1 00:00:00,500 --> 00:00:02,830 The following content is provided under a Creative 2 00:00:02,830 --> 00:00:04,370 Commons license. 3 00:00:04,370 --> 00:00:06,670 Your support will help MIT OpenCourseWare 4 00:00:06,670 --> 00:00:11,030 continue to offer high-quality educational resources for free. 5 00:00:11,030 --> 00:00:13,660 To make a donation or view additional materials 6 00:00:13,660 --> 00:00:17,610 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,610 --> 00:00:18,540 at ocw.mit.edu. 8 00:00:25,860 --> 00:00:29,160 JOANNE STUBBE: The last time, we were finishing the first part 9 00:00:29,160 --> 00:00:33,120 of the reactive oxygen species module, focused 10 00:00:33,120 --> 00:00:40,500 on how we as humans fight bacterial or viral infections 11 00:00:40,500 --> 00:00:44,190 using neutrophils, the white blood cells. 12 00:00:44,190 --> 00:00:47,670 And I introduced you to this cartoon. 13 00:00:47,670 --> 00:00:50,700 So here's our neutrophil, all of this blue stuff. 14 00:00:50,700 --> 00:00:52,590 It has an unusual-looking nucleus. 15 00:00:52,590 --> 00:00:55,830 Neutrophils have weird nuclei. 16 00:00:55,830 --> 00:00:59,910 They somehow sense the bacteria you saw, the bacteria getting 17 00:00:59,910 --> 00:01:02,790 chased by the neutrophil. 18 00:01:02,790 --> 00:01:07,050 And then somebody asked me this question. 19 00:01:07,050 --> 00:01:09,240 Somebody asked me this question last time 20 00:01:09,240 --> 00:01:14,840 about where the NOX2 proteins end up. 21 00:01:14,840 --> 00:01:16,980 And let me reiterate again that they can 22 00:01:16,980 --> 00:01:20,250 be found in multiple membranes. 23 00:01:20,250 --> 00:01:22,620 The predominant membrane in neutrophils 24 00:01:22,620 --> 00:01:26,010 is in these little vesicles within the cell, 25 00:01:26,010 --> 00:01:29,610 OK, but the bacteria out here, so they need 26 00:01:29,610 --> 00:01:31,740 to somehow engulf the bacteria. 27 00:01:31,740 --> 00:01:37,710 So they can also be found in the plasma membrane. 28 00:01:37,710 --> 00:01:41,760 And they need to engulf the bacteria to form phagosomes. 29 00:01:41,760 --> 00:01:43,380 And so here you see them again. 30 00:01:43,380 --> 00:01:50,460 So this is the phagosome with the NOX2 protein, 31 00:01:50,460 --> 00:01:56,050 with this predisposition where the NADPH is in the cytosol. 32 00:01:56,050 --> 00:01:56,550 OK? 33 00:01:56,550 --> 00:02:00,690 So you see that the location here, the NADPH 34 00:02:00,690 --> 00:02:03,390 is also in the cytosol. 35 00:02:03,390 --> 00:02:05,700 And so you need to think about where you're located. 36 00:02:05,700 --> 00:02:09,360 And we'll see in a few minutes that there 37 00:02:09,360 --> 00:02:11,388 are lots of different kinds of NOX proteins, 38 00:02:11,388 --> 00:02:12,930 and they all have different locations 39 00:02:12,930 --> 00:02:15,180 and all have different factors that 40 00:02:15,180 --> 00:02:18,840 control the regulation of all of these things. 41 00:02:18,840 --> 00:02:24,870 OK, so what I want to do today is start out by looking, 42 00:02:24,870 --> 00:02:27,570 talking about the NOX complex, what the chemistry is, 43 00:02:27,570 --> 00:02:31,020 because it's not only involved in killing bacteria, 44 00:02:31,020 --> 00:02:33,030 but we'll see-- and you've already seen in last 45 00:02:33,030 --> 00:02:34,380 week's recitation-- 46 00:02:34,380 --> 00:02:36,120 in the signaling process. 47 00:02:36,120 --> 00:02:38,070 It's the same protein. 48 00:02:38,070 --> 00:02:41,610 And so once the bacteria are engulfed, 49 00:02:41,610 --> 00:02:44,880 you now have a phagosome within the neutrophil. 50 00:02:44,880 --> 00:02:47,560 And here's the NOX complex. 51 00:02:47,560 --> 00:02:50,730 Remember, it's composed of two proteins, 52 00:02:50,730 --> 00:02:55,830 the 91-kilodalton glycoprotein and a 22-kilodalton protein, 53 00:02:55,830 --> 00:02:56,790 both membrane-bound. 54 00:02:56,790 --> 00:02:59,400 I drew those on the board last time. 55 00:02:59,400 --> 00:03:03,420 And the NADPH/NADP is in the cytosol. 56 00:03:03,420 --> 00:03:06,030 But all the chemistry is going to happen 57 00:03:06,030 --> 00:03:08,310 in the lumen of the phagosome. 58 00:03:08,310 --> 00:03:13,140 OK, so somehow the reducing equivalents from the NADPH 59 00:03:13,140 --> 00:03:19,080 need to be transferred into the lumen, where oxygen is going 60 00:03:19,080 --> 00:03:21,150 to be reduced to superoxide. 61 00:03:21,150 --> 00:03:24,630 So I want to talk a little bit about how that happens first, 62 00:03:24,630 --> 00:03:28,770 given what we know about the cofactors in NOX2 that 63 00:03:28,770 --> 00:03:30,840 are essential for this process. 64 00:03:30,840 --> 00:03:34,800 And then what we'll do is look a little bit at how 65 00:03:34,800 --> 00:03:37,800 the superoxide that's generated in the phagosome 66 00:03:37,800 --> 00:03:39,660 gets converted to hydrogen peroxide, 67 00:03:39,660 --> 00:03:42,000 and ultimately with another protein 68 00:03:42,000 --> 00:03:45,270 we're briefly going to discuss, a heme-dependent protein, 69 00:03:45,270 --> 00:03:49,710 myeloperoxidase, which uses chloride, 70 00:03:49,710 --> 00:03:51,300 can form hypochlorous acid. 71 00:03:51,300 --> 00:03:51,800 OK? 72 00:03:51,800 --> 00:03:53,910 So that's where we're going. 73 00:03:53,910 --> 00:03:56,460 This is a cartoon I drew on the board 74 00:03:56,460 --> 00:03:59,460 the last time, where you can see that we 75 00:03:59,460 --> 00:04:04,320 have a flavin domain, a flavin domain that's 76 00:04:04,320 --> 00:04:05,760 located in the cytosol. 77 00:04:05,760 --> 00:04:08,120 And then you have two hemes. 78 00:04:08,120 --> 00:04:13,680 And the unusual part about this system is that these hemes-- 79 00:04:13,680 --> 00:04:16,304 we've seen heme before with reversible binding of oxygen 80 00:04:16,304 --> 00:04:19,950 in hemoglobin-- they're both hexacoordinate. 81 00:04:19,950 --> 00:04:22,079 So there's no binding site for oxygen. 82 00:04:22,079 --> 00:04:26,040 So the chemistry is not happening by reversible binding 83 00:04:26,040 --> 00:04:28,900 of oxygen to the heme. 84 00:04:28,900 --> 00:04:32,580 In fact, you know, how close this is located to the surface, 85 00:04:32,580 --> 00:04:34,080 we don't have any structure of this, 86 00:04:34,080 --> 00:04:36,600 but somehow this reduction has to occur 87 00:04:36,600 --> 00:04:40,590 by an electron transfer process that 88 00:04:40,590 --> 00:04:44,670 probably occurs through the edge of the perforin system. 89 00:04:44,670 --> 00:04:46,590 So what you have here-- and I think 90 00:04:46,590 --> 00:04:48,240 this is an important teaching point, 91 00:04:48,240 --> 00:04:52,080 because I think there are only two ways inside the cell 92 00:04:52,080 --> 00:04:54,450 that you control all the redox balance. 93 00:04:54,450 --> 00:04:57,810 The redox balance within NADPH and flavins 94 00:04:57,810 --> 00:05:00,270 really play a central role in everything, 95 00:05:00,270 --> 00:05:03,360 so you really need to understand how these cofactors work, 96 00:05:03,360 --> 00:05:05,280 and how they're controlled. 97 00:05:05,280 --> 00:05:07,740 And so if you have a flavin-- 98 00:05:07,740 --> 00:05:10,890 and this is also written-- you don't have to write this down. 99 00:05:10,890 --> 00:05:12,450 This is written on the next handout, 100 00:05:12,450 --> 00:05:15,590 so you don't have to draw all this stuff out. 101 00:05:15,590 --> 00:05:22,800 OK, so I'm going to draw the business end of the flavin. 102 00:05:22,800 --> 00:05:27,580 OK, so this can be flavin adenine dinucleotide. 103 00:05:27,580 --> 00:05:30,490 So this is the oxidized state. 104 00:05:30,490 --> 00:05:33,850 And the two important places where redox chem-- 105 00:05:33,850 --> 00:05:35,500 redox chemistry, but look at flavin. 106 00:05:35,500 --> 00:05:37,833 If you don't know anything about heterocyclic chemistry, 107 00:05:37,833 --> 00:05:39,010 it's confusing. 108 00:05:39,010 --> 00:05:42,310 But in fact, we know a huge amount about flavin chemistry 109 00:05:42,310 --> 00:05:46,030 from studying model reactions in organic chemistry. 110 00:05:46,030 --> 00:05:49,150 Decades ago, Tom Bruce did that. 111 00:05:49,150 --> 00:05:54,000 And so the two places where the chemistry in general happen 112 00:05:54,000 --> 00:05:55,470 are either the inside-- 113 00:05:55,470 --> 00:05:59,680 so this is 1, 2, 3, 4. 114 00:05:59,680 --> 00:06:01,060 This is the N5-- 115 00:06:01,060 --> 00:06:04,693 or the C4A position, and this is the C4A position here. 116 00:06:04,693 --> 00:06:06,610 And I'm not going to go through this in detail 117 00:06:06,610 --> 00:06:09,490 but this is the oxidized form, and what 118 00:06:09,490 --> 00:06:12,880 we have is the reaction of this oxidized form with the reduced 119 00:06:12,880 --> 00:06:15,760 form of NADPH. 120 00:06:15,760 --> 00:06:19,510 And I'm not going to draw out the whole structure here 121 00:06:19,510 --> 00:06:21,310 either. 122 00:06:21,310 --> 00:06:27,820 But hopefully you all know now that NADPH and general 123 00:06:27,820 --> 00:06:30,880 works by hydride transfer, so it's almost always a two 124 00:06:30,880 --> 00:06:31,840 electron transfer. 125 00:06:31,840 --> 00:06:34,540 The one electron chemistry is really 126 00:06:34,540 --> 00:06:37,180 outside the realm where it would normally 127 00:06:37,180 --> 00:06:39,700 happen inside the cell. 128 00:06:39,700 --> 00:06:42,670 So basically the chemistry involves 129 00:06:42,670 --> 00:06:44,590 transfer of a hydride, a hydrogen 130 00:06:44,590 --> 00:06:48,910 with a pair of electrons to the N5 position. 131 00:06:48,910 --> 00:06:54,790 And so you go from the oxidized state to the reduced state. 132 00:06:54,790 --> 00:06:58,540 And so again I'm not going to draw out the whole flavin. 133 00:06:58,540 --> 00:06:59,380 The rest of the-- 134 00:06:59,380 --> 00:07:01,380 I guess I don't have a picture there but-- 135 00:07:01,380 --> 00:07:03,490 well, let me just show you where we're going. 136 00:07:03,490 --> 00:07:07,150 So the key is, which is interesting about this, 137 00:07:07,150 --> 00:07:09,140 we need to get across a membrane. 138 00:07:09,140 --> 00:07:10,680 So how do you get across a membrane? 139 00:07:10,680 --> 00:07:15,070 So the flavin domain is way over here, 140 00:07:15,070 --> 00:07:16,530 and we need to have two hemes. 141 00:07:16,530 --> 00:07:20,350 Remember you can do electron transfer over 10 to 15 axioms 142 00:07:20,350 --> 00:07:22,810 with very fast rate constants. 143 00:07:22,810 --> 00:07:27,100 Somehow these reducing equivalents from NADPH 144 00:07:27,100 --> 00:07:29,920 need to be transferred to the flavin. 145 00:07:29,920 --> 00:07:33,490 And the major function of the flavin inside the cell 146 00:07:33,490 --> 00:07:40,870 is to mediate two electron one electron chemistry. 147 00:07:40,870 --> 00:07:42,220 And here's an example that. 148 00:07:42,220 --> 00:07:45,400 The two-electron chemistry is being provided 149 00:07:45,400 --> 00:07:48,010 by the NADPH ass a hydride. 150 00:07:48,010 --> 00:07:53,740 But what we have to do is the hemes in this system are 151 00:07:53,740 --> 00:07:56,800 in the plus 3 oxidation state, so what 152 00:07:56,800 --> 00:07:59,800 we need to do is be able to convert-- ultimately 153 00:07:59,800 --> 00:08:02,920 we want to reduce oxygen to super oxides, 154 00:08:02,920 --> 00:08:04,810 so we need an electron. 155 00:08:04,810 --> 00:08:07,930 So that electron is coming from NADPH. 156 00:08:07,930 --> 00:08:11,750 So we need to have an electron transfer-- 157 00:08:11,750 --> 00:08:15,470 a single electron transfer to the heme because it only can-- 158 00:08:15,470 --> 00:08:19,040 iron can only be reduced by one electron. 159 00:08:19,040 --> 00:08:25,940 So we end up then with this system. 160 00:08:25,940 --> 00:08:34,179 So this is the reduced state of the flavin. 161 00:08:34,179 --> 00:08:37,179 And you can draw a resonance structure. 162 00:08:37,179 --> 00:08:40,510 This is deep-- you can draw all kinds of resonance structures 163 00:08:40,510 --> 00:08:41,179 with flavins. 164 00:08:41,179 --> 00:08:43,450 That's why I can they can do one-electron chemistry. 165 00:08:43,450 --> 00:08:47,200 So one-electron chemistry, you can make the one electron 166 00:08:47,200 --> 00:08:48,715 oxidized a reduced state depending 167 00:08:48,715 --> 00:08:50,090 on which state you're starting in 168 00:08:50,090 --> 00:08:51,760 and the electrons are delocalized. 169 00:08:51,760 --> 00:08:53,650 They turn out to be blue, or they turn out 170 00:08:53,650 --> 00:08:56,590 to be red depending on the prognation states. 171 00:08:56,590 --> 00:08:58,920 So what you can then do is-- 172 00:08:58,920 --> 00:09:00,850 let me just write that over here. 173 00:09:00,850 --> 00:09:02,950 So I'm just drawing a resonance structure of that 174 00:09:02,950 --> 00:09:04,150 so you can see-- 175 00:09:04,150 --> 00:09:06,840 let me not. 176 00:09:06,840 --> 00:09:10,850 And again let me just show you that that's there so you 177 00:09:10,850 --> 00:09:12,100 don't need to write that down. 178 00:09:12,100 --> 00:09:13,517 You don't need to write this down. 179 00:09:13,517 --> 00:09:14,380 It's all there. 180 00:09:14,380 --> 00:09:16,810 So just pay attention to me. 181 00:09:16,810 --> 00:09:19,960 So you're going to do an electron transfer to reduce 182 00:09:19,960 --> 00:09:21,910 an iron 3 to an iron 2. 183 00:09:21,910 --> 00:09:23,800 Then you've got another iron 3 because we got 184 00:09:23,800 --> 00:09:25,240 to get through this membrane. 185 00:09:25,240 --> 00:09:28,960 So that heme-- and again, this is where the redox potentials 186 00:09:28,960 --> 00:09:30,790 become critical-- 187 00:09:30,790 --> 00:09:35,860 can transfer an electron because now in the reduced state 188 00:09:35,860 --> 00:09:39,610 to the other heme so that becomes in the iron 2 state. 189 00:09:39,610 --> 00:09:41,920 So the one out here is in the iron 2 the state. 190 00:09:41,920 --> 00:09:45,730 Now it can transfer electron from oxygen 191 00:09:45,730 --> 00:09:47,560 to form super oxide. 192 00:09:47,560 --> 00:09:49,930 But at the same time during this process, 193 00:09:49,930 --> 00:09:52,180 we're transferring the two electrons 194 00:09:52,180 --> 00:09:57,190 that the Flavin received from the NADPH one at a time, 195 00:09:57,190 --> 00:09:59,350 so then we can repeat that process. 196 00:09:59,350 --> 00:10:01,450 And that's why you get the stochiometry 197 00:10:01,450 --> 00:10:02,500 of the overall reaction. 198 00:10:02,500 --> 00:10:06,260 You get two super oxides produced. 199 00:10:06,260 --> 00:10:11,300 So you have a resonance form of this. 200 00:10:11,300 --> 00:10:11,830 Let me see. 201 00:10:11,830 --> 00:10:13,247 I think I need to write over here. 202 00:10:13,247 --> 00:10:16,690 So anyhow you have a resonance form of this where you-- 203 00:10:25,640 --> 00:10:28,460 And now we're ready to do-- so this is the same as-- 204 00:10:28,460 --> 00:10:29,940 so this is a resonance form. 205 00:10:29,940 --> 00:10:31,250 These are the same structures. 206 00:10:37,060 --> 00:10:40,580 In the flavin, this is attached either to adenine 207 00:10:40,580 --> 00:10:45,140 or to a ribose biphosphate FMN versus FAD. 208 00:10:45,140 --> 00:10:47,795 And now what you're ready to do is you have the heme. 209 00:10:50,690 --> 00:10:53,730 And so the heme again is embedded in the membrane. 210 00:10:53,730 --> 00:10:57,350 And so now what you're doing is electron transfer. 211 00:10:57,350 --> 00:11:00,590 So you do an electron transfer reaction 212 00:11:00,590 --> 00:11:10,410 and you get this structure. 213 00:11:10,410 --> 00:11:12,525 So make a big dot for the radical. 214 00:11:20,380 --> 00:11:25,710 And now we've reduced one of the hemes to iron 2. 215 00:11:25,710 --> 00:11:27,460 And if you look at the redoxx potentials-- 216 00:11:27,460 --> 00:11:29,350 I haven't ever read these original papers-- 217 00:11:29,350 --> 00:11:32,530 but they're close to being matched 218 00:11:32,530 --> 00:11:34,290 in terms of redox potentials. 219 00:11:34,290 --> 00:11:36,955 I haven't read how they measured these kinds of things, 220 00:11:36,955 --> 00:11:38,830 but the system needs to be set up so that you 221 00:11:38,830 --> 00:11:41,800 can do transfer the electrons across the membrane 222 00:11:41,800 --> 00:11:46,760 and ultimately reduce the super oxide over here. 223 00:11:46,760 --> 00:11:49,520 And so now what happens so you've gotten to this stage. 224 00:11:49,520 --> 00:11:54,250 So now you have a semi quinone form of the flavin. 225 00:11:54,250 --> 00:11:59,860 this guy can then be re-oxidized by the next heme, 226 00:11:59,860 --> 00:12:04,930 generating the iron 2 form and regenerating the iron 3 form. 227 00:12:04,930 --> 00:12:07,330 So this guy then-- 228 00:12:07,330 --> 00:12:09,352 so let's-- to distinguish between them-- 229 00:12:09,352 --> 00:12:11,560 again I don't think this-- hopefully most of you were 230 00:12:11,560 --> 00:12:16,300 seeing this, but you have another one, 231 00:12:16,300 --> 00:12:21,220 so it donates an electron to covert the iron 3 form so we 232 00:12:21,220 --> 00:12:27,150 have an iron 3 form to the iron to form. 233 00:12:27,150 --> 00:12:33,340 And it itself becomes re-oxidized. 234 00:12:33,340 --> 00:12:35,060 And so now what's happening is you're 235 00:12:35,060 --> 00:12:38,840 set up to do another electron transfer where this is going 236 00:12:38,840 --> 00:12:43,430 to go to the iron 3, transfer it again, and in the end 237 00:12:43,430 --> 00:12:47,210 this guy is now in the lumen-- 238 00:12:47,210 --> 00:12:48,157 adjacent to the lumen. 239 00:12:48,157 --> 00:12:49,490 I don't know where it's located. 240 00:12:49,490 --> 00:12:51,950 We don't have a structure but this guy 241 00:12:51,950 --> 00:12:56,990 is probably through the hemage going to convert oxygen 242 00:12:56,990 --> 00:13:00,080 into super oxide. 243 00:13:00,080 --> 00:13:01,790 So is everybody following that? 244 00:13:01,790 --> 00:13:05,660 The main point here is you all hear about the flavin 245 00:13:05,660 --> 00:13:08,195 being the major mediator between one-electron chemistry 246 00:13:08,195 --> 00:13:09,320 and two-electron chemistry. 247 00:13:09,320 --> 00:13:12,710 Most of the time people don't draw out the details of this, 248 00:13:12,710 --> 00:13:16,090 but these things can all be observed spectroscopically 249 00:13:16,090 --> 00:13:17,090 because they're colored. 250 00:13:17,090 --> 00:13:17,590 Yeah. 251 00:13:17,590 --> 00:13:19,227 The second iron reduction happens 252 00:13:19,227 --> 00:13:20,420 from the semi [INAUDIBLE]? 253 00:13:20,420 --> 00:13:22,340 JOANNE STUBBE: The second iron-- yeah. 254 00:13:22,340 --> 00:13:23,400 Happens from the semi-- 255 00:13:23,400 --> 00:13:23,705 AUDIENCE: So you generate your process. 256 00:13:23,705 --> 00:13:24,630 JOANNE STUBBE: Right. 257 00:13:24,630 --> 00:13:25,130 Right. 258 00:13:25,130 --> 00:13:29,660 So you regenerate the oxidized form, so in the end over here, 259 00:13:29,660 --> 00:13:31,540 you go all the way through this. 260 00:13:31,540 --> 00:13:34,160 And again I haven't looked at the kinetics in the paper 261 00:13:34,160 --> 00:13:36,320 very carefully, but it very efficiently 262 00:13:36,320 --> 00:13:39,348 does this and shuttles the electron across. 263 00:13:39,348 --> 00:13:40,640 So you're doing the same thing. 264 00:13:40,640 --> 00:13:42,765 You're reorganizing the reduced form of the flavin, 265 00:13:42,765 --> 00:13:44,570 but you're doing it one electron at a time. 266 00:13:44,570 --> 00:13:49,250 So here's the key take home message. 267 00:13:49,250 --> 00:13:52,040 So all of this then happens in the phagosome. 268 00:13:55,545 --> 00:13:59,940 And so what you're generating then is superoxide, OK? 269 00:13:59,940 --> 00:14:01,770 Now what happens to superoxide? 270 00:14:01,770 --> 00:14:03,960 So superoxide could potentially do chemistry, 271 00:14:03,960 --> 00:14:06,030 but we talked about, last time, what are 272 00:14:06,030 --> 00:14:08,550 the properties of superoxide? 273 00:14:08,550 --> 00:14:12,075 It's not all that reactive, and frankly having 274 00:14:12,075 --> 00:14:13,950 read a lot of papers, I think we don't really 275 00:14:13,950 --> 00:14:18,120 understand all the details of how the bacteria die when 276 00:14:18,120 --> 00:14:21,410 they're engulfed by the phagosome-- 277 00:14:21,410 --> 00:14:26,460 but a key player in all in this overall process. 278 00:14:26,460 --> 00:14:28,680 And it's certainly not the only player, 279 00:14:28,680 --> 00:14:32,370 because you can actually wipe out my myloperoxidase, 280 00:14:32,370 --> 00:14:34,170 and you can still kill bacteria. 281 00:14:34,170 --> 00:14:36,420 So it's much more complicated than what 282 00:14:36,420 --> 00:14:39,420 I'm telling you, but a key player in when 283 00:14:39,420 --> 00:14:44,400 most people describe this is myloperoxidase, 284 00:14:44,400 --> 00:14:45,480 which is a heme protein. 285 00:14:45,480 --> 00:14:47,100 I'll show you that in a minute. 286 00:14:47,100 --> 00:14:50,310 But it turns out that these myloperoxidases 287 00:14:50,310 --> 00:14:53,700 exist in little granules. 288 00:14:53,700 --> 00:14:57,750 Just like you saw the little vesicle with the NOX2 289 00:14:57,750 --> 00:15:01,650 it, that was predominantly sitting inside the neutrophil, 290 00:15:01,650 --> 00:15:03,800 you also have little vesicles. 291 00:15:03,800 --> 00:15:07,320 And the vesicles are stuffed with myloperoxidase. 292 00:15:07,320 --> 00:15:11,400 And somehow there's a signal, and the myloperoxidase then 293 00:15:11,400 --> 00:15:13,080 fuses with the phagosome. 294 00:15:13,080 --> 00:15:18,310 So this is a phagosome, and you have a huge amount of protein 295 00:15:18,310 --> 00:15:18,810 in there. 296 00:15:18,810 --> 00:15:22,320 It gets dumped into the phagosome, 297 00:15:22,320 --> 00:15:23,900 so you have a heme protein. 298 00:15:23,900 --> 00:15:25,500 And that one's dumped in here. 299 00:15:25,500 --> 00:15:28,510 Inside the cell, you generated a gradient, 300 00:15:28,510 --> 00:15:33,810 so there's some complicated independent reactions. 301 00:15:33,810 --> 00:15:37,860 You need to sort of neutralize the pH, which happens. 302 00:15:37,860 --> 00:15:41,220 But once you get inside the cell of the myloperoxidase, 303 00:15:41,220 --> 00:15:44,310 the protonation state is such that you can rapidly 304 00:15:44,310 --> 00:15:47,580 protinate superoxide to hydrogen peroxide, 305 00:15:47,580 --> 00:15:49,800 and we'll see that hydrogen peroxide reacts 306 00:15:49,800 --> 00:15:55,180 with myloperoxidase, which then reacts with chloride 307 00:15:55,180 --> 00:15:57,110 which is also present. 308 00:15:57,110 --> 00:16:00,920 In the hypochlorus acid, we'll see is a key player, 309 00:16:00,920 --> 00:16:02,100 and how can you tell that? 310 00:16:02,100 --> 00:16:04,830 Because if you isolate the proteins 311 00:16:04,830 --> 00:16:08,460 that come out of the phagosome, they're all chlorinated. 312 00:16:08,460 --> 00:16:10,650 So you generate-- if you go back, 313 00:16:10,650 --> 00:16:14,520 and you look at the little sheet I showed you about reactivity-- 314 00:16:14,520 --> 00:16:17,430 hypochlorus acid is really reactive. 315 00:16:17,430 --> 00:16:19,980 It's reactive, very reactive kinetically 316 00:16:19,980 --> 00:16:22,350 and also thermodynamically. 317 00:16:22,350 --> 00:16:26,790 OK, so the myloperoxidase-- so once we 318 00:16:26,790 --> 00:16:28,950 get-- so we've gotten our superoxide, 319 00:16:28,950 --> 00:16:30,450 so now we're in the phagosome. 320 00:16:33,010 --> 00:16:37,200 And now we want to look at myloperoxidase, 321 00:16:37,200 --> 00:16:43,260 and so that catalyzes the reaction of hydrogen peroxide 322 00:16:43,260 --> 00:16:47,820 and chloride to form hypochlorus acid. 323 00:16:47,820 --> 00:16:53,430 And this is myloperoxidase, and it's a heme dependent protein. 324 00:16:56,070 --> 00:16:58,920 And so the question is how does this work? 325 00:16:58,920 --> 00:17:04,079 And so what do we know about myloperoxidase? 326 00:17:04,079 --> 00:17:06,050 People have been studying this for decades, 327 00:17:06,050 --> 00:17:08,280 and you're going to see the chemistry is actually 328 00:17:08,280 --> 00:17:09,240 quite complicated. 329 00:17:09,240 --> 00:17:11,670 It's very important, so people are always 330 00:17:11,670 --> 00:17:14,010 trying to figure out the details of the chemistry. 331 00:17:14,010 --> 00:17:16,980 But the devil is in the kinetics in the environment 332 00:17:16,980 --> 00:17:20,655 of the phagosome, so it's not so easy to sort all this out. 333 00:17:20,655 --> 00:17:22,530 But if you look at the structure, number one, 334 00:17:22,530 --> 00:17:23,760 you see this is-- 335 00:17:23,760 --> 00:17:25,440 this is from an X-ray structure. 336 00:17:25,440 --> 00:17:28,710 It's bent, so it has an unusual structure. 337 00:17:28,710 --> 00:17:32,220 It has an axial ligand that's a histidine, 338 00:17:32,220 --> 00:17:36,150 and there's no second axial ligand. 339 00:17:36,150 --> 00:17:39,120 And it's covalently bound in two places. 340 00:17:39,120 --> 00:17:41,147 There are parts in the heme that are hanging off 341 00:17:41,147 --> 00:17:43,230 the protoporphyrin IX, where it's covalently bound 342 00:17:43,230 --> 00:17:46,020 to the protein, and the covalent attachment's 343 00:17:46,020 --> 00:17:47,820 distinct from most of the heme. 344 00:17:47,820 --> 00:17:52,560 So that's all you need to know, in terms of what we're 345 00:17:52,560 --> 00:17:54,120 going to be talking about. 346 00:17:54,120 --> 00:17:57,990 So you have a heme protein, and most 347 00:17:57,990 --> 00:18:00,180 of the time I don't talk about the heme systems. 348 00:18:00,180 --> 00:18:01,470 But I think the heme systems-- 349 00:18:01,470 --> 00:18:03,660 you guys ought to know something about hemes. 350 00:18:03,660 --> 00:18:06,990 We've talked about hemes with reversible oxygen binding. 351 00:18:06,990 --> 00:18:10,140 You've seen hemes in cholesterol biosynthesis. 352 00:18:10,140 --> 00:18:15,420 In many of the natural products, biosynthetic pathways, 353 00:18:15,420 --> 00:18:17,520 you have hemes that do hydroxylation reactions 354 00:18:17,520 --> 00:18:19,350 or epoxidation reactions. 355 00:18:19,350 --> 00:18:24,010 Hemes play a central role in many reactions inside the cell. 356 00:18:24,010 --> 00:18:27,120 And this one, the general reaction, I think 357 00:18:27,120 --> 00:18:30,240 is pretty straightforward to understand. 358 00:18:30,240 --> 00:18:34,140 So what you have-- and so again this straight line 359 00:18:34,140 --> 00:18:39,240 is protoporphyrin IX, so I'll just write protoporphyrin IX. 360 00:18:39,240 --> 00:18:43,013 And again, it's ligated to histidine, 361 00:18:43,013 --> 00:18:44,305 so this is part of the protein. 362 00:18:47,310 --> 00:18:52,260 And there's no second axial ligand. 363 00:18:52,260 --> 00:18:58,190 You take the superoxide which rapidly disproportionates, 364 00:18:58,190 --> 00:19:02,000 so this can be rapidly disproportionated 365 00:19:02,000 --> 00:19:05,210 in the presence of protons to form hydrogen 366 00:19:05,210 --> 00:19:07,820 peroxide and oxygen gas. 367 00:19:07,820 --> 00:19:10,460 And we've already gone through that reaction. 368 00:19:10,460 --> 00:19:13,640 And so now what happens is the peroxide 369 00:19:13,640 --> 00:19:16,430 is going to bind to the heme, and so this 370 00:19:16,430 --> 00:19:17,930 is the key to the reaction. 371 00:19:17,930 --> 00:19:26,750 So you lose a proton, and the oxygen binds to the heme. 372 00:19:29,260 --> 00:19:31,910 And you generate that species. 373 00:19:31,910 --> 00:19:33,740 Now this species-- and again, this 374 00:19:33,740 --> 00:19:36,410 is dependent-- this is where it becomes distinct 375 00:19:36,410 --> 00:19:38,450 when trying to think about all the chemistry 376 00:19:38,450 --> 00:19:39,810 that hemes can do. 377 00:19:39,810 --> 00:19:42,640 You need to look at the two axial ligands, 378 00:19:42,640 --> 00:19:44,710 and what the environment is around the ligand. 379 00:19:44,710 --> 00:19:45,710 So that's another thing. 380 00:19:45,710 --> 00:19:48,740 I've tried to stress how important these ligands are-- 381 00:19:48,740 --> 00:19:53,160 and the second coordination sphere around the system. 382 00:19:53,160 --> 00:19:55,160 So what happens now, in this system, 383 00:19:55,160 --> 00:20:00,050 is in some way the enzyme catalyzes heterolytic cleavage 384 00:20:00,050 --> 00:20:06,140 of the oxygen oxygen bond, and forms what 385 00:20:06,140 --> 00:20:09,650 is formerly an iron IV species. 386 00:20:09,650 --> 00:20:14,480 But it's not an iron five species, 387 00:20:14,480 --> 00:20:17,240 so we've lost a molecule water. 388 00:20:17,240 --> 00:20:19,850 Somehow this leaves as water, so we 389 00:20:19,850 --> 00:20:22,130 have some groups in the active site that 390 00:20:22,130 --> 00:20:24,230 can facilitate that cleavage. 391 00:20:24,230 --> 00:20:32,570 And this is formerly an iron V species, 392 00:20:32,570 --> 00:20:35,810 so we're using electrons from the iron porphyrin system 393 00:20:35,810 --> 00:20:39,270 to facilitate cleavage of that bond. 394 00:20:39,270 --> 00:20:40,603 Now how do we know this? 395 00:20:40,603 --> 00:20:42,020 We know this because we know a lot 396 00:20:42,020 --> 00:20:47,600 about the spectroscopy of hemes and of the iron in the hemes, 397 00:20:47,600 --> 00:20:52,010 and we can actually look at all of these intermediates. 398 00:20:52,010 --> 00:20:53,510 And so what does this mean here? 399 00:20:53,510 --> 00:20:55,840 What happens is-- remember your porphyrin. 400 00:20:55,840 --> 00:20:58,340 Well, you can see the porphyrin, but you have this. 401 00:20:58,340 --> 00:21:01,160 You have all these pyrroles. 402 00:21:01,160 --> 00:21:04,280 And so iron V is a hot oxygen. Nobody's 403 00:21:04,280 --> 00:21:07,080 ever seen the iron V in any of these systems, 404 00:21:07,080 --> 00:21:08,930 so what you see-- 405 00:21:08,930 --> 00:21:14,270 spectroscopically, you see an iron IV and one electron 406 00:21:14,270 --> 00:21:15,770 oxidized porphyrin ring. 407 00:21:15,770 --> 00:21:17,210 OK, so that's what this is. 408 00:21:17,210 --> 00:21:21,560 This is a one electron oxidized porphyrin ring. 409 00:21:29,120 --> 00:21:31,010 So now what do you want to do? 410 00:21:31,010 --> 00:21:33,320 In the normal reaction, what you want to do-- 411 00:21:33,320 --> 00:21:38,300 the one that forms hypochlorus acid is a two-electron 412 00:21:38,300 --> 00:21:42,950 reaction, and the chloride can come in-- 413 00:21:42,950 --> 00:21:45,620 and you're going to form hypochlorus acid. 414 00:21:45,620 --> 00:21:48,590 So the chloride comes in and attacks, 415 00:21:48,590 --> 00:21:50,630 and one of the electrons goes back to the iron. 416 00:21:50,630 --> 00:21:52,460 And the other goes back to the porphyrin. 417 00:21:52,460 --> 00:21:54,890 So these one-headed arrows means you're 418 00:21:54,890 --> 00:21:59,930 doing one-electron transfers, and so what you've generated 419 00:21:59,930 --> 00:22:02,150 then is hypochlorus acid. 420 00:22:02,150 --> 00:22:05,840 The pKa of this is 7.4. 421 00:22:05,840 --> 00:22:10,220 And then you've generated back your iron III porphyrin, 422 00:22:10,220 --> 00:22:14,830 so you're ready to start again. 423 00:22:14,830 --> 00:22:17,140 So you're doing a two-electron process, 424 00:22:17,140 --> 00:22:21,010 and we know that the driving force for this reaction 425 00:22:21,010 --> 00:22:21,770 is large. 426 00:22:21,770 --> 00:22:27,630 It's 1.16 volts, so this is a very favorable reaction. 427 00:22:27,630 --> 00:22:31,560 Now if any of you have thought about heme-dependent systems-- 428 00:22:31,560 --> 00:22:35,550 before, if you have an iron oxo, this is a hot oxidant. 429 00:22:35,550 --> 00:22:37,070 It's dying to be reduced. 430 00:22:37,070 --> 00:22:39,270 It can be two-electron reduced, but it can also 431 00:22:39,270 --> 00:22:42,030 be one-electron reduced, depending 432 00:22:42,030 --> 00:22:45,480 on what small molecules are around here. 433 00:22:45,480 --> 00:22:49,170 And myloperoxidase does both kinds of chemistry. 434 00:22:49,170 --> 00:22:50,628 The predominant chemistry-- so this 435 00:22:50,628 --> 00:22:52,128 is what you need to look at the rate 436 00:22:52,128 --> 00:22:53,280 constants for the reaction. 437 00:22:53,280 --> 00:22:55,950 The predominant chemistry is thought to be this, 438 00:22:55,950 --> 00:22:57,690 but I will show you a slide where 439 00:22:57,690 --> 00:23:00,600 we know it can catalyze a lot of other chemistries 440 00:23:00,600 --> 00:23:03,180 by one-electron transfers, as well. 441 00:23:03,180 --> 00:23:06,800 And what's happening in the phagosome with the cell, 442 00:23:06,800 --> 00:23:10,500 if you want to look at that, the [INAUDIBLE] review article 443 00:23:10,500 --> 00:23:13,290 I gave you spent a lot of time thinking 444 00:23:13,290 --> 00:23:15,670 about these kinds of reactions. 445 00:23:15,670 --> 00:23:17,850 And sort of think it's beyond the scope of what 446 00:23:17,850 --> 00:23:19,500 we need to talk about. 447 00:23:19,500 --> 00:23:24,450 So here what we have happening-- so here now I'm just going to-- 448 00:23:24,450 --> 00:23:28,050 I'll tell you what R is in a minute, 449 00:23:28,050 --> 00:23:32,970 but what we're going to do is have one-electron transfer. 450 00:23:32,970 --> 00:23:36,660 And so instead of reducing this two electrons at a time, 451 00:23:36,660 --> 00:23:40,410 we're going to do two one-electron transfers, OK? 452 00:23:40,410 --> 00:23:41,970 So the system has to be set up. 453 00:23:41,970 --> 00:23:43,890 You have to have the right RH. 454 00:23:43,890 --> 00:23:47,100 You need to know what the redox potentials of these are-- 455 00:23:47,100 --> 00:23:49,410 determined by the ligands. 456 00:23:49,410 --> 00:23:52,230 All of that stuff, you need to think about. 457 00:23:52,230 --> 00:23:54,360 So now we're doing one-electron chemistry, 458 00:23:54,360 --> 00:23:56,910 so this is another possibility. 459 00:23:56,910 --> 00:24:01,590 And this again depends on how much chloride you have around. 460 00:24:01,590 --> 00:24:06,408 It's a potent oxidant, so it can be rapidly reduced, 461 00:24:06,408 --> 00:24:08,700 depending on whether you have an RH around that's going 462 00:24:08,700 --> 00:24:11,190 to actually do the reduction. 463 00:24:11,190 --> 00:24:14,310 And so what you generate then-- 464 00:24:14,310 --> 00:24:24,530 is you reduce the cation radical, and you form an R dot. 465 00:24:27,420 --> 00:24:29,400 And then the next step, which you can do, 466 00:24:29,400 --> 00:24:31,770 is a second one-electron reduction, 467 00:24:31,770 --> 00:24:35,070 so you're doing two one-electron reductions. 468 00:24:35,070 --> 00:24:37,560 And this driving force is not as large-- 469 00:24:37,560 --> 00:24:43,230 0.97 volts-- but again it's one-electron. 470 00:24:43,230 --> 00:24:45,930 And you're back then to iron III in water. 471 00:24:48,636 --> 00:24:51,910 I'm being sloppy about where the protons have come from. 472 00:24:54,430 --> 00:24:57,550 And you produced another R dot. 473 00:24:57,550 --> 00:25:00,130 So what could these R dots be? 474 00:25:00,130 --> 00:25:04,900 One of these R dots is ascorbate, vitamin C. So one 475 00:25:04,900 --> 00:25:07,760 of the RH's, which can-- 476 00:25:07,760 --> 00:25:09,100 the ascorbate can form radicals. 477 00:25:09,100 --> 00:25:11,410 I'm not going to go through this in any detail. 478 00:25:14,530 --> 00:25:18,130 Another RH could be tyrosines. 479 00:25:18,130 --> 00:25:21,680 So this is the amino acid tyrosine, 480 00:25:21,680 --> 00:25:24,530 and you form tyrosyl radicals. 481 00:25:24,530 --> 00:25:27,170 Has anybody ever seen that before in our department? 482 00:25:29,930 --> 00:25:32,300 Anybody seen use of this before? 483 00:25:32,300 --> 00:25:32,967 AUDIENCE: Apex? 484 00:25:32,967 --> 00:25:33,800 JOANNE STUBBE: Yeah. 485 00:25:33,800 --> 00:25:35,330 So, Apex. 486 00:25:35,330 --> 00:25:37,850 So this is the technology that is the basis. 487 00:25:37,850 --> 00:25:41,150 She doesn't use myloperoxidase, but King's Lab-- 488 00:25:41,150 --> 00:25:43,130 it uses a ascorbate peroxidase, which 489 00:25:43,130 --> 00:25:45,380 catalyzes a similar reaction. 490 00:25:45,380 --> 00:25:52,820 So your R dot then becomes a phenoxide radical. 491 00:25:52,820 --> 00:25:55,070 And that can then do for the chemistry. 492 00:25:55,070 --> 00:25:57,980 Anyhow, so what you're generating, 493 00:25:57,980 --> 00:26:02,120 most people believe the key bad player in all of this, 494 00:26:02,120 --> 00:26:04,880 but I'm just telling you it's more complicated than that, 495 00:26:04,880 --> 00:26:08,330 is the hypochlorus acid, which clearly gets formed, and can 496 00:26:08,330 --> 00:26:11,060 be evidenced by the chlorination reactions you 497 00:26:11,060 --> 00:26:12,690 see of all your proteins. 498 00:26:12,690 --> 00:26:17,670 So you can isolate chlorinated aromatics out of the phagosome. 499 00:26:17,670 --> 00:26:21,140 So if we go through here-- 500 00:26:21,140 --> 00:26:22,940 let me just give you this one first. 501 00:26:22,940 --> 00:26:26,070 So I've written this out for you in some detail. 502 00:26:26,070 --> 00:26:29,530 Again, it's two electrons, one electron, 503 00:26:29,530 --> 00:26:32,120 HOCl is two electrons. 504 00:26:32,120 --> 00:26:35,210 And that's thought to be the predominant species, 505 00:26:35,210 --> 00:26:38,290 but let me just tell you that-- 506 00:26:38,290 --> 00:26:40,730 so this is the one we're talking about in the handout. 507 00:26:40,730 --> 00:26:43,850 This is the major reaction, but you can 508 00:26:43,850 --> 00:26:45,595 do a lot of other reactions. 509 00:26:45,595 --> 00:26:48,220 And so what you need to look at to see if these are important-- 510 00:26:48,220 --> 00:26:51,740 just like with superoxide, you need to look at the kinetics 511 00:26:51,740 --> 00:26:55,130 of the reaction under the conditions where these 512 00:26:55,130 --> 00:26:57,230 molecules find themselves-- 513 00:26:57,230 --> 00:26:59,030 to figure out what's really going on, 514 00:26:59,030 --> 00:27:02,690 and how much these other pathways contribute. 515 00:27:02,690 --> 00:27:05,120 [INAUDIBLE] actually just-- there's 516 00:27:05,120 --> 00:27:07,890 a paper online in the annual reviews of biochemistry, where 517 00:27:07,890 --> 00:27:11,750 she talks about the neutrophils in a lot of detail, 518 00:27:11,750 --> 00:27:14,180 and the complexity of all this radical chemistry. 519 00:27:14,180 --> 00:27:16,970 So what I want you guys to take home from this 520 00:27:16,970 --> 00:27:22,820 is that we're working pretty hard with the NADPH oxidases, 521 00:27:22,820 --> 00:27:24,470 to engulf a bacteria. 522 00:27:24,470 --> 00:27:27,830 We're using reactive oxygen species, superoxide 523 00:27:27,830 --> 00:27:30,370 and hypochlorus acid to try to do 524 00:27:30,370 --> 00:27:35,600 in the bacteria inside the phagosome. 525 00:27:35,600 --> 00:27:40,630 So that's all I want to say about this section 526 00:27:40,630 --> 00:27:44,810 of reactive oxygen species, and now what I want to do 527 00:27:44,810 --> 00:27:47,060 is talk about something we've already talked about, 528 00:27:47,060 --> 00:27:49,280 because we've gone over this-- 529 00:27:49,280 --> 00:27:54,020 because we've gone over this in recitation. 530 00:27:54,020 --> 00:27:58,400 We spent a couple of recitations on the Carrol paper. 531 00:27:58,400 --> 00:28:00,950 And so I told you-- 532 00:28:00,950 --> 00:28:04,125 when I was introducing this, I gave you an outline 533 00:28:04,125 --> 00:28:05,000 of where we're going. 534 00:28:05,000 --> 00:28:09,830 We're going to reactive out of control versus controlled 535 00:28:09,830 --> 00:28:13,820 hydrogen peroxide superoxide production and signaling. 536 00:28:13,820 --> 00:28:16,400 So again, it's this thing all about-- it's 537 00:28:16,400 --> 00:28:18,170 all about homeostasis. 538 00:28:23,267 --> 00:28:24,850 Just need to get my act together here. 539 00:28:24,850 --> 00:28:26,860 So where are we going with this? 540 00:28:26,860 --> 00:28:29,140 This is the outline of where we're going to go, 541 00:28:29,140 --> 00:28:31,900 and so I'm not going to write the outline, because it 542 00:28:31,900 --> 00:28:32,860 will take me too long. 543 00:28:32,860 --> 00:28:34,540 And I really want to get-- you can read 544 00:28:34,540 --> 00:28:37,180 the outline on my PowerPoint. 545 00:28:37,180 --> 00:28:40,480 But what I want to do very briefly 546 00:28:40,480 --> 00:28:46,630 is give you an overview of how these reactive oxygen 547 00:28:46,630 --> 00:28:51,910 species are thought, in general, to play a role in signaling. 548 00:28:51,910 --> 00:28:53,560 A lot of people working on this-- 549 00:28:53,560 --> 00:28:55,750 there are many, many proteins involved. 550 00:28:55,750 --> 00:28:57,910 We're only looking at one of these proteins, 551 00:28:57,910 --> 00:29:01,630 the epidermal growth factor receptor, which we talked about 552 00:29:01,630 --> 00:29:03,400 in recitation. 553 00:29:03,400 --> 00:29:07,390 I also want to sort of give you an overview of the importance 554 00:29:07,390 --> 00:29:11,950 of cysteines in general in the proteome, 555 00:29:11,950 --> 00:29:15,250 and the role they play in this signaling process. 556 00:29:15,250 --> 00:29:15,890 We'll see. 557 00:29:15,890 --> 00:29:20,320 We are looking at one small modification, sulfenylation, 558 00:29:20,320 --> 00:29:24,310 but we'll see that there are many other modifications. 559 00:29:24,310 --> 00:29:26,560 And so I think one of the things for the future 560 00:29:26,560 --> 00:29:30,220 is figuring out, like the Carrol paper did, 561 00:29:30,220 --> 00:29:33,940 how biologically important are all these modifications 562 00:29:33,940 --> 00:29:37,750 that we can now identify because of the amazing power of Mass 563 00:29:37,750 --> 00:29:40,570 Spec and the creativity of chemists 564 00:29:40,570 --> 00:29:43,990 to figure out how to generate reagents. 565 00:29:43,990 --> 00:29:46,240 And so then I'll specifically introduce you 566 00:29:46,240 --> 00:29:51,790 very quickly to NOX and growth factors in NOX2-- 567 00:29:51,790 --> 00:29:54,130 the big family of NOX2. 568 00:29:54,130 --> 00:29:57,220 And then I'm going to talk about the general principles 569 00:29:57,220 --> 00:30:00,020 of signaling, what's required. 570 00:30:00,020 --> 00:30:02,710 And this is true of all signaling, not just with NOX, 571 00:30:02,710 --> 00:30:04,900 but I'll use NOX as an example. 572 00:30:04,900 --> 00:30:07,150 And then I will probably spend no time on this at all. 573 00:30:07,150 --> 00:30:09,370 The last part is how do we know all of this? 574 00:30:09,370 --> 00:30:11,500 We spent two recitations on this, 575 00:30:11,500 --> 00:30:14,190 so I'll tell you the key things I want you to remember. 576 00:30:14,190 --> 00:30:16,090 But you've now read papers. 577 00:30:16,090 --> 00:30:17,380 You've thought about this. 578 00:30:17,380 --> 00:30:19,672 And hopefully you can go back and think about it again, 579 00:30:19,672 --> 00:30:22,620 and it will all sort of now make more sense to you. 580 00:30:22,620 --> 00:30:27,330 OK, so where are we going? 581 00:30:27,330 --> 00:30:31,030 And so what I want you to see is sort of the big picture-- 582 00:30:31,030 --> 00:30:33,805 so again, the overview. 583 00:30:38,180 --> 00:30:42,800 And the overview now is not of the bad radicals 584 00:30:42,800 --> 00:30:45,050 that we're talking about, but they are still 585 00:30:45,050 --> 00:30:49,460 the bad radicals-- but controlling them in a way. 586 00:30:49,460 --> 00:30:51,433 So in the radical systems we're going 587 00:30:51,433 --> 00:30:52,850 to be looking at-- we're not going 588 00:30:52,850 --> 00:30:54,650 to be looking at all of them. 589 00:30:54,650 --> 00:30:57,600 But the signaling agents that we're going to be looking at-- 590 00:30:57,600 --> 00:31:01,398 so this is an overview of signaling. 591 00:31:01,398 --> 00:31:03,440 The signaling agents we're going to be looking at 592 00:31:03,440 --> 00:31:08,775 are superoxide, hydrogen peroxide, and NO. 593 00:31:08,775 --> 00:31:10,400 And we've already talked about the fact 594 00:31:10,400 --> 00:31:14,570 that we're not discussing NO, but in our department 595 00:31:14,570 --> 00:31:19,130 in biomedical engineering, Tannibaum's lab 596 00:31:19,130 --> 00:31:22,310 has been a major player in figuring out 597 00:31:22,310 --> 00:31:26,800 how to look at the modifications of cysteines by NO. 598 00:31:26,800 --> 00:31:27,710 It's not by NO. 599 00:31:27,710 --> 00:31:32,870 It's by a metabolite of NO that then reacts with the cysteines. 600 00:31:32,870 --> 00:31:35,210 So this is a very-- and he does that by Mass Spec, 601 00:31:35,210 --> 00:31:39,350 so this is a very active area of research. 602 00:31:39,350 --> 00:31:44,760 So now we're looking at signaling, not bad stuff. 603 00:31:44,760 --> 00:31:48,230 And one of the things that-- where have we seen this before? 604 00:31:48,230 --> 00:31:51,770 We've seen, although I don't think we realized it-- 605 00:31:51,770 --> 00:31:53,990 we were looking at iron homeostasis. 606 00:31:59,850 --> 00:32:02,350 This is what happens when you get up at 4:00 in the morning. 607 00:32:02,350 --> 00:32:04,670 OK, homeostasis. 608 00:32:04,670 --> 00:32:05,614 Homeostasis. 609 00:32:09,520 --> 00:32:12,670 And we have two proteins, the iron responsive binding protein 610 00:32:12,670 --> 00:32:13,840 one and two. 611 00:32:13,840 --> 00:32:17,020 And what do we know about iron responsive binding protein one? 612 00:32:17,020 --> 00:32:19,660 It has an iron sulfur cluster. 613 00:32:19,660 --> 00:32:22,300 And remember it has to go from the apostate 614 00:32:22,300 --> 00:32:27,370 to the iron cluster state, so we have IRP 1, 615 00:32:27,370 --> 00:32:33,980 and we go from the apo to the 4 iron 4 sulfur cluster. 616 00:32:33,980 --> 00:32:36,700 And it's believed, but it has not been very well studied, 617 00:32:36,700 --> 00:32:40,510 that this can be a sensor of oxidative stress, 618 00:32:40,510 --> 00:32:44,680 and so both NO and reactive oxygen 619 00:32:44,680 --> 00:32:49,990 species, such as hydrogen peroxide, 620 00:32:49,990 --> 00:32:53,950 can cause the metal center to be destroyed. 621 00:32:53,950 --> 00:32:56,420 And somehow you get to the apo state, 622 00:32:56,420 --> 00:32:59,770 and that's an active area of research. 623 00:32:59,770 --> 00:33:02,320 And then we know what the signals are. 624 00:33:02,320 --> 00:33:06,340 You have translational control by iron responsive elements 625 00:33:06,340 --> 00:33:08,710 at the five prime or the three prime end. 626 00:33:08,710 --> 00:33:11,020 The same thing happens with IRP 2. 627 00:33:11,020 --> 00:33:12,980 So I'm just trying to tie things together, 628 00:33:12,980 --> 00:33:18,070 but does anybody remember what the sensor was with IRP 2? 629 00:33:18,070 --> 00:33:20,364 Anybody remember what it did? 630 00:33:20,364 --> 00:33:21,690 AUDIENCE: Ubiquitin ligase? 631 00:33:21,690 --> 00:33:22,760 JOANNE STUBBE: The what? 632 00:33:22,760 --> 00:33:24,720 AUDIENCE: Is it ubiquitin ligase? 633 00:33:24,720 --> 00:33:27,180 JOANNE STUBBE: Yeah, so we had a little ubiquitin ligase 634 00:33:27,180 --> 00:33:30,310 remain with the sensor that binds in ways we still 635 00:33:30,310 --> 00:33:31,750 don't understand, so again this is 636 00:33:31,750 --> 00:33:34,990 something that's very much an active area of research. 637 00:33:34,990 --> 00:33:38,970 You have iron in oxygen-- 638 00:33:38,970 --> 00:33:44,900 sorry, LBX L5, leucine-5, domain of the ubiquitin ligase. 639 00:33:44,900 --> 00:33:47,290 I'm not going to draw all of that out. 640 00:33:47,290 --> 00:33:49,300 So again, these are all tied-- with what 641 00:33:49,300 --> 00:33:51,010 happens with iron and oxygen, it's 642 00:33:51,010 --> 00:33:57,370 all tied to these reactive oxygen species. 643 00:33:57,370 --> 00:33:59,260 So what we've already talked about-- so 644 00:33:59,260 --> 00:34:03,160 this is the major focus, our growth factors. 645 00:34:09,239 --> 00:34:11,909 And I'll show you using PowerPoints, 646 00:34:11,909 --> 00:34:17,659 but this kind of signal is signaling is also 647 00:34:17,659 --> 00:34:26,830 involved in cell proliferation and cell differentiation. 648 00:34:26,830 --> 00:34:35,117 So it's widely used in the example that we chose to use, 649 00:34:35,117 --> 00:34:36,659 because it was one of the ones that's 650 00:34:36,659 --> 00:34:42,679 been most carefully and recently characterized as EGF receptor. 651 00:34:42,679 --> 00:34:44,458 EGF receptor is also of great interest, 652 00:34:44,458 --> 00:34:45,375 because it's a target. 653 00:34:48,449 --> 00:34:52,139 It's the target for successful drugs used clinically 654 00:34:52,139 --> 00:34:54,570 in the treatment of cancer. 655 00:34:54,570 --> 00:34:56,159 So we then have another. 656 00:34:56,159 --> 00:35:02,580 So there are two other important signaling pathways that 657 00:35:02,580 --> 00:35:04,900 are proposed to be involved. 658 00:35:04,900 --> 00:35:09,950 One is called the antioxidant pathway, 659 00:35:09,950 --> 00:35:11,550 and there is a transcription factor. 660 00:35:11,550 --> 00:35:13,008 Some of you might have heard of it. 661 00:35:13,008 --> 00:35:15,290 Has anybody heard of NRF2? 662 00:35:15,290 --> 00:35:15,790 No. 663 00:35:15,790 --> 00:35:19,530 So if you're more biological or-- 664 00:35:19,530 --> 00:35:21,390 have you heard of the antioxidant? 665 00:35:21,390 --> 00:35:22,350 You're biological. 666 00:35:22,350 --> 00:35:23,478 Have you heard of NRF2? 667 00:35:23,478 --> 00:35:24,020 AUDIENCE: No. 668 00:35:24,020 --> 00:35:24,480 JOANNE STUBBE: No. 669 00:35:24,480 --> 00:35:25,080 OK. 670 00:35:25,080 --> 00:35:34,520 So anyhow, we have an antioxidant pathway, 671 00:35:34,520 --> 00:35:42,140 and NRF2 is a transcription factor. 672 00:35:42,140 --> 00:35:43,080 And it turns out-- 673 00:35:43,080 --> 00:35:45,080 I'm not going to go through the details of this, 674 00:35:45,080 --> 00:35:49,400 but for those of you who want to read about the details of this, 675 00:35:49,400 --> 00:35:52,970 this is a cell signaling review article published, where 676 00:35:52,970 --> 00:35:56,990 they go into all of the proposed mechanisms of how 677 00:35:56,990 --> 00:36:00,800 these reactive oxygen species connect to signaling. 678 00:36:00,800 --> 00:36:03,470 We're going to focus on epidermal growth factor 679 00:36:03,470 --> 00:36:05,360 receptor, but I think you need to know 680 00:36:05,360 --> 00:36:06,920 the picture is much bigger. 681 00:36:06,920 --> 00:36:12,410 It turns out that NRF2 is in a complex with an E3 ubiquitin 682 00:36:12,410 --> 00:36:15,710 ligase, and part of that ligase-- 683 00:36:15,710 --> 00:36:18,440 it's a multi enzyme complex-- 684 00:36:18,440 --> 00:36:19,370 is keap. 685 00:36:19,370 --> 00:36:22,340 Keap has a huge number of cysteines on it. 686 00:36:22,340 --> 00:36:26,690 These cysteines get oxidized by some kind of reactive oxygen 687 00:36:26,690 --> 00:36:27,320 species. 688 00:36:27,320 --> 00:36:31,910 In that one, you want to turn on your antioxidant defense. 689 00:36:31,910 --> 00:36:36,560 And so what happens when Keap cysteines get oxidized. 690 00:36:36,560 --> 00:36:41,660 It dissociates from NRF2, and NRF2 can go into the nucleus, 691 00:36:41,660 --> 00:36:44,745 and it turns on a whole bunch of genes. 692 00:36:44,745 --> 00:36:46,370 So I mean you're just getting the idea. 693 00:36:46,370 --> 00:36:48,810 I'm not going to go through any details, 694 00:36:48,810 --> 00:36:50,720 but it plays a central role. 695 00:36:50,720 --> 00:36:52,527 And this system, if you google it, 696 00:36:52,527 --> 00:36:54,860 you'll find there are hundreds of papers on this system. 697 00:36:54,860 --> 00:36:56,780 This is a very interesting system. 698 00:36:56,780 --> 00:36:59,390 I keep waiting for them to get to some stage 699 00:36:59,390 --> 00:37:02,030 where I can really talk about the biochemistry, 700 00:37:02,030 --> 00:37:05,240 but we aren't at that stage yet, in my opinion. 701 00:37:05,240 --> 00:37:09,605 And then the other thing is DNA damage and repair. 702 00:37:16,920 --> 00:37:19,230 And if you have DNA damage, or you're 703 00:37:19,230 --> 00:37:20,958 starting to get oxidative stress, 704 00:37:20,958 --> 00:37:22,500 and these things are out of control-- 705 00:37:22,500 --> 00:37:26,310 hydroxide radical is reacting with your nucleic acids-- 706 00:37:26,310 --> 00:37:27,760 you need to do something. 707 00:37:27,760 --> 00:37:30,060 So you turn on a signaling pathway, 708 00:37:30,060 --> 00:37:32,910 and this is controlled by a kinase. 709 00:37:32,910 --> 00:37:35,730 I'm not going to go into this in detail, but some of you 710 00:37:35,730 --> 00:37:37,110 might have heard of this. 711 00:37:37,110 --> 00:37:40,440 This is called the ATM pathway, and what you see-- actually, 712 00:37:40,440 --> 00:37:42,990 you'll see, I think, in the next five years if you remain 713 00:37:42,990 --> 00:37:44,100 biochemist-- 714 00:37:44,100 --> 00:37:47,020 all these acronyms for all these pathways, 715 00:37:47,020 --> 00:37:49,950 you're going to get it, because now we're seeing them 716 00:37:49,950 --> 00:37:51,510 over, and over, and over again. 717 00:37:51,510 --> 00:37:54,940 And at first, it was hard to see how this all fits together, 718 00:37:54,940 --> 00:37:56,040 but we're getting there. 719 00:37:56,040 --> 00:37:59,110 It's all fitting together, I think, in an interesting way. 720 00:37:59,110 --> 00:38:01,110 So all I want you to get out of this-- 721 00:38:01,110 --> 00:38:03,150 we're going to be talking about growth matters, 722 00:38:03,150 --> 00:38:05,730 but this is also a huge area, just like we just 723 00:38:05,730 --> 00:38:10,440 saw with oxidative stress trying to kill our bacteria. 724 00:38:10,440 --> 00:38:15,008 OK, so the next slide is one I took out of this article. 725 00:38:15,008 --> 00:38:16,800 I'm not going to go through this in detail, 726 00:38:16,800 --> 00:38:19,360 but we've already been through the iron 727 00:38:19,360 --> 00:38:21,100 responsive binding protein. 728 00:38:21,100 --> 00:38:23,100 So this is a summary of that, and we spent a lot 729 00:38:23,100 --> 00:38:24,900 of time talking about this. 730 00:38:24,900 --> 00:38:27,690 This is what we're going to talk about now-- 731 00:38:27,690 --> 00:38:32,350 the role of sulfenylation, in controlling kinase activity, 732 00:38:32,350 --> 00:38:33,960 and phosphatase activity. 733 00:38:33,960 --> 00:38:36,280 That's what we spent the last two recitation 734 00:38:36,280 --> 00:38:38,340 sections talking about. 735 00:38:38,340 --> 00:38:41,130 This is a generic approach to that, 736 00:38:41,130 --> 00:38:43,650 and I'm going to show you there are many, many growth 737 00:38:43,650 --> 00:38:48,090 factors that are thought to go through the same pathway. 738 00:38:48,090 --> 00:38:52,110 And so I just want you to remember that. 739 00:38:52,110 --> 00:38:56,100 So this is a big area, and so now the next thing 740 00:38:56,100 --> 00:38:58,320 is-- so there's another sort of overview picture 741 00:38:58,320 --> 00:38:59,580 I want you to get. 742 00:38:59,580 --> 00:39:01,350 So there's a second overview picture 743 00:39:01,350 --> 00:39:05,910 that I think is also important, and that cysteines really 744 00:39:05,910 --> 00:39:09,910 are playing a major role in all of these modifications. 745 00:39:09,910 --> 00:39:13,470 They are the easiest to oxidize, and so I 746 00:39:13,470 --> 00:39:21,430 think cysteine modifications are important. 747 00:39:27,180 --> 00:39:29,130 And there are many, many modifications. 748 00:39:29,130 --> 00:39:33,630 The question is, do they happen inside the cell? 749 00:39:33,630 --> 00:39:35,820 Do they happen inside the cell in a way 750 00:39:35,820 --> 00:39:40,935 that we can connect them to some interesting biology? 751 00:39:40,935 --> 00:39:42,060 And then what triggers off? 752 00:39:42,060 --> 00:39:45,780 Ultimately, what triggers off these modifications? 753 00:39:45,780 --> 00:39:51,210 So we've been talking about the kinases, 754 00:39:51,210 --> 00:39:55,430 and we've been talking about sulfenylation. 755 00:39:55,430 --> 00:39:58,070 That's what we spent two recitations on, 756 00:39:58,070 --> 00:40:00,360 so that's one important thing. 757 00:40:00,360 --> 00:40:02,465 But somehow, we're going to see that one 758 00:40:02,465 --> 00:40:03,840 of the important things that I've 759 00:40:03,840 --> 00:40:06,570 tried to stress in recitations was these reactions need 760 00:40:06,570 --> 00:40:12,300 to be reversible, so ultimately sulfenylation-- some way, 761 00:40:12,300 --> 00:40:13,200 come to this later. 762 00:40:13,200 --> 00:40:16,110 But it's going to be able to be converted back 763 00:40:16,110 --> 00:40:18,960 into the reduced state. 764 00:40:18,960 --> 00:40:25,230 We've seen that you can form sulfonic acids. 765 00:40:25,230 --> 00:40:28,860 This is also-- if you look at the Carrol paper carefully-- 766 00:40:28,860 --> 00:40:31,160 we didn't talk about this very much-- 767 00:40:31,160 --> 00:40:32,970 it's also reversible. 768 00:40:32,970 --> 00:40:34,290 There are sets of enzymes. 769 00:40:34,290 --> 00:40:36,570 Hydrogen peroxide can do the oxidation-- 770 00:40:36,570 --> 00:40:39,120 the back reaction that people have discovered an enzyme. 771 00:40:39,120 --> 00:40:41,610 They can do the back reaction. 772 00:40:41,610 --> 00:40:44,730 And then there's an irreversible step. 773 00:40:44,730 --> 00:40:48,960 So people don't know, but because it's irreversible 774 00:40:48,960 --> 00:40:53,850 this is likely not physiologically important. 775 00:41:02,010 --> 00:41:05,370 So in addition to these states, if you 776 00:41:05,370 --> 00:41:08,550 start reading the literature, or you read any literature now, 777 00:41:08,550 --> 00:41:10,620 we have glutathione that we've talked about. 778 00:41:10,620 --> 00:41:15,900 Glutathione is this tripeptide with glutamylcysteine glysine. 779 00:41:15,900 --> 00:41:20,910 It is able to convert the sulfenic acid 780 00:41:20,910 --> 00:41:25,300 into a glutathionlyated protein. 781 00:41:25,300 --> 00:41:27,720 We're seeing these all over the place. 782 00:41:27,720 --> 00:41:29,070 Is this the signaling pathway? 783 00:41:29,070 --> 00:41:29,970 How is it controlled? 784 00:41:29,970 --> 00:41:31,120 What's going on with these? 785 00:41:31,120 --> 00:41:33,030 I think we don't know the answer to that. 786 00:41:33,030 --> 00:41:40,590 So you can actually have glutathione react to give SSG. 787 00:41:43,410 --> 00:41:47,520 You can also have other kinds of proteins that I think-- 788 00:41:47,520 --> 00:41:49,110 so here they have RSH. 789 00:41:49,110 --> 00:41:51,390 This could be a thioredoxin protein, 790 00:41:51,390 --> 00:41:54,150 which if we get to deoxy nucleotide formation, 791 00:41:54,150 --> 00:41:55,860 there are hundreds of thioredoxins 792 00:41:55,860 --> 00:41:59,710 inside the cell that do the same kind of thing. 793 00:41:59,710 --> 00:42:03,450 And so one can also go from here, 794 00:42:03,450 --> 00:42:10,810 so you can have a little protein called thioredoxin, 795 00:42:10,810 --> 00:42:14,560 and it has two cysteines. 796 00:42:14,560 --> 00:42:19,840 And it can convert this back into the SH, 797 00:42:19,840 --> 00:42:23,770 and it itself can become oxidized. 798 00:42:23,770 --> 00:42:26,710 And there's a way of cycling the thioredoxin. 799 00:42:26,710 --> 00:42:29,440 So you're getting the idea, OK. 800 00:42:29,440 --> 00:42:31,480 Over here in this model, we're not 801 00:42:31,480 --> 00:42:33,970 going to talk about this, because I decided not to talk 802 00:42:33,970 --> 00:42:36,910 about reactive-- not on nitrogen species, 803 00:42:36,910 --> 00:42:47,230 but nitrous oxide can get converted into peroxynitrite. 804 00:42:47,230 --> 00:42:50,620 Peroxynitrite is able to actually catalyze 805 00:42:50,620 --> 00:42:52,780 formation of molecules like this, which is thought 806 00:42:52,780 --> 00:42:54,340 to be involved in signaling. 807 00:42:54,340 --> 00:42:59,560 They're also controlled reversibly by thioredoxins, 808 00:42:59,560 --> 00:43:03,820 and again this is what the Tenenbaum Lab studies. 809 00:43:03,820 --> 00:43:07,900 And these things can also cyclize 810 00:43:07,900 --> 00:43:10,460 to form these kinds of structures. 811 00:43:10,460 --> 00:43:12,702 So these are called sulfenamides. 812 00:43:12,702 --> 00:43:13,660 People have found them. 813 00:43:13,660 --> 00:43:15,160 They have X-ray structures of them. 814 00:43:15,160 --> 00:43:17,230 Are they important in signaling? 815 00:43:17,230 --> 00:43:21,462 I don't know, but you can see that you have many, many kinds 816 00:43:21,462 --> 00:43:22,170 of modifications. 817 00:43:22,170 --> 00:43:23,890 That's the takehome lesson from this, 818 00:43:23,890 --> 00:43:26,450 and then the big question is how important 819 00:43:26,450 --> 00:43:29,530 are these in terms of controlling homeostasis? 820 00:43:33,070 --> 00:43:37,516 So what I want to do now is briefly look at the players. 821 00:43:37,516 --> 00:43:38,905 I think I'm going to raise this. 822 00:43:38,905 --> 00:43:40,780 But briefly look at the players we've already 823 00:43:40,780 --> 00:43:43,540 started to look at, and make a few points, 824 00:43:43,540 --> 00:43:49,450 and make the general points about the signaling process, 825 00:43:49,450 --> 00:43:53,080 using epidermal growth factor receptor as an example. 826 00:43:57,293 --> 00:43:59,210 And so I'm hitting you over the head with this 827 00:43:59,210 --> 00:44:00,752 again, because we have already looked 828 00:44:00,752 --> 00:44:02,250 at this a couple of times. 829 00:44:02,250 --> 00:44:15,210 So we have an overview of EGFR and NOX. 830 00:44:15,210 --> 00:44:16,420 So we just looked at NOX. 831 00:44:16,420 --> 00:44:17,850 This is also NOX2. 832 00:44:17,850 --> 00:44:19,560 It's the same protein. 833 00:44:19,560 --> 00:44:21,480 They're found in different places. 834 00:44:21,480 --> 00:44:24,210 And so remember with NOX2 we had all these factors 835 00:44:24,210 --> 00:44:26,820 that I told you, if we're involved in the phagosome, 836 00:44:26,820 --> 00:44:31,260 you had a GTPase, you had phagosome oxidase. 837 00:44:31,260 --> 00:44:33,750 Now you have, in some cases, similar factors-- 838 00:44:33,750 --> 00:44:36,000 in other cases, additional factors-- 839 00:44:36,000 --> 00:44:38,340 that play a role in these multi enzyme complexes 840 00:44:38,340 --> 00:44:40,950 that allow it to do something else, OK? 841 00:44:40,950 --> 00:44:45,010 So nature reuses, over and over again, these different factors. 842 00:44:45,010 --> 00:44:50,550 So this is the cartoon picture you guys have seen before. 843 00:44:50,550 --> 00:44:52,020 We use this in recitation. 844 00:44:52,020 --> 00:44:58,980 This is where I started to have you think about recitation 11, 845 00:44:58,980 --> 00:45:01,500 when we started this, to try to introduce you to the system 846 00:45:01,500 --> 00:45:02,725 again. 847 00:45:02,725 --> 00:45:05,100 And so I just want to make a couple of points about this, 848 00:45:05,100 --> 00:45:09,120 but here's our epidermal growth factor receptor, which you all 849 00:45:09,120 --> 00:45:11,460 know now is a tyrosine kinase. 850 00:45:11,460 --> 00:45:16,690 Here is the NOX protein, and here you can see we have Rac1. 851 00:45:16,690 --> 00:45:19,560 If you go back, and you look at your notes from last time Rac1 852 00:45:19,560 --> 00:45:20,700 is a GTPase. 853 00:45:20,700 --> 00:45:24,150 You can control its activity with little proteins that 854 00:45:24,150 --> 00:45:26,300 can bind to it and inhibit it. 855 00:45:26,300 --> 00:45:28,050 And the funny thing about this, and people 856 00:45:28,050 --> 00:45:29,700 were asking me questions about this, 857 00:45:29,700 --> 00:45:33,240 is that the chemistry, the tyrosine kinase, 858 00:45:33,240 --> 00:45:37,900 is in the inside of the cell. 859 00:45:37,900 --> 00:45:44,420 NOX1, the NADPH-- and going to NADP is in the inside 860 00:45:44,420 --> 00:45:47,390 of the cell just like we just talked about-- 861 00:45:47,390 --> 00:45:50,540 but where is-- because of the predisposition 862 00:45:50,540 --> 00:45:56,120 of the flavin and the two hemes, where is superoxide produced? 863 00:45:56,120 --> 00:45:58,010 On the outside of the cell. 864 00:45:58,010 --> 00:45:59,480 That's rather bizarre. 865 00:45:59,480 --> 00:46:01,730 This is still rather bizarre to me. 866 00:46:01,730 --> 00:46:04,040 This is the model people have in the literature, 867 00:46:04,040 --> 00:46:09,200 but you're reducing equivalents from NADPH 868 00:46:09,200 --> 00:46:15,760 to convert oxygen to superoxide, which rapidly disproportionates 869 00:46:15,760 --> 00:46:17,660 to form hydrogen peroxide. 870 00:46:17,660 --> 00:46:19,430 And so then the question is we're 871 00:46:19,430 --> 00:46:22,580 saying hydrogen peroxide is the key signaler that's 872 00:46:22,580 --> 00:46:24,110 doing sulfenylation. 873 00:46:24,110 --> 00:46:26,450 How does it get into the cell? 874 00:46:26,450 --> 00:46:29,150 So the model then is it gets into the cell 875 00:46:29,150 --> 00:46:31,910 through an aquaporin. 876 00:46:31,910 --> 00:46:35,660 And is this aquaporin just moving around in the membrane, 877 00:46:35,660 --> 00:46:39,150 or is this some organization within the membrane? 878 00:46:39,150 --> 00:46:40,530 So this is going to be useful. 879 00:46:40,530 --> 00:46:41,990 We've already talked about the fact 880 00:46:41,990 --> 00:46:46,040 that hydrogen peroxide is not very reactive. 881 00:46:46,040 --> 00:46:50,000 So one way you can get something to be more reactive 882 00:46:50,000 --> 00:46:52,850 is by increasing its concentration. 883 00:46:52,850 --> 00:46:55,500 So nature does this all the time. 884 00:46:55,500 --> 00:46:59,300 So if you can somehow stick things together and generate 885 00:46:59,300 --> 00:47:02,630 something, and it's generated right adjacent to where you're 886 00:47:02,630 --> 00:47:06,380 going to react, it has a greater probability of reacting here 887 00:47:06,380 --> 00:47:07,220 than over here. 888 00:47:07,220 --> 00:47:09,500 And where have you seen that before? 889 00:47:09,500 --> 00:47:11,390 Any of you thought about that? 890 00:47:11,390 --> 00:47:13,776 Graduate students should know this. 891 00:47:13,776 --> 00:47:15,430 AUDIENCE: DNA templated synthesis? 892 00:47:15,430 --> 00:47:16,430 JOANNE STUBBE: DNA what? 893 00:47:16,430 --> 00:47:17,510 AUDIENCE: DNA templated synthesis? 894 00:47:17,510 --> 00:47:17,890 JOANNE STUBBE: No. 895 00:47:17,890 --> 00:47:18,420 So, yeah. 896 00:47:18,420 --> 00:47:18,920 No. 897 00:47:18,920 --> 00:47:22,220 So you do, but I mean in terms of all of these reactive oxygen 898 00:47:22,220 --> 00:47:23,980 species. 899 00:47:23,980 --> 00:47:26,000 The Ting Lab, that's what she does. 900 00:47:26,000 --> 00:47:28,760 She generates these things in the middle of the cell, 901 00:47:28,760 --> 00:47:31,350 and it's all dependent-- how long can this go? 902 00:47:31,350 --> 00:47:33,530 Remember we talked about this diffusion question. 903 00:47:33,530 --> 00:47:37,370 How far does it go before it actually reacts? 904 00:47:37,370 --> 00:47:38,810 So the idea, and the question you 905 00:47:38,810 --> 00:47:41,780 need to ask yourself is, if you generate this, 906 00:47:41,780 --> 00:47:43,410 are these organized? 907 00:47:43,410 --> 00:47:45,200 Do you remember from the recitation? 908 00:47:45,200 --> 00:47:46,370 Are these guys organized? 909 00:47:50,970 --> 00:47:53,231 What did we learn the last time in recitation? 910 00:47:56,460 --> 00:47:59,018 So we talked about-- we had this cartoon, 911 00:47:59,018 --> 00:48:00,060 and we talked about this. 912 00:48:00,060 --> 00:48:01,690 You looked at the data. 913 00:48:01,690 --> 00:48:03,090 What did the data tell you? 914 00:48:03,090 --> 00:48:05,380 Are these guys organized in some way, 915 00:48:05,380 --> 00:48:09,070 so that this hydrogen peroxide can actually 916 00:48:09,070 --> 00:48:12,393 do sulfenylation reactions? 917 00:48:12,393 --> 00:48:13,810 So what was the evidence for that? 918 00:48:13,810 --> 00:48:14,772 Does anybody remember? 919 00:48:14,772 --> 00:48:17,230 I mean, so if you don't remember this, you need to go back, 920 00:48:17,230 --> 00:48:20,410 and you need to read the paper again, OK? 921 00:48:20,410 --> 00:48:22,330 And I have all of this stuff on a PowerPoint, 922 00:48:22,330 --> 00:48:23,530 but I'm not going to go through it again. 923 00:48:23,530 --> 00:48:23,830 Yeah? 924 00:48:23,830 --> 00:48:25,247 AUDIENCE: There was colocalization 925 00:48:25,247 --> 00:48:26,320 between, like, the NOX. 926 00:48:26,320 --> 00:48:27,310 JOANNE STUBBE: Right. 927 00:48:27,310 --> 00:48:31,790 So there was colocalization between the NOX2 928 00:48:31,790 --> 00:48:34,730 in the growth factor, epidermal growth factor. 929 00:48:34,730 --> 00:48:38,890 And what else was there colocalization from? 930 00:48:38,890 --> 00:48:41,950 It's not shown here, but there was also colocalization 931 00:48:41,950 --> 00:48:44,533 of a phosphatase-- which that's not shown in here, 932 00:48:44,533 --> 00:48:46,450 but it's going to be shown in the next slide-- 933 00:48:46,450 --> 00:48:49,930 plays a key role in controlling the phosphorylation state. 934 00:48:49,930 --> 00:48:51,968 OK, so this idea of-- 935 00:48:51,968 --> 00:48:54,010 I'm going to write this down because I think this 936 00:48:54,010 --> 00:48:55,840 is a central idea in biology-- 937 00:48:55,840 --> 00:48:59,410 is how you localize things to make them more reactive. 938 00:48:59,410 --> 00:49:01,690 Whether this makes it reactive enough-- 939 00:49:01,690 --> 00:49:04,000 it does make it reactive enough, because we can clearly 940 00:49:04,000 --> 00:49:07,300 sulfenylate, but are we missing something on top of it, 941 00:49:07,300 --> 00:49:11,830 to make it reactive enough to be able to do what we need to do? 942 00:49:11,830 --> 00:49:18,130 So what we see with this system is EGF, the Growth Factor, 943 00:49:18,130 --> 00:49:26,400 causes EGF dimerization. 944 00:49:29,552 --> 00:49:31,760 That's what I've shown you in the cartoon over there. 945 00:49:31,760 --> 00:49:33,430 I'm not going to draw out the cartoon, 946 00:49:33,430 --> 00:49:36,910 because you've seen this cartoon a bunch of times. 947 00:49:36,910 --> 00:49:39,060 And what does that do? 948 00:49:39,060 --> 00:49:50,820 The tyrosine kinase activates itself by phosphorylation, 949 00:49:50,820 --> 00:49:52,420 and we're going to come back to this. 950 00:49:52,420 --> 00:49:55,780 So one way again, everybody has seen phosphorylations. 951 00:49:55,780 --> 00:49:59,620 Whether they activate or inactivate, you need to study. 952 00:49:59,620 --> 00:50:02,110 So here's the tyrosine kinase domain. 953 00:50:02,110 --> 00:50:05,200 When they come together, it has activity 954 00:50:05,200 --> 00:50:06,770 that it can phosphorylate itself, 955 00:50:06,770 --> 00:50:09,310 so you get into this form which then 956 00:50:09,310 --> 00:50:11,335 triggers signaling cascades. 957 00:50:15,820 --> 00:50:19,480 So the other thing we need to think about in this paper 958 00:50:19,480 --> 00:50:23,110 is-- so here we are going from the tyrosine 959 00:50:23,110 --> 00:50:24,640 to the phosphorylated tyrosine. 960 00:50:24,640 --> 00:50:27,910 You should write down that this is active. 961 00:50:27,910 --> 00:50:30,580 How do you activate a phosphorylated tyrosine 962 00:50:30,580 --> 00:50:31,990 if this is active? 963 00:50:31,990 --> 00:50:34,270 You use a phosphatase. 964 00:50:34,270 --> 00:50:37,820 So this, also, we saw in this paper. 965 00:50:37,820 --> 00:50:40,900 And in this paper, this Carrol paper, she identified, 966 00:50:40,900 --> 00:50:42,460 or she claims to have identified-- 967 00:50:42,460 --> 00:50:44,710 you can make your own judgment on that now-- 968 00:50:44,710 --> 00:50:47,920 the phosphatase, in the cell type that she looked at, 969 00:50:47,920 --> 00:50:53,020 that played a key role in the tyrosine kinase activity. 970 00:50:53,020 --> 00:50:55,330 So you're converting it from an active form 971 00:50:55,330 --> 00:50:56,890 to an inactive form, which is what 972 00:50:56,890 --> 00:51:01,138 you see by phosphorylation, dephosphorylation all the time. 973 00:51:01,138 --> 00:51:02,930 I'm going to come back to this in a minute. 974 00:51:02,930 --> 00:51:06,350 So here's a protein tyrosine phosphatase. 975 00:51:06,350 --> 00:51:12,165 So we have a protein tyrosine phosphatase. 976 00:51:22,160 --> 00:51:24,380 And so while I didn't write that out, these ends, 977 00:51:24,380 --> 00:51:26,840 there are lots of different kinds of phosphatases. 978 00:51:26,840 --> 00:51:33,990 But they have a thiolate in their active site, 979 00:51:33,990 --> 00:51:46,530 and these things is attached to a tyrosine on a protein. 980 00:51:46,530 --> 00:51:51,210 So here's our tyrosine kinase. 981 00:51:51,210 --> 00:51:54,660 So what happens again is you use covalent catalysis 982 00:51:54,660 --> 00:51:56,640 in two steps. 983 00:51:56,640 --> 00:52:01,710 So you phosphorylate, and then you hydrolyze. 984 00:52:01,710 --> 00:52:08,090 So you phosphorylate, and then you hydrolyze. 985 00:52:08,090 --> 00:52:17,840 So you end up then with your tyrosine and the kinase, 986 00:52:17,840 --> 00:52:20,780 and you end up back with ES minus. 987 00:52:23,330 --> 00:52:25,310 So again, there are many different kinds 988 00:52:25,310 --> 00:52:28,400 of phosphatases, but all the ones involved apparently 989 00:52:28,400 --> 00:52:30,680 in these signaling processes-- if you go back and look 990 00:52:30,680 --> 00:52:32,900 at the Carrol paper-- 991 00:52:32,900 --> 00:52:34,820 all have cysteines in their active site. 992 00:52:34,820 --> 00:52:37,520 You've seen this, covalent catalysis with cysteine, 993 00:52:37,520 --> 00:52:41,420 over, and over, and over again now at this stage. 994 00:52:41,420 --> 00:52:44,150 So this is going to be a key control. 995 00:52:44,150 --> 00:52:49,970 This is the active state, so this form is the active state. 996 00:52:49,970 --> 00:52:55,730 And over here-- sorry. 997 00:52:55,730 --> 00:52:58,400 So if you take this now, and you treat 998 00:52:58,400 --> 00:53:03,680 this with hydrogen peroxide, this becomes sulfenylated, 999 00:53:03,680 --> 00:53:05,480 and this becomes the inactive state. 1000 00:53:11,330 --> 00:53:16,610 So sulfenylation, just like with the tyrosine kinase 1001 00:53:16,610 --> 00:53:19,400 that we talked about in recitation, 1002 00:53:19,400 --> 00:53:20,980 can become activated. 1003 00:53:20,980 --> 00:53:24,170 The sulfenylation in this case becomes inactivated, 1004 00:53:24,170 --> 00:53:26,030 so that's what it's all about in these post 1005 00:53:26,030 --> 00:53:28,520 translational modifications. 1006 00:53:28,520 --> 00:53:31,880 And the question is-- 1007 00:53:31,880 --> 00:53:33,330 I'm sorry, I'm over again. 1008 00:53:33,330 --> 00:53:36,080 But the question is are these models correct? 1009 00:53:36,080 --> 00:53:38,840 So what we'll do next time is spend a little bit 1010 00:53:38,840 --> 00:53:43,130 of time talking about the six general principles of post 1011 00:53:43,130 --> 00:53:44,840 translational modification, in general, 1012 00:53:44,840 --> 00:53:47,850 and what the expectations are using what you've already-- 1013 00:53:47,850 --> 00:53:50,720 which we've already seen in recitations 11 and 12. 1014 00:53:50,720 --> 00:53:53,780 And then we're going to move on to the last module 1015 00:53:53,780 --> 00:53:57,970 on nucleotide metabolism.