1 00:00:00,500 --> 00:00:02,820 The following content is provided under a Creative 2 00:00:02,820 --> 00:00:04,360 Commons license. 3 00:00:04,360 --> 00:00:06,660 Your support will help MIT OpenCourseWare 4 00:00:06,660 --> 00:00:11,020 continue to offer high quality educational resources for free. 5 00:00:11,020 --> 00:00:13,650 To make a donation or view additional materials 6 00:00:13,650 --> 00:00:17,600 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,600 --> 00:00:18,530 at ocw.mit.edu. 8 00:00:25,197 --> 00:00:26,780 JOANNE STUBBE: What I want to do today 9 00:00:26,780 --> 00:00:33,230 is finish up module 7 on reactive oxygen species 10 00:00:33,230 --> 00:00:37,760 and then move on into the last module, which we are obviously 11 00:00:37,760 --> 00:00:39,370 not going to get completely through. 12 00:00:39,370 --> 00:00:41,420 We're going to be focused mostly on purines 13 00:00:41,420 --> 00:00:43,070 and maybe some pyrimidines. 14 00:00:43,070 --> 00:00:44,840 And I'll give you a big overview of what 15 00:00:44,840 --> 00:00:47,270 I think the things are you need to think 16 00:00:47,270 --> 00:00:50,780 about in nucleotide and deoxynucleotide metabolism 17 00:00:50,780 --> 00:00:51,800 as a starting point. 18 00:00:51,800 --> 00:00:52,580 OK. 19 00:00:52,580 --> 00:00:59,720 So we've been talking about module 7 and, in this section, 20 00:00:59,720 --> 00:01:02,920 how you control reactive oxygen species for signaling. 21 00:01:02,920 --> 00:01:05,209 We were going through the generic overview. 22 00:01:05,209 --> 00:01:08,660 And at the end of the last lecture, 23 00:01:08,660 --> 00:01:10,820 this is the system we were talking 24 00:01:10,820 --> 00:01:14,600 about using epidermal growth factor 25 00:01:14,600 --> 00:01:18,830 receptor, which we've now looked at quite a bit as an example. 26 00:01:18,830 --> 00:01:21,590 But what I wanted to point out is 27 00:01:21,590 --> 00:01:25,280 that it's not limited to epidermal growth factor 28 00:01:25,280 --> 00:01:25,910 receptor. 29 00:01:25,910 --> 00:01:30,860 So you have insulin growth factor receptor, 30 00:01:30,860 --> 00:01:37,450 nerve growth factor signaling, VEGF, IL-1, IL-4, et cetera. 31 00:01:37,450 --> 00:01:40,610 And all of these things are all distinct. 32 00:01:40,610 --> 00:01:43,670 They all have different signaling cascades. 33 00:01:43,670 --> 00:01:45,890 But the generic approach that we've 34 00:01:45,890 --> 00:01:50,240 been looking at in the Kate Carroll paper is also, 35 00:01:50,240 --> 00:01:53,210 I think, applicable to these other systems. 36 00:01:53,210 --> 00:01:56,570 And so what I wanted to do was just 37 00:01:56,570 --> 00:01:59,900 make one more point with this, and then what I'm going to do 38 00:01:59,900 --> 00:02:03,950 is summarize the general principles 39 00:02:03,950 --> 00:02:05,750 of post-translational modification 40 00:02:05,750 --> 00:02:09,050 by anything-- we're using post-translational modification 41 00:02:09,050 --> 00:02:13,220 by sulfenylation and then briefly come back 42 00:02:13,220 --> 00:02:14,360 to the methods used. 43 00:02:14,360 --> 00:02:17,480 But we spent a lot of time in recitations in 11 and 12 44 00:02:17,480 --> 00:02:18,980 focused on methods, so I'm not going 45 00:02:18,980 --> 00:02:20,313 to spend very much time on that. 46 00:02:20,313 --> 00:02:23,300 It's also in your PowerPoint handouts. 47 00:02:23,300 --> 00:02:27,950 So the key thing here is the general-- 48 00:02:27,950 --> 00:02:31,710 is we have EGF, OK, so that's Epidermal Growth Factor 49 00:02:31,710 --> 00:02:33,200 in the membrane. 50 00:02:33,200 --> 00:02:36,620 We have epidermal growth factor receptor, which you all 51 00:02:36,620 --> 00:02:42,020 know has to dimerize and you all know, at this stage, 52 00:02:42,020 --> 00:02:43,495 is a tyrosine kinase. 53 00:02:46,010 --> 00:02:48,350 And the key thing we're going to be focused on 54 00:02:48,350 --> 00:02:51,290 is if we modify these proteins, what is 55 00:02:51,290 --> 00:02:53,420 the biological consequence, OK? 56 00:02:53,420 --> 00:02:55,340 Do you have any biological consequence? 57 00:02:55,340 --> 00:02:58,940 And if you don't, it's probably just an artifact of the fact 58 00:02:58,940 --> 00:03:02,330 that cysteines react rapidly with hydrogen-- not rapidly, 59 00:03:02,330 --> 00:03:03,980 but they react with hydrogen peroxide 60 00:03:03,980 --> 00:03:07,050 at some level to give you modification. 61 00:03:07,050 --> 00:03:08,870 So this is all, I'm just going to say, 62 00:03:08,870 --> 00:03:10,580 tyrosine kinase activity. 63 00:03:10,580 --> 00:03:13,650 We've already gone through that. 64 00:03:13,650 --> 00:03:19,250 And what happens is you activate the NOX proteins. 65 00:03:19,250 --> 00:03:23,880 And in this case, it's the NOX2 isozymes. 66 00:03:23,880 --> 00:03:32,570 And this is outside, and this is inside the cell. 67 00:03:32,570 --> 00:03:36,830 And NOX2 can generate superoxide-- 68 00:03:36,830 --> 00:03:40,790 OK, so let's just put this in parentheses-- 69 00:03:40,790 --> 00:03:44,240 which can rapidly generate hydrogen peroxide. 70 00:03:44,240 --> 00:03:47,780 And so the issue is that the superoxide and all 71 00:03:47,780 --> 00:03:49,610 of the hydrogen peroxide needs to come 72 00:03:49,610 --> 00:03:51,950 from the outside of the cell to the inside of the cell. 73 00:03:51,950 --> 00:03:53,030 OK. 74 00:03:53,030 --> 00:03:54,650 So we have hydrogen peroxide. 75 00:03:54,650 --> 00:03:56,450 And what is hydrogen peroxide doing? 76 00:03:56,450 --> 00:03:58,250 So the model is-- 77 00:03:58,250 --> 00:04:00,590 and this is what we've been focusing on-- 78 00:04:00,590 --> 00:04:04,280 that the hydrogen peroxide can modify the cysteine 79 00:04:04,280 --> 00:04:06,140 by sulfenylation, OK? 80 00:04:06,140 --> 00:04:10,490 So we can go from SH to SOH. 81 00:04:10,490 --> 00:04:15,060 And in the case of the tyrosine kinase 82 00:04:15,060 --> 00:04:17,899 and in the paper you had to read, 83 00:04:17,899 --> 00:04:21,140 it turns out that tyrosine kinase by activity assays 84 00:04:21,140 --> 00:04:22,550 was more active. 85 00:04:22,550 --> 00:04:23,750 So it's phosphorylated. 86 00:04:23,750 --> 00:04:25,040 It's sulfenylated. 87 00:04:25,040 --> 00:04:26,690 That leads to higher activity. 88 00:04:26,690 --> 00:04:31,100 That means it's potentially biologically interesting. 89 00:04:31,100 --> 00:04:33,680 And we also, in the Kate Carroll paper, 90 00:04:33,680 --> 00:04:36,770 looked not only at the activity, but we looked downstream 91 00:04:36,770 --> 00:04:38,480 at the signaling pathways, and we 92 00:04:38,480 --> 00:04:42,560 saw signaling as defined by phosphorylation events. 93 00:04:42,560 --> 00:04:44,270 We saw more signaling. 94 00:04:44,270 --> 00:04:46,940 So those are the kinds of peak criteria 95 00:04:46,940 --> 00:04:51,390 people are looking at for being biologically interesting. 96 00:04:51,390 --> 00:04:53,660 Now, what we also have is a key control, 97 00:04:53,660 --> 00:04:59,420 and, in these cascades, like over there, we also have PTP. 98 00:04:59,420 --> 00:05:05,610 And that's Protein Tyrosine Phosphatase. 99 00:05:05,610 --> 00:05:10,460 And these proteins all have a cysteine at the active site. 100 00:05:10,460 --> 00:05:12,800 We talked about this before. 101 00:05:12,800 --> 00:05:17,180 And the cysteine at the active site, what can it do? 102 00:05:17,180 --> 00:05:21,050 It can really sort of dephosphorylate 103 00:05:21,050 --> 00:05:23,330 the tyrosine kinase. 104 00:05:23,330 --> 00:05:27,510 And if you remove the phosphate, the activity is lowered. 105 00:05:27,510 --> 00:05:28,017 OK. 106 00:05:28,017 --> 00:05:29,850 So again, you have something that activates, 107 00:05:29,850 --> 00:05:32,010 something that removes it. 108 00:05:32,010 --> 00:05:36,530 But what we also know-- so this is the active form, 109 00:05:36,530 --> 00:05:40,050 and this is the key in all these signaling events. 110 00:05:40,050 --> 00:05:42,270 And so what we also have-- so let me 111 00:05:42,270 --> 00:05:45,420 go over here, since I didn't leave quite enough room. 112 00:05:45,420 --> 00:05:54,050 So we have PTP that can also react with hydrogen peroxide 113 00:05:54,050 --> 00:05:56,360 to become sulfenylated. 114 00:05:56,360 --> 00:05:58,170 That's the inactive form. 115 00:05:58,170 --> 00:06:04,550 So when it's in this state, basically, you put a roadblock 116 00:06:04,550 --> 00:06:05,490 in this pathway. 117 00:06:05,490 --> 00:06:08,190 So this is inactive. 118 00:06:08,190 --> 00:06:08,690 OK. 119 00:06:08,690 --> 00:06:12,170 And the Carroll paper spent a lot of time trying to define-- 120 00:06:12,170 --> 00:06:14,570 there are lots of protein tyrosine phosphatases 121 00:06:14,570 --> 00:06:18,030 inside the cell-- not anywhere near as many as kinases. 122 00:06:18,030 --> 00:06:20,900 So one protein tyrosine phosphatase 123 00:06:20,900 --> 00:06:24,230 services many proteins. 124 00:06:24,230 --> 00:06:32,000 But both of these guys are regulated by sulfenylation. 125 00:06:32,000 --> 00:06:34,790 And there's one third thing, and so this is just 126 00:06:34,790 --> 00:06:37,430 giving us the big picture now. 127 00:06:37,430 --> 00:06:40,310 If you have hydrogen peroxide in the cell, 128 00:06:40,310 --> 00:06:42,410 I've already told you that there are enzymes that 129 00:06:42,410 --> 00:06:44,750 can degrade hydrogen peroxide-- 130 00:06:44,750 --> 00:06:46,560 peroxiredoxins. 131 00:06:46,560 --> 00:06:49,110 And so that removes the hydrogen peroxide, 132 00:06:49,110 --> 00:06:51,810 which then prevents these things from happening. 133 00:06:51,810 --> 00:06:56,640 So you have peroxiredoxins, which I already talked about. 134 00:06:56,640 --> 00:06:59,600 And so the hydrogen peroxide concentration goes down. 135 00:06:59,600 --> 00:07:02,720 So that's another mechanism of control. 136 00:07:02,720 --> 00:07:03,470 OK. 137 00:07:03,470 --> 00:07:08,010 So the take-home message is shown in this slide. 138 00:07:08,010 --> 00:07:09,980 It's shown in the papers you had to read. 139 00:07:09,980 --> 00:07:12,380 And there are many proteins that have 140 00:07:12,380 --> 00:07:14,030 some variation on this theme, and this 141 00:07:14,030 --> 00:07:15,920 is a really active area of research 142 00:07:15,920 --> 00:07:17,970 to look at this in more detail. 143 00:07:17,970 --> 00:07:19,940 OK. 144 00:07:19,940 --> 00:07:21,180 Yeah? 145 00:07:21,180 --> 00:07:25,878 AUDIENCE: The tyrosine kinase activity, [INAUDIBLE] 160% 146 00:07:25,878 --> 00:07:26,420 or something. 147 00:07:26,420 --> 00:07:28,710 I was just wondering how they actually 148 00:07:28,710 --> 00:07:30,480 classified that as [INAUDIBLE]. 149 00:07:30,480 --> 00:07:31,410 JOANNE STUBBE: Active? 150 00:07:31,410 --> 00:07:34,768 So, I mean, in biology, that's a huge effect. 151 00:07:34,768 --> 00:07:35,310 AUDIENCE: OK. 152 00:07:35,310 --> 00:07:36,893 JOANNE STUBBE: So, I mean, to somebody 153 00:07:36,893 --> 00:07:42,150 that's doing something in the test tube, a factor of two 154 00:07:42,150 --> 00:07:43,140 is nothing. 155 00:07:43,140 --> 00:07:45,390 In biology, that's all it takes. 156 00:07:45,390 --> 00:07:47,200 So the question is, is it enough? 157 00:07:47,200 --> 00:07:49,000 And you should always ask that question. 158 00:07:49,000 --> 00:07:51,000 And then you've got to look at the consequences, 159 00:07:51,000 --> 00:07:52,167 and you do more experiments. 160 00:07:52,167 --> 00:07:55,703 If you hadn't seen any effect, well, maybe you 161 00:07:55,703 --> 00:07:57,370 didn't have the right proteins in there, 162 00:07:57,370 --> 00:07:59,328 and you need five more proteins to assay, which 163 00:07:59,328 --> 00:08:00,710 would give you a bigger effect. 164 00:08:00,710 --> 00:08:01,210 OK. 165 00:08:01,210 --> 00:08:03,660 So that's the issue with all of these problems. 166 00:08:03,660 --> 00:08:06,450 That particular experiment, if you go back and look at it, 167 00:08:06,450 --> 00:08:08,700 was done in crude extracts, OK. 168 00:08:08,700 --> 00:08:11,610 And the activity is extremely low. 169 00:08:11,610 --> 00:08:16,140 They had to use a luciferase assay 170 00:08:16,140 --> 00:08:18,810 to be able to measure this and amplify the signal, OK, 171 00:08:18,810 --> 00:08:21,300 which probably has a lot of issues with-- 172 00:08:21,300 --> 00:08:22,960 can have a lot of issues. 173 00:08:22,960 --> 00:08:25,170 So if you're not happy with that, 174 00:08:25,170 --> 00:08:27,150 then you're going to have trouble in biology. 175 00:08:30,060 --> 00:08:33,419 So the question is, what is the baseline? 176 00:08:33,419 --> 00:08:34,860 How much slop do you have? 177 00:08:34,860 --> 00:08:37,450 And then you have to do the experiments many, many times. 178 00:08:37,450 --> 00:08:40,110 It's all a question of statistics. 179 00:08:40,110 --> 00:08:41,780 And then do you believe it? 180 00:08:41,780 --> 00:08:44,580 And does the rest of the community believe it? 181 00:08:44,580 --> 00:08:46,217 So that's a good question. 182 00:08:46,217 --> 00:08:48,300 But if that's what they saw, that's what they saw. 183 00:08:48,300 --> 00:08:51,480 And their interpretation was, based on this and other-- 184 00:08:51,480 --> 00:08:53,280 they did a lot of experiments in the paper, 185 00:08:53,280 --> 00:08:56,240 and that's why we chose that paper-- 186 00:08:56,240 --> 00:08:59,310 suggested this is a good working hypothesis. 187 00:08:59,310 --> 00:09:01,140 So I'm one of these people, you always 188 00:09:01,140 --> 00:09:03,300 start out with the simplest working hypothesis. 189 00:09:03,300 --> 00:09:04,770 You do experiments. 190 00:09:04,770 --> 00:09:07,260 It always gets more complicated, always. 191 00:09:07,260 --> 00:09:09,297 And then you expand it, or you change it. 192 00:09:09,297 --> 00:09:10,630 There's nothing wrong with that. 193 00:09:10,630 --> 00:09:13,200 That's what science is all about. 194 00:09:13,200 --> 00:09:15,420 So NOX. 195 00:09:15,420 --> 00:09:16,680 We've been talking about NOX. 196 00:09:16,680 --> 00:09:20,190 We talked about it for the last couple lectures. 197 00:09:20,190 --> 00:09:21,750 And I had already told you that there 198 00:09:21,750 --> 00:09:25,020 were seven isozymes of NOX. 199 00:09:25,020 --> 00:09:25,560 OK. 200 00:09:25,560 --> 00:09:30,180 We had focused on this guy in the phagosome. 201 00:09:30,180 --> 00:09:33,570 We now are focusing on NOX2 again. 202 00:09:33,570 --> 00:09:37,320 And this guy is also important, but this guy is not. 203 00:09:37,320 --> 00:09:39,330 That's the phagasome oxidase. 204 00:09:39,330 --> 00:09:41,880 So you're changing the factors inside the cell 205 00:09:41,880 --> 00:09:43,710 that govern what happens. 206 00:09:43,710 --> 00:09:46,000 And so each one of these guys-- 207 00:09:46,000 --> 00:09:49,662 you Google it, you find another 100 papers on this. 208 00:09:49,662 --> 00:09:51,870 People are trying to understand the details of what's 209 00:09:51,870 --> 00:09:54,100 going on with these systems. 210 00:09:54,100 --> 00:09:54,600 OK. 211 00:09:54,600 --> 00:09:58,200 So that sort of just shows you, again, with generic, 212 00:09:58,200 --> 00:10:01,860 it affects a lot of growth factors, or a lot of cytokines 213 00:10:01,860 --> 00:10:05,130 can use these signaling pathways that NOX is important in. 214 00:10:05,130 --> 00:10:08,310 Many of them, in the model, sulfenylation 215 00:10:08,310 --> 00:10:09,300 is also important. 216 00:10:09,300 --> 00:10:12,810 But in many cases, that remains to be established. 217 00:10:12,810 --> 00:10:13,440 OK. 218 00:10:13,440 --> 00:10:15,150 So what I want to briefly do, then, 219 00:10:15,150 --> 00:10:22,200 is look at the general principles of regulation. 220 00:10:26,270 --> 00:10:26,800 OK. 221 00:10:26,800 --> 00:10:30,400 And I'm just going to briefly outline these. 222 00:10:30,400 --> 00:10:34,840 And we've gone through each one of these examples in the two 223 00:10:34,840 --> 00:10:36,350 recitation sections. 224 00:10:36,350 --> 00:10:41,140 So I'm just sort of reviewing this and making a point. 225 00:10:41,140 --> 00:10:48,180 So is post-translational modification important? 226 00:10:48,180 --> 00:10:49,390 OK. 227 00:10:49,390 --> 00:10:52,690 And I think your generation needs to think about this, 228 00:10:52,690 --> 00:10:56,610 because as the methods become more and more sophisticated, 229 00:10:56,610 --> 00:11:01,180 OK, we've got really amazing mass spec methods if you 230 00:11:01,180 --> 00:11:03,440 can figure out how to do them correctly. 231 00:11:03,440 --> 00:11:06,490 Everything, almost any metabolite in the cells, 232 00:11:06,490 --> 00:11:07,540 can modify a protein. 233 00:11:07,540 --> 00:11:10,280 Acetyl-CoA, it acetylates things. 234 00:11:10,280 --> 00:11:13,330 S-Adenosyl methionine, the universal methylating agent, 235 00:11:13,330 --> 00:11:14,480 methylates things. 236 00:11:14,480 --> 00:11:14,980 OK. 237 00:11:14,980 --> 00:11:19,170 So you have hundreds of modifications on your protein, 238 00:11:19,170 --> 00:11:22,300 OK, and it is, in fact, related to, 239 00:11:22,300 --> 00:11:25,270 in part, I think, the metabolites interacting 240 00:11:25,270 --> 00:11:27,610 with the proteins not enzymatically. 241 00:11:27,610 --> 00:11:30,100 The question is, is it interesting? 242 00:11:30,100 --> 00:11:31,690 So I want you to think about that. 243 00:11:31,690 --> 00:11:33,440 So that's why the general principle is, 244 00:11:33,440 --> 00:11:35,065 what are you going to use as a control? 245 00:11:35,065 --> 00:11:36,590 You can see it. 246 00:11:36,590 --> 00:11:38,530 Are you going to spend five years of your life 247 00:11:38,530 --> 00:11:39,490 chasing this? 248 00:11:39,490 --> 00:11:40,892 Or is it not interesting? 249 00:11:40,892 --> 00:11:42,600 So you need to think about that question. 250 00:11:42,600 --> 00:11:43,930 It's not an easy question. 251 00:11:43,930 --> 00:11:46,510 And that's what everybody is into now. 252 00:11:46,510 --> 00:11:48,800 That's the future for the next five years. 253 00:11:48,800 --> 00:11:52,240 So one of the things we see is that-- 254 00:11:52,240 --> 00:11:54,430 and I told you this 20 times-- 255 00:11:54,430 --> 00:11:58,240 it needs to be reversible. 256 00:11:58,240 --> 00:11:58,770 OK. 257 00:11:58,770 --> 00:12:02,290 So in our case, it doesn't matter 258 00:12:02,290 --> 00:12:07,480 whether it's phosphorylation, dephosphorylation, acetylation, 259 00:12:07,480 --> 00:12:11,800 deacetylation, methylation, lots of the methyl group. 260 00:12:11,800 --> 00:12:13,090 OK. 261 00:12:13,090 --> 00:12:15,880 Ubiquitination, deubiquitination. 262 00:12:15,880 --> 00:12:17,770 We seen many examples of this. 263 00:12:17,770 --> 00:12:19,960 It needs to be reversible. 264 00:12:19,960 --> 00:12:23,770 In our case-- and this is related to, again, 265 00:12:23,770 --> 00:12:24,715 oxidative stress-- 266 00:12:29,170 --> 00:12:30,850 so this is forward-- 267 00:12:30,850 --> 00:12:32,920 how do you reverse this? 268 00:12:32,920 --> 00:12:35,080 So you need a reductant. 269 00:12:35,080 --> 00:12:39,160 And this could be any one of a number of things. 270 00:12:39,160 --> 00:12:41,960 There are lots of reductants inside the cells. 271 00:12:41,960 --> 00:12:44,820 So I'm just going to say reductant. 272 00:12:44,820 --> 00:12:48,640 I've used thioredoxin here, but this has not 273 00:12:48,640 --> 00:12:52,720 been identified in the case of the NOX2 system 274 00:12:52,720 --> 00:12:57,020 in the epidermal growth factor receptor. 275 00:12:57,020 --> 00:12:58,630 We've already looked at this. 276 00:12:58,630 --> 00:13:01,870 You can convert this back to the cysteine. 277 00:13:01,870 --> 00:13:02,740 This is reduced. 278 00:13:02,740 --> 00:13:04,240 Something else needs to be oxidized. 279 00:13:04,240 --> 00:13:05,170 OK. 280 00:13:05,170 --> 00:13:08,530 So that's a basic thing that you need to think about. 281 00:13:08,530 --> 00:13:09,537 OK. 282 00:13:09,537 --> 00:13:11,620 The second thing, which I think is very important, 283 00:13:11,620 --> 00:13:14,380 and I think this is a general principle used 284 00:13:14,380 --> 00:13:16,960 in biology over and over and over again, 285 00:13:16,960 --> 00:13:19,580 is increasing the effective molarity. 286 00:13:19,580 --> 00:13:20,080 OK. 287 00:13:20,080 --> 00:13:32,080 And so we say increasing effective molarity. 288 00:13:32,080 --> 00:13:34,310 And why is that important? 289 00:13:34,310 --> 00:13:36,340 Because if you have two things reacting-- 290 00:13:36,340 --> 00:13:38,860 here, we have the things reacting. 291 00:13:38,860 --> 00:13:41,290 Here, we have two things reacting, hydrogen peroxide 292 00:13:41,290 --> 00:13:42,640 and a protein. 293 00:13:42,640 --> 00:13:46,215 So if we can generate them right next to each other, 294 00:13:46,215 --> 00:13:47,590 the concentration is much higher. 295 00:13:47,590 --> 00:13:49,680 The rate of the reaction has to be faster. 296 00:13:49,680 --> 00:13:50,980 OK. 297 00:13:50,980 --> 00:13:55,210 And so how do you increase the effective molarity 298 00:13:55,210 --> 00:13:57,970 in the case of the epidermal growth factor receptor? 299 00:13:57,970 --> 00:13:59,140 We looked at this. 300 00:13:59,140 --> 00:14:03,190 These guys were in the membrane, and NOX interacted 301 00:14:03,190 --> 00:14:05,110 with the epidermal growth factor receptor 302 00:14:05,110 --> 00:14:07,720 by the immunoprecipitation experiments 303 00:14:07,720 --> 00:14:10,360 that we looked at in the recitation. 304 00:14:10,360 --> 00:14:14,200 So an example is, I'll just say, NOX 305 00:14:14,200 --> 00:14:20,120 dot EGF receptor immunoprecipitation. 306 00:14:20,120 --> 00:14:20,980 OK. 307 00:14:20,980 --> 00:14:23,060 So another way that-- 308 00:14:23,060 --> 00:14:25,510 and we're talking about signaling-- 309 00:14:25,510 --> 00:14:28,720 nature has used over and over again is, lots of times, 310 00:14:28,720 --> 00:14:32,830 we have these little G proteins, GTPases. 311 00:14:35,920 --> 00:14:39,040 And these GTPases can be in the cytosol. 312 00:14:39,040 --> 00:14:41,890 But a lot of the time, they do all of the signaling 313 00:14:41,890 --> 00:14:42,640 at the membrane. 314 00:14:42,640 --> 00:14:44,140 How do you get them to the membrane? 315 00:14:44,140 --> 00:14:46,900 Anybody got any idea? 316 00:14:46,900 --> 00:14:49,420 So these things move around inside the cell. 317 00:14:49,420 --> 00:14:51,550 Localization becomes really key. 318 00:14:51,550 --> 00:14:53,590 How would you get a little, soluble G 319 00:14:53,590 --> 00:14:56,617 protein to the membrane? 320 00:14:56,617 --> 00:14:58,117 AUDIENCE: Through post-translational 321 00:14:58,117 --> 00:15:00,890 modification, like a GPI anchor. 322 00:15:00,890 --> 00:15:03,140 JOANNE STUBBE: Yeah, so you would put an anchor on it. 323 00:15:03,140 --> 00:15:05,000 And what do you use as anchors? 324 00:15:05,000 --> 00:15:06,510 You can use isoprenes. 325 00:15:06,510 --> 00:15:10,610 So farnesylated, geranylated are frequently used. 326 00:15:10,610 --> 00:15:14,420 You've seen that in the first module, module 5, 327 00:15:14,420 --> 00:15:15,920 of the second half. 328 00:15:15,920 --> 00:15:18,340 And you also can put a fatty acid on there. 329 00:15:18,340 --> 00:15:20,790 It's used over and over again. 330 00:15:20,790 --> 00:15:23,000 So the prenylation reaction, people 331 00:15:23,000 --> 00:15:24,800 have been looking for the reversibility 332 00:15:24,800 --> 00:15:25,923 of that for a long time. 333 00:15:25,923 --> 00:15:27,590 And as far as I know, no one's found it. 334 00:15:27,590 --> 00:15:31,840 But the fatty acid, which is put on usually as a thioester 335 00:15:31,840 --> 00:15:34,820 or as an amide, you can hydrolyze it off. 336 00:15:34,820 --> 00:15:37,940 So what you can do, then, is have-- let's just use 337 00:15:37,940 --> 00:15:40,280 fatty acid whatever. 338 00:15:40,280 --> 00:15:44,750 So you have CoA fatty acid. 339 00:15:44,750 --> 00:15:47,760 And so this gets modified. 340 00:15:47,760 --> 00:15:49,880 And then this goes to the membrane. 341 00:15:49,880 --> 00:15:53,270 So what that does, then, is it takes it out 342 00:15:53,270 --> 00:15:55,238 of solution under a certain set of conditions. 343 00:15:55,238 --> 00:15:56,780 So you've modified your protein, just 344 00:15:56,780 --> 00:15:59,090 like we had by sulfenylation. 345 00:15:59,090 --> 00:16:01,460 And you bring it to wherever the proteins 346 00:16:01,460 --> 00:16:03,590 are it's interacting with. 347 00:16:03,590 --> 00:16:06,990 So you're increasing the effective molarity. 348 00:16:06,990 --> 00:16:07,490 OK. 349 00:16:07,490 --> 00:16:11,210 So this happens all the time. 350 00:16:11,210 --> 00:16:14,750 Putting that into a big picture related 351 00:16:14,750 --> 00:16:17,720 to what I'm going to say next is, I think, 352 00:16:17,720 --> 00:16:20,610 incredibly important. 353 00:16:20,610 --> 00:16:23,420 So this is a general principle, but one 354 00:16:23,420 --> 00:16:28,830 that needs to be studied or described in a lot more detail. 355 00:16:28,830 --> 00:16:35,090 So the third thing is the post-translational modification 356 00:16:35,090 --> 00:16:37,520 must have a biological phenotype. 357 00:16:48,250 --> 00:16:50,990 So this is the question that Shiva was just asking. 358 00:16:50,990 --> 00:16:54,140 If this is increased by whatever, 50%, 359 00:16:54,140 --> 00:16:55,080 is it interesting? 360 00:16:55,080 --> 00:16:55,980 Is it important? 361 00:16:55,980 --> 00:16:56,610 OK. 362 00:16:56,610 --> 00:16:59,910 So you need to do additional experiments if you don't think, 363 00:16:59,910 --> 00:17:03,100 based on what you know about the system, that that's true. 364 00:17:03,100 --> 00:17:07,260 And so in the case of the NOX system, what do we use? 365 00:17:07,260 --> 00:17:09,869 Remember, we talked about this. 366 00:17:09,869 --> 00:17:10,900 We did two things. 367 00:17:10,900 --> 00:17:19,490 We have increased activity of the tyrosine kinase. 368 00:17:19,490 --> 00:17:28,550 And then we also had increased downstream signaling. 369 00:17:31,260 --> 00:17:32,750 And how did they look at that? 370 00:17:32,750 --> 00:17:36,680 We looked at that by phosphorylation. 371 00:17:36,680 --> 00:17:40,690 So we used antibodies to serine phosphate. 372 00:17:40,690 --> 00:17:41,750 OK. 373 00:17:41,750 --> 00:17:46,700 So by those two criteria, the NOX system in the Carroll paper 374 00:17:46,700 --> 00:17:47,720 was interesting. 375 00:17:47,720 --> 00:17:53,810 And then the fourth thing that I think is also really important 376 00:17:53,810 --> 00:17:55,910 is relating to this one. 377 00:17:55,910 --> 00:17:59,900 Whatever the signaling agent is, if you 378 00:17:59,900 --> 00:18:03,330 have ways of removing it, you then decrease the signaling. 379 00:18:03,330 --> 00:18:03,830 OK. 380 00:18:03,830 --> 00:18:06,050 And so this is frequently observed 381 00:18:06,050 --> 00:18:08,370 in many of these systems. 382 00:18:08,370 --> 00:18:22,780 So enzymes can modulate the concentrations 383 00:18:22,780 --> 00:18:23,950 of the signaling agent. 384 00:18:31,010 --> 00:18:35,190 And the example I used up there with the NOX system-- 385 00:18:35,190 --> 00:18:37,440 so we're looking at hydrogen peroxide, 386 00:18:37,440 --> 00:18:41,130 and I'm not going to draw the structures out, 387 00:18:41,130 --> 00:18:43,590 because I've already done this before, the peroxiredoxins. 388 00:18:43,590 --> 00:18:46,230 We've gone through that two lectures ago. 389 00:18:46,230 --> 00:18:49,030 Something can remove that signaling agent. 390 00:18:49,030 --> 00:18:49,920 OK. 391 00:18:49,920 --> 00:18:52,620 So to me, these are the key things 392 00:18:52,620 --> 00:19:00,070 you need to think about if you're looking at whether you 393 00:19:00,070 --> 00:19:02,630 think your post-translational modification is interesting 394 00:19:02,630 --> 00:19:03,130 or not. 395 00:19:03,130 --> 00:19:05,680 And a lot of people are doing that. 396 00:19:05,680 --> 00:19:08,170 So we see lots of modifications because 397 00:19:08,170 --> 00:19:09,850 of the power of mass spec. 398 00:19:09,850 --> 00:19:12,160 The question is, are they interesting? 399 00:19:12,160 --> 00:19:15,070 And so finally, the only other thing I wanted to say here 400 00:19:15,070 --> 00:19:17,630 is in the last little section. 401 00:19:17,630 --> 00:19:20,750 And I'm not going to look at this in any detail, either. 402 00:19:20,750 --> 00:19:25,150 But if you look at methods-- so this is the last-- 403 00:19:25,150 --> 00:19:27,100 how do you look at this? 404 00:19:27,100 --> 00:19:29,790 So what you saw in the Carroll case-- 405 00:19:29,790 --> 00:19:33,310 and again, it's not unique to the Carroll case-- 406 00:19:33,310 --> 00:19:35,860 is you need to develop a reagent that's 407 00:19:35,860 --> 00:19:37,570 specific for the post-translational 408 00:19:37,570 --> 00:19:38,740 modification. 409 00:19:38,740 --> 00:19:43,900 So number one, you need to develop 410 00:19:43,900 --> 00:19:54,700 a reagent specific for post-translational 411 00:19:54,700 --> 00:19:57,010 modification. 412 00:19:57,010 --> 00:20:01,390 It needs to be specific. 413 00:20:01,390 --> 00:20:03,490 It needs to be fast. 414 00:20:03,490 --> 00:20:07,510 So the kinetics are important under physiological conditions. 415 00:20:11,550 --> 00:20:14,070 And it needs to be cell permeable. 416 00:20:17,660 --> 00:20:18,640 OK. 417 00:20:18,640 --> 00:20:21,760 Because ultimately, with something like hydrogen 418 00:20:21,760 --> 00:20:26,620 peroxide or NO or many of the other signaling agents, 419 00:20:26,620 --> 00:20:29,140 these guys are really reactive. 420 00:20:29,140 --> 00:20:31,810 And you crack open the cells, and you do things out, 421 00:20:31,810 --> 00:20:34,690 and you add more oxygen. You can change 422 00:20:34,690 --> 00:20:37,640 the levels of modification all over the place. 423 00:20:37,640 --> 00:20:41,380 So you really want to look at this contained within the cell 424 00:20:41,380 --> 00:20:44,860 under controlled growth conditions. 425 00:20:44,860 --> 00:20:51,200 And this is what the two papers we looked at by Carroll 426 00:20:51,200 --> 00:20:52,200 were focused on. 427 00:20:52,200 --> 00:20:54,390 OK. 428 00:20:54,390 --> 00:20:56,980 So you have a reagent. 429 00:20:56,980 --> 00:20:59,340 Hopefully, you believe dimedone was a good reagent. 430 00:20:59,340 --> 00:20:59,840 OK. 431 00:20:59,840 --> 00:21:01,450 So I'm not going to-- 432 00:21:01,450 --> 00:21:07,170 but NOX for NOX, sulfenylation, we use dimedone. 433 00:21:07,170 --> 00:21:07,670 OK. 434 00:21:07,670 --> 00:21:09,070 We discussed this. 435 00:21:09,070 --> 00:21:11,110 We've discussed the mechanism. 436 00:21:11,110 --> 00:21:16,480 And then what we looked at is MS analysis 437 00:21:16,480 --> 00:21:19,220 and how you had modified the reagent 438 00:21:19,220 --> 00:21:23,740 so it worked effectively inside the cells so you can enrich. 439 00:21:23,740 --> 00:21:25,360 And then use modern methods. 440 00:21:25,360 --> 00:21:28,780 We break down the protein into peptides and sequence 441 00:21:28,780 --> 00:21:30,772 doing this MS, MS. OK. 442 00:21:30,772 --> 00:21:32,230 So I'm not going to talk about that 443 00:21:32,230 --> 00:21:35,230 more, because we had two whole recitations on these topics. 444 00:21:35,230 --> 00:21:36,250 OK. 445 00:21:36,250 --> 00:21:38,950 So that's what I wanted to say in this module 446 00:21:38,950 --> 00:21:42,130 on reactive oxygen species. 447 00:21:42,130 --> 00:21:44,590 Reactive oxygen species, I think, are front and center. 448 00:21:44,590 --> 00:21:47,220 You can't pick up any journals or even 449 00:21:47,220 --> 00:21:49,330 listen on the radio or newspapers, 450 00:21:49,330 --> 00:21:52,630 if you read newspapers, without seeing reactive oxygen, 451 00:21:52,630 --> 00:21:54,040 reactive nitrogen species. 452 00:21:54,040 --> 00:21:56,950 I think you now know what you need to think about. 453 00:21:56,950 --> 00:21:58,060 And here's an example-- 454 00:21:58,060 --> 00:22:01,310 reactive oxygen species can modify cysteines. 455 00:22:01,310 --> 00:22:03,340 Cysteines, you've seen over and over and over 456 00:22:03,340 --> 00:22:07,270 again, play central roles in enzymatic reactions 457 00:22:07,270 --> 00:22:09,910 and control of signaling pathways. 458 00:22:09,910 --> 00:22:12,850 And I think the growth factor receptor 459 00:22:12,850 --> 00:22:15,190 is a good example of that, of the kinds of things 460 00:22:15,190 --> 00:22:17,320 you need to do to try to determine 461 00:22:17,320 --> 00:22:20,380 whether these modifications, which are everywhere, 462 00:22:20,380 --> 00:22:21,930 are really, in fact, real. 463 00:22:21,930 --> 00:22:22,900 OK. 464 00:22:22,900 --> 00:22:27,170 So that's what I wanted you to get out of this little module. 465 00:22:27,170 --> 00:22:31,530 What I want to do now is move into the next module. 466 00:22:31,530 --> 00:22:35,470 And the next module, last module, module 8, 467 00:22:35,470 --> 00:22:38,860 is going to be on nucleotide metabolism. 468 00:22:43,880 --> 00:22:45,240 How bad am I? 469 00:22:45,240 --> 00:22:45,740 Oh, good. 470 00:22:45,740 --> 00:22:46,657 I've got lots of time. 471 00:22:46,657 --> 00:22:48,180 All right. 472 00:22:48,180 --> 00:22:48,760 OK. 473 00:22:48,760 --> 00:22:54,800 So let me just erase something so we have some place to start. 474 00:22:54,800 --> 00:22:58,520 So nucleotide metabolism is something 475 00:22:58,520 --> 00:23:01,400 that, in our introductory course, 476 00:23:01,400 --> 00:23:04,460 we don't talk about at all, because we just 477 00:23:04,460 --> 00:23:09,620 don't have time, and we just focus on glycolysis, sugar 478 00:23:09,620 --> 00:23:15,470 biosynthesis and degradation, fatty acid biosynthesis 479 00:23:15,470 --> 00:23:16,260 and degradation. 480 00:23:16,260 --> 00:23:18,235 But you all know, and I'll show you 481 00:23:18,235 --> 00:23:20,090 that, nucleotides are everywhere. 482 00:23:20,090 --> 00:23:24,440 And so, in my opinion, nucleotides had their heyday 483 00:23:24,440 --> 00:23:27,860 when I was your age. 484 00:23:27,860 --> 00:23:29,540 Everybody and his brother was focused 485 00:23:29,540 --> 00:23:30,800 on nucleotide metabolism. 486 00:23:30,800 --> 00:23:32,210 The data is really old. 487 00:23:32,210 --> 00:23:36,240 We learned how to make nucleotides back in those days. 488 00:23:36,240 --> 00:23:38,540 But we didn't have any of the tools we have now. 489 00:23:38,540 --> 00:23:42,350 We used a T60 rather than an 800 megahertz machine 490 00:23:42,350 --> 00:23:43,650 to look at [INAUDIBLE]. 491 00:23:43,650 --> 00:23:45,650 I mean, you had to take the spectra 20 times 492 00:23:45,650 --> 00:23:47,390 to remove the spinning sidebands. 493 00:23:47,390 --> 00:23:50,210 Anyhow, we didn't have any of the modern methods. 494 00:23:50,210 --> 00:23:51,920 But everything back in those days 495 00:23:51,920 --> 00:23:54,050 was correct, because people really 496 00:23:54,050 --> 00:23:56,450 cared about the truth back in those days, 497 00:23:56,450 --> 00:23:59,280 as opposed to publishing in Nature, Cell, and Science. 498 00:23:59,280 --> 00:23:59,840 OK. 499 00:23:59,840 --> 00:24:02,053 So that data, if you want reproducibility 500 00:24:02,053 --> 00:24:03,470 and you go back in the literature, 501 00:24:03,470 --> 00:24:05,690 is absolutely going to be reproducible. 502 00:24:05,690 --> 00:24:06,190 OK. 503 00:24:06,190 --> 00:24:08,300 So I'm going to show you where we are. 504 00:24:08,300 --> 00:24:11,170 But I would say, in the next decade, 505 00:24:11,170 --> 00:24:14,200 it's going to be the era of nucleotides. 506 00:24:14,200 --> 00:24:16,400 But what we need is ways of looking 507 00:24:16,400 --> 00:24:18,720 at nucleotides inside the cell. 508 00:24:18,720 --> 00:24:20,720 And I'll show you the complexity of this. 509 00:24:20,720 --> 00:24:22,500 But nucleotides are everywhere. 510 00:24:22,500 --> 00:24:24,600 They control everything. 511 00:24:24,600 --> 00:24:25,280 OK. 512 00:24:25,280 --> 00:24:30,050 And we really don't know that much about regulation. 513 00:24:30,050 --> 00:24:33,682 And to understand regulation, you need to be inside the cell. 514 00:24:33,682 --> 00:24:35,390 I can tell you what all these enzymes do. 515 00:24:35,390 --> 00:24:37,370 I know a lot about the enzymes. 516 00:24:37,370 --> 00:24:39,780 But the question is, how do they work inside the cell? 517 00:24:39,780 --> 00:24:40,940 And how are they regulated? 518 00:24:40,940 --> 00:24:46,130 So I'm going to try to give you sort of a picture of what 519 00:24:46,130 --> 00:24:50,540 the issues are and teach you something about pathways, 520 00:24:50,540 --> 00:24:54,761 because a purine pathway, to me, is sort of an amazing-- 521 00:24:54,761 --> 00:24:58,370 it's not erasing-- it's sort of an amazing pathway. 522 00:24:58,370 --> 00:25:02,900 And in fact, one of my heroes, when I first moved to MIT, 523 00:25:02,900 --> 00:25:08,390 is Jack Buchanan, whose picture is on the first slide. 524 00:25:08,390 --> 00:25:10,500 He was still here. 525 00:25:10,500 --> 00:25:13,650 And I just remember talking to other people. 526 00:25:13,650 --> 00:25:14,620 He was older than me. 527 00:25:14,620 --> 00:25:17,340 I think he was probably 75. 528 00:25:17,340 --> 00:25:19,140 And he was just my hero. 529 00:25:19,140 --> 00:25:20,730 I mean, if you read his papers, it's 530 00:25:20,730 --> 00:25:23,820 totally mind boggling what the guy did with what he had. 531 00:25:23,820 --> 00:25:24,600 OK. 532 00:25:24,600 --> 00:25:26,580 And everybody was dumping on him, 533 00:25:26,580 --> 00:25:30,240 because he had moved into the state of the art back 534 00:25:30,240 --> 00:25:31,060 in those days. 535 00:25:31,060 --> 00:25:31,560 OK. 536 00:25:31,560 --> 00:25:35,480 But if you took what he did in perspective, 537 00:25:35,480 --> 00:25:37,230 he'd done so much more than all the people 538 00:25:37,230 --> 00:25:38,130 that were dumping on him. 539 00:25:38,130 --> 00:25:38,890 It drove me nuts. 540 00:25:38,890 --> 00:25:40,515 So I used to have fights with everybody 541 00:25:40,515 --> 00:25:43,200 when I got here, telling everybody what 542 00:25:43,200 --> 00:25:45,818 a great scientist this guy was. 543 00:25:45,818 --> 00:25:47,360 And I'll try to point out why I think 544 00:25:47,360 --> 00:25:53,160 he was such a great scientist when we look at the pathway. 545 00:25:53,160 --> 00:25:59,340 Anyhow, the purine biosynthetic pathway, we'll see, 546 00:25:59,340 --> 00:26:00,870 was elucidated in pigeons. 547 00:26:00,870 --> 00:26:05,610 He used to catch the pigeons in the Boston Common. 548 00:26:05,610 --> 00:26:08,490 And then I'll tell you why. 549 00:26:08,490 --> 00:26:12,390 They have a different metabolism of excretion than humans do, 550 00:26:12,390 --> 00:26:14,400 and so you could feed the pigeons N15. 551 00:26:14,400 --> 00:26:17,160 This was back in the 1940s, 1950s. 552 00:26:17,160 --> 00:26:21,150 You could feed them stable isotopically labeled 553 00:26:21,150 --> 00:26:22,590 nitrogen stuff. 554 00:26:22,590 --> 00:26:25,690 And we'll see purine's got nitrogens all over the place. 555 00:26:25,690 --> 00:26:29,850 And then you isolate the poop and then characterized it. 556 00:26:29,850 --> 00:26:31,930 And that's how we unraveled the pathway. 557 00:26:31,930 --> 00:26:32,770 OK. 558 00:26:32,770 --> 00:26:33,270 All right. 559 00:26:33,270 --> 00:26:35,410 So where am I? 560 00:26:35,410 --> 00:26:35,910 All right. 561 00:26:35,910 --> 00:26:37,493 I just want to make sure I'm in order. 562 00:26:37,493 --> 00:26:39,600 So reading. 563 00:26:39,600 --> 00:26:43,620 So what I've assigned you to read in 5.07, 564 00:26:43,620 --> 00:26:46,340 people haven't done nucleotide metabolism. 565 00:26:46,340 --> 00:26:49,350 So we put it online for the chapter 566 00:26:49,350 --> 00:26:51,600 on nucleotide metabolism from Voet and Voet. 567 00:26:51,600 --> 00:26:53,700 There's a lot of stuff in there that's not right, 568 00:26:53,700 --> 00:26:57,480 but it gives you sort of an overview. 569 00:26:57,480 --> 00:27:01,140 And you can take it out of any book if you use Stryer 570 00:27:01,140 --> 00:27:02,190 or if you use whatever. 571 00:27:02,190 --> 00:27:03,510 You can use any book you want. 572 00:27:03,510 --> 00:27:06,540 It just sort of gives you a big picture. 573 00:27:06,540 --> 00:27:08,490 And the picture keeps changing, and the books 574 00:27:08,490 --> 00:27:10,430 don't keep up to date. 575 00:27:10,430 --> 00:27:11,470 OK. 576 00:27:11,470 --> 00:27:14,370 I gave you an article to read by Benkovic, which 577 00:27:14,370 --> 00:27:17,885 is a review not just focused on the papers 578 00:27:17,885 --> 00:27:19,260 that we've talked about and we'll 579 00:27:19,260 --> 00:27:21,900 talk about today in recitation. 580 00:27:21,900 --> 00:27:26,580 And so what I want to do is, after introducing you 581 00:27:26,580 --> 00:27:29,130 to the nomenclature, I'm going to give you 582 00:27:29,130 --> 00:27:32,190 a general overview of nucleotide metabolism, 583 00:27:32,190 --> 00:27:35,370 focus a little bit on the biology of purines. 584 00:27:35,370 --> 00:27:37,590 Then we'll talk about the pathway 585 00:27:37,590 --> 00:27:42,000 and why I think the pathway is interesting. 586 00:27:42,000 --> 00:27:44,760 And we were going to close with this section, which 587 00:27:44,760 --> 00:27:46,327 is what we're doing on today. 588 00:27:46,327 --> 00:27:47,910 One of the reasons I talked about this 589 00:27:47,910 --> 00:27:52,800 is because I think this idea of purinosomes, complexes 590 00:27:52,800 --> 00:27:56,160 of transiently interacting proteins, 591 00:27:56,160 --> 00:28:00,120 has captured people's attention for decades. 592 00:28:00,120 --> 00:28:02,280 And when this paper came out in 2008, 593 00:28:02,280 --> 00:28:06,690 it was one of the first examples where people thought they might 594 00:28:06,690 --> 00:28:09,730 have gotten evidence inside tissue culture cells-- 595 00:28:09,730 --> 00:28:12,150 so it's still in vitro-- 596 00:28:12,150 --> 00:28:16,640 to show that these transient interactions of pathways 597 00:28:16,640 --> 00:28:20,890 play another regulatory mechanism inside cells. 598 00:28:20,890 --> 00:28:21,570 OK. 599 00:28:21,570 --> 00:28:23,790 So that's where we're going. 600 00:28:23,790 --> 00:28:24,290 OK. 601 00:28:24,290 --> 00:28:25,740 So nomenclature. 602 00:28:25,740 --> 00:28:26,240 OK. 603 00:28:26,240 --> 00:28:30,800 So many of you probably have seen this 604 00:28:30,800 --> 00:28:35,500 before if you took 7.05 instead of 5.07. 605 00:28:35,500 --> 00:28:37,170 I guess they taught in-- 606 00:28:37,170 --> 00:28:40,280 did they teach you in 5.07 nucleotides? 607 00:28:40,280 --> 00:28:43,760 Any of you have Ting and Klibanov? 608 00:28:43,760 --> 00:28:46,700 Didn't they teach you about nucleotide metabolism? 609 00:28:46,700 --> 00:28:49,037 I thought they taught about DNA replication. 610 00:28:49,037 --> 00:28:50,870 AUDIENCE: They talked about DNA replication. 611 00:28:50,870 --> 00:28:53,328 JOANNE STUBBE: Well, how can you talk about DNA replication 612 00:28:53,328 --> 00:28:55,040 without knowing what a nucleotide is? 613 00:28:55,040 --> 00:28:55,790 Sorry. 614 00:28:55,790 --> 00:28:56,540 All right. 615 00:28:56,540 --> 00:28:59,863 So anyhow, I'm not going to draw. 616 00:28:59,863 --> 00:29:02,030 I'm not going to draw these structures on the board. 617 00:29:02,030 --> 00:29:04,070 But this is like the amino acids. 618 00:29:04,070 --> 00:29:06,590 I think you should know the nucleotides, OK? 619 00:29:06,590 --> 00:29:08,600 People hate me for the amino acid side chains, 620 00:29:08,600 --> 00:29:12,410 and the pKa is something else you can dislike me for. 621 00:29:12,410 --> 00:29:15,290 But anyhow, these are the bases. 622 00:29:15,290 --> 00:29:18,290 The names are not so easy to remember. 623 00:29:18,290 --> 00:29:21,843 But, I mean, it's central to all of genetic material. 624 00:29:21,843 --> 00:29:23,510 So it's pretty darn important, no matter 625 00:29:23,510 --> 00:29:26,360 what kind of a biologist, biochemist you are. 626 00:29:26,360 --> 00:29:28,880 So we're going to be looking at the purines-- 627 00:29:28,880 --> 00:29:30,500 adenine and guanine. 628 00:29:30,500 --> 00:29:32,320 So these are the bases-- 629 00:29:32,320 --> 00:29:35,990 thymine, cytosine, and uracil. 630 00:29:35,990 --> 00:29:37,460 OK. 631 00:29:37,460 --> 00:29:42,270 And if you take the base and stick on a sugar-- 632 00:29:42,270 --> 00:29:44,870 OK, so this sugar is ribose-- 633 00:29:44,870 --> 00:29:47,190 you now have the nucleoside. 634 00:29:47,190 --> 00:29:47,690 OK. 635 00:29:47,690 --> 00:29:49,065 And this is in the introductory-- 636 00:29:49,065 --> 00:29:51,523 if you don't know this, you should read the first few pages 637 00:29:51,523 --> 00:29:53,330 of Voet and Voet, and they'll introduce you 638 00:29:53,330 --> 00:29:54,590 to this nomenclature again. 639 00:29:54,590 --> 00:29:56,690 But you can come back to your notes. 640 00:29:56,690 --> 00:29:58,400 So I've redone these notes again, 641 00:29:58,400 --> 00:30:00,080 and I will repost them again-- whoops-- 642 00:30:00,080 --> 00:30:03,440 putting in more detail, because I didn't really 643 00:30:03,440 --> 00:30:07,380 know what your backgrounds are. 644 00:30:07,380 --> 00:30:10,240 So this is something that I think-- 645 00:30:10,240 --> 00:30:14,580 so we have adenosine, cytidine, guanosine, uridine. 646 00:30:14,580 --> 00:30:17,378 What about thymidine? 647 00:30:17,378 --> 00:30:18,670 Why don't I have that up there? 648 00:30:22,400 --> 00:30:24,950 So this is a take-home message from the next few lectures. 649 00:30:28,942 --> 00:30:32,440 AUDIENCE: Because they're [INAUDIBLE].. 650 00:30:32,440 --> 00:30:34,450 JOANNE STUBBE: So these all have [INAUDIBLE],, 651 00:30:34,450 --> 00:30:36,790 two prime, three prime sys hydroxyls. 652 00:30:36,790 --> 00:30:39,480 There is no ribothymidine. 653 00:30:39,480 --> 00:30:40,140 OK. 654 00:30:40,140 --> 00:30:41,590 You only have deoxy. 655 00:30:41,590 --> 00:30:42,090 OK. 656 00:30:42,090 --> 00:30:44,340 So thymidine, some people write "deoxy." 657 00:30:44,340 --> 00:30:45,650 That's redundant. 658 00:30:45,650 --> 00:30:46,710 It is deoxy. 659 00:30:46,710 --> 00:30:49,830 Thymidine is deoxy. 660 00:30:49,830 --> 00:30:53,380 So this hydroxyl is replaced with a hydrogen, 661 00:30:53,380 --> 00:30:56,290 OK, on thymidine. 662 00:30:56,290 --> 00:31:00,640 So that becomes really important in connecting 663 00:31:00,640 --> 00:31:04,050 nucleotide and deoxynucleotide metabolism, 664 00:31:04,050 --> 00:31:07,360 because you have to get from the nucleoside 665 00:31:07,360 --> 00:31:09,190 to the deoxynucleoside. 666 00:31:09,190 --> 00:31:10,990 And it's not straightforward. 667 00:31:10,990 --> 00:31:11,500 OK. 668 00:31:11,500 --> 00:31:12,650 There are many, many steps. 669 00:31:12,650 --> 00:31:16,480 The metabolism is complicated. 670 00:31:16,480 --> 00:31:17,860 And I'll show you one of them. 671 00:31:17,860 --> 00:31:20,760 But every organism is slightly different. 672 00:31:20,760 --> 00:31:21,280 OK. 673 00:31:21,280 --> 00:31:24,237 So one of the things I want you to remember is you have bases, 674 00:31:24,237 --> 00:31:25,570 and you have bases in the sugar. 675 00:31:25,570 --> 00:31:26,660 Those are the nucleosides. 676 00:31:26,660 --> 00:31:28,420 These are the bases. 677 00:31:28,420 --> 00:31:33,910 And in DNA, you have T, or, as in RNA, you have U. 678 00:31:33,910 --> 00:31:36,670 So you need both uridine and you need 679 00:31:36,670 --> 00:31:42,100 thymidine in DNA as the building blocks for DNA biosynthesis. 680 00:31:42,100 --> 00:31:42,850 OK. 681 00:31:42,850 --> 00:31:46,960 And what we're going to do-- 682 00:31:46,960 --> 00:31:51,310 and this was, again, developed mostly 683 00:31:51,310 --> 00:31:55,810 from the work of Jack Buchanan's lab a long time ago. 684 00:31:55,810 --> 00:31:59,330 And you don't need to remember this. 685 00:31:59,330 --> 00:32:03,235 But what pigeons excrete is uric acid. 686 00:32:07,630 --> 00:32:21,530 And so this is the molecule they isolated from pigeon poop, OK, 687 00:32:21,530 --> 00:32:23,660 which allowed them to tell, ultimately-- 688 00:32:23,660 --> 00:32:26,915 which is the key to these isotopic labeling experiments-- 689 00:32:30,230 --> 00:32:37,230 the source of all of the different atoms in purines. 690 00:32:37,230 --> 00:32:37,730 OK. 691 00:32:37,730 --> 00:32:38,880 And we're going to come back to this. 692 00:32:38,880 --> 00:32:40,580 But what I want you to see-- this 693 00:32:40,580 --> 00:32:42,740 is true in both purines and pyrimidines. 694 00:32:42,740 --> 00:32:46,490 And what we're focusing on, what we're going to be focusing on, 695 00:32:46,490 --> 00:32:50,120 is de novo purine biosynthesis. 696 00:32:50,120 --> 00:32:51,650 But what I'm going to also show you, 697 00:32:51,650 --> 00:32:53,930 of course, is you have salvage. 698 00:32:53,930 --> 00:32:58,850 So you can get purines from the diet your DNA breaks down, 699 00:32:58,850 --> 00:33:00,500 your RNA breaks down. 700 00:33:00,500 --> 00:33:04,260 So all of that stuff can then be used, as well. 701 00:33:04,260 --> 00:33:06,470 And so it's a question of de novo, 702 00:33:06,470 --> 00:33:08,450 and it's a question of salvage. 703 00:33:08,450 --> 00:33:11,390 I think it's really underappreciated how important 704 00:33:11,390 --> 00:33:14,270 salvage pathways are. 705 00:33:14,270 --> 00:33:17,150 And now, with mass spec and isotopic labeling, 706 00:33:17,150 --> 00:33:19,460 we can actually figure that out fairly recently. 707 00:33:19,460 --> 00:33:26,480 And people interested in making chemotherapeutics 708 00:33:26,480 --> 00:33:29,330 are finding, really, sort of things nobody ever expected 709 00:33:29,330 --> 00:33:32,840 in terms of how much comes from salvage versus how much comes 710 00:33:32,840 --> 00:33:34,050 from de novo. 711 00:33:34,050 --> 00:33:34,550 OK. 712 00:33:34,550 --> 00:33:36,247 And the salvage is easy to understand. 713 00:33:36,247 --> 00:33:36,830 I'll show you. 714 00:33:36,830 --> 00:33:38,460 That's chemically simple. 715 00:33:38,460 --> 00:33:41,260 The de novo is much more complicated. 716 00:33:41,260 --> 00:33:42,140 OK. 717 00:33:42,140 --> 00:33:46,520 So anyhow, it's these labeling-- 718 00:33:46,520 --> 00:33:48,330 we'll come back to this in a minute. 719 00:33:48,330 --> 00:33:51,500 But I think this is important. 720 00:33:51,500 --> 00:33:56,050 All of these atoms come from simple building blocks. 721 00:33:56,050 --> 00:33:58,050 And you'll see that when we look at the pathway. 722 00:33:58,050 --> 00:33:59,750 So glutamine. 723 00:33:59,750 --> 00:34:03,740 Glutamine is the major source of ammonia 724 00:34:03,740 --> 00:34:04,905 in all metabolic pathways. 725 00:34:04,905 --> 00:34:05,780 How does that happen? 726 00:34:05,780 --> 00:34:06,697 I'm going to show you. 727 00:34:06,697 --> 00:34:09,139 That will be one of the generic reactions I talk about, 728 00:34:09,139 --> 00:34:12,679 because the same approach is used over and over again 729 00:34:12,679 --> 00:34:13,280 by nature. 730 00:34:13,280 --> 00:34:17,449 And the nitrogens play a key role 731 00:34:17,449 --> 00:34:21,105 in these heterocyclic purines and pyrimidines. 732 00:34:21,105 --> 00:34:22,250 Glycine. 733 00:34:22,250 --> 00:34:24,699 We'll see where glycine comes from. 734 00:34:24,699 --> 00:34:28,120 Aspartic acid, formate, and bicarbonate. 735 00:34:28,120 --> 00:34:28,620 OK. 736 00:34:28,620 --> 00:34:31,070 So you can't get much simpler than that. 737 00:34:31,070 --> 00:34:34,159 And most of you probably know these all self assemble, 738 00:34:34,159 --> 00:34:36,273 allowing you to maybe think about the evolution 739 00:34:36,273 --> 00:34:36,940 of this process. 740 00:34:36,940 --> 00:34:38,607 You can throw them all together, and you 741 00:34:38,607 --> 00:34:41,150 can get a purine out the other side 742 00:34:41,150 --> 00:34:44,520 with varying degrees of success. 743 00:34:44,520 --> 00:34:45,020 OK. 744 00:34:45,020 --> 00:34:47,280 So that's a purine. 745 00:34:47,280 --> 00:34:50,480 So what I want to do now is sort of give you an overview. 746 00:34:54,570 --> 00:34:56,638 So I've introduce you to the nomenclature 747 00:34:56,638 --> 00:34:58,180 and what the purines are going to be. 748 00:34:58,180 --> 00:35:00,150 But I want to give you an overview 749 00:35:00,150 --> 00:35:02,950 to nucleotide metabolism in general. 750 00:35:02,950 --> 00:35:03,450 OK. 751 00:35:04,910 --> 00:35:08,280 There's a lot of stuff, so the way I'm going to do this 752 00:35:08,280 --> 00:35:09,310 is up and down. 753 00:35:09,310 --> 00:35:09,810 OK. 754 00:35:09,810 --> 00:35:12,480 So you need a piece of paper, if you're writing this down, that 755 00:35:12,480 --> 00:35:13,630 goes up and down. 756 00:35:13,630 --> 00:35:14,520 OK. 757 00:35:14,520 --> 00:35:22,380 So what's central to everything is 758 00:35:22,380 --> 00:35:24,534 phosphoribosyl pyrophosphate. 759 00:35:34,440 --> 00:35:34,940 OK. 760 00:35:34,940 --> 00:35:36,410 So this is a central player. 761 00:35:39,140 --> 00:35:40,090 So this is PRPP. 762 00:35:42,850 --> 00:35:45,340 And in your recitation and also in your handout, 763 00:35:45,340 --> 00:35:47,170 I've given you the horrible names 764 00:35:47,170 --> 00:35:50,298 that are involved with the purine pathway. 765 00:35:50,298 --> 00:35:52,340 If we have a test, I will give you the structures 766 00:35:52,340 --> 00:35:53,257 of the purine pathway. 767 00:35:53,257 --> 00:35:55,943 I don't expect you to remember the details 768 00:35:55,943 --> 00:35:56,860 of the purine pathway. 769 00:35:56,860 --> 00:36:00,520 It's complicated, and I'm not sure I would have designed it 770 00:36:00,520 --> 00:36:01,920 that way to start with. 771 00:36:01,920 --> 00:36:03,760 So it's not like it's so logical, 772 00:36:03,760 --> 00:36:06,860 like some of the other pathways, which are straightforward. 773 00:36:06,860 --> 00:36:07,360 OK. 774 00:36:07,360 --> 00:36:11,110 So where do you think phosphoribosyl pyrophosphate 775 00:36:11,110 --> 00:36:11,620 comes from? 776 00:36:11,620 --> 00:36:12,745 Does anybody have any idea? 777 00:36:12,745 --> 00:36:16,910 What did you do learn from basic metabolism? 778 00:36:16,910 --> 00:36:21,770 This is something that's covered in most introductory courses. 779 00:36:21,770 --> 00:36:26,084 Where does PRPP most likely come from? 780 00:36:26,084 --> 00:36:28,197 AUDIENCE: Out of the pentose phosphate pathway? 781 00:36:28,197 --> 00:36:30,280 JOANNE STUBBE: Yeah, out of the pentose phosphate. 782 00:36:30,280 --> 00:36:33,200 So the two things that play a really critical role 783 00:36:33,200 --> 00:36:38,733 in nucleotide metabolism are the oxidative and non-oxidative 784 00:36:38,733 --> 00:36:40,400 pentose phosphate pathway where you form 785 00:36:40,400 --> 00:36:43,320 ribose biphosphate and NADPH. 786 00:36:43,320 --> 00:36:43,820 OK. 787 00:36:43,820 --> 00:36:47,220 So over here, we have ribose biphosphate. 788 00:36:55,670 --> 00:36:59,890 And for phosphate, from now on, and for pyrophosphate, 789 00:36:59,890 --> 00:37:02,240 from now on, I'm going to abbreviate it 790 00:37:02,240 --> 00:37:04,040 so I don't have to draw the structures out. 791 00:37:04,040 --> 00:37:05,660 But the chargers are important, so you 792 00:37:05,660 --> 00:37:08,540 need to remember the structures that are charged. 793 00:37:08,540 --> 00:37:12,200 So this is ribose biphosphate. 794 00:37:12,200 --> 00:37:17,570 1 prime, 2 prime, 3 prime, 4 prime, 5 prime. 795 00:37:17,570 --> 00:37:18,600 OK. 796 00:37:18,600 --> 00:37:19,920 Let me ask this question. 797 00:37:19,920 --> 00:37:23,730 Why do you think this is the major form of ribose 798 00:37:23,730 --> 00:37:26,508 inside the cell? 799 00:37:26,508 --> 00:37:28,300 I don't know if they teach you this or not, 800 00:37:28,300 --> 00:37:31,200 but I think it's important. 801 00:37:31,200 --> 00:37:33,930 Why is ribose always phosphorylated inside the cell? 802 00:37:37,460 --> 00:37:38,870 AUDIENCE: To keep it in the cell? 803 00:37:38,870 --> 00:37:39,770 JOANNE STUBBE: To keep it in the cell. 804 00:37:39,770 --> 00:37:41,728 But what happens if you don't phosphorylate it? 805 00:37:44,740 --> 00:37:45,980 Yeah, to keep it in the cell. 806 00:37:45,980 --> 00:37:49,190 Phosphates keep things inside cells. 807 00:37:49,190 --> 00:37:52,422 What happens to that structure when it's not phosphorylated? 808 00:37:55,020 --> 00:37:58,280 AUDIENCE: The [INAUDIBLE] it can open. 809 00:37:58,280 --> 00:37:59,530 JOANNE STUBBE: So it can open. 810 00:37:59,530 --> 00:38:01,258 Is that what you see inside the cell? 811 00:38:01,258 --> 00:38:01,800 AUDIENCE: No. 812 00:38:01,800 --> 00:38:02,550 JOANNE STUBBE: No. 813 00:38:02,550 --> 00:38:04,210 What do you see? 814 00:38:04,210 --> 00:38:08,250 What kind of a sugar is this if you look at-- it's 815 00:38:08,250 --> 00:38:09,620 a five-membered ring sugar. 816 00:38:09,620 --> 00:38:10,960 What's that? 817 00:38:10,960 --> 00:38:14,235 Anybody remember that? 818 00:38:14,235 --> 00:38:15,110 This is what happens. 819 00:38:15,110 --> 00:38:18,120 I digress, and then we don't get to finish the course. 820 00:38:18,120 --> 00:38:19,460 So it's a furanose. 821 00:38:19,460 --> 00:38:20,650 OK. 822 00:38:20,650 --> 00:38:24,000 If you ring open this thing, then it can close. 823 00:38:24,000 --> 00:38:27,700 It either forms a five-membered ring or a six-membered ring. 824 00:38:27,700 --> 00:38:30,610 It forms the six-membered ring almost all the time. 825 00:38:30,610 --> 00:38:31,750 That's a pyranose. 826 00:38:31,750 --> 00:38:33,310 So it's not in the right state. 827 00:38:33,310 --> 00:38:35,283 So then you have to have your enzymes. 828 00:38:35,283 --> 00:38:36,700 And there are enzymes that do this 829 00:38:36,700 --> 00:38:39,430 that can catalyze back into conversion 830 00:38:39,430 --> 00:38:40,450 into the ribose form. 831 00:38:40,450 --> 00:38:44,380 So phosphorylation plays-- it keeps it inside the cell, which 832 00:38:44,380 --> 00:38:46,270 is incredibly important. 833 00:38:46,270 --> 00:38:50,620 But it also controls the state with which you want 834 00:38:50,620 --> 00:38:52,710 to deal in metabolic pathways. 835 00:38:52,710 --> 00:38:53,210 OK. 836 00:39:00,500 --> 00:39:02,700 And we're going to be talking about-- hopefully, 837 00:39:02,700 --> 00:39:03,510 we'll get this far. 838 00:39:03,510 --> 00:39:04,200 I'm not sure. 839 00:39:04,200 --> 00:39:07,650 But if we start with glutamine, then it'll 840 00:39:07,650 --> 00:39:09,350 abbreviate like this. 841 00:39:09,350 --> 00:39:16,310 Bicarbonate and aspartate. 842 00:39:16,310 --> 00:39:17,520 OK. 843 00:39:17,520 --> 00:39:19,110 So those are the same three things 844 00:39:19,110 --> 00:39:22,750 I just told you were involved in making the purine ring. 845 00:39:22,750 --> 00:39:25,500 These are also involved in making the pyrimidine ring. 846 00:39:25,500 --> 00:39:35,450 So what we're looking at now is de novo pyrimidine pathway. 847 00:39:38,963 --> 00:39:40,340 OK. 848 00:39:40,340 --> 00:39:44,940 And what we'll see if we do this is that we have-- 849 00:39:49,360 --> 00:39:50,720 skipped a number. 850 00:39:50,720 --> 00:39:51,780 So there are four steps. 851 00:39:55,070 --> 00:39:59,000 And you make the molecule called orotate. 852 00:40:02,374 --> 00:40:03,340 OK. 853 00:40:03,340 --> 00:40:06,700 And what we're doing now, which is 854 00:40:06,700 --> 00:40:10,270 going to be completely distinct from the purine pathway, 855 00:40:10,270 --> 00:40:11,750 is you make the base. 856 00:40:11,750 --> 00:40:12,250 OK. 857 00:40:12,250 --> 00:40:14,530 So you make your nucleotide base. 858 00:40:14,530 --> 00:40:15,250 Let me go back. 859 00:40:19,110 --> 00:40:23,370 You make your nucleotide base first. 860 00:40:23,370 --> 00:40:26,780 And then you're going to stick on the ribose biphosphate. 861 00:40:26,780 --> 00:40:30,420 In purine biosynthesis, you make the base 862 00:40:30,420 --> 00:40:32,010 on the ribose biphosphate. 863 00:40:32,010 --> 00:40:34,780 So the strategy is distinct. 864 00:40:34,780 --> 00:40:35,280 OK. 865 00:40:35,280 --> 00:40:37,830 So here, we have no base on it. 866 00:40:37,830 --> 00:40:41,340 So what happens here is it interacts 867 00:40:41,340 --> 00:40:43,060 with the phosphoribosyl pyrophosphate. 868 00:40:43,060 --> 00:40:45,270 We'll talk about this reaction, because it's 869 00:40:45,270 --> 00:40:49,115 a major way you use salvage proteins if you get a base. 870 00:40:49,115 --> 00:40:50,490 How do you put them back together 871 00:40:50,490 --> 00:40:52,950 to form the nucleoside? 872 00:40:52,950 --> 00:40:54,300 It allows you to form OMP. 873 00:40:57,070 --> 00:40:57,570 OK. 874 00:40:57,570 --> 00:41:00,880 So OMP, we're not there yet. 875 00:41:00,880 --> 00:41:04,580 OMP, we'll see, can get converted. 876 00:41:04,580 --> 00:41:05,870 It loses CO2. 877 00:41:05,870 --> 00:41:07,030 We'll look a little bit. 878 00:41:07,030 --> 00:41:10,500 The chemistry in this pathway is really pretty simple. 879 00:41:10,500 --> 00:41:14,420 So this is enzyme five. 880 00:41:14,420 --> 00:41:15,720 This is enzyme six. 881 00:41:15,720 --> 00:41:20,300 So you lose CO2, and you form UMP. 882 00:41:20,300 --> 00:41:20,990 OK. 883 00:41:20,990 --> 00:41:29,390 So UMP is one of the nucleotides we need to actually make RNA. 884 00:41:34,130 --> 00:41:38,240 To make DNA, we need deoxythymidine. 885 00:41:38,240 --> 00:41:39,200 OK. 886 00:41:39,200 --> 00:41:43,110 And we also need deoxycytidine. 887 00:41:43,110 --> 00:41:50,000 So this pathway does not give us cytidine. 888 00:41:50,000 --> 00:41:55,280 And so the way we go from UMP to the cytidine monophosphate 889 00:41:55,280 --> 00:41:56,130 is complicated. 890 00:41:56,130 --> 00:41:56,630 OK. 891 00:41:56,630 --> 00:42:00,050 So you're going to see there's a couple little-- 892 00:42:00,050 --> 00:42:02,550 central to everything is sort of straightforward. 893 00:42:02,550 --> 00:42:05,655 But then you'll see it's going to be organism specific. 894 00:42:05,655 --> 00:42:07,280 And there's a lot of messing around you 895 00:42:07,280 --> 00:42:10,670 have to do with kinases and hydrolases 896 00:42:10,670 --> 00:42:13,130 to get you into the right stage to get 897 00:42:13,130 --> 00:42:19,400 you all of the building blocks required for RNA and DNA 898 00:42:19,400 --> 00:42:20,130 biosynthesis. 899 00:42:20,130 --> 00:42:20,630 OK. 900 00:42:20,630 --> 00:42:22,850 So I'm going to go over here. 901 00:42:25,450 --> 00:42:31,520 And I'm going to say many steps. 902 00:42:31,520 --> 00:42:34,655 And we'll look at this to form CTP. 903 00:42:37,570 --> 00:42:40,350 And this does not go through CMP. 904 00:42:40,350 --> 00:42:40,850 OK. 905 00:42:40,850 --> 00:42:42,770 So there are many steps here. 906 00:42:42,770 --> 00:42:46,040 And so let's just put a question mark there. 907 00:42:46,040 --> 00:42:53,180 Also, we need to have TTP. 908 00:42:53,180 --> 00:42:57,426 And again, there are many steps. 909 00:42:57,426 --> 00:43:00,400 And we're going to have to figure out how to do this. 910 00:43:00,400 --> 00:43:00,950 OK. 911 00:43:00,950 --> 00:43:07,680 So it's not simple to get from UMP to CMP or deoxy TMP. 912 00:43:07,680 --> 00:43:08,180 OK. 913 00:43:08,180 --> 00:43:09,680 So I'm just telling you where you're 914 00:43:09,680 --> 00:43:12,675 going to see the complexity in the end. 915 00:43:12,675 --> 00:43:13,850 OK. 916 00:43:13,850 --> 00:43:19,160 So phosphoribosyl pyrophosphate is central in what it does. 917 00:43:19,160 --> 00:43:21,110 I'm not going to have enough room to do this. 918 00:43:21,110 --> 00:43:22,505 But anyhow, there are 10 steps. 919 00:43:26,090 --> 00:43:29,070 And you've already seen this if you had recitation on Thursday. 920 00:43:29,070 --> 00:43:30,630 Or hopefully, you read the paper. 921 00:43:30,630 --> 00:43:36,240 This is the purine pathway de novo. 922 00:43:36,240 --> 00:43:38,990 OK. 923 00:43:38,990 --> 00:43:43,440 And so what we're doing is we have the sugar. 924 00:43:43,440 --> 00:43:48,030 And so in every single step in the pathway, what you're doing 925 00:43:48,030 --> 00:43:49,850 is you're building up the base. 926 00:43:49,850 --> 00:43:50,350 OK. 927 00:43:50,350 --> 00:43:51,190 So you're adding it. 928 00:43:51,190 --> 00:43:53,070 So that's why there are so many steps. 929 00:43:53,070 --> 00:43:56,850 And I showed you whatever on the first slide or maybe 930 00:43:56,850 --> 00:44:00,360 the second one where all of the pieces come from. 931 00:44:00,360 --> 00:44:03,360 So again, let me just emphasize this. 932 00:44:03,360 --> 00:44:05,310 These all come from small building blocks. 933 00:44:05,310 --> 00:44:06,990 Let me do that over here. 934 00:44:06,990 --> 00:44:15,430 So you have glycine, bicarbonate, aspartic acid, 935 00:44:15,430 --> 00:44:17,988 and formate. 936 00:44:17,988 --> 00:44:20,400 OK. 937 00:44:20,400 --> 00:44:25,200 So the other thing from PRPP is salvage. 938 00:44:25,200 --> 00:44:29,070 And the salvage pathways are really important when 939 00:44:29,070 --> 00:44:33,600 you're scarfing up bases that are provided by the diet 940 00:44:33,600 --> 00:44:35,980 or from breakdown of DNA and RNA. 941 00:44:35,980 --> 00:44:37,410 So you have the salvage pathways. 942 00:44:40,450 --> 00:44:40,980 OK. 943 00:44:40,980 --> 00:44:44,490 And so this can come from the diet 944 00:44:44,490 --> 00:44:52,670 or from nucleoside or tide, tide being having a phosphate on it 945 00:44:52,670 --> 00:44:53,625 break down. 946 00:44:58,080 --> 00:44:59,810 And why is this important? 947 00:44:59,810 --> 00:45:02,320 It's important because many organisms like parasites, 948 00:45:02,320 --> 00:45:05,650 like malaria parasites, don't make purines. 949 00:45:05,650 --> 00:45:08,440 The only way they can get purines for anything 950 00:45:08,440 --> 00:45:09,770 is from salvage pathways. 951 00:45:09,770 --> 00:45:15,420 It's a major target focusing on anti-malarial 952 00:45:15,420 --> 00:45:18,500 and, in some cases, antiviral systems. 953 00:45:18,500 --> 00:45:20,840 So here, we have 10 steps. 954 00:45:20,840 --> 00:45:22,420 And at the bottom of this, I'm not 955 00:45:22,420 --> 00:45:24,850 going to draw the structure out. 956 00:45:24,850 --> 00:45:26,918 We don't get to AMP and GMP, which 957 00:45:26,918 --> 00:45:28,960 is what we were looking at in the previous slide. 958 00:45:28,960 --> 00:45:31,020 We get to IMP. 959 00:45:31,020 --> 00:45:31,520 OK. 960 00:45:31,520 --> 00:45:38,140 And then IMP, that's a branch point. 961 00:45:38,140 --> 00:45:42,630 IMP can get converted either to GMP and AMP. 962 00:45:42,630 --> 00:45:47,460 So those are the two purine nucleotides 963 00:45:47,460 --> 00:45:51,630 that we need as building blocks to make both RNA and DNA. 964 00:45:51,630 --> 00:45:57,950 So we end up over here with AMP and GMP. 965 00:45:57,950 --> 00:45:59,800 OK. 966 00:45:59,800 --> 00:46:02,290 So when you get that down, there's 967 00:46:02,290 --> 00:46:05,530 one other thing I want to say up on the top board. 968 00:46:05,530 --> 00:46:08,590 And that's to introduce you to a co-factor that many of you 969 00:46:08,590 --> 00:46:10,450 probably haven't thought about before, 970 00:46:10,450 --> 00:46:14,305 which I plan to talk about. 971 00:46:14,305 --> 00:46:15,055 And that's folate. 972 00:46:19,390 --> 00:46:22,320 So any of you think about chemo therapeutics, 973 00:46:22,320 --> 00:46:24,790 folates have been around for decades. 974 00:46:24,790 --> 00:46:28,665 And it's a major target, successful target, 975 00:46:28,665 --> 00:46:30,040 of drugs that are used clinically 976 00:46:30,040 --> 00:46:34,540 in the treatment of a wide range of cancers. 977 00:46:34,540 --> 00:46:39,340 So folate, this is a key co-factor. 978 00:46:39,340 --> 00:46:44,260 And what I will show you is that it can do chemistry. 979 00:46:44,260 --> 00:46:47,615 It does one carbon transfer, so one carbon at a time. 980 00:46:47,615 --> 00:46:49,240 And what's really interesting about it, 981 00:46:49,240 --> 00:46:51,100 and I'll draw the mechanisms out-- 982 00:46:51,100 --> 00:46:53,440 it can transfer the methyl-- it can transfer 983 00:46:53,440 --> 00:46:56,500 one carbon in an all oxidation state in the methyl 984 00:46:56,500 --> 00:46:59,620 state, the aldehyde state, and the acid state. 985 00:46:59,620 --> 00:47:05,710 So for example, in the purine pathway over here-- 986 00:47:05,710 --> 00:47:07,750 I'm just going to draw this out-- 987 00:47:07,750 --> 00:47:10,990 you need it in this state, the acid state. 988 00:47:10,990 --> 00:47:12,610 OK. 989 00:47:12,610 --> 00:47:14,740 In this state, what you're going to see 990 00:47:14,740 --> 00:47:19,570 is you need it in this state. 991 00:47:19,570 --> 00:47:23,110 We'll come back and look at this again. 992 00:47:23,110 --> 00:47:25,410 Sorry. 993 00:47:25,410 --> 00:47:28,740 Methylenetetrahydrofolate, which is a key player. 994 00:47:33,940 --> 00:47:39,900 So this is going to end up being the methyl group and thymidine. 995 00:47:39,900 --> 00:47:40,400 OK. 996 00:47:42,910 --> 00:47:46,825 The only interesting co-factor chemistry in the purine pathway 997 00:47:46,825 --> 00:47:47,560 is folate. 998 00:47:47,560 --> 00:47:53,670 And folate plays a central role in therapeutic design. 999 00:47:53,670 --> 00:47:54,430 OK. 1000 00:47:54,430 --> 00:47:57,040 So then we're down here, and we still haven't gotten finished. 1001 00:47:57,040 --> 00:47:58,630 How are we doing? 1002 00:47:58,630 --> 00:48:01,510 All right, I'm over. 1003 00:48:01,510 --> 00:48:02,590 So just let me say this. 1004 00:48:02,590 --> 00:48:05,410 So now, you're into kinases. 1005 00:48:05,410 --> 00:48:06,263 OK. 1006 00:48:06,263 --> 00:48:07,930 And there are lots of different kinases. 1007 00:48:07,930 --> 00:48:09,970 So the kinase story gets complicated, 1008 00:48:09,970 --> 00:48:12,730 but it's extremely important. 1009 00:48:12,730 --> 00:48:15,970 So if you're going to make deoxynucleotides, 1010 00:48:15,970 --> 00:48:18,370 you have to have it in the diphosphate stage. 1011 00:48:18,370 --> 00:48:21,910 So there are kinases that can convert these guys and also 1012 00:48:21,910 --> 00:48:27,600 the pyrimidines from over there into NDPs. 1013 00:48:27,600 --> 00:48:29,150 OK. 1014 00:48:29,150 --> 00:48:32,900 So we're going to have to think about kinases. 1015 00:48:32,900 --> 00:48:35,050 And in all organisms-- 1016 00:48:35,050 --> 00:48:37,440 again, this is de novo-- 1017 00:48:37,440 --> 00:48:42,890 deoxy NDPs are made by ribonucleotide reductases. 1018 00:48:42,890 --> 00:48:43,600 OK. 1019 00:48:43,600 --> 00:48:49,470 So this is the only way, de novo, 1020 00:48:49,470 --> 00:48:52,870 that you could make deoxynucleotides. 1021 00:48:52,870 --> 00:48:56,790 If you think about the substrates for DNA replication 1022 00:48:56,790 --> 00:48:59,130 and repair, they need to be triphosphate. 1023 00:48:59,130 --> 00:49:00,690 So again, you need kinases again. 1024 00:49:00,690 --> 00:49:02,670 So I'm going to stop here. 1025 00:49:02,670 --> 00:49:05,820 I will finish off the last half to get 1026 00:49:05,820 --> 00:49:07,500 this to go back together. 1027 00:49:07,500 --> 00:49:10,410 And we will talk about folate metabolism, 1028 00:49:10,410 --> 00:49:12,210 introductory and folate metabolism, 1029 00:49:12,210 --> 00:49:13,808 so I don't sort of digress. 1030 00:49:13,808 --> 00:49:16,350 And then we're going to look at the purine pathway and things 1031 00:49:16,350 --> 00:49:19,580 that I think are interesting about the purine pathway.