:23:12,970 --> 00:23:18,260 You have the deuterated methyl groups. 488 00:23:18,260 --> 00:23:21,150 And that means you have a sulfenic acid that you've 489 00:23:21,150 --> 00:23:24,000 modified because she's shown that it's specific 490 00:23:24,000 --> 00:23:28,950 versus the methylated, which reacts with thiols only. 491 00:23:28,950 --> 00:23:32,460 So that's the basis of the assay. 492 00:23:32,460 --> 00:23:34,980 And again, it's not easy to find a reagent that 493 00:23:34,980 --> 00:23:35,940 allows you to do this. 494 00:23:35,940 --> 00:23:40,090 And this is where we were last time at the end of the class. 495 00:23:40,090 --> 00:23:42,660 I didn't get this far with all the classes, 496 00:23:42,660 --> 00:23:45,540 and I can't remember who was in the class. 497 00:23:45,540 --> 00:23:47,370 But we were looking at this. 498 00:23:47,370 --> 00:23:49,650 This was just proof of concept. 499 00:23:49,650 --> 00:23:52,980 So we have a glutathione peroxidase, which 500 00:23:52,980 --> 00:23:56,590 was also in the current paper. 501 00:23:56,590 --> 00:23:59,220 And what do we know about the glutathione peroxidase 502 00:23:59,220 --> 00:24:00,960 in this paper? 503 00:24:00,960 --> 00:24:03,210 If you read the paper, did this ring a bell 504 00:24:03,210 --> 00:24:05,500 from the previous paper? 505 00:24:05,500 --> 00:24:10,312 So what was unique about glutathione peroxidase? 506 00:24:10,312 --> 00:24:12,222 AUDIENCE: It has an active site cysteine 507 00:24:12,222 --> 00:24:13,280 JOANNE STUBBE: Yeah, it has an active site. 508 00:24:13,280 --> 00:24:15,450 AUDIENCE: --that they can use to validate their approach. 509 00:24:15,450 --> 00:24:17,491 JOANNE STUBBE: So it has an active site cysteine, 510 00:24:17,491 --> 00:24:18,930 which can get modified. 511 00:24:18,930 --> 00:24:20,690 There's something reactive about that. 512 00:24:20,690 --> 00:24:22,780 And it catalyzes. 513 00:24:22,780 --> 00:24:24,500 It has peroxidase activity. 514 00:24:24,500 --> 00:24:26,930 So it plays a very important role 515 00:24:26,930 --> 00:24:29,790 in controlling these reactive oxygen species. 516 00:24:29,790 --> 00:24:34,590 So it's a small protein that's been very well characterized. 517 00:24:34,590 --> 00:24:36,350 And so if you look here, what do you see? 518 00:24:36,350 --> 00:24:38,654 This is where we were at the end of the lecture. 519 00:24:38,654 --> 00:24:39,320 What do you see? 520 00:24:39,320 --> 00:24:42,250 So this is an in vitro experiment, 521 00:24:42,250 --> 00:24:44,040 not an in vivo experiment. 522 00:24:44,040 --> 00:24:45,430 So we're in the test tube. 523 00:24:45,430 --> 00:24:46,760 And so what do you see? 524 00:24:46,760 --> 00:24:51,995 So they treated it either here with dimedone, 525 00:24:51,995 --> 00:24:57,200 OK, which labels sulfenic acids, or they treated it 526 00:24:57,200 --> 00:24:59,700 with iodo-dimedone. 527 00:24:59,700 --> 00:25:00,950 And so what did that tell you? 528 00:25:00,950 --> 00:25:02,750 This is where we were last time. 529 00:25:02,750 --> 00:25:05,570 When you looked at that, did that say anything to you 530 00:25:05,570 --> 00:25:08,257 without looking at the analysis out the other side? 531 00:25:08,257 --> 00:25:09,590 So you're looking at the figure. 532 00:25:09,590 --> 00:25:12,950 What did you think when you looked at the figure? 533 00:25:12,950 --> 00:25:14,138 What's your name? 534 00:25:14,138 --> 00:25:14,846 AUDIENCE: Nicole. 535 00:25:14,846 --> 00:25:18,720 JOANNE STUBBE: Nicole, what do you think? 536 00:25:18,720 --> 00:25:21,395 AUDIENCE: You can see that, as hydrogen peroxide 537 00:25:21,395 --> 00:25:25,940 increases with the dimedone, the levels increase. 538 00:25:25,940 --> 00:25:29,760 But with the iod0-dimedone, the levels decrease. 539 00:25:29,760 --> 00:25:31,320 JOANNE STUBBE: OK, so that's good. 540 00:25:31,320 --> 00:25:33,730 And then you can say one more thing. 541 00:25:33,730 --> 00:25:37,830 And what you could say is, in this lane-- 542 00:25:37,830 --> 00:25:43,198 so this is where they're looking at the iodo-dimedone, 543 00:25:43,198 --> 00:25:45,500 and there's no hydrogen peroxide. 544 00:25:45,500 --> 00:25:47,540 What does that tell you? 545 00:25:47,540 --> 00:25:49,640 So that's an extra piece of information 546 00:25:49,640 --> 00:25:53,420 that was more subtle out of this. 547 00:25:53,420 --> 00:25:58,050 So if you look at this, you could think about it. 548 00:25:58,050 --> 00:26:00,490 You could do it. 549 00:26:00,490 --> 00:26:05,590 So look at this, so we're increasing hydrogen peroxide. 550 00:26:05,590 --> 00:26:07,750 And you saw it increases. 551 00:26:07,750 --> 00:26:11,790 Here we have just cysteine. 552 00:26:11,790 --> 00:26:14,850 Of the 100%, we're hitting it with something 553 00:26:14,850 --> 00:26:17,750 that reacts with sulfhydryl groups, the iodo. 554 00:26:17,750 --> 00:26:19,800 And look at this compared to this. 555 00:26:19,800 --> 00:26:22,134 What do you see? 556 00:26:22,134 --> 00:26:23,879 What does it look like? 557 00:26:23,879 --> 00:26:25,962 AUDIENCE: It looks like there's more [INAUDIBLE].. 558 00:26:25,962 --> 00:26:27,210 JOANNE STUBBE: So that's exactly it. 559 00:26:27,210 --> 00:26:28,380 And it's the eyeball method. 560 00:26:28,380 --> 00:26:30,310 So you can't tell anything by the eyeball method. 561 00:26:30,310 --> 00:26:31,520 You have to have a way of quantity-- 562 00:26:31,520 --> 00:26:32,900 we talked about phosphorimaging. 563 00:26:32,900 --> 00:26:34,370 That's what people do. 564 00:26:34,370 --> 00:26:36,950 But what this tells you is, if you looked again 565 00:26:36,950 --> 00:26:39,650 at the details, you know, they use 566 00:26:39,650 --> 00:26:42,510 50 micromolar of the protein. 567 00:26:42,510 --> 00:26:46,280 And they went to I think with 100 micromolar. 568 00:26:46,280 --> 00:26:49,130 And even when they went to 100 micromolar, 569 00:26:49,130 --> 00:26:51,260 they didn't inactivate. 570 00:26:51,260 --> 00:26:58,290 They didn't modify all of the GPx-3. 571 00:26:58,290 --> 00:26:59,689 OK, now would you expect them to? 572 00:26:59,689 --> 00:27:00,230 I don't know. 573 00:27:00,230 --> 00:27:03,290 They probably tried a lot of different conditions. 574 00:27:03,290 --> 00:27:04,790 I mean it's concentration dependent. 575 00:27:04,790 --> 00:27:07,370 It's time dependent. 576 00:27:07,370 --> 00:27:09,380 And that wasn't given in the details. 577 00:27:09,380 --> 00:27:12,350 OK, but this tells you then-- 578 00:27:12,350 --> 00:27:15,710 that takes you to the next one of the sets of data 579 00:27:15,710 --> 00:27:20,110 here, which you could have gotten in some form 580 00:27:20,110 --> 00:27:22,600 by looking at that data. 581 00:27:22,600 --> 00:27:27,520 So now what you're looking at is they're looking at a ratio of, 582 00:27:27,520 --> 00:27:30,810 you know, what's sulfenylated versus what's 583 00:27:30,810 --> 00:27:33,690 a sulfhydryl plus sulfenylated. 584 00:27:33,690 --> 00:27:34,850 And what do you see? 585 00:27:34,850 --> 00:27:38,300 Cysteine 36 is known to be at the active site, 586 00:27:38,300 --> 00:27:40,600 but they showed that in this experiment. 587 00:27:40,600 --> 00:27:43,810 And you see that you don't reach 100% labeling. 588 00:27:43,810 --> 00:27:48,010 OK, so that you saw in the previous set of data. 589 00:27:48,010 --> 00:27:50,905 You couldn't tell what the ratio was. 590 00:27:50,905 --> 00:27:54,970 And this is sort of you're just looking at this is the mass. 591 00:27:54,970 --> 00:27:57,670 OK, and if you know what the protein is, 592 00:27:57,670 --> 00:28:00,747 remember, and you cleave the protein down with trypsin, 593 00:28:00,747 --> 00:28:02,830 you're going to get all these little peptides out. 594 00:28:02,830 --> 00:28:04,960 That's what we talked about last time. 595 00:28:04,960 --> 00:28:07,000 And we're looking at charge-to-mass. 596 00:28:07,000 --> 00:28:10,390 We're looking for charge-to-mass differences in ratios 597 00:28:10,390 --> 00:28:16,480 depending on what the charge is for deuterium versus protons 598 00:28:16,480 --> 00:28:18,310 to tell whether it's sulfenylated, 599 00:28:18,310 --> 00:28:21,740 or it's just an SH group. 600 00:28:21,740 --> 00:28:25,780 And so what they found was this charge-to-mass of 541 601 00:28:25,780 --> 00:28:28,310 versus 554. 602 00:28:28,310 --> 00:28:30,880 And that told them-- if you look at the sequence 603 00:28:30,880 --> 00:28:34,340 that I told you in the computer can you analyze all this, 604 00:28:34,340 --> 00:28:38,980 it told you you were looking at peptide 36 to 43. 605 00:28:38,980 --> 00:28:43,900 And we know cysteine 36 is within that peptide. 606 00:28:43,900 --> 00:28:48,670 So that didn't tell us that the modification 607 00:28:48,670 --> 00:28:52,060 is based out that cysteine. 608 00:28:52,060 --> 00:28:54,740 But then you could go back in, and you could sequence. 609 00:28:54,740 --> 00:28:56,508 Yeah? 610 00:28:56,508 --> 00:28:59,335 AUDIENCE: Why is the m/z different by three? 611 00:28:59,335 --> 00:29:00,960 JOANNE STUBBE: Because you have charge. 612 00:29:00,960 --> 00:29:02,540 Because of the charge of the system. 613 00:29:02,540 --> 00:29:03,460 AUDIENCE: [INAUDIBLE] 614 00:29:03,460 --> 00:29:06,180 JOANNE STUBBE: Yeah, so you can get this at every-- 615 00:29:06,180 --> 00:29:08,430 you know, depending on how many masses you see, 616 00:29:08,430 --> 00:29:10,380 you can still pick up the data, but the number 617 00:29:10,380 --> 00:29:11,200 will be different. 618 00:29:11,200 --> 00:29:13,650 OK, so you would like to have six, 619 00:29:13,650 --> 00:29:15,600 but you might not be in that mass range. 620 00:29:15,600 --> 00:29:17,190 I mean here you would be. 621 00:29:17,190 --> 00:29:20,370 So you could have looked at that to have a difference of six. 622 00:29:24,040 --> 00:29:25,710 So this tells you that you're not 623 00:29:25,710 --> 00:29:28,080 getting complete sulfenylation. 624 00:29:28,080 --> 00:29:30,360 So then the question is did any of you 625 00:29:30,360 --> 00:29:32,490 recognize that when you looked at the paper. 626 00:29:32,490 --> 00:29:34,400 Or did it just go by you? 627 00:29:34,400 --> 00:29:36,180 What could be going on? 628 00:29:38,904 --> 00:29:40,732 So we already talked about the fact 629 00:29:40,732 --> 00:29:42,690 that maybe they didn't do the experiment right. 630 00:29:42,690 --> 00:29:44,220 They didn't have high enough concentrations. 631 00:29:44,220 --> 00:29:45,690 They didn't let it go long enough. 632 00:29:45,690 --> 00:29:50,300 Let's ignore that, OK, because they're good scientists. 633 00:29:50,300 --> 00:29:53,440 With some people, you might not want to ignore that. 634 00:29:53,440 --> 00:29:55,080 But then the question is what could 635 00:29:55,080 --> 00:29:58,000 be accounting for this result. 636 00:29:58,000 --> 00:29:59,530 AUDIENCE: Antibodies they used. 637 00:29:59,530 --> 00:30:00,530 JOANNE STUBBE: The what? 638 00:30:00,530 --> 00:30:01,988 AUDIENCE: They antibodies they used 639 00:30:01,988 --> 00:30:03,912 may not have the [INAUDIBLE]. 640 00:30:03,912 --> 00:30:07,166 So maybe we should [INAUDIBLE]. 641 00:30:07,166 --> 00:30:09,580 JOANNE STUBBE: OK, I mean, you know, 642 00:30:09,580 --> 00:30:11,980 you're going to use enough antibodies to be able-- you've 643 00:30:11,980 --> 00:30:12,920 go to check that out. 644 00:30:12,920 --> 00:30:14,420 So that would be another control you 645 00:30:14,420 --> 00:30:17,155 would need to do to make sure everything is under control. 646 00:30:20,362 --> 00:30:24,160 OK, so what we talked about last time in addition 647 00:30:24,160 --> 00:30:31,430 is hydrogen peroxide can oxidize this to these things as well. 648 00:30:31,430 --> 00:30:33,130 It's much slower. 649 00:30:33,130 --> 00:30:35,860 And so you don't know how would you look for this. 650 00:30:35,860 --> 00:30:39,814 So one explanation is that you have some competing rates-- 651 00:30:39,814 --> 00:30:41,480 again, we don't know anything about it-- 652 00:30:41,480 --> 00:30:45,910 but that you had changed it and that the dimedone no longer 653 00:30:45,910 --> 00:30:50,120 reacts with these forms based on the mechanism we described. 654 00:30:50,120 --> 00:30:51,820 So that's a possibility. 655 00:30:51,820 --> 00:30:56,080 And in fact, if you read the supplementary information, 656 00:30:56,080 --> 00:30:59,230 how might you distinguish between this and this? 657 00:31:04,230 --> 00:31:06,500 Say you thought that this was happening. 658 00:31:06,500 --> 00:31:09,740 I mean we're missing a lot of stuff. 659 00:31:09,740 --> 00:31:11,490 So there's a number of explanations. 660 00:31:11,490 --> 00:31:12,800 And that's just one of them. 661 00:31:16,034 --> 00:31:18,191 So what method are we using? 662 00:31:18,191 --> 00:31:19,024 AUDIENCE: Mass spec. 663 00:31:19,024 --> 00:31:21,600 JOANNE STUBBE: Yeah, how can you distinguish this from this? 664 00:31:21,600 --> 00:31:22,391 AUDIENCE: Per mass. 665 00:31:22,391 --> 00:31:24,030 JOANNE STUBBE: Yeah, by mass. 666 00:31:24,030 --> 00:31:26,550 And so if you look at that, you're 667 00:31:26,550 --> 00:31:30,840 going to have a different mass, right, on your little peptide 668 00:31:30,840 --> 00:31:31,340 species. 669 00:31:31,340 --> 00:31:32,673 You'll have the peptide species. 670 00:31:32,673 --> 00:31:34,470 And if you look at the supplementary data, 671 00:31:34,470 --> 00:31:38,370 they show mass in both of these states. 672 00:31:38,370 --> 00:31:42,990 OK, so that suggests that that might provide an explanation 673 00:31:42,990 --> 00:31:44,070 for what's going on. 674 00:31:46,590 --> 00:31:49,790 Maybe you could think of a lot of other explanations. 675 00:31:49,790 --> 00:31:51,220 Here I won't go through this. 676 00:31:51,220 --> 00:31:53,740 But they looked at mutants. 677 00:31:53,740 --> 00:31:56,440 And they wondered if all the cysteines 678 00:31:56,440 --> 00:32:00,820 could be modified, OK, because cysteines are reactive. 679 00:32:00,820 --> 00:32:04,420 Although, the ones in the active site often have lower pKa's. 680 00:32:04,420 --> 00:32:06,640 So this all-- there are thiolates. 681 00:32:06,640 --> 00:32:09,470 So you might expect the reaction to be faster. 682 00:32:09,470 --> 00:32:11,620 But again, the reaction, even with the thiolate, 683 00:32:11,620 --> 00:32:13,503 with hydrogen peroxide, is slow. 684 00:32:13,503 --> 00:32:17,660 OK, so that's not the whole story. 685 00:32:17,660 --> 00:32:22,750 And in fact, I think it was in the Nature Chemical Biology 686 00:32:22,750 --> 00:32:25,300 paper, there are some proteins they 687 00:32:25,300 --> 00:32:28,070 look at that all the cysteines can be modified. 688 00:32:28,070 --> 00:32:31,870 So this should start making you nervous in terms 689 00:32:31,870 --> 00:32:34,180 of how important sulfenylation is 690 00:32:34,180 --> 00:32:36,790 if it contributes a lot, if all of these things 691 00:32:36,790 --> 00:32:38,710 are heavily sulfenylated. 692 00:32:38,710 --> 00:32:41,680 And in this experiment, they made a few mutants, 693 00:32:41,680 --> 00:32:43,360 and then they repeat the experiments. 694 00:32:43,360 --> 00:32:47,330 And they see that they don't see any modification. 695 00:32:47,330 --> 00:32:52,150 So that suggests that the cysteine 36 is the one. 696 00:32:52,150 --> 00:32:55,510 In the case of in the test tube, glutathione peroxidase 697 00:32:55,510 --> 00:32:59,080 is the one that's interesting and is being modified. 698 00:32:59,080 --> 00:33:01,182 So it is selective. 699 00:33:01,182 --> 00:33:02,890 It doesn't say anything about the biology 700 00:33:02,890 --> 00:33:06,090 because they're not looking at the biology of GPx-3. 701 00:33:06,090 --> 00:33:10,680 And so then this is the method that they used. 702 00:33:10,680 --> 00:33:13,950 So once you identify that peptide, 703 00:33:13,950 --> 00:33:20,090 now you can have a second mass spectrometer 704 00:33:20,090 --> 00:33:25,120 and use a method to do collision-induced dissociation, 705 00:33:25,120 --> 00:33:28,030 which allows you to sequence the peptide. 706 00:33:28,030 --> 00:33:30,820 And now you ought to be able to-- it depends 707 00:33:30,820 --> 00:33:33,680 on the stability and the fragmentation patterns, 708 00:33:33,680 --> 00:33:38,290 but we know peptide bonds can fragment in a number of ways. 709 00:33:38,290 --> 00:33:40,870 And people have figured this out over the years, 710 00:33:40,870 --> 00:33:43,320 namely Klaus Biemann figured this out 711 00:33:43,320 --> 00:33:49,660 that we have B and Y. And so one reports 712 00:33:49,660 --> 00:33:51,370 on the C-terminus of the peptide, 713 00:33:51,370 --> 00:33:55,120 the other on the N-terminus of the peptide. 714 00:33:55,120 --> 00:33:57,340 And those are the fragmentation patterns that 715 00:33:57,340 --> 00:33:58,990 are observed most frequently. 716 00:33:58,990 --> 00:34:01,120 And lots of times, one side, you don't 717 00:34:01,120 --> 00:34:02,950 see both with equal intensity. 718 00:34:02,950 --> 00:34:04,680 They fly differently. 719 00:34:04,680 --> 00:34:07,820 But that allows you then to sequence, 720 00:34:07,820 --> 00:34:11,920 and that's what they did in these papers. 721 00:34:11,920 --> 00:34:14,230 And this just is an example of-- this 722 00:34:14,230 --> 00:34:17,650 isn't from glutathione peroxidase, 723 00:34:17,650 --> 00:34:19,300 but you can see you can now assign. 724 00:34:19,300 --> 00:34:20,674 If you wanted to go through this, 725 00:34:20,674 --> 00:34:22,150 you can walk through all of this. 726 00:34:22,150 --> 00:34:25,690 And you can see B2, B3, B4, et cetera, 727 00:34:25,690 --> 00:34:27,520 and you can see Y1, Y2, Y3. 728 00:34:27,520 --> 00:34:29,080 And the two of them should add up 729 00:34:29,080 --> 00:34:31,780 to give you your peptide fragment back. 730 00:34:31,780 --> 00:34:33,969 I mean once you get used to looking at these things 731 00:34:33,969 --> 00:34:36,980 and then understand the mechanism of fragmentation, 732 00:34:36,980 --> 00:34:39,239 which is what Biemann worked out, 733 00:34:39,239 --> 00:34:43,540 you then have a picture of sequencing by mass spec. 734 00:34:43,540 --> 00:34:45,880 And this is just another example that, again, this 735 00:34:45,880 --> 00:34:48,650 is not related to this particular problem. 736 00:34:48,650 --> 00:34:51,460 But you should see all these B fragments, all these Y 737 00:34:51,460 --> 00:34:52,060 fragments. 738 00:34:52,060 --> 00:34:56,350 And the two of them should sum to give you the total mass 739 00:34:56,350 --> 00:34:58,710 of whatever your peptide is. 740 00:34:58,710 --> 00:35:04,010 OK, so they then looked at glyceraldehyde 3-phosphate 741 00:35:04,010 --> 00:35:08,130 dehydrogenase is another control in the last paper. 742 00:35:08,130 --> 00:35:10,910 That's an enzyme in the glycolysis pathway that 743 00:35:10,910 --> 00:35:13,730 has a cysteine in the active site that plays 744 00:35:13,730 --> 00:35:15,950 an essential role in catalysis. 745 00:35:15,950 --> 00:35:18,770 And they asked the question does it get sulfenylated. 746 00:35:18,770 --> 00:35:22,130 So it's implicated in all kinds of regulatory mechanisms 747 00:35:22,130 --> 00:35:22,830 as well. 748 00:35:22,830 --> 00:35:24,290 And they did the same experiment. 749 00:35:24,290 --> 00:35:26,240 And what did they see here? 750 00:35:26,240 --> 00:35:28,550 So this is, again, the same ratio, 751 00:35:28,550 --> 00:35:32,060 deuterium over the sum of deuterium and protonated. 752 00:35:32,060 --> 00:35:34,180 And here they see one. 753 00:35:34,180 --> 00:35:38,560 OK, so this one, they are able to titrate stoichiometrically 754 00:35:38,560 --> 00:35:40,350 on this active site. 755 00:35:40,350 --> 00:35:41,810 So again, it's a question. 756 00:35:41,810 --> 00:35:44,750 What that's telling you, which is an issue in the end, 757 00:35:44,750 --> 00:35:47,560 is, you know, they all have different reactivities 758 00:35:47,560 --> 00:35:48,260 is what-- 759 00:35:48,260 --> 00:35:49,460 yeah? 760 00:35:49,460 --> 00:35:55,330 AUDIENCE: In the other slide, was there only one cysteine 761 00:35:55,330 --> 00:35:57,569 in the protein that they sent, like the fragment 762 00:35:57,569 --> 00:35:59,735 of the protein that they sent through the mass spec? 763 00:35:59,735 --> 00:36:01,610 JOANNE STUBBE: No, so they actually-- whoops. 764 00:36:05,155 --> 00:36:07,070 Where is it? 765 00:36:07,070 --> 00:36:08,860 Yeah, so this one-- 766 00:36:08,860 --> 00:36:12,170 no, so they were looking at-- 767 00:36:12,170 --> 00:36:15,120 so they mutated this so they now had 768 00:36:15,120 --> 00:36:17,550 two cysteines, the one in the active site, 769 00:36:17,550 --> 00:36:20,240 and they had another cysteine, cysteine 64. 770 00:36:20,240 --> 00:36:22,308 AUDIENCE: So was it still measured by-- 771 00:36:22,308 --> 00:36:24,575 you said it reacts initially with-- 772 00:36:24,575 --> 00:36:25,950 JOANNE STUBBE: Well, you react it 773 00:36:25,950 --> 00:36:28,920 with both dimedone-- you react it with hydrogen peroxide, 774 00:36:28,920 --> 00:36:30,930 and then you react it either with dimedone 775 00:36:30,930 --> 00:36:33,520 or with iodo-dimedone. 776 00:36:33,520 --> 00:36:36,630 And what they found is they saw no-- 777 00:36:36,630 --> 00:36:38,320 so they found fragments. 778 00:36:38,320 --> 00:36:41,490 OK, so the fragment that they were interested in, 779 00:36:41,490 --> 00:36:47,130 since that was the cysteine, is 64, OK, with 58 through 67. 780 00:36:47,130 --> 00:36:49,650 So that's the mass charge 639. 781 00:36:49,650 --> 00:36:52,040 So that's what they focused on. 782 00:36:52,040 --> 00:36:55,800 OK, and so then they asked the question do you see D6. 783 00:36:55,800 --> 00:37:00,030 OK, D6 would be indicative of sulfenylation, 784 00:37:00,030 --> 00:37:02,010 and they saw no sulfenylation. 785 00:37:02,010 --> 00:37:03,810 So they can get modification. 786 00:37:03,810 --> 00:37:06,400 And they pulled out that cysteine, 787 00:37:06,400 --> 00:37:08,520 but they get no sulfenylation. 788 00:37:08,520 --> 00:37:12,532 Only the 36 was sulfenylated. 789 00:37:12,532 --> 00:37:15,480 So they did a whole bunch of experiments like this. 790 00:37:15,480 --> 00:37:17,320 This is again just proof of concept, 791 00:37:17,320 --> 00:37:19,740 but I think this data, just comparing 792 00:37:19,740 --> 00:37:22,590 the two proteins they looked at, shows you 793 00:37:22,590 --> 00:37:24,540 that you have different reactivities, which 794 00:37:24,540 --> 00:37:28,410 I think is part of the issues with these labeling methods 795 00:37:28,410 --> 00:37:29,880 that you're trying to use in cells. 796 00:37:29,880 --> 00:37:30,180 Yeah? 797 00:37:30,180 --> 00:37:32,138 AUDIENCE: So in the method where they were just 798 00:37:32,138 --> 00:37:36,340 looking at the cysteine 36, they got 50% sulfenylation. 799 00:37:36,340 --> 00:37:38,843 JOANNE STUBBE: So 50% sulfenylation, right. 800 00:37:38,843 --> 00:37:40,980 AUDIENCE: Yeah, and there were no other cysteines 801 00:37:40,980 --> 00:37:42,112 in that fragment because-- 802 00:37:42,112 --> 00:37:44,570 JOANNE STUBBE: There were no other cysteines in that frame. 803 00:37:44,570 --> 00:37:46,800 So if you look at the sequence, you know, 804 00:37:46,800 --> 00:37:48,210 I mean this is a small protein. 805 00:37:48,210 --> 00:37:51,250 And so you know-- 806 00:37:51,250 --> 00:37:58,860 OK, so and now let's move into today or the precursor 807 00:37:58,860 --> 00:37:59,980 to today's paper. 808 00:37:59,980 --> 00:38:02,329 But this was the major focus. 809 00:38:02,329 --> 00:38:03,870 So what they wanted to be able to do, 810 00:38:03,870 --> 00:38:05,520 they had proof of concept. 811 00:38:05,520 --> 00:38:11,510 And now the question is how do we show that this can-- 812 00:38:11,510 --> 00:38:13,390 can we do this inside the cell? 813 00:38:13,390 --> 00:38:16,490 OK, so what are the issues inside the cell? 814 00:38:16,490 --> 00:38:17,690 I've labeled some here. 815 00:38:17,690 --> 00:38:21,210 But have you guys done any thinking about this? 816 00:38:21,210 --> 00:38:22,230 So we want this thing. 817 00:38:22,230 --> 00:38:24,780 What do we need of any reagent we're going to use? 818 00:38:24,780 --> 00:38:26,010 And what you see-- 819 00:38:26,010 --> 00:38:29,490 I think they've probably tried 20 or 30 reagents. 820 00:38:29,490 --> 00:38:32,530 I think these are the ones that came closest to working. 821 00:38:32,530 --> 00:38:34,890 But if you look at this, there was also-- 822 00:38:34,890 --> 00:38:36,922 it probably was in supplementary information. 823 00:38:36,922 --> 00:38:37,630 I don't remember. 824 00:38:40,770 --> 00:38:42,000 There it is maybe. 825 00:38:42,000 --> 00:38:47,070 Let me-- does anybody remember looking at a figure where 826 00:38:47,070 --> 00:38:48,180 they address this issue? 827 00:38:59,640 --> 00:39:01,354 Nobody remembers. 828 00:39:01,354 --> 00:39:02,270 AUDIENCE: Which issue? 829 00:39:02,270 --> 00:39:03,380 Yeah, which issue are you referring to? 830 00:39:03,380 --> 00:39:05,629 JOANNE STUBBE: Oh, the issue is whether you're getting 831 00:39:05,629 --> 00:39:07,570 sulfenylation inside the cell. 832 00:39:07,570 --> 00:39:11,830 Yeah, OK, and which reagent? 833 00:39:11,830 --> 00:39:13,600 So the first thing you want to ask 834 00:39:13,600 --> 00:39:15,250 are you getting sulfenylation. 835 00:39:15,250 --> 00:39:17,950 And which reagent works best? 836 00:39:17,950 --> 00:39:19,074 AUDIENCE: It's too deep. 837 00:39:19,074 --> 00:39:20,990 JOANNE STUBBE: OK, so it's definitely in here. 838 00:39:20,990 --> 00:39:22,960 I don't remember which one it is. 839 00:39:22,960 --> 00:39:24,445 AUDIENCE: They talked about it in the supplementary 840 00:39:24,445 --> 00:39:24,940 [INAUDIBLE]. 841 00:39:24,940 --> 00:39:25,814 JOANNE STUBBE: Is it? 842 00:39:25,814 --> 00:39:27,610 OK, so let see if it's 2D. 843 00:39:27,610 --> 00:39:28,950 So I have this on a slide. 844 00:39:28,950 --> 00:39:30,500 I can go forward. 845 00:39:30,500 --> 00:39:33,420 So the question is which one of these guys do you want to use. 846 00:39:33,420 --> 00:39:34,580 AUDIENCE: Alkyne. 847 00:39:34,580 --> 00:39:36,302 JOANNE STUBBE: OK, why? 848 00:39:36,302 --> 00:39:38,385 AUDIENCE: In one of their preliminary experiments, 849 00:39:38,385 --> 00:39:40,066 it worked the best. 850 00:39:40,066 --> 00:39:41,940 JOANNE STUBBE: OK, so this is the experiment. 851 00:39:41,940 --> 00:39:42,915 So what is this? 852 00:39:42,915 --> 00:39:44,250 This is the experiment. 853 00:39:44,250 --> 00:39:46,902 So somebody want to describe this experiment to me? 854 00:39:46,902 --> 00:39:48,360 So these are the kinds of questions 855 00:39:48,360 --> 00:39:49,590 they're asking in this paper. 856 00:39:49,590 --> 00:39:52,890 OK, so this is the same. 857 00:39:52,890 --> 00:39:54,510 This is the same in vitro. 858 00:39:54,510 --> 00:39:56,230 That's what we just did with the GPx. 859 00:39:56,230 --> 00:39:57,605 That's what we just went through. 860 00:39:57,605 --> 00:39:58,460 And what conclusion? 861 00:39:58,460 --> 00:40:00,000 You know it's really clean. 862 00:40:00,000 --> 00:40:03,230 And so you're looking at an antibody to dimedone. 863 00:40:03,230 --> 00:40:05,590 OK, that's what they're using. 864 00:40:05,590 --> 00:40:09,810 And so what conclusion can you draw from this with respect 865 00:40:09,810 --> 00:40:15,820 to these three systems, these three reagents? 866 00:40:15,820 --> 00:40:17,570 AUDIENCE: That they're comparing basically 867 00:40:17,570 --> 00:40:22,970 if you label with the N3 on the guy 868 00:40:22,970 --> 00:40:25,540 that you're trying to look at or with the alkyne. 869 00:40:25,540 --> 00:40:26,600 And in vitro, they-- 870 00:40:26,600 --> 00:40:30,090 JOANNE STUBBE: Right, not only N3, but two different N3's-- 871 00:40:30,090 --> 00:40:32,000 you know, what is the nature of the linker? 872 00:40:32,000 --> 00:40:33,920 OK, that's something you need to pay attention to. 873 00:40:33,920 --> 00:40:35,836 AUDIENCE: I think that's in the supplementary. 874 00:40:35,836 --> 00:40:37,332 This is just with one linker. 875 00:40:37,332 --> 00:40:39,290 Yeah, they didn't do this experiment with the-- 876 00:40:39,290 --> 00:40:42,380 JOANNE STUBBE: OK, so here they have DAZ2 and DYN. 877 00:40:42,380 --> 00:40:43,530 So here they have-- 878 00:40:43,530 --> 00:40:44,030 whoops. 879 00:40:44,030 --> 00:40:45,481 I don't know where DYN2 is. 880 00:40:45,481 --> 00:40:46,980 AUDIENCE: So that's with the alkyne. 881 00:40:46,980 --> 00:40:48,890 JOANNE STUBBE: OK, so that's in-- so they're 882 00:40:48,890 --> 00:40:49,700 the same linker? 883 00:40:49,700 --> 00:40:50,660 AUDIENCE: Yeah. 884 00:40:50,660 --> 00:40:51,770 JOANNE STUBBE: OK, because I don't remember that. 885 00:40:51,770 --> 00:40:53,490 So that's what they did here. 886 00:40:53,490 --> 00:40:54,630 And then they clicked it. 887 00:40:54,630 --> 00:40:56,000 OK, we were going to talk about that. 888 00:40:56,000 --> 00:40:57,000 We haven't gotten there. 889 00:40:57,000 --> 00:41:02,455 And what did they end up seeing in this particular reaction? 890 00:41:02,455 --> 00:41:03,570 AUDIENCE: [INAUDIBLE]. 891 00:41:03,570 --> 00:41:06,130 Yeah, like they really that equal in vitro. 892 00:41:06,130 --> 00:41:10,100 JOANNE STUBBE: So you see it here, hydrogen peroxide. 893 00:41:10,100 --> 00:41:11,380 So they sulfenylated. 894 00:41:11,380 --> 00:41:14,650 OK, so without sulfenylation, without hydrogen peroxide, 895 00:41:14,650 --> 00:41:15,740 they don't see anything. 896 00:41:15,740 --> 00:41:17,480 So that's good. 897 00:41:17,480 --> 00:41:19,840 And then here, they have hydrogen peroxide. 898 00:41:19,840 --> 00:41:21,180 So they sulfenylated. 899 00:41:21,180 --> 00:41:22,420 This is with-- 900 00:41:22,420 --> 00:41:23,130 AUDIENCE: Alkyne. 901 00:41:23,130 --> 00:41:24,296 JOANNE STUBBE: --the alkyne. 902 00:41:24,296 --> 00:41:26,134 And this is with-- 903 00:41:26,134 --> 00:41:26,800 AUDIENCE: Azide. 904 00:41:26,800 --> 00:41:27,925 JOANNE STUBBE: --the azide. 905 00:41:27,925 --> 00:41:29,680 So both of these is seeing something. 906 00:41:29,680 --> 00:41:32,920 OK, so now what they do over here 907 00:41:32,920 --> 00:41:36,300 is do the same experiment inside these cells. 908 00:41:36,300 --> 00:41:38,950 OK, and so inside the cells, what are you 909 00:41:38,950 --> 00:41:41,437 going to end up seeing when you look at this? 910 00:41:41,437 --> 00:41:43,270 AUDIENCE: More stuff that gets sulfenylated. 911 00:41:43,270 --> 00:41:44,436 JOANNE STUBBE: A mess, yeah. 912 00:41:44,436 --> 00:41:45,580 So you see a mess. 913 00:41:45,580 --> 00:41:48,910 But what do you see here? 914 00:41:48,910 --> 00:41:51,310 So again, they're doing the same kind of thing. 915 00:41:51,310 --> 00:41:54,010 We haven't talked about the reaction 916 00:41:54,010 --> 00:41:56,240 yet of how you pull these out. 917 00:41:56,240 --> 00:41:58,600 But which one is most heavily modified? 918 00:41:58,600 --> 00:42:04,231 This one, and this is the one where you're using-- 919 00:42:04,231 --> 00:42:06,000 AUDIENCE: Where you label with alkyne-- 920 00:42:06,000 --> 00:42:07,450 JOANNE STUBBE: --alkyne and click with-- 921 00:42:07,450 --> 00:42:07,690 AUDIENCE: --and azide. 922 00:42:07,690 --> 00:42:08,648 JOANNE STUBBE: --azide. 923 00:42:08,648 --> 00:42:11,230 So this is the one that-- is this the one they use? 924 00:42:11,230 --> 00:42:12,190 This is the one they should have used. 925 00:42:12,190 --> 00:42:13,065 AUDIENCE: [INAUDIBLE] 926 00:42:13,065 --> 00:42:16,492 JOANNE STUBBE: Yeah, so if you go back-- 927 00:42:16,492 --> 00:42:17,440 whoops. 928 00:42:17,440 --> 00:42:20,050 So if you go back over here, we're 929 00:42:20,050 --> 00:42:22,220 trying to decide which one we want to use. 930 00:42:22,220 --> 00:42:24,010 And so that's the kind of experiment 931 00:42:24,010 --> 00:42:25,720 they did to try to design. 932 00:42:25,720 --> 00:42:29,320 So what are you concerned about in this experiment? 933 00:42:29,320 --> 00:42:32,090 The first thing is what has to happen with this molecule. 934 00:42:32,090 --> 00:42:33,904 I needs to get in the cell. 935 00:42:33,904 --> 00:42:37,650 OK, so I guess they know it gets into the cell, 936 00:42:37,650 --> 00:42:41,430 but they didn't really do any experiments to determine 937 00:42:41,430 --> 00:42:43,041 how much got into the cell. 938 00:42:43,041 --> 00:42:44,790 And it could be one that could have gotten 939 00:42:44,790 --> 00:42:46,510 into the cell much more than the other, 940 00:42:46,510 --> 00:42:49,320 which would have given you the same result. Presumably, 941 00:42:49,320 --> 00:42:50,830 they looked at that. 942 00:42:50,830 --> 00:42:53,640 So it is cell permeable. 943 00:42:53,640 --> 00:42:57,275 The key thing, I think, is in the cellular milieu, 944 00:42:57,275 --> 00:42:59,780 you've got to be able to do all these reactions. 945 00:42:59,780 --> 00:43:04,710 OK, but what is the issue in the cellular milieu doing 946 00:43:04,710 --> 00:43:05,460 these reactions? 947 00:43:05,460 --> 00:43:09,710 How do you decide how to do the experiment? 948 00:43:09,710 --> 00:43:11,250 What do you have to worry about? 949 00:43:11,250 --> 00:43:11,940 AUDIENCE: You want to have-- 950 00:43:11,940 --> 00:43:13,460 I mean both of these are the case. 951 00:43:13,460 --> 00:43:14,580 You want to have some sort of chemistry 952 00:43:14,580 --> 00:43:16,860 that's orthogonal to all the processes in the cells. 953 00:43:16,860 --> 00:43:18,660 JOANNE STUBBE: So we want to do that. 954 00:43:18,660 --> 00:43:19,524 So that's important. 955 00:43:19,524 --> 00:43:21,690 But OK, so that's one thing you have to worry about. 956 00:43:21,690 --> 00:43:24,210 We think that they have that under control. 957 00:43:24,210 --> 00:43:26,775 What else do you need to worry about? 958 00:43:26,775 --> 00:43:29,300 AUDIENCE: That this doesn't alter the sulfenylation profile 959 00:43:29,300 --> 00:43:29,810 in any way. 960 00:43:29,810 --> 00:43:31,560 JOANNE STUBBE: Well, I mean you have to be 961 00:43:31,560 --> 00:43:33,660 able to sulfenylate first. 962 00:43:33,660 --> 00:43:40,770 OK, what governs sulfenylation if you look at these, 963 00:43:40,770 --> 00:43:42,210 if you look at these molecules? 964 00:43:44,820 --> 00:43:46,280 So you sulfenylate. 965 00:43:46,280 --> 00:43:47,970 Let's assume you can sulfenylate. 966 00:43:47,970 --> 00:43:52,400 We can't control that if you've got hydrogen peroxide in there, 967 00:43:52,400 --> 00:43:54,180 if you've generated hydrogen peroxide. 968 00:43:54,180 --> 00:43:56,750 So they've somehow got to do that. 969 00:43:56,750 --> 00:43:57,650 And you sulfenylate. 970 00:43:57,650 --> 00:43:58,650 Or maybe they don't. 971 00:43:58,650 --> 00:44:02,390 They do EGF stimulated reaction. 972 00:44:02,390 --> 00:44:03,890 Then what do you have to worry about 973 00:44:03,890 --> 00:44:05,489 with respect to these analogs? 974 00:44:05,489 --> 00:44:07,780 AUDIENCE: How well those react with the sulfenyl group. 975 00:44:07,780 --> 00:44:08,930 JOANNE STUBBE: Right, and so what 976 00:44:08,930 --> 00:44:10,055 do you have to think about? 977 00:44:10,055 --> 00:44:12,696 AUDIENCE: What the time scale is with the sulfenylation 978 00:44:12,696 --> 00:44:14,765 equilibrium versus like this reaction 979 00:44:14,765 --> 00:44:15,990 reacting with that species. 980 00:44:15,990 --> 00:44:17,870 JOANNE STUBBE: And so that's all bimolecular. 981 00:44:17,870 --> 00:44:21,290 And the rate-- and it's bimolecular meaning something 982 00:44:21,290 --> 00:44:23,000 might be there in nanomolar. 983 00:44:23,000 --> 00:44:25,280 Something might be there in micromolar. 984 00:44:25,280 --> 00:44:27,500 The rate of the reaction is automatically a factor 985 00:44:27,500 --> 00:44:29,360 of 1,000 fold difference. 986 00:44:29,360 --> 00:44:32,030 So how long do you let this go? 987 00:44:32,030 --> 00:44:35,620 If you let it sit for a very long period of time, 988 00:44:35,620 --> 00:44:37,970 sulfenylation, we're talking about the importance 989 00:44:37,970 --> 00:44:39,490 of reversibility. 990 00:44:39,490 --> 00:44:42,020 So are there redox systems? 991 00:44:42,020 --> 00:44:44,820 There are that can remove the sulfenyl group. 992 00:44:44,820 --> 00:44:48,140 So designing the experiment to be informative 993 00:44:48,140 --> 00:44:50,880 is, in my opinion, not trivial. 994 00:44:50,880 --> 00:44:52,657 So looking at the details is key. 995 00:44:52,657 --> 00:44:54,740 And you need to know a lot about the system, which 996 00:44:54,740 --> 00:44:57,110 I don't know anything about. 997 00:44:57,110 --> 00:44:59,190 And then, you know, this has going 998 00:44:59,190 --> 00:45:02,290 to be able to get into the active site. 999 00:45:02,290 --> 00:45:04,860 OK, so the active sites have got to be big enough. 1000 00:45:04,860 --> 00:45:08,225 We know it can do that in glutathione peroxidase and GAP 1001 00:45:08,225 --> 00:45:09,170 dehydrogenase. 1002 00:45:09,170 --> 00:45:11,430 We looked at that. 1003 00:45:11,430 --> 00:45:15,090 But this needs to be long enough so, 1004 00:45:15,090 --> 00:45:19,580 wherever the active site is, you can do some chemistry out here. 1005 00:45:19,580 --> 00:45:21,830 So all of these things are issues 1006 00:45:21,830 --> 00:45:24,320 that you have to deal with. 1007 00:45:24,320 --> 00:45:27,340 And then the thing we we were facing before, 1008 00:45:27,340 --> 00:45:30,380 if we only labeled with this, we have no way 1009 00:45:30,380 --> 00:45:32,090 to tell where the label is. 1010 00:45:32,090 --> 00:45:34,630 And so again, the idea is that you 1011 00:45:34,630 --> 00:45:37,820 use these things to click it to something 1012 00:45:37,820 --> 00:45:40,840 that's going to inform you where the label is. 1013 00:45:40,840 --> 00:45:41,770 OK, yeah? 1014 00:45:41,770 --> 00:45:44,670 AUDIENCE: Was it synthetically driven that the [INAUDIBLE] 1015 00:45:44,670 --> 00:45:46,855 linker they install at the like [INAUDIBLE] position 1016 00:45:46,855 --> 00:45:48,410 of the reactive site versus-- 1017 00:45:48,410 --> 00:45:51,240 JOANNE STUBBE: My guess is yes, but I didn't read the papers 1018 00:45:51,240 --> 00:45:52,490 carefully enough to know that. 1019 00:45:52,490 --> 00:45:55,220 So in the paper that you were assigned, 1020 00:45:55,220 --> 00:45:57,050 they had a lot of synthetic chemistry. 1021 00:45:57,050 --> 00:46:01,364 So if you read the details, which is key, 1022 00:46:01,364 --> 00:46:02,780 I'm sure a lot of these things are 1023 00:46:02,780 --> 00:46:04,970 going to be driven by what's easiest to make. 1024 00:46:04,970 --> 00:46:08,600 You need to be able to make large amounts of it. 1025 00:46:08,600 --> 00:46:10,550 And these are now all commercially available. 1026 00:46:10,550 --> 00:46:12,502 OK, so this is-- 1027 00:46:12,502 --> 00:46:14,390 so yeah, I think it is. 1028 00:46:14,390 --> 00:46:16,422 But to me, you would do an experiment-- 1029 00:46:16,422 --> 00:46:17,380 AUDIENCE: To show that? 1030 00:46:17,380 --> 00:46:19,850 JOANNE STUBBE: --at different positions because one of them 1031 00:46:19,850 --> 00:46:21,350 might be much, much more efficient 1032 00:46:21,350 --> 00:46:23,390 at much lower concentrations. 1033 00:46:23,390 --> 00:46:24,980 And you might be much better off. 1034 00:46:24,980 --> 00:46:26,180 I don't know. 1035 00:46:26,180 --> 00:46:29,210 Or you certainly might want to use more than one reagent 1036 00:46:29,210 --> 00:46:33,440 because of the issues of trying to get this thing to react. 1037 00:46:33,440 --> 00:46:35,900 And then you have this question of, 1038 00:46:35,900 --> 00:46:39,230 even if you get these things, how do they react. 1039 00:46:39,230 --> 00:46:43,820 And most of you have probably heard about click chemistry 1040 00:46:43,820 --> 00:46:45,640 since it was invented by-- 1041 00:46:45,640 --> 00:46:49,760 it wasn't invented by Barry, but Barry popularized it. 1042 00:46:49,760 --> 00:46:54,530 And it's copper-catalyzed in most reactions. 1043 00:46:54,530 --> 00:46:56,540 But to do that, you do it in cell 1044 00:46:56,540 --> 00:47:00,100 extracts, OK, because copper is really toxic to cells. 1045 00:47:00,100 --> 00:47:05,280 So it's not useful for looking at this inside the cell. 1046 00:47:05,280 --> 00:47:11,780 And so the Bertozzi lab made a strained alkyne with a fluorine 1047 00:47:11,780 --> 00:47:15,350 on it-- actually, Jeremy did that who was an Alice Ting 1048 00:47:15,350 --> 00:47:17,220 undergraduate here-- 1049 00:47:17,220 --> 00:47:18,170 that makes it click. 1050 00:47:18,170 --> 00:47:20,960 But it's still not good in my opinion. 1051 00:47:20,960 --> 00:47:23,400 This still needs a lot of work. 1052 00:47:23,400 --> 00:47:28,050 And so I think the best methods actually 1053 00:47:28,050 --> 00:47:33,550 are the new methods that are coming out of this guy's lab 1054 00:47:33,550 --> 00:47:36,820 where he makes these tetrazine analogs. 1055 00:47:36,820 --> 00:47:39,380 And then he's made cyclopropenes. 1056 00:47:39,380 --> 00:47:43,460 And they react much, much faster under mild conditions. 1057 00:47:43,460 --> 00:47:47,830 And people are using these to put on fluorescent probes. 1058 00:47:47,830 --> 00:47:52,750 So anybody who is interested in that, you can read about-- 1059 00:47:52,750 --> 00:47:55,310 this guy, he's done a lot of creative science. 1060 00:47:55,310 --> 00:47:57,620 He's a young guy about Brad's age. 1061 00:47:57,620 --> 00:47:59,706 But I read all his papers because I 1062 00:47:59,706 --> 00:48:00,830 think there so interesting. 1063 00:48:00,830 --> 00:48:02,829 There aren't many young people that I do get to. 1064 00:48:02,829 --> 00:48:04,340 Anyhow, this guy is good. 1065 00:48:07,010 --> 00:48:11,790 OK, so the issue now is how did how does this happen. 1066 00:48:11,790 --> 00:48:13,430 And how do you do the analysis? 1067 00:48:13,430 --> 00:48:16,310 And so now that we're finished, none of you 1068 00:48:16,310 --> 00:48:17,902 have come to the board yet. 1069 00:48:17,902 --> 00:48:22,740 OK, all right, so the reagents used, 1070 00:48:22,740 --> 00:48:24,999 you need to think about the reagents used. 1071 00:48:24,999 --> 00:48:26,290 So I'm going back to the model. 1072 00:48:26,290 --> 00:48:28,500 I already gave you the model. 1073 00:48:28,500 --> 00:48:30,980 All right, so one of the reagents they used 1074 00:48:30,980 --> 00:48:31,820 is this guy-- 1075 00:48:34,508 --> 00:48:37,660 dihydrochlorofluoroescein. 1076 00:48:37,660 --> 00:48:39,236 What did they use that for? 1077 00:48:39,236 --> 00:48:40,940 AUDIENCE: [INAUDIBLE] 1078 00:48:40,940 --> 00:48:42,890 JOANNE STUBBE: For what? 1079 00:48:42,890 --> 00:48:45,465 AUDIENCE: For hydrogen peroxide detection. 1080 00:48:45,465 --> 00:48:47,771 JOANNE STUBBE: OK, but is it hydrogen peroxide? 1081 00:48:50,928 --> 00:48:52,680 Do you know how it works? 1082 00:48:52,680 --> 00:48:53,831 AUDIENCE: No. 1083 00:48:53,831 --> 00:48:56,080 JOANNE STUBBE: So to me, the first thing you should do 1084 00:48:56,080 --> 00:48:58,180 is you should have googled it. 1085 00:48:58,180 --> 00:49:00,511 And so it's dihydro. 1086 00:49:00,511 --> 00:49:03,137 So this is in the reduced state. 1087 00:49:03,137 --> 00:49:04,720 I mean, I could draw this structure up 1088 00:49:04,720 --> 00:49:07,360 on the blackboard for you, but the importance 1089 00:49:07,360 --> 00:49:09,726 is that it's in the reduced state. 1090 00:49:09,726 --> 00:49:13,092 And furthermore, what does the DA stand for? 1091 00:49:15,804 --> 00:49:17,460 So this is where the reagent-- 1092 00:49:17,460 --> 00:49:19,169 you know, to me, I didn't have a clue 1093 00:49:19,169 --> 00:49:21,210 either because I don't work on fluorescent probes 1094 00:49:21,210 --> 00:49:22,330 inside the cell. 1095 00:49:22,330 --> 00:49:24,274 So I immediately went and looked it up 1096 00:49:24,274 --> 00:49:25,690 because I didn't know what it was, 1097 00:49:25,690 --> 00:49:27,540 and I didn't know what I was looking at. 1098 00:49:27,540 --> 00:49:31,080 And so here was one whole figure with this reagent. 1099 00:49:31,080 --> 00:49:32,580 And so how could you understand what 1100 00:49:32,580 --> 00:49:36,270 the reagent was telling you if you didn't know what it was? 1101 00:49:36,270 --> 00:49:38,550 And so what it's got-- 1102 00:49:38,550 --> 00:49:40,140 I can't remember the whole structure. 1103 00:49:40,140 --> 00:49:41,400 You can google it. 1104 00:49:41,400 --> 00:49:44,270 But it's got a couple of hydroxyl groups. 1105 00:49:44,270 --> 00:49:50,050 And the hydroxyl groups are acetylated. 1106 00:49:50,050 --> 00:49:52,096 Why might they want to acetylate? 1107 00:49:54,892 --> 00:49:58,158 Why would they use the diacetate? 1108 00:49:58,158 --> 00:49:59,741 AUDIENCE: It's not fluorescent, right? 1109 00:49:59,741 --> 00:50:00,741 JOANNE STUBBE: It's not. 1110 00:50:00,741 --> 00:50:03,070 The reduced form is not fluorescent. 1111 00:50:09,310 --> 00:50:12,504 AUDIENCE: So in order for it to-- well, 1112 00:50:12,504 --> 00:50:13,920 in order for it to be fluorescent, 1113 00:50:13,920 --> 00:50:15,778 you're going to have to take off the acetates, which will happen 1114 00:50:15,778 --> 00:50:16,380 in the cell [INAUDIBLE]. 1115 00:50:16,380 --> 00:50:18,421 JOANNE STUBBE: Right, so the first thing happens. 1116 00:50:18,421 --> 00:50:22,720 Number one, but how do you get the reagent into the cell? 1117 00:50:22,720 --> 00:50:26,510 It turns out so it depends on the pKa of this hydroxyl. 1118 00:50:26,510 --> 00:50:29,410 And so the acetate groups facilitate uptake 1119 00:50:29,410 --> 00:50:30,490 into the cell. 1120 00:50:30,490 --> 00:50:34,330 Then it gets inside the cell, and the acetate groups 1121 00:50:34,330 --> 00:50:35,920 get hydrolyzed off. 1122 00:50:35,920 --> 00:50:38,060 It's in the reduced state. 1123 00:50:38,060 --> 00:50:39,949 And so this molecule in the reduced state-- 1124 00:50:39,949 --> 00:50:40,990 AUDIENCE: Is fluorescent. 1125 00:50:40,990 --> 00:50:42,910 JOANNE STUBBE: --reacts with, quote, 1126 00:50:42,910 --> 00:50:46,090 "reactive oxygen species," unquote, 1127 00:50:46,090 --> 00:50:48,310 to get into the oxidized state. 1128 00:50:48,310 --> 00:50:50,680 And that's what becomes fluorescent. 1129 00:50:50,680 --> 00:50:53,245 So it starts out in the reduced state. 1130 00:50:57,030 --> 00:51:04,360 And it has two acetate groups, sorry, two acetate groups. 1131 00:51:04,360 --> 00:51:09,830 OK, and so this gets into the cell. 1132 00:51:09,830 --> 00:51:11,150 And then you have-- 1133 00:51:11,150 --> 00:51:14,820 you still have the reduced state. 1134 00:51:14,820 --> 00:51:17,200 And now you have a hydroxyl. 1135 00:51:17,200 --> 00:51:20,910 And now this with reactive oxygen species-- 1136 00:51:20,910 --> 00:51:25,170 so this is still non-fluorescent. 1137 00:51:25,170 --> 00:51:27,530 And we're going to talk about fluorescence next time. 1138 00:51:27,530 --> 00:51:28,370 And then it gets in. 1139 00:51:28,370 --> 00:51:32,450 It gets oxidized by reactive oxygen species 1140 00:51:32,450 --> 00:51:34,250 and becomes fluorescent. 1141 00:51:34,250 --> 00:51:36,770 So that's the assay, but it doesn't just 1142 00:51:36,770 --> 00:51:38,570 react with hydrogen peroxide. 1143 00:51:38,570 --> 00:51:40,430 It can react with a lot of molecules. 1144 00:51:40,430 --> 00:51:43,310 And in fact, the Collins paper that I talked about in class 1145 00:51:43,310 --> 00:51:45,750 where I made some snide comment on it-- 1146 00:51:45,750 --> 00:51:48,440 anyhow, I mean the problem was, in the original paper, 1147 00:51:48,440 --> 00:51:50,240 people just used these things blindly 1148 00:51:50,240 --> 00:51:53,030 thinking they're reacting with specific molecules. 1149 00:51:53,030 --> 00:51:55,075 And in the last five years, there's 1150 00:51:55,075 --> 00:51:56,450 been a huge number of people that 1151 00:51:56,450 --> 00:51:59,210 have focused on making sensors specific 1152 00:51:59,210 --> 00:52:00,840 for each reactive species. 1153 00:52:00,840 --> 00:52:03,290 And that's really what you need to do 1154 00:52:03,290 --> 00:52:05,390 if you're going to make a sweeping generalization 1155 00:52:05,390 --> 00:52:06,770 about something like this. 1156 00:52:06,770 --> 00:52:10,470 So yeah, we're at the end of our time. 1157 00:52:10,470 --> 00:52:12,740 But what you should do is go back, 1158 00:52:12,740 --> 00:52:16,984 and there are a whole bunch of reagents in this paper 1159 00:52:16,984 --> 00:52:18,650 that, if you didn't know what they were, 1160 00:52:18,650 --> 00:52:20,910 there's no way you can understand the data. 1161 00:52:20,910 --> 00:52:23,026 OK, so the first thing you did-- or when 1162 00:52:23,026 --> 00:52:24,650 I looked at this, the first thing I did 1163 00:52:24,650 --> 00:52:26,450 is I made a list of these reagents 1164 00:52:26,450 --> 00:52:28,880 because I understand what was going on. 1165 00:52:28,880 --> 00:52:30,560 You know, like I never heard of-- 1166 00:52:30,560 --> 00:52:32,900 what is it-- apocynin. 1167 00:52:32,900 --> 00:52:36,050 That's a specific inhibitor of NOX2 isozymes. 1168 00:52:36,050 --> 00:52:38,270 So there are a bunch of different isozymes. 1169 00:52:38,270 --> 00:52:41,090 People are really interested in these therapeutically. 1170 00:52:41,090 --> 00:52:43,770 So people have developed specific inhibitors. 1171 00:52:43,770 --> 00:52:47,430 If you look at these guys, which they call them the wrong thing. 1172 00:52:47,430 --> 00:52:49,610 They have a longer name, but these 1173 00:52:49,610 --> 00:52:52,220 are covalent and irreversible inhibitors 1174 00:52:52,220 --> 00:52:55,550 of epidermal-derived growth factor receptor. 1175 00:52:55,550 --> 00:52:58,700 OK, so you need to know that to be 1176 00:52:58,700 --> 00:53:01,550 able to look at each one of these panels 1177 00:53:01,550 --> 00:53:04,880 to figure out what the data tells you. 1178 00:53:04,880 --> 00:53:08,280 OK, so now I would suggest you go-- you might see this again. 1179 00:53:08,280 --> 00:53:11,610 Maybe you'll see this on the final exam, some of this data. 1180 00:53:11,610 --> 00:53:14,664 Anyhow, you should go back, and you should look at the data. 1181 00:53:14,664 --> 00:53:16,830 This is what we've been trying to get you to do over 1182 00:53:16,830 --> 00:53:19,560 this course is, you know, it takes a lot of energy 1183 00:53:19,560 --> 00:53:21,182 to read a paper. 1184 00:53:21,182 --> 00:53:23,560 that's one of the take-home messages from the course. 1185 00:53:23,560 --> 00:53:26,070 OK, so we have finished.