1 00:00:16,379 --> 00:00:18,810 BARBARA IMPERIALI: Now, I want to talk today 2 00:00:18,810 --> 00:00:22,620 about one small thing before we move on to signaling, 3 00:00:22,620 --> 00:00:24,480 because it really kind of completes 4 00:00:24,480 --> 00:00:28,900 the work that we talked about with respect to trafficking. 5 00:00:28,900 --> 00:00:32,520 So I popped this question up last time, 6 00:00:32,520 --> 00:00:35,430 and it seemed like there weren't quite enough sort of people 7 00:00:35,430 --> 00:00:36,900 leaping to give me an answer. 8 00:00:36,900 --> 00:00:39,990 But let's just take a look at the big picture of things, 9 00:00:39,990 --> 00:00:41,670 as it's always good to do. 10 00:00:41,670 --> 00:00:44,940 Because this will also get me to one other topic, 11 00:00:44,940 --> 00:00:47,910 which is protein misfolding. 12 00:00:47,910 --> 00:00:50,940 So at the end of the day, what really 13 00:00:50,940 --> 00:00:55,100 defines where a protein is, what it does, 14 00:00:55,100 --> 00:00:57,870 is defined by its sequence. 15 00:00:57,870 --> 00:01:00,990 But you always want to remember that a protein sequence is 16 00:01:00,990 --> 00:01:03,090 defined by its messenger. 17 00:01:03,090 --> 00:01:06,690 The messenger is defined by the pre-messenger. 18 00:01:06,690 --> 00:01:09,510 Yes, there may be splicing events that really 19 00:01:09,510 --> 00:01:12,990 cause changes in localization. 20 00:01:12,990 --> 00:01:16,410 But the pre-messenger includes the content. 21 00:01:16,410 --> 00:01:19,480 And then what defines that is the DNA. 22 00:01:19,480 --> 00:01:22,550 There's certain aspects of regulation 23 00:01:22,550 --> 00:01:26,100 at the epigenetic level that we don't talk about barely 24 00:01:26,100 --> 00:01:27,220 in this course. 25 00:01:27,220 --> 00:01:29,940 But I want you to make sure that you realize 26 00:01:29,940 --> 00:01:33,720 at the end of the day, what the protein is, 27 00:01:33,720 --> 00:01:37,140 how it folds, is defined originally 28 00:01:37,140 --> 00:01:40,560 by the sequence of the DNA, although a long way along here. 29 00:01:40,560 --> 00:01:44,190 The post-translational modifications that we started 30 00:01:44,190 --> 00:01:48,060 talking about last time are defined by the protein 31 00:01:48,060 --> 00:01:52,140 sequence, which all the way is defined by the DNA-- 32 00:01:52,140 --> 00:01:54,610 so, so much of protein function. 33 00:01:54,610 --> 00:01:57,660 And there's one more aspect of proteins 34 00:01:57,660 --> 00:02:00,330 that's defined by the DNA sequence, 35 00:02:00,330 --> 00:02:06,690 and that's whether a protein folds well, or perhaps, 36 00:02:06,690 --> 00:02:08,550 in some cases, misfolds. 37 00:02:08,550 --> 00:02:11,370 And that's the thing I want to talk about very briefly today. 38 00:02:11,370 --> 00:02:14,310 Because I think that this captures the picture. 39 00:02:14,310 --> 00:02:23,640 So let's just go over here and write misfolded proteins, 40 00:02:23,640 --> 00:02:26,370 which, just like everything else, 41 00:02:26,370 --> 00:02:30,270 largely end up being dictated by the DNA. 42 00:02:30,270 --> 00:02:33,450 Because whether a protein folds faithfully 43 00:02:33,450 --> 00:02:36,240 into a good structure or misfolds 44 00:02:36,240 --> 00:02:38,610 can be a function of the protein sequence. 45 00:02:38,610 --> 00:02:44,940 So there could be mutations in the protein that ultimately 46 00:02:44,940 --> 00:02:48,960 end up that the protein misfolds and forms 47 00:02:48,960 --> 00:02:53,220 either a misfolded tertiary structure, or even worse, 48 00:02:53,220 --> 00:02:56,820 adopts an aggregated form that causes a lot of damage 49 00:02:56,820 --> 00:02:59,160 within cells and outsides of cells. 50 00:02:59,160 --> 00:03:02,760 So I want to talk just briefly about the processes that we 51 00:03:02,760 --> 00:03:04,740 have-- it's just one slide-- 52 00:03:04,740 --> 00:03:07,570 to deal with misfolded proteins. 53 00:03:07,570 --> 00:03:11,760 So when a protein is translated, it almost 54 00:03:11,760 --> 00:03:15,570 starts folding straight away, especially large proteins. 55 00:03:15,570 --> 00:03:18,480 A fair amount of a protein may have already emerged 56 00:03:18,480 --> 00:03:21,750 from the ribosome and started folding, even 57 00:03:21,750 --> 00:03:24,120 when the whole protein isn't made. 58 00:03:24,120 --> 00:03:31,110 The sequence ultimately ends you up with a well-folded protein. 59 00:03:31,110 --> 00:03:34,740 But if the protein does not fold fast enough, 60 00:03:34,740 --> 00:03:36,930 or there is a mistake in this, which 61 00:03:36,930 --> 00:03:40,170 might be caused intrinsically by the primary sequence, 62 00:03:40,170 --> 00:03:41,880 if there's a mistake in that-- 63 00:03:41,880 --> 00:03:48,770 so slow folding or incorrect folding-- 64 00:03:56,810 --> 00:03:59,810 then you will end up with a protein that's partially folded 65 00:03:59,810 --> 00:04:01,850 within the context of a cell. 66 00:04:01,850 --> 00:04:05,150 We especially encounter misfolded proteins 67 00:04:05,150 --> 00:04:07,610 when we are overexpressing proteins 68 00:04:07,610 --> 00:04:10,730 in cells, because you're just making one of a type of protein 69 00:04:10,730 --> 00:04:13,160 really quickly, and it doesn't have a chance 70 00:04:13,160 --> 00:04:15,680 to adopt its faithful structure. 71 00:04:15,680 --> 00:04:20,430 So there are proteins within the cell that 72 00:04:20,430 --> 00:04:23,700 helped sort of protect the folding process early 73 00:04:23,700 --> 00:04:29,010 on, to allow the protein to have enough time in singular, not 74 00:04:29,010 --> 00:04:32,730 with a lot of copies of itself around that are misfolded, 75 00:04:32,730 --> 00:04:34,740 to adopt a folded structure. 76 00:04:34,740 --> 00:04:36,770 And these proteins are called chaperones. 77 00:04:40,290 --> 00:04:42,940 I don't know if you guys are familiar with the term 78 00:04:42,940 --> 00:04:44,020 chaperone. 79 00:04:44,020 --> 00:04:45,790 It was a term that was heavily used 80 00:04:45,790 --> 00:04:49,280 in the sort of 18th and 19th century. 81 00:04:49,280 --> 00:04:53,620 A chaperone used to be an aunt or someone who 82 00:04:53,620 --> 00:04:56,080 you would send out with your beautiful young daughter 83 00:04:56,080 --> 00:05:00,040 to chaperone her, to protect her so she didn't get bothered 84 00:05:00,040 --> 00:05:02,140 by those mean men out there. 85 00:05:02,140 --> 00:05:05,920 So chaperones were-- that was the original definition 86 00:05:05,920 --> 00:05:07,000 of the chaperone. 87 00:05:07,000 --> 00:05:10,610 And it's kind of interesting that the chaperones are now 88 00:05:10,610 --> 00:05:22,690 proteins that help folding or protect against misfolding. 89 00:05:32,500 --> 00:05:34,420 How do they do this? 90 00:05:34,420 --> 00:05:37,930 Generally, a protein will fold poorly 91 00:05:37,930 --> 00:05:40,780 if it's very, very-- if it's quite hydrophobic. 92 00:05:40,780 --> 00:05:44,500 And hydrophobic patches are exposed in aggregate. 93 00:05:44,500 --> 00:05:46,610 So let's say, you have a protein, 94 00:05:46,610 --> 00:05:49,810 and there's a lot of copies, but they're not folded. 95 00:05:49,810 --> 00:05:54,700 If you have things that are hydrophobic that would normally 96 00:05:54,700 --> 00:05:58,630 end up tucked inside the protein, 97 00:05:58,630 --> 00:06:01,760 if the protein hasn't folded in its good time, 98 00:06:01,760 --> 00:06:06,610 these will just start to form aggregates, sort 99 00:06:06,610 --> 00:06:08,600 of associating with each other. 100 00:06:08,600 --> 00:06:10,240 It's just a physical phenomenon. 101 00:06:10,240 --> 00:06:13,780 If you put something that's got a lot of hydrophobic faces 102 00:06:13,780 --> 00:06:16,930 on the outside, this will start forming 103 00:06:16,930 --> 00:06:21,400 an aggregated bundle, and not a nicely folded protein at all. 104 00:06:21,400 --> 00:06:25,330 What the chaperones may do is in part 105 00:06:25,330 --> 00:06:29,650 hold the partially folded protein. 106 00:06:29,650 --> 00:06:32,920 So let's just think of this big jelly bean as a chaperone 107 00:06:32,920 --> 00:06:36,850 until things start to adopt a favorable state. 108 00:06:36,850 --> 00:06:38,710 But sometimes it's just too much. 109 00:06:38,710 --> 00:06:42,410 The chaperone cannot handle the flux of protein. 110 00:06:42,410 --> 00:06:45,940 So the protein ends up being recognized as misfolded. 111 00:06:45,940 --> 00:06:49,660 And then it gets tagged as a misfolded protein, 112 00:06:49,660 --> 00:06:53,060 and it gets taken to a place in the cell for disposal. 113 00:06:53,060 --> 00:07:01,050 So if you are unable to fold, there is a tagging process. 114 00:07:01,050 --> 00:07:02,580 And I mentioned it last time. 115 00:07:02,580 --> 00:07:04,770 It's a process known as ubiquitination. 116 00:07:12,170 --> 00:07:15,080 This is also a post-translational 117 00:07:15,080 --> 00:07:17,780 modification, but it's one that occurs 118 00:07:17,780 --> 00:07:20,070 on poorly folded proteins. 119 00:07:20,070 --> 00:07:22,370 And I'm going to describe to you that system, 120 00:07:22,370 --> 00:07:26,000 because the ubiquitination is the flag, the signal, 121 00:07:26,000 --> 00:07:30,170 or the tag, to take this protein to the great shredder, 122 00:07:30,170 --> 00:07:31,230 basically. 123 00:07:31,230 --> 00:07:34,160 And so what does the-- what's a paper shredder-- 124 00:07:34,160 --> 00:07:36,440 I like the analogy with a paper shredder. 125 00:07:36,440 --> 00:07:41,015 So here's a fellow who's got too much on in his inbox. 126 00:07:41,015 --> 00:07:42,890 So he just sends it straight to the shredder. 127 00:07:42,890 --> 00:07:46,580 It's a little bit about too much misfolded protein being made. 128 00:07:46,580 --> 00:07:50,450 So instead of sort of waiting to deal with the paperwork, 129 00:07:50,450 --> 00:07:52,280 you just send it straight to the shredder. 130 00:07:52,280 --> 00:07:55,940 And the proteasome is the cellular shredder 131 00:07:55,940 --> 00:07:59,570 that actually breaks proteins up into small chunks, 132 00:07:59,570 --> 00:08:01,590 and then digests them out. 133 00:08:01,590 --> 00:08:17,350 So think of the proteasome as a shredder, 134 00:08:17,350 --> 00:08:27,170 which chops up proteins into small pieces, 135 00:08:27,170 --> 00:08:35,240 mostly into short peptides that are 8 to 14 136 00:08:35,240 --> 00:08:36,919 amino acids in length-- 137 00:08:36,919 --> 00:08:38,480 fairly small. 138 00:08:38,480 --> 00:08:41,630 Short peptides won't cause a problem in aggregation, 139 00:08:41,630 --> 00:08:43,669 and will then be further digested. 140 00:08:43,669 --> 00:08:46,580 Now, if you've got this shredder sitting around in the cell, 141 00:08:46,580 --> 00:08:49,940 it's like having a paper shredder on all the time. 142 00:08:49,940 --> 00:08:52,400 You've got a-- there's a risk things may end up in there 143 00:08:52,400 --> 00:08:53,960 without meaning to be. 144 00:08:53,960 --> 00:08:56,690 So for things to be tagged for shredding, 145 00:08:56,690 --> 00:09:01,110 they go through what's known as the ubiquitin system. 146 00:09:01,110 --> 00:09:04,200 So the first thing to get into that is-- 147 00:09:09,390 --> 00:09:11,670 and it's only then that proteins are 148 00:09:11,670 --> 00:09:15,930 tagged for shredding up or chopping up by the proteasome. 149 00:09:15,930 --> 00:09:19,190 So as you can sort of tell by its name, 150 00:09:19,190 --> 00:09:23,610 it's got protease function, but it's 151 00:09:23,610 --> 00:09:26,730 a large, macromolecular protease, 152 00:09:26,730 --> 00:09:29,280 with lots and lots of subunits that 153 00:09:29,280 --> 00:09:32,580 are important to cut that polypeptide into smaller 154 00:09:32,580 --> 00:09:33,890 pieces. 155 00:09:33,890 --> 00:09:37,020 But because many of your proteins 156 00:09:37,020 --> 00:09:40,530 may be partially folded or misfolded, 157 00:09:40,530 --> 00:09:42,900 they first have to be unfolded. 158 00:09:42,900 --> 00:09:48,500 So the ubiquitin is the signal to send proteins 159 00:09:48,500 --> 00:09:52,020 to the proteasome, where the second action is 160 00:09:52,020 --> 00:09:55,800 protease activity, and the first action is unfolding. 161 00:09:58,960 --> 00:10:00,840 So what I show you on this picture 162 00:10:00,840 --> 00:10:05,010 is the barrel structure of a proteasome. 163 00:10:05,010 --> 00:10:07,410 Let me explain the components of it. 164 00:10:07,410 --> 00:10:10,530 The red component of the proteasome 165 00:10:10,530 --> 00:10:14,760 is a multimeric ring that uses ATP and starts 166 00:10:14,760 --> 00:10:19,290 tugging apart the protein that you need to destroy. 167 00:10:19,290 --> 00:10:22,110 But it will only do that if the protein 168 00:10:22,110 --> 00:10:26,390 becomes labeled for destruction by the ubiquitin system. 169 00:10:26,390 --> 00:10:30,060 And I am showing you here a massively simplified version. 170 00:10:30,060 --> 00:10:32,700 Let's say this is a misfolded protein. 171 00:10:32,700 --> 00:10:35,400 It gets tagged with another protein. 172 00:10:35,400 --> 00:10:38,310 It's a really little protein known as ubiquitin. 173 00:10:38,310 --> 00:10:40,410 I've shown you the three-dimensional structure 174 00:10:40,410 --> 00:10:41,250 here. 175 00:10:41,250 --> 00:10:44,220 And using ATP, you end up managing 176 00:10:44,220 --> 00:10:47,310 to put a ubiquitin chain on the protein that's 177 00:10:47,310 --> 00:10:49,020 going to be destroyed. 178 00:10:49,020 --> 00:10:51,870 That is a post-translational modification that 179 00:10:51,870 --> 00:10:54,210 is a tagging for destruction. 180 00:10:54,210 --> 00:10:56,670 If the protein is not tagged, then it's 181 00:10:56,670 --> 00:10:58,170 not going to be chewed up. 182 00:10:58,170 --> 00:10:59,040 That makes sense. 183 00:10:59,040 --> 00:11:01,500 You don't want to be chopping up proteins 184 00:11:01,500 --> 00:11:04,350 in a cell with wild abandon. 185 00:11:04,350 --> 00:11:07,410 Once the ubiquitin chain is on here, 186 00:11:07,410 --> 00:11:11,160 the protein will bind to the unfoldase part 187 00:11:11,160 --> 00:11:13,800 of the proteasome, and with ATP, it 188 00:11:13,800 --> 00:11:18,120 will just stop tugging the rest of the residual structure apart 189 00:11:18,120 --> 00:11:24,355 to thread the protein down into the blue part of the barrel. 190 00:11:24,355 --> 00:11:25,980 It's a little hard to see it like this, 191 00:11:25,980 --> 00:11:29,010 but it's literally, the proteasome, 192 00:11:29,010 --> 00:11:31,560 are four concentric rings. 193 00:11:31,560 --> 00:11:32,460 Let me see. 194 00:11:32,460 --> 00:11:34,590 I hope my artwork is going to be good enough. 195 00:11:38,620 --> 00:11:39,860 Well, that's an unfoldase. 196 00:11:43,312 --> 00:11:44,760 And so is that. 197 00:11:44,760 --> 00:11:50,770 And then in the center, there is a protease. 198 00:11:50,770 --> 00:11:53,760 And each of these components is multimeric, 199 00:11:53,760 --> 00:11:56,790 having six or seven subunits. 200 00:11:56,790 --> 00:11:59,850 So it's a huge structure. 201 00:11:59,850 --> 00:12:03,060 It has a sedimentation coefficient 202 00:12:03,060 --> 00:12:06,540 of 20S, that entire structure. 203 00:12:06,540 --> 00:12:09,600 I don't know if you remember when I talked about ribosomes, 204 00:12:09,600 --> 00:12:13,350 they were so big we didn't tend to talk about them by size. 205 00:12:13,350 --> 00:12:16,760 We talked about them by sedimentation coefficient. 206 00:12:16,760 --> 00:12:19,930 And the large and small subunits of the ribosome, 207 00:12:19,930 --> 00:12:32,310 the eukaryotic one, just to remind you, were 40S and 60S. 208 00:12:32,310 --> 00:12:36,690 So just remember that S stands for Svedberg. 209 00:12:36,690 --> 00:12:40,150 It's a sedimentation coefficient unit. 210 00:12:40,150 --> 00:12:44,340 It describes how fast approaching precipitates. 211 00:12:44,340 --> 00:12:47,250 So once the protein has been labeled with ubiquitin, 212 00:12:47,250 --> 00:12:49,260 it binds to the unfoldase. 213 00:12:49,260 --> 00:12:53,880 And then the single strand feeds into the center core, which 214 00:12:53,880 --> 00:12:57,360 is two sections of protease. 215 00:12:57,360 --> 00:12:59,280 So it's feeding in here. 216 00:12:59,280 --> 00:13:03,850 It sees the protease activity. 217 00:13:03,850 --> 00:13:08,190 And then it's just short pieces of protein 218 00:13:08,190 --> 00:13:10,940 are spit out of the proteasome. 219 00:13:14,490 --> 00:13:17,640 Once these are really little pieces of peptide, 220 00:13:17,640 --> 00:13:21,490 they're readily digested by proteases within the cell. 221 00:13:21,490 --> 00:13:23,880 And you can recycle the amino acids, 222 00:13:23,880 --> 00:13:27,600 or you can do other things with these small pieces of peptide. 223 00:13:27,600 --> 00:13:31,440 They actually end up sometimes being sent for presentation 224 00:13:31,440 --> 00:13:34,590 on the surface of the cell by the immune system. 225 00:13:34,590 --> 00:13:37,340 And you may hear a little bit more about that later. 226 00:13:37,340 --> 00:13:40,175 So the proteasome-- oh, I apologize-- 227 00:13:40,175 --> 00:13:41,300 this should have been 26S-- 228 00:13:44,980 --> 00:13:47,350 has a molecular weight that's very large-- 229 00:13:47,350 --> 00:13:49,240 2,000 kilodaltons. 230 00:13:49,240 --> 00:13:53,950 That's why we refer to it by its sedimentation coefficient. 231 00:13:53,950 --> 00:13:56,500 So this machinery is very important 232 00:13:56,500 --> 00:14:01,300 to get rid of misfolded or aggregated proteins 233 00:14:01,300 --> 00:14:02,470 to destroy them. 234 00:14:02,470 --> 00:14:05,860 Now, does-- are people aware of the sorts of diseases that can 235 00:14:05,860 --> 00:14:08,540 result from misfolded proteins? 236 00:14:08,540 --> 00:14:10,360 Has anyone been reading the news much 237 00:14:10,360 --> 00:14:15,520 about certain types of diseases, particularly in neurobiology? 238 00:14:15,520 --> 00:14:17,140 Anyone aware of those? 239 00:14:17,140 --> 00:14:17,902 Yeah. 240 00:14:17,902 --> 00:14:19,510 AUDIENCE: Was it mad cow disease? 241 00:14:19,510 --> 00:14:19,810 BARBARA IMPERIALI: Which one? 242 00:14:19,810 --> 00:14:20,770 AUDIENCE: Mad cow. 243 00:14:20,770 --> 00:14:21,070 BARBARA IMPERIALI: Yes. 244 00:14:21,070 --> 00:14:21,670 Mad cow. 245 00:14:21,670 --> 00:14:25,540 So there are a variety of neurological disorders, 246 00:14:25,540 --> 00:14:27,070 and mad cow is one of them. 247 00:14:31,130 --> 00:14:34,710 Creutz U-T-Z feldt-Jakob. 248 00:14:38,530 --> 00:14:40,830 But Alzheimer's disease is another one. 249 00:14:44,170 --> 00:14:46,150 Pick's disease is another. 250 00:14:46,150 --> 00:14:49,750 There are a wide variety of neurological disorders 251 00:14:49,750 --> 00:14:54,080 that result from misfolded proteins, both inside the cell 252 00:14:54,080 --> 00:14:57,880 and in the extracellular matrix, forming these tangles that 253 00:14:57,880 --> 00:15:01,900 are toxic to the neurons, causing them to no longer 254 00:15:01,900 --> 00:15:04,090 function, and then resulting in many 255 00:15:04,090 --> 00:15:06,550 of these neurological disorders. 256 00:15:06,550 --> 00:15:09,640 The ones I've described to you, I've mentioned to you here. 257 00:15:09,640 --> 00:15:13,420 I know many of you are familiar with Alzheimer's disease. 258 00:15:13,420 --> 00:15:19,000 Mad cow disease is a variant of a particular protein misfolding 259 00:15:19,000 --> 00:15:21,810 disease that was first noted in cattle. 260 00:15:21,810 --> 00:15:25,870 And they basically just fell down, dropped down. 261 00:15:25,870 --> 00:15:29,670 And it was in some cases ascribed to-- 262 00:15:29,670 --> 00:15:34,930 the contagion with the disease is ascribed not to a virus 263 00:15:34,930 --> 00:15:37,960 or to a microorganism, but literally, 264 00:15:37,960 --> 00:15:41,080 to misfolded proteins causing the formation 265 00:15:41,080 --> 00:15:43,330 of more misfolded proteins. 266 00:15:43,330 --> 00:15:49,820 So these are all collectively designated as prion diseases. 267 00:15:49,820 --> 00:15:52,030 I think you'll have read that term. 268 00:15:52,030 --> 00:15:54,130 And it's a particular kind of disease 269 00:15:54,130 --> 00:15:59,020 that the infectious agent isn't a living system-- 270 00:15:59,020 --> 00:16:02,000 not a virus, not a microbe, a fungus, 271 00:16:02,000 --> 00:16:05,170 a protozoan, but rather a protein, 272 00:16:05,170 --> 00:16:08,860 where it's misfolded structure nucleates 273 00:16:08,860 --> 00:16:11,470 the formation of more misfolded structure 274 00:16:11,470 --> 00:16:13,340 that leads to the disease. 275 00:16:13,340 --> 00:16:16,190 So I grew up in England during the years 276 00:16:16,190 --> 00:16:18,550 where there was a lot of mad cow disease in England. 277 00:16:18,550 --> 00:16:22,210 And even though I'm a vegetarian in the US for 30 years, 278 00:16:22,210 --> 00:16:25,420 I can't give blood in the US, because I lived in England 279 00:16:25,420 --> 00:16:28,930 during the time when there was a lot of mad cow disease. 280 00:16:28,930 --> 00:16:31,690 And this can be dormant for a long, long time 281 00:16:31,690 --> 00:16:33,710 before it suddenly takes over. 282 00:16:33,710 --> 00:16:38,230 So there's restrictions on blood donation in certain cases. 283 00:16:38,230 --> 00:16:40,630 And it's because it's not something 284 00:16:40,630 --> 00:16:42,370 you can treat with an antibiotic, 285 00:16:42,370 --> 00:16:44,140 you can treat with an antiviral. 286 00:16:44,140 --> 00:16:48,400 It's literally traces of badly folded protein 287 00:16:48,400 --> 00:16:52,390 that can nucleate the formation of more badly folded protein, 288 00:16:52,390 --> 00:16:55,480 that can lead to the diseases. 289 00:16:55,480 --> 00:17:00,400 These were-- there's particular instances of some 290 00:17:00,400 --> 00:17:04,480 of these diseases in tribes where there's 291 00:17:04,480 --> 00:17:07,060 pretty serious cannibalism, and eating 292 00:17:07,060 --> 00:17:10,270 your sort of senior relative's brains 293 00:17:10,270 --> 00:17:14,950 was considered to be something-- an important act of respect. 294 00:17:14,950 --> 00:17:17,260 And there was this transfer of some 295 00:17:17,260 --> 00:17:20,980 of these prion-type diseases through cannibalism as well. 296 00:17:20,980 --> 00:17:25,099 So eating contaminated meat, be it a cow, 297 00:17:25,099 --> 00:17:28,487 be it your grandparents, whatever, 298 00:17:28,487 --> 00:17:30,070 it's something that actually is-- it's 299 00:17:30,070 --> 00:17:33,220 a serious transmissible disease. 300 00:17:33,220 --> 00:17:36,490 And it's really-- in the situations 301 00:17:36,490 --> 00:17:40,390 where it can be sort of related back to contaminated meat are 302 00:17:40,390 --> 00:17:41,360 one thing. 303 00:17:41,360 --> 00:17:45,700 But there are variations in the case of Alzheimer's, where 304 00:17:45,700 --> 00:17:50,620 the sequence of proteins may dictate that they don't fold 305 00:17:50,620 --> 00:17:54,010 well, or they're not post-translationally modified 306 00:17:54,010 --> 00:17:57,760 properly, so they end up as misfolded proteins. 307 00:17:57,760 --> 00:18:01,450 So these are often genetically linked disorders, 308 00:18:01,450 --> 00:18:03,520 some of the things like Alzheimer's. 309 00:18:03,520 --> 00:18:06,250 And once again, remember that goes all the way back 310 00:18:06,250 --> 00:18:09,400 to the DNA, which might, in some cases, 311 00:18:09,400 --> 00:18:11,300 trigger the misfolded disease. 312 00:18:11,300 --> 00:18:14,950 So it's a fascinating area, and there's a tremendous amount 313 00:18:14,950 --> 00:18:16,150 to be studied. 314 00:18:16,150 --> 00:18:20,220 Because of the aging population, these diseases are piling up, 315 00:18:20,220 --> 00:18:25,360 and we need to mitigate the causes of the disease, 316 00:18:25,360 --> 00:18:28,930 and find ways, for example, to slow down. 317 00:18:28,930 --> 00:18:33,070 If there are these fibrils of protein that are misfolded, 318 00:18:33,070 --> 00:18:36,520 can we maybe inhibit that formation 319 00:18:36,520 --> 00:18:39,100 with some kind of small molecule inhibitor 320 00:18:39,100 --> 00:18:42,300 to mitigate the symptoms of the disease? 321 00:18:42,300 --> 00:18:44,350 So it's a very, very active area, 322 00:18:44,350 --> 00:18:48,460 because almost every-- many, many neurological disorders 323 00:18:48,460 --> 00:18:50,755 seem to be coming down to misfolded proteins. 324 00:18:54,140 --> 00:18:56,050 So let's move on now to signaling. 325 00:19:04,400 --> 00:19:05,170 All right. 326 00:19:16,770 --> 00:19:18,780 So we're going to spend two lectures 327 00:19:18,780 --> 00:19:22,320 on-- the remainder of this lecture plus the next lecture. 328 00:19:22,320 --> 00:19:24,540 And what I want to do in this lecture 329 00:19:24,540 --> 00:19:29,020 is introduce you to some of the paradigms, the nuts and bolts, 330 00:19:29,020 --> 00:19:31,470 the mechanics of protein signaling. 331 00:19:31,470 --> 00:19:33,380 And then in the next lecture, I'm 332 00:19:33,380 --> 00:19:35,310 going to show you examples of how 333 00:19:35,310 --> 00:19:39,030 all the characteristics that we define signaling by 334 00:19:39,030 --> 00:19:42,700 get represented in signaling pathways within cells. 335 00:19:42,700 --> 00:19:44,940 So I'm going to give you all the moving parts, 336 00:19:44,940 --> 00:19:48,180 and then we'll move forward to see how the moving parts might 337 00:19:48,180 --> 00:19:53,070 function in a physiological action, such as a response 338 00:19:53,070 --> 00:19:57,180 to something particularly scary, or as a trigger to do-- 339 00:19:57,180 --> 00:19:59,110 for the cells to do something different. 340 00:19:59,110 --> 00:20:01,500 So let me take you, first of all, 341 00:20:01,500 --> 00:20:04,510 to a cartoon-like image of a cell. 342 00:20:04,510 --> 00:20:07,530 And we're going to just take from the very simplest 343 00:20:07,530 --> 00:20:08,400 beginning. 344 00:20:08,400 --> 00:20:11,790 But then this topic will get quite complex, as you see. 345 00:20:11,790 --> 00:20:14,640 But that's why I think it's important to reduce 346 00:20:14,640 --> 00:20:19,620 the process of protein signaling down to simple aspects of it 347 00:20:19,620 --> 00:20:22,410 that we can really recognize, even in much 348 00:20:22,410 --> 00:20:24,760 more complicated pathways. 349 00:20:24,760 --> 00:20:26,950 So in protein cellular signaling, 350 00:20:26,950 --> 00:20:30,000 this is a complex system of communication 351 00:20:30,000 --> 00:20:33,310 that governs all basic activities of the cell. 352 00:20:33,310 --> 00:20:35,670 There are no cells that don't do signaling. 353 00:20:35,670 --> 00:20:38,550 Bacteria and eukaryotic cells may do 354 00:20:38,550 --> 00:20:40,980 signaling slightly differently. 355 00:20:40,980 --> 00:20:46,890 But they still do have an integrated correlated system 356 00:20:46,890 --> 00:20:49,380 that's responsible for triggering 357 00:20:49,380 --> 00:20:53,910 functions of the cell through a series of discrete steps. 358 00:20:53,910 --> 00:20:56,520 So protein signaling can be dissected 359 00:20:56,520 --> 00:21:00,570 into three basic steps, where you, first of all, 360 00:21:00,570 --> 00:21:01,410 receive a signal. 361 00:21:06,500 --> 00:21:09,930 And we're going to talk about what that signal is. 362 00:21:09,930 --> 00:21:11,780 What's the nature of that signal, 363 00:21:11,780 --> 00:21:13,680 is it small molecule, large molecule? 364 00:21:13,680 --> 00:21:15,240 Where is the signal? 365 00:21:15,240 --> 00:21:17,220 Where does it act? 366 00:21:17,220 --> 00:21:20,115 Then the next step is to transduce the signal. 367 00:21:26,000 --> 00:21:28,750 And finally, you have an outcome, which is a response. 368 00:21:32,090 --> 00:21:34,750 So we're going to talk about each of these components 369 00:21:34,750 --> 00:21:38,320 in order to understand flux through cellular signaling 370 00:21:38,320 --> 00:21:40,930 pathways, and how they work to give you 371 00:21:40,930 --> 00:21:44,160 a rapid response to a necessary signal. 372 00:21:44,160 --> 00:21:44,660 All right. 373 00:21:44,660 --> 00:21:49,060 So in this cartoon, let's just, for example, think about what 374 00:21:49,060 --> 00:21:51,460 if we want to trigger cell division? 375 00:21:51,460 --> 00:21:55,210 We might have a signal, which is the yellow molecule-- 376 00:21:55,210 --> 00:21:57,280 a small molecule, large molecule. 377 00:21:57,280 --> 00:21:58,930 We'll get to that later. 378 00:21:58,930 --> 00:22:02,800 There's a cell here, where on the surface of the cell 379 00:22:02,800 --> 00:22:04,980 is a receptor. 380 00:22:04,980 --> 00:22:08,140 And that would be the entity that receives the signal. 381 00:22:08,140 --> 00:22:11,150 So in the first step in the process, 382 00:22:11,150 --> 00:22:14,050 there's a buildup of a concentration of a signal. 383 00:22:14,050 --> 00:22:16,480 And it occupies the receptors on the surface 384 00:22:16,480 --> 00:22:19,510 of the cell, and in some cases, inside the cell. 385 00:22:19,510 --> 00:22:22,150 We'll talk about a bifurcation there. 386 00:22:22,150 --> 00:22:25,210 But really, a lot of cellular signaling 387 00:22:25,210 --> 00:22:28,450 is dominated by cells coming-- by signals 388 00:22:28,450 --> 00:22:30,880 coming from outside the cell. 389 00:22:30,880 --> 00:22:35,950 What happens upon this binding event is the transduction. 390 00:22:35,950 --> 00:22:39,070 If you bind to something on the outside of the cell, 391 00:22:39,070 --> 00:22:42,670 as a consequence, you might have a change on that structure. 392 00:22:42,670 --> 00:22:44,860 If it crosses the membrane, you might 393 00:22:44,860 --> 00:22:48,310 have a change on that same molecule structure that's 394 00:22:48,310 --> 00:22:50,180 on the inside of the cell. 395 00:22:50,180 --> 00:22:52,600 So that's why it's called transduction. 396 00:22:52,600 --> 00:22:54,850 You're transducing a soluble signal 397 00:22:54,850 --> 00:22:58,840 from outside, binding that signal to the cell surface 398 00:22:58,840 --> 00:22:59,710 receptor. 399 00:22:59,710 --> 00:23:03,160 And the cell surface receptor is responding in some way. 400 00:23:03,160 --> 00:23:06,010 And there are two principle ways in which 401 00:23:06,010 --> 00:23:08,470 we respond to extracellular signals, 402 00:23:08,470 --> 00:23:10,960 and we'll cover them both. 403 00:23:10,960 --> 00:23:13,000 The next event that might happen is 404 00:23:13,000 --> 00:23:16,120 through the change that happens to the intracellular 405 00:23:16,120 --> 00:23:18,100 component of the receptor. 406 00:23:18,100 --> 00:23:21,460 There might be a change, a binding event, 407 00:23:21,460 --> 00:23:24,320 another step occur within the cell. 408 00:23:24,320 --> 00:23:26,630 And as a function of that, you get a response. 409 00:23:26,630 --> 00:23:27,130 All right? 410 00:23:27,130 --> 00:23:31,300 So it's really-- thinking it in these three components 411 00:23:31,300 --> 00:23:33,760 is a good way to kind of dissect out 412 00:23:33,760 --> 00:23:35,925 the beginnings of the complication. 413 00:23:35,925 --> 00:23:37,300 And then what we'll be able to do 414 00:23:37,300 --> 00:23:41,800 is really start to see what kinds of molecules come in? 415 00:23:41,800 --> 00:23:43,390 How are they received? 416 00:23:43,390 --> 00:23:45,430 How is the signal transduced? 417 00:23:45,430 --> 00:23:49,990 And what's the ultimate outcome with respect to a response? 418 00:23:49,990 --> 00:23:51,840 Everyone OK with that? 419 00:23:51,840 --> 00:23:52,390 All right. 420 00:23:52,390 --> 00:23:55,460 Now this is what you have to look forward to. 421 00:23:55,460 --> 00:23:57,610 So we give you something with three moving parts, 422 00:23:57,610 --> 00:23:59,800 and suddenly we show you something with sort 423 00:23:59,800 --> 00:24:03,100 of, you know, 100 moving parts. 424 00:24:03,100 --> 00:24:05,860 And cell biologists very, very frequently 425 00:24:05,860 --> 00:24:08,370 look at these maps of cells, where 426 00:24:08,370 --> 00:24:10,120 what they're looking at with each 427 00:24:10,120 --> 00:24:13,990 of these sort of little acronyms or names, all of these 428 00:24:13,990 --> 00:24:17,620 are proteins, where they have been mapped out 429 00:24:17,620 --> 00:24:20,860 through cell biology and cellular biochemistry 430 00:24:20,860 --> 00:24:24,150 to be existing in certain components of the cell. 431 00:24:24,150 --> 00:24:26,980 And what has also been mapped out very frequently 432 00:24:26,980 --> 00:24:28,990 is, who talks to who? 433 00:24:28,990 --> 00:24:33,440 So the fact that JAK S might interact with STAT 35 434 00:24:33,440 --> 00:24:34,270 and so on. 435 00:24:34,270 --> 00:24:37,840 So much of this was worked out through cell biology 436 00:24:37,840 --> 00:24:40,940 and biochemistry, and also by genetics. 437 00:24:40,940 --> 00:24:42,610 So Professor Martin has talked to you 438 00:24:42,610 --> 00:24:47,210 about identifying a player in a complex system by genetics. 439 00:24:47,210 --> 00:24:50,050 Let's say you have a cell that fails to divide. 440 00:24:50,050 --> 00:24:54,010 You might perhaps screen or divides unevenly, 441 00:24:54,010 --> 00:24:56,390 or has some defect in cell division. 442 00:24:56,390 --> 00:24:59,770 You might be able to pick out a particular player. 443 00:24:59,770 --> 00:25:04,150 Now, the key thing I want to point out to you with this cell 444 00:25:04,150 --> 00:25:08,410 is what's on the outside of the cell 445 00:25:08,410 --> 00:25:10,780 and runs across the membrane, and might 446 00:25:10,780 --> 00:25:12,940 have the chance, the opportunity, 447 00:25:12,940 --> 00:25:17,020 to have both an extracellular receptor and an intracellular 448 00:25:17,020 --> 00:25:18,100 function. 449 00:25:18,100 --> 00:25:22,270 And those key proteins are things like receptor tyrosine 450 00:25:22,270 --> 00:25:22,840 kinases. 451 00:25:22,840 --> 00:25:26,230 And we're going to talk about all of these in a moment. 452 00:25:26,230 --> 00:25:30,340 G protein-coupled receptors, and various other cell surface 453 00:25:30,340 --> 00:25:31,270 receptors-- 454 00:25:31,270 --> 00:25:32,560 so all of these-- 455 00:25:32,560 --> 00:25:42,800 anything that spans a membrane has the opportunity 456 00:25:42,800 --> 00:25:45,965 to be an important component of a signaling pathway. 457 00:25:48,590 --> 00:25:50,960 Because what you're routinely trying to do 458 00:25:50,960 --> 00:25:55,250 is have your signal recognized on the outside of the cell 459 00:25:55,250 --> 00:25:57,770 by something that spans the membrane. 460 00:25:57,770 --> 00:26:01,340 The signal will bind to that. 461 00:26:01,340 --> 00:26:04,100 And then you will have an intracellular response. 462 00:26:04,100 --> 00:26:05,340 So that's breaking it down. 463 00:26:05,340 --> 00:26:08,030 That's why proteins that are made 464 00:26:08,030 --> 00:26:10,310 through the secretory pathway that we talked 465 00:26:10,310 --> 00:26:12,440 about in the last lecture, that go 466 00:26:12,440 --> 00:26:14,780 through that endomembrane system, 467 00:26:14,780 --> 00:26:17,810 and end up being parked in the plasma membrane 468 00:26:17,810 --> 00:26:19,550 are so important. 469 00:26:19,550 --> 00:26:23,720 Other proteins that actually get secreted through that pathway 470 00:26:23,720 --> 00:26:24,630 are also important. 471 00:26:24,630 --> 00:26:27,300 What do you think they may be important for? 472 00:26:27,300 --> 00:26:29,988 Let's say you've made a protein within the cell. 473 00:26:29,988 --> 00:26:31,280 It goes through all the system. 474 00:26:31,280 --> 00:26:34,370 It doesn't stay parked in the cell membrane. 475 00:26:34,370 --> 00:26:36,400 It actually gets released from the cell. 476 00:26:36,400 --> 00:26:39,210 What might that be doing? 477 00:26:39,210 --> 00:26:40,085 AUDIENCE: [INAUDIBLE] 478 00:26:40,085 --> 00:26:41,085 BARBARA IMPERIALI: Yeah. 479 00:26:41,085 --> 00:26:41,600 Exactly. 480 00:26:41,600 --> 00:26:44,180 So that endomembrane system that I 481 00:26:44,180 --> 00:26:48,350 described to you, that pathway is great for making receivers. 482 00:26:48,350 --> 00:26:50,480 And it's great for making signals. 483 00:26:50,480 --> 00:26:55,450 And that's really what can sort of fuel the functions of cells. 484 00:26:55,450 --> 00:26:56,120 OK. 485 00:26:56,120 --> 00:26:59,240 So in systems biology, you may have heard this term 486 00:26:59,240 --> 00:27:00,530 quite frequently. 487 00:27:00,530 --> 00:27:04,790 Systems biology is research that helps 488 00:27:04,790 --> 00:27:07,940 us understand the underlying structure of signaling 489 00:27:07,940 --> 00:27:09,170 networks. 490 00:27:09,170 --> 00:27:11,420 So a lot of people who have common interests 491 00:27:11,420 --> 00:27:15,920 in engineering, computational analysis and cell biology, 492 00:27:15,920 --> 00:27:18,920 might bring in data to allow them 493 00:27:18,920 --> 00:27:21,440 to make models of cellular systems, 494 00:27:21,440 --> 00:27:24,890 to understand flux through signaling pathways. 495 00:27:24,890 --> 00:27:27,470 So they may make fundamental measurements 496 00:27:27,470 --> 00:27:31,430 about the concentrations of some components within the cell. 497 00:27:31,430 --> 00:27:34,580 And then try to say, OK, I know based on everything 498 00:27:34,580 --> 00:27:39,350 I've measured that this is a dominant pathway for gene 499 00:27:39,350 --> 00:27:40,560 regulation. 500 00:27:40,560 --> 00:27:44,330 And I could control this pathway by different-- 501 00:27:44,330 --> 00:27:48,140 by sort of different types of interactions. 502 00:27:48,140 --> 00:27:51,630 In this cellular system, I also show you another component, 503 00:27:51,630 --> 00:27:53,350 which is the nucleus. 504 00:27:53,350 --> 00:27:56,660 And when we discuss and describe specific cell 505 00:27:56,660 --> 00:27:59,270 signaling networks, in some cases 506 00:27:59,270 --> 00:28:02,030 the signaling network may involve 507 00:28:02,030 --> 00:28:06,650 receiving a signal, undergoing a variety of changes 508 00:28:06,650 --> 00:28:09,350 in the cytoplasm, but then a change that 509 00:28:09,350 --> 00:28:14,060 eventually results in a protein going to the nucleus. 510 00:28:14,060 --> 00:28:17,770 And oftentimes, those proteins that run into the nucleus 511 00:28:17,770 --> 00:28:23,270 are transcription factors that then trigger DNA replication 512 00:28:23,270 --> 00:28:24,830 or transcription. 513 00:28:24,830 --> 00:28:27,180 And then promote activities. 514 00:28:27,180 --> 00:28:28,790 So this is how you think about it. 515 00:28:28,790 --> 00:28:32,150 When you think of cellular signaling, it's really about, 516 00:28:32,150 --> 00:28:34,040 what does the signal need to do? 517 00:28:34,040 --> 00:28:39,090 And what's the pathway that I follow to get there? 518 00:28:39,090 --> 00:28:40,830 So all of those are membrane proteins. 519 00:28:40,830 --> 00:28:44,310 So now let's look at the canonical aspects 520 00:28:44,310 --> 00:28:46,240 of signal transduction. 521 00:28:46,240 --> 00:28:49,320 So the first-- and I'm going to rely on these little cartoons. 522 00:28:49,320 --> 00:28:53,430 But I want, both in this lecture and the next, 523 00:28:53,430 --> 00:28:57,200 to really show you where these recur in so many systems. 524 00:28:57,200 --> 00:29:02,150 So to that purpose, I want to talk about the characteristics. 525 00:29:08,290 --> 00:29:11,560 So the first critical characteristic 526 00:29:11,560 --> 00:29:14,440 is a signal and it's specificity. 527 00:29:26,150 --> 00:29:28,150 So a signal will be something that 528 00:29:28,150 --> 00:29:29,740 comes from outside of the cell. 529 00:29:29,740 --> 00:29:32,800 It could be a hormone that's produced in the hypothalamus 530 00:29:32,800 --> 00:29:34,570 and sent to another organ. 531 00:29:34,570 --> 00:29:37,710 But the most important thing about the signal 532 00:29:37,710 --> 00:29:42,190 is that that signal, which binds to a receptor in a cell 533 00:29:42,190 --> 00:29:47,190 membrane, is specific for a particular receptor, 534 00:29:47,190 --> 00:29:50,080 and the different signal won't blind to the same receptor. 535 00:29:50,080 --> 00:29:53,950 You have to have faithful signal specificity 536 00:29:53,950 --> 00:29:55,760 to trigger the right function. 537 00:29:55,760 --> 00:29:59,530 So if it's a hormone, it's got to be the hormone that you want 538 00:29:59,530 --> 00:30:02,710 to trigger the receptor, not a related but different-looking 539 00:30:02,710 --> 00:30:03,640 structure. 540 00:30:03,640 --> 00:30:05,410 If it's a small protein, you want 541 00:30:05,410 --> 00:30:09,597 it to be the exact one that binds with high specificity 542 00:30:09,597 --> 00:30:10,180 to a receptor. 543 00:30:16,890 --> 00:30:21,480 So what that means is if something is binding-- 544 00:30:21,480 --> 00:30:23,850 if a small molecule is binding to a protein 545 00:30:23,850 --> 00:30:27,150 on the surface of a cell with high specificity 546 00:30:27,150 --> 00:30:31,350 and high affinity, it means that even at a low concentration, 547 00:30:31,350 --> 00:30:34,140 it will make that binding contact. 548 00:30:34,140 --> 00:30:37,890 But all the other small molecules that are around 549 00:30:37,890 --> 00:30:41,220 won't crosstalk into that triggering that interaction. 550 00:30:41,220 --> 00:30:46,290 So we have high specificity, and we gain that specificity 551 00:30:46,290 --> 00:30:48,180 through macromolecular interactions, 552 00:30:48,180 --> 00:30:51,450 just like the ones we talked about in biochemistry. 553 00:30:51,450 --> 00:30:53,970 So if we have a small molecule or a protein bind 554 00:30:53,970 --> 00:30:58,680 to the receptor, it's making all those hydrogen bonding, 555 00:30:58,680 --> 00:31:01,920 electrostatic, noncovalent types of interactions 556 00:31:01,920 --> 00:31:07,740 with high specificity, so that a low concentration of the signal 557 00:31:07,740 --> 00:31:14,430 molecule is efficient for binding to the receptor 558 00:31:14,430 --> 00:31:16,410 to trigger the function. 559 00:31:16,410 --> 00:31:22,220 The next characteristic is amplification. 560 00:31:28,052 --> 00:31:31,000 Now let's put some lines between these guys. 561 00:31:35,450 --> 00:31:37,520 Now, with all the signaling pathways 562 00:31:37,520 --> 00:31:39,560 that you're going to see, we're going 563 00:31:39,560 --> 00:31:43,730 to be looking where in a pathway you get amplification. 564 00:31:43,730 --> 00:31:46,400 Very commonly, you might have a response 565 00:31:46,400 --> 00:31:50,740 that's just the result of a single molecule binding 566 00:31:50,740 --> 00:31:52,390 a single receptor. 567 00:31:52,390 --> 00:31:55,300 But at the end of the day, you might want a large response. 568 00:31:55,300 --> 00:31:57,640 You might want to make a lot of ATP. 569 00:31:57,640 --> 00:32:01,420 Or you might want to replicate all of the genome. 570 00:32:01,420 --> 00:32:04,390 So you need some kind of amplification where in a sense 571 00:32:04,390 --> 00:32:06,910 you're turning up the volume on your signal. 572 00:32:06,910 --> 00:32:08,920 And you need to do that rapidly. 573 00:32:08,920 --> 00:32:11,800 So frequently in signaling pathways, 574 00:32:11,800 --> 00:32:15,040 you go through a cascade of reactions 575 00:32:15,040 --> 00:32:17,890 where the signal might affect an enzyme. 576 00:32:17,890 --> 00:32:20,290 But once you make that enzyme active, 577 00:32:20,290 --> 00:32:23,590 it might work on many, many copies of another enzyme. 578 00:32:23,590 --> 00:32:26,660 And then each of those may work on even more copies. 579 00:32:26,660 --> 00:32:28,930 So that's what I mean by amplification, 580 00:32:28,930 --> 00:32:30,670 where at some stage you've generated 581 00:32:30,670 --> 00:32:36,380 a molecule that can result in the cascade of a reaction. 582 00:32:36,380 --> 00:32:41,990 So we often refer to these as cascades. 583 00:32:41,990 --> 00:32:44,450 So if you're Spanish-speaking, cascada. 584 00:32:44,450 --> 00:32:47,270 You want to think about a waterfall coming from just 585 00:32:47,270 --> 00:32:49,160 a single molecule of water. 586 00:32:49,160 --> 00:32:52,400 You're getting a large increase in your signal 587 00:32:52,400 --> 00:32:54,740 as a result of amplification. 588 00:32:54,740 --> 00:32:58,730 The next feature or characteristic of signaling 589 00:32:58,730 --> 00:32:59,280 is feedback. 590 00:33:06,290 --> 00:33:10,760 At the end of the day, if you're signaling, 591 00:33:10,760 --> 00:33:12,230 I got to make some ATP. 592 00:33:12,230 --> 00:33:14,210 I got to run out of the woods. 593 00:33:14,210 --> 00:33:15,410 I'm getting chased. 594 00:33:15,410 --> 00:33:17,360 At a certain stage, you need to stop 595 00:33:17,360 --> 00:33:19,550 all of the process occurring. 596 00:33:19,550 --> 00:33:23,390 So feedback is just a negative feedback loop 597 00:33:23,390 --> 00:33:27,800 that might slow down some of those steps that 598 00:33:27,800 --> 00:33:29,420 are involved in amplification. 599 00:33:29,420 --> 00:33:33,290 So for a pathway, you only want the pathway turned on 600 00:33:33,290 --> 00:33:35,390 for a prescribed amount of time. 601 00:33:35,390 --> 00:33:37,820 And then you want to be able to say, 602 00:33:37,820 --> 00:33:39,550 I'm done with that whole pathway. 603 00:33:39,550 --> 00:33:42,840 I don't need to keep churning through all those enzymes. 604 00:33:42,840 --> 00:33:43,970 It's time to stop that. 605 00:33:43,970 --> 00:33:47,270 And that usually occurs through negative feedback. 606 00:33:47,270 --> 00:33:49,850 And remember, we talked about negative feedback 607 00:33:49,850 --> 00:33:52,910 when we were talking about enzyme catalyzed pathways. 608 00:33:52,910 --> 00:33:55,850 So feedback is very often some kind 609 00:33:55,850 --> 00:34:02,180 of negative feedback, which suppresses 610 00:34:02,180 --> 00:34:04,760 a series of transformations, perhaps 611 00:34:04,760 --> 00:34:07,940 through a product of those transformations acting 612 00:34:07,940 --> 00:34:10,370 as an inhibitor on an early step. 613 00:34:10,370 --> 00:34:13,250 And then finally, the other component 614 00:34:13,250 --> 00:34:14,850 of a signaling network-- 615 00:34:14,850 --> 00:34:16,850 if you think of signaling networks 616 00:34:16,850 --> 00:34:20,520 as electronic structures, you have integration. 617 00:34:20,520 --> 00:34:23,495 So that's the last characteristic feature. 618 00:34:31,804 --> 00:34:38,830 And let me go back to that big circuit 619 00:34:38,830 --> 00:34:43,159 diagram quickly to show you an example of integration. 620 00:34:43,159 --> 00:34:45,489 So if you look at this signaling pathway, 621 00:34:45,489 --> 00:34:48,340 all these signaling steps are not single. 622 00:34:48,340 --> 00:34:51,260 You just have a signal come in, and you end up, 623 00:34:51,260 --> 00:34:52,989 for example, in the nucleus. 624 00:34:52,989 --> 00:34:56,500 But rather, other components may have crosstalk 625 00:34:56,500 --> 00:34:59,140 within one pathway, and start out 626 00:34:59,140 --> 00:35:01,510 either amplifying or turning down 627 00:35:01,510 --> 00:35:03,520 a particular signaling pathway. 628 00:35:03,520 --> 00:35:04,780 So these are networks. 629 00:35:04,780 --> 00:35:06,610 They're not pathways. 630 00:35:06,610 --> 00:35:09,910 They're networks that interact and communicate, 631 00:35:09,910 --> 00:35:12,730 all to amplify signals or turn down signals. 632 00:35:12,730 --> 00:35:16,870 So integration is an important part of signaling, 633 00:35:16,870 --> 00:35:20,500 because you're often dealing with the integrated function 634 00:35:20,500 --> 00:35:25,060 of a number of pathways to get a particular response. 635 00:35:25,060 --> 00:35:28,450 And that actually ends up being one of the situations where 636 00:35:28,450 --> 00:35:31,000 sometimes a particular enzyme may 637 00:35:31,000 --> 00:35:35,510 look like a perfect target for a therapeutic agent. 638 00:35:35,510 --> 00:35:38,830 But if you don't take into account the integration steps, 639 00:35:38,830 --> 00:35:40,540 you may be trying to deal with a-- 640 00:35:40,540 --> 00:35:43,150 you may think you're dealing with a single pathway, 641 00:35:43,150 --> 00:35:45,520 but you're, rather, dealing with crosstalk 642 00:35:45,520 --> 00:35:47,740 with a lot of other pathways. 643 00:35:47,740 --> 00:35:49,900 And what often happens in a cell is 644 00:35:49,900 --> 00:35:52,910 there's compensation from other pathways. 645 00:35:52,910 --> 00:35:54,570 Is everybody following? 646 00:35:54,570 --> 00:35:57,380 Any questions here about this? 647 00:35:57,380 --> 00:35:59,320 So what I want you to think about 648 00:35:59,320 --> 00:36:03,400 is that it's just amazing what is orchestrated 649 00:36:03,400 --> 00:36:06,350 to have even the simplest functions in the cell, 650 00:36:06,350 --> 00:36:10,510 how many interacting components there may be. 651 00:36:10,510 --> 00:36:15,880 Specificity, amplification, feedback, and integration-- 652 00:36:15,880 --> 00:36:19,300 all right, so let's talk briefly about types of signals 653 00:36:19,300 --> 00:36:22,300 and how we name them, where they come from, in order 654 00:36:22,300 --> 00:36:24,970 to make sure we're all on the same page with respect 655 00:36:24,970 --> 00:36:27,550 to the language that's used. 656 00:36:27,550 --> 00:36:38,760 So now, signals may take different molecular forms. 657 00:36:38,760 --> 00:36:47,140 They may be small molecules, for example, an amino acid 658 00:36:47,140 --> 00:36:49,990 or a phospholipid-- 659 00:36:49,990 --> 00:36:51,370 just something little. 660 00:36:51,370 --> 00:36:53,880 Alternatively, they may be proteins. 661 00:36:57,240 --> 00:37:01,060 They may be carbohydrates. 662 00:37:01,060 --> 00:37:03,520 They might take different forms in terms 663 00:37:03,520 --> 00:37:05,440 of their molecular structure. 664 00:37:05,440 --> 00:37:09,653 But we tend to describe signals by where they come from. 665 00:37:09,653 --> 00:37:11,320 So what I've shown you here is a picture 666 00:37:11,320 --> 00:37:13,510 from the book that just describes how 667 00:37:13,510 --> 00:37:17,200 we refer to certain signals. 668 00:37:17,200 --> 00:37:19,345 So there are four different terms-- 669 00:37:22,780 --> 00:37:36,920 autocrine, juxtacrine-- and I'm going to just give you a little 670 00:37:36,920 --> 00:37:41,570 hint to how to remember these terms-- 671 00:37:41,570 --> 00:37:43,220 paracrine, endocrine. 672 00:37:47,380 --> 00:37:48,380 OK. 673 00:37:48,380 --> 00:37:52,940 So these don't tell you anything about the molecule. 674 00:37:52,940 --> 00:37:55,760 They tell you about where it's come from. 675 00:37:55,760 --> 00:37:58,640 So an autocrine signal is a signal 676 00:37:58,640 --> 00:38:03,440 that may come from a cell, but it's signaling to itself. 677 00:38:03,440 --> 00:38:07,910 So it may produce a component that's released. 678 00:38:07,910 --> 00:38:11,510 And so it's producing this through a secretory pathway. 679 00:38:11,510 --> 00:38:14,850 It's a release, and it stays in the vicinity of the cell. 680 00:38:14,850 --> 00:38:17,180 So the self is self-signaling. 681 00:38:17,180 --> 00:38:22,040 So whenever you see something auto, 682 00:38:22,040 --> 00:38:23,960 you just want to say, oh, that means 683 00:38:23,960 --> 00:38:27,900 it's coming from the same cell where the signal occurs. 684 00:38:27,900 --> 00:38:31,790 Let's move to the next one, which is paracrine. 685 00:38:31,790 --> 00:38:33,380 I'm going to talk about jux-- 686 00:38:33,380 --> 00:38:36,020 and that's usually from a nearby cell, 687 00:38:36,020 --> 00:38:38,240 not a cell that's in contact-- 688 00:38:38,240 --> 00:38:39,630 definitely a different cell. 689 00:38:39,630 --> 00:38:45,460 So paracrine is-- we would always call nearby. 690 00:38:45,460 --> 00:38:48,740 And endocrine is completely from somewhere else, so 691 00:38:48,740 --> 00:38:52,160 perhaps coming through the circulatory system. 692 00:38:52,160 --> 00:38:56,330 One cell may release an endocrine signal. 693 00:38:56,330 --> 00:38:59,930 It may weave its way through the vascular system, 694 00:38:59,930 --> 00:39:02,860 and then target a cell. 695 00:39:02,860 --> 00:39:04,985 So endocrine is always from a distance. 696 00:39:08,640 --> 00:39:11,520 And juxtacrine is the only one that's a little odd. 697 00:39:11,520 --> 00:39:14,400 It's really from cells that actually 698 00:39:14,400 --> 00:39:16,690 are in contact with each other. 699 00:39:16,690 --> 00:39:19,840 So it's not self-signaling within a cell. 700 00:39:19,840 --> 00:39:23,730 It's not a cell that's nearby but pretty close. 701 00:39:23,730 --> 00:39:26,590 It's actually physically making a contact. 702 00:39:26,590 --> 00:39:29,280 And so that's the last terminology there. 703 00:39:29,280 --> 00:39:32,700 So hopefully, I can get this calcium wave to show you. 704 00:39:32,700 --> 00:39:40,310 This is just a video of juxtacrine to signaling. 705 00:39:40,310 --> 00:39:42,640 I just want you to sort of keep an eye on things. 706 00:39:42,640 --> 00:39:44,230 It's usually a cell. 707 00:39:44,230 --> 00:39:46,960 What you're observing here is a dye 708 00:39:46,960 --> 00:39:49,810 that lights up in the presence of calcium flux. 709 00:39:49,810 --> 00:39:51,610 It's called Fura-2. 710 00:39:51,610 --> 00:39:55,780 And so when you stare at these for long enough, what 711 00:39:55,780 --> 00:39:59,800 you can notice is that when one signal will often 712 00:39:59,800 --> 00:40:02,920 come from an adjacent cell right near it-- 713 00:40:02,920 --> 00:40:04,540 so there are long prostheses. 714 00:40:04,540 --> 00:40:07,000 You're not looking at the entire cell, 715 00:40:07,000 --> 00:40:09,280 but they're definitely-- for example, this little duo 716 00:40:09,280 --> 00:40:11,890 down here, they keep signaling to each other. 717 00:40:11,890 --> 00:40:14,680 And that's just a juxtacrine signaling, because the cells 718 00:40:14,680 --> 00:40:16,540 are in the contact. 719 00:40:16,540 --> 00:40:18,730 So that just shows you the difference there. 720 00:40:18,730 --> 00:40:22,570 If it was autocrine, you just have a single cell responding. 721 00:40:22,570 --> 00:40:25,180 If it's paracrine, they would be at more of a distance 722 00:40:25,180 --> 00:40:26,980 to each other. 723 00:40:26,980 --> 00:40:33,730 I hope that imagery-- this is from a website in the Smith Lab 724 00:40:33,730 --> 00:40:36,700 at Stanford. 725 00:40:36,700 --> 00:40:37,450 OK. 726 00:40:37,450 --> 00:40:40,750 And then the last thing I want to give you an example, 727 00:40:40,750 --> 00:40:43,600 there are many, many hormones in the body that 728 00:40:43,600 --> 00:40:49,180 undergo endocrine signaling, and so one example I thought 729 00:40:49,180 --> 00:40:51,670 I would tell you about, you all know that insulin 730 00:40:51,670 --> 00:40:54,160 is made in the pancreas. 731 00:40:54,160 --> 00:40:58,300 It's an important hormone for regulating glucose levels. 732 00:40:58,300 --> 00:41:02,650 And it's actually-- functions at the muscle level. 733 00:41:02,650 --> 00:41:06,340 So insulin is an example of an endocrine signal, 734 00:41:06,340 --> 00:41:08,830 because it travels a distance from where 735 00:41:08,830 --> 00:41:13,165 it's made in the body to where it functions in the body. 736 00:41:13,165 --> 00:41:14,620 All right. 737 00:41:14,620 --> 00:41:19,520 Now-- so we've talked about the types of signals. 738 00:41:19,520 --> 00:41:23,090 Let's now move to the types of receptors. 739 00:41:28,660 --> 00:41:40,160 Now, we cover both the intracellular 740 00:41:40,160 --> 00:41:42,200 and the cell surface receptors. 741 00:41:48,200 --> 00:41:52,130 But we really will focus a lot on the cell surface receptors. 742 00:41:52,130 --> 00:41:55,700 I just want to give you a clue that not all signaling is 743 00:41:55,700 --> 00:41:56,610 cell surface. 744 00:41:56,610 --> 00:41:59,030 So what I've shown you here is a cartoon 745 00:41:59,030 --> 00:42:01,850 where you see signaling, where a signal 746 00:42:01,850 --> 00:42:03,830 comes from outside the cell. 747 00:42:03,830 --> 00:42:06,830 It goes into the cell and triggers a change. 748 00:42:06,830 --> 00:42:09,120 And then the majority of the time 749 00:42:09,120 --> 00:42:10,910 we'll talk about these receptors that 750 00:42:10,910 --> 00:42:13,250 are in the plasma membrane. 751 00:42:13,250 --> 00:42:17,000 And they have an outside place where the signal binds, 752 00:42:17,000 --> 00:42:19,490 and they trigger a response inside. 753 00:42:19,490 --> 00:42:22,190 And it's only very specific signals 754 00:42:22,190 --> 00:42:26,990 that are able to signal intracellularly, 755 00:42:26,990 --> 00:42:31,490 that is, to cross the membrane to get inside the cytoplasm 756 00:42:31,490 --> 00:42:32,570 to do the triggering. 757 00:42:32,570 --> 00:42:35,675 What kinds of molecules can cross the membrane easily? 758 00:42:38,510 --> 00:42:40,160 We talked about that before, when 759 00:42:40,160 --> 00:42:43,435 we talked about getting across that barrier. 760 00:42:46,150 --> 00:42:47,620 Yeah? 761 00:42:47,620 --> 00:42:48,460 Nonpolar. 762 00:42:48,460 --> 00:42:49,060 OK. 763 00:42:49,060 --> 00:42:51,950 So you can look at a-- you can stare at a molecule, 764 00:42:51,950 --> 00:42:55,070 and if it's very polar or pretty large, 765 00:42:55,070 --> 00:42:57,550 it's not going to be able to sneak through a membrane. 766 00:42:57,550 --> 00:43:00,580 So something like a steroid molecule, 767 00:43:00,580 --> 00:43:03,370 a large, greasy molecule, can definitely 768 00:43:03,370 --> 00:43:04,870 make that transition. 769 00:43:04,870 --> 00:43:07,810 And so those are the only types of signals that we can really 770 00:43:07,810 --> 00:43:12,590 do inside the cell, because they can get across the cell. 771 00:43:12,590 --> 00:43:16,090 Many, many other signals have to go through this-- 772 00:43:16,090 --> 00:43:19,010 bind to the outside of a cell, and transduce a signal 773 00:43:19,010 --> 00:43:20,710 to the inside of the cell. 774 00:43:20,710 --> 00:43:28,060 So one very typical signal that can bind to an intracellular 775 00:43:28,060 --> 00:43:30,280 receptor is a steroid. 776 00:43:30,280 --> 00:43:34,810 So remember when I talked to you about these lipidic molecules, 777 00:43:34,810 --> 00:43:37,480 things like testosterone and cortisol. 778 00:43:37,480 --> 00:43:40,390 These are very hydrophobic molecules. 779 00:43:40,390 --> 00:43:44,110 So they literally can cross from the outside of the cell 780 00:43:44,110 --> 00:43:45,980 without a transporter. 781 00:43:45,980 --> 00:43:48,390 So for example, the hormone cortisol. 782 00:43:48,390 --> 00:43:51,060 And when that functions, it just-- 783 00:43:51,060 --> 00:43:53,720 an amount of it becomes available, 784 00:43:53,720 --> 00:43:55,450 for example, in the bloodstream. 785 00:43:55,450 --> 00:43:57,970 It crosses the cell, and it binds 786 00:43:57,970 --> 00:44:01,100 to an intracellular receptor. 787 00:44:01,100 --> 00:44:04,090 Once it binds to that intracellular receptor, 788 00:44:04,090 --> 00:44:08,140 this disengages a different kind of chaperone protein 789 00:44:08,140 --> 00:44:10,030 that's keeping it stable. 790 00:44:10,030 --> 00:44:14,920 Once it's found, it can then go into the nucleus and trigger 791 00:44:14,920 --> 00:44:16,220 transcription. 792 00:44:16,220 --> 00:44:19,930 So this is the one example of an intracellular receptor 793 00:44:19,930 --> 00:44:21,197 that we'll talk about. 794 00:44:21,197 --> 00:44:22,780 I just wanted to show you a little bit 795 00:44:22,780 --> 00:44:24,860 about the steroid receptors. 796 00:44:24,860 --> 00:44:28,660 These are molecules that are very-- 797 00:44:28,660 --> 00:44:33,650 these are macromolecules, proteins that are very-- 798 00:44:33,650 --> 00:44:35,230 quite a complex structure. 799 00:44:35,230 --> 00:44:37,480 But they can literally-- and I'll show you the picture 800 00:44:37,480 --> 00:44:40,330 at the beginning of the talk next time-- 801 00:44:40,330 --> 00:44:43,930 they can literally engulf these proteins. 802 00:44:43,930 --> 00:44:46,090 So once the steroid is bound to that, 803 00:44:46,090 --> 00:44:48,190 it completely changes shape. 804 00:44:48,190 --> 00:44:50,500 And that's what enables the change for it 805 00:44:50,500 --> 00:44:54,770 to be triggered and sent to the nucleus. 806 00:44:54,770 --> 00:44:58,810 Now, the key types of receptors that we'll focus on, though, 807 00:44:58,810 --> 00:45:01,060 are the cell surface receptors. 808 00:45:01,060 --> 00:45:03,430 And there are three basic classes 809 00:45:03,430 --> 00:45:06,910 of molecules that occur in the plasma membrane that 810 00:45:06,910 --> 00:45:09,130 are critical for cellular signaling. 811 00:45:09,130 --> 00:45:12,490 They are the G protein-coupled receptors, 812 00:45:12,490 --> 00:45:15,010 the receptor tyrosine kinases, and then 813 00:45:15,010 --> 00:45:19,720 you will talk in the lecture 22 about ion channels, 814 00:45:19,720 --> 00:45:22,720 and how they perform a receptor function. 815 00:45:22,720 --> 00:45:25,010 So the membrane proteins, first of all, 816 00:45:25,010 --> 00:45:27,390 I want to underscore their importance. 817 00:45:27,390 --> 00:45:33,310 50%-- they comprise 50% of drug targets, 818 00:45:33,310 --> 00:45:36,850 the receptor tyrosine kinases and the G protein-coupled 819 00:45:36,850 --> 00:45:38,080 receptors. 820 00:45:38,080 --> 00:45:44,350 The G protein-coupled receptors have this 7 transmembrane helix 821 00:45:44,350 --> 00:45:48,170 structure, which spans a membrane. 822 00:45:48,170 --> 00:45:53,700 This would be the outside of the cell, 823 00:45:53,700 --> 00:45:56,430 and the inside of the cells-- so there's 824 00:45:56,430 --> 00:45:58,320 signals going across there. 825 00:45:58,320 --> 00:46:02,280 The receptor tyrosine kinases are another important type 826 00:46:02,280 --> 00:46:03,330 of receptor. 827 00:46:03,330 --> 00:46:06,180 They are dimeric proteins that in the presence 828 00:46:06,180 --> 00:46:10,320 of a ligand dimerize, and then cause intracellular signaling. 829 00:46:10,320 --> 00:46:13,800 Once again, they cross the plasma membrane 830 00:46:13,800 --> 00:46:15,900 from the outside to the cytosol. 831 00:46:15,900 --> 00:46:19,410 And then lastly, there are the ion channels, which also 832 00:46:19,410 --> 00:46:21,360 may cross the plasma membrane. 833 00:46:21,360 --> 00:46:24,370 And when you think about these classes of proteins, 834 00:46:24,370 --> 00:46:27,060 there's a tremendous amount to be learned with respect 835 00:46:27,060 --> 00:46:28,440 to their functions. 836 00:46:28,440 --> 00:46:30,600 And they are so important to understand 837 00:46:30,600 --> 00:46:33,630 their physical functions in the body, 838 00:46:33,630 --> 00:46:37,920 because they really represent the place, the nexus, where 839 00:46:37,920 --> 00:46:40,030 signaling happens in the cell. 840 00:46:40,030 --> 00:46:44,520 So I want to briefly show you a picture of a GPCR. 841 00:46:44,520 --> 00:46:48,180 It's a 7 transmembrane helix structure. 842 00:46:48,180 --> 00:46:50,250 You can see it here. 843 00:46:50,250 --> 00:46:54,180 There are about 30% of modern drugs actually target 844 00:46:54,180 --> 00:46:55,710 the GPCRs. 845 00:46:55,710 --> 00:46:59,490 And here, I'm just going to show you the structure of a GPCR. 846 00:46:59,490 --> 00:47:02,490 Those are the 7 transmembrane helices. 847 00:47:02,490 --> 00:47:06,300 If you stretch them out, that's about the width of a membrane. 848 00:47:06,300 --> 00:47:08,400 That's typical of a signal that would 849 00:47:08,400 --> 00:47:11,620 bind to that kind of receptor. 850 00:47:11,620 --> 00:47:13,330 This is a chemokine. 851 00:47:13,330 --> 00:47:15,360 It's a small protein receptor. 852 00:47:15,360 --> 00:47:17,520 So you can see that structure and how 853 00:47:17,520 --> 00:47:22,470 it would go from one side of the membrane to the other. 854 00:47:22,470 --> 00:47:27,210 In it's bound state, the chemokine 855 00:47:27,210 --> 00:47:31,860 binds to the 7 transmembrane helix receptor 856 00:47:31,860 --> 00:47:33,600 through kind of a clamping action. 857 00:47:33,600 --> 00:47:35,610 The magenta is the chemokine. 858 00:47:35,610 --> 00:47:40,710 The blue and the green space filled parts are actually 859 00:47:40,710 --> 00:47:44,190 what holds the chemokine. 860 00:47:44,190 --> 00:47:46,620 And if you look at it where the membrane would be, 861 00:47:46,620 --> 00:47:48,780 you can see how you can transduce 862 00:47:48,780 --> 00:47:52,320 a signal from one side of the membrane to the other, 863 00:47:52,320 --> 00:47:54,960 by the binding of the magenta molecule 864 00:47:54,960 --> 00:47:59,640 to the outside of the cell, to those loops outside the cell. 865 00:47:59,640 --> 00:48:02,220 That would have a significant perturbation 866 00:48:02,220 --> 00:48:06,690 to the biology and chemistry of what's going on on the inside. 867 00:48:06,690 --> 00:48:09,540 So next class, we'll talk about pathways 868 00:48:09,540 --> 00:48:13,710 that are initiated by these G protein-coupled receptors, 869 00:48:13,710 --> 00:48:16,810 and what that terminology means. 870 00:48:16,810 --> 00:48:18,360 OK.