Introduction to Apoptosis

Part 1: Introduction to Apoptosis

00:00:04.18 Hi. My name is Xiaodong Wang. I work at Howard Hughes Medical Institute at UT-Southwestern Medical Center in Dallas, Texas.
00:00:14.12 And today, we are going to talk about apoptosis, which is a form of programmed cell death,
00:00:21.25 that, a death that executed by an intrinsic biochemical program inside our cells.
00:00:29.07 Apoptosis actually plays a very important for the well-being of a whole organism, including us.
00:00:40.02 And in here, I've listed several examples the function of apoptosis.
00:00:47.17 During normal development, apoptosis play a very critical role in shaping up the shape of our organs and bodies.
00:00:58.20 I'm going to give you a couple more visual examples in the next few slides.
00:01:04.24 And also for the immune system function. After our body encounters a foreign antigen,
00:01:12.00 either from a bacteria or virus, we will generate specific T-cells, lots of T-cells and B-cells to counter that antigen.
00:01:21.22 But once that antigen is eliminated, these cells need to be eliminated from the body as well, and they undergo apoptosis.
00:01:32.03 Also, tissue homeostasis and we know once we reach adult size,
00:01:40.06 our, the size of the organs will stay the same. But, in some of our organs, like skin and liver,
00:01:48.02 the cells continue to divide. And these newly generated cells have to be balanced by the apoptosis of some of the old ones.
00:01:56.21 And also apoptosis plays a critical role in eliminating the damaged cells in our body.
00:02:04.03 For example, the sunburn cells, when we walk outdoors under Texas sun for five minutes, for example, in the summertime,
00:02:13.08 we will get sunburned. That's because the cells in our skin will respond to the damage caused by the UV light.
00:02:22.05 They will activate their apoptotic program and die and that's how get sunburns.
00:02:28.14 Also, the chemical and the radiation and the virus damages cells, needs to be eliminated by apoptosis.
00:02:37.04 And if they cannot be eliminated by apoptosis properly, then these damages may cause human disease.
00:02:46.23 Since apoptosis are very important for many of these physiological functions of a whole organism,
00:02:54.22 if something is wrong with apoptosis, then there are many forms of human disease that can arise.
00:03:02.19 And for example, in case of cancer or autoimmune disease, the apoptotic program cannot be executed properly
00:03:12.24 and that will result in too many cells in these cases and that contributes to the disease of cancer and autoimmune disease.
00:03:22.20 On the other side, the flip side of the coin, such as radiation damage, or ischemia reperfusion during stroke or heart attack,
00:03:33.29 and the cells undergo apoptosis improperly and prematurely, and that will cause permanent damage.
00:03:44.13 So, as you can see, the proper balance of apoptosis is very, very important for the well-beings of a whole organism.
00:03:55.23 And if there are something wrong with the program, there are many disease can arise.
00:04:03.04 So, one purpose of study apoptosis and trying to understand the molecular details of the apoptotic program
00:04:13.28 is to understand what's wrong in these disease states and hopefully to come out with strategies and to correct these defects
00:04:24.18 and battle these diseases. And in then end of my talk, I'm going to give you two specific examples and how we might be able to do that.
00:04:33.10 So, here is just two visual demonstrations that if apoptosis cannot happen properly during development, what will happen.
00:04:45.26 And in this mouse, has a gene that is required for apoptosis knocked out from their genome.
00:04:54.02 As a result, this mouse has too much brain tissue, so they grow out of the skull.
00:04:59.28 And you may already know that more than half of neurons that were generated has to die by apoptosis even before we are born.
00:05:11.17 And if that process cannot happen properly and you are not going to get a normal animal.
00:05:18.28 Another well-known example is the recession of the web, interdigital web,
00:05:25.09 and the cells without this web have to undergo apoptosis at a particular time to give us this nice fingers and toes.
00:05:34.19 So, we know apoptosis is important and we really want to study the molecular details about apoptosis,
00:05:46.20 how does this all start? Apoptosis, the initial description of apoptosis is mostly based on its morphological changes
00:05:59.17 associated with this form of cell death.
00:06:02.02 And these morphological changes will include the cell shrinkage,
00:06:06.13 the membrane blebbing, meaning that in normal cells, the membrane is relatively smooth and rigid,
00:06:14.02 but during apoptosis, the membrane start waving and we call that membrane blebbing.
00:06:18.29 And some of the organelles in a cell, such as nuclear membrane, will break down during apoptosis.
00:06:25.29 The chromatin will condense and also finally, you will form the small membrane bound vesicles that we call apoptotic bodies
00:06:37.25 that literally slip off the cell and which will be rapidly phagocytosed by the macrophages in neighboring cells.
00:06:47.03 So, to give a visual example what is apoptosis really means, I usually say that apoptosis is basically you start with a cell that looks like a melon,
00:07:00.26 and after apoptosis you end up like hundreds of small grapes.
00:07:07.28 This form of cell death, the key feature for this form of cell death is the cellular content
00:07:17.11 are not leaked out and although the cell dies, the cellular content remains inside the cell in membrane bound vesicle,
00:07:26.00 which will frequently, rapidly phagocytose by other cells, so the cellular content never spills out into the bloodstream,
00:07:34.09 that way it will not cause the inflammation response.
00:07:38.21 So, these characteristic morphological changes are caused by specific biochemical reactions
00:07:51.11 and here are several examples of specific biochemical changes that are associated with apoptosis.
00:08:00.26 For example, the chromatin condensation. So, if you stain the DNA of apoptotic cells,
00:08:09.17 you can see the DNA now staying like a half moon. That's because, first of all, the nuclear membrane breaks down,
00:08:17.03 you don't have the nice, round shape of the nucleus staining, and also the chromatin condensed into this very bright stain
00:08:28.15 that gives you this typical half-moon shape. This is one of the easiest and quick ways to measure apoptosis in tissue culture,
00:08:38.27 is just to simply stain DNA with a Hoechst dye, or DAPI.
00:08:44.23 Another characteristic biochemical changes for apoptosis is the flipping of the PS, phosphatidyl serine,
00:08:55.24 which is a phospholipid on a cell membrane. You know a cell membrane has two leaflets,
00:09:04.20 and in living cells, most of the PS is in inner leaflets, but during apoptosis, the PS will flip out,
00:09:12.15 and appears on the outside of the cell and that can be specifically detected because PS has this protein, annexia-V,
00:09:23.25 has specificity for the PS and when you conjugate a dye, a fluorescent dye, with annexia-V, and you add this dye to a cell,
00:09:40.17 and the apoptotic cells, now, because their PS is flipped outside, can be stained by annexia-V.
00:09:48.15 And here is actually the first biochemical marker described for apoptosis, DNA fragmentation.
00:09:58.12 Meaning that, if you take DNA out of apoptotic cells and you analyze this DNA on an agarose gel,
00:10:06.10 and you see the DNA appears as a ladder. And with, fragmented into these fragments.
00:10:14.04 With each fragment, with this interval of about a hundred forty nucleotides.
00:10:20.11 Which is the size of the DNA wrapped up around the nucleosome octamer.
00:10:26.14 And caspase activation, and that's the biochemical markers of apoptosis.
00:10:34.11 These caspases are a group of intracellular proteases and their active site is cysteine,
00:10:42.16 and they cleave their substrate after aspartic acid, is how they get its name, cysteine aparase, or caspase.
00:10:50.12 And these proteases are in living cells, they are made as a zymogen,
00:10:57.19 they are not active. But when a cell is undergoing apoptosis, they will become activated.
00:11:03.17 and the activation is represented by the cleavage of its precursor form, or zymogen form,
00:11:14.07 into the active form, which is usually about 20 and 10 kilo Daltons.
00:11:23.13 And we will talk a lot more about caspase activation in the later part of my talk.
00:11:29.02 And the caspase activity is known to be responsible for the most biochemical and the morphological changes associated with apoptosis.
00:11:41.14 So the true molecular nature of apoptosis is first demonstrated in the study of worm, C. elegans, by John Sulston and Bob Horowitz.
00:11:57.24 The one unique feature about C. elegans is during development, all the cell lineage can be traced.
00:12:11.03 And, meaning that the cells, the number of cells, and the position of the cells,
00:12:19.21 and the fate of the cells, is constant during development.
00:12:26.00 So, in this way, all the cell fates can be traced during the entire development.
00:12:34.23 Of course this all is relatively simple, the adult only has about a thousand cells.
00:12:44.05 And during this lineage analysis, these two scientists discovered that some of the cells that they are generated,
00:12:54.15 but they also disappear, they never made it to the final adult stage.
00:13:01.27 And these cells die and they die at a particular developmental time and space in the body.
00:13:12.05 So, because of this property, these scientists are able to mutagenize the worm,
00:13:18.21 and found mutants that were unable to properly execute this form of cell death and the cells that normally will die,
00:13:28.20 during development, now lived.
00:13:31.27 And based on these genetic analysis, these scientists were able to clone genes and mapped a genetic pathway for apoptosis,
00:13:42.08 which is shown in the next slides. And this is the core pathway of apoptosis in C. elegans.
00:13:52.12 These are the important genes that play critical role and I like to point out that in this pathway,
00:14:01.01 you have in the top of the pathway, you have Egl-3, Egl-1, sorry,
00:14:06.15 it will negatively regulate a protein called Ced-9, and the Ced-9 will negatively regulate a protein called Ced-4,
00:14:15.27 which will positively regulate a protein called Ced-3.
00:14:20.22 And the Ced-3 and Ced-4 are required for the apoptosis during development in C. elegans.
00:14:30.05 If you have loss of function of these two genes,
00:14:33.03 and the C. elegans will have more cells in the adult.
00:14:41.14 I think another important features for this apoptotic pathway in C. elegans,
00:14:49.07 all these genes have homologues in, all the way to mammals, including human.
00:15:00.10 And the, for example, the Ced-3 is a gene that encodes a C. elegans caspase,
00:15:10.08 I already mentioned that caspases are critical enzymes in activating apoptosis.
00:15:15.23 And also Apaf-1 protein is homologous to Ced-4 and the Bcl-2 family of proteins is homologous to Ced-9
00:15:23.22 and the BH3-only protein is homologous to Egl-1 and we are going to discuss in details how these proteins function.
00:15:33.04 Another important experimental breakthrough for our understanding of the molecular details of apoptosis
00:15:44.24 is actually come out from pediatric oncologist study in these childhood leukemia, like follicular lymphoma.
00:15:57.08 In these cases, it's the B-cell lymphoma, and Janet Rowley in the 1970's realized that there are chromosomal translocations
00:16:18.19 in this follicular lymphoma and the translocation happened between chromosome 18 and 14,
00:16:26.22 and as a result, this protein, this gene, Bcl-2, now is translocated and in chromosome 14,
00:16:37.17 and now is under the control of IgG promoter. As you know, these B-cells are specialized cells, so they're making antibodies,
00:16:46.22 the IgG promoter is very strong in these cells.
00:16:50.02 But now, instead of making IgG heavy chain, this Igg promoter now controls the Bcl-2 gene.
00:16:58.14 As a result, this B-cell will make a lot more Bcl-2 than normal.
00:17:04.24 And as a result, that these B-cells become follicular lymphomas.
00:17:09.25 The key function for the Bcl-2 family proteins is actually derived from this particular experiment,
00:17:18.28 done by David Vaux, Suzanne Cory and Jerry Adams in the early, in the late 80's.
00:17:26.19 And in that experiment, they cultured normal B-cells, as well as B-cells from follicular lymphoma cells
00:17:36.01 and in order to culture the B-cell in vitro, you have to add a specific growth hormone, IL-3, interleukin-3.
00:17:45.28 And if you take this IL-3 out of the tissue culture medium,
00:17:50.26 the normal B-cells will die, will undergo apoptosis.
00:17:54.20 But the B-cells from follicular lymphoma, that overexpress BCL-2, will not die,
00:18:03.15 were resistant to death when you take out the IL-3 out of the tissue culture media.
00:18:09.25 So, that means that the BCL-2, which is an oncogene, that overexpression will cause tumor, in this case, follicular lymphoma,
00:18:20.09 and that its function is to make the cells resistant to apoptosis and you know, previous to this observation,
00:18:31.15 and the general ideas for the function of oncogenes is that they promote cell growth and proliferation,
00:18:39.06 but this experiment demonstrated that the oncogene could also be anti-cell death.
00:18:44.24 The Bcl-2 family has many members and now that we know that in addition to the Bcl-2,
00:18:56.06 which is the founding member of this family, there are quite a few proteins in human genomes that show homology with Bcl-2.
00:19:07.05 And based on these homology alignments, we can divide the Bcl-2 protein into four domains.
00:19:17.02 BH one to four, the BH domain meaning the Bcl-2 homologous domain,
00:19:23.08 and there are also, we can, three subgroups in this family.
00:19:30.12 And we, on top of the family, are these proteins that are very homologous to Bcl-2, such as Bcl-SL or Mcl-1, and a few others.
00:19:40.00 And these proteins showed, the same homologous domains, BH 1 to 4,
00:19:47.08 compared to Bcl-2. At the C-terminal of the protein, and they also have this transmembrane domain,
00:19:55.08 that will anchor these proteins to mitochondria as well as other organelles.
00:20:02.12 So, these Bcl-2 family proteins, like Bcl-SL and Mcl-1, has a similar function as Bcl-2, that they are anti-apoptosis,
00:20:14.22 they counter, they can protect cells from apoptosis if overexpressed in cells.
00:20:22.14 And here is a couple of proteins, like Bax and Bak, that we called pro-apoptotic Bcl-2 family proteins.
00:20:36.00 And these proteins show homology with Bcl-2 at BH 1 to 3 domain, but they don't have a BH4 domain,
00:20:46.01 and these proteins contrast to the Bcl-2 family genes, Bcl-2, that their function is to be pro-apoptosis.
00:20:57.16 And the detailed function we're also going to talk about a little bit more.
00:21:01.08 And then there are also another group of Bcl-2 homologous proteins that we call the BH3 only
00:21:07.21 and I mentioned that in C. elegans, the Egl-1 protein is one of the BH3 only proteins.
00:21:14.08 The reason we call them BH3 only is because in the primary sequence of these proteins,
00:21:19.28 the BH3 domain is the only recognized domain that is homologous to Bcl-2.
00:21:26.19 So, the key enzyme that drives apoptosis is this enzyme we now called caspase.
00:21:41.00 These proteins, I mentioned before, that they are cysteine protease, there, the active site is cysteine,
00:21:50.06 And they're made in the cell, in the living cell, as a zymogen.
00:21:54.08 They are made, but they are not active.
00:21:56.27 So, during apoptosis, this zymogen needs to be activated.
00:22:01.29 And the activation process is also through proteolysis. It's usually by the cut, about two thirds part of the protein,
00:22:12.23 and this cut will activate the zymogen, which will also cut off the pro-domain at their first substrate.
00:22:23.08 So, the active enzyme is a hetero tetramer, it has four subunits in the enzyme, it has two symmetric active sites,
00:22:32.16 as demonstrated by this red ball, they're all generated from the precursor dimeric protein,
00:22:40.08 and it seems that the caspase, the enzyme that drives apoptosis,
00:22:47.05 and also executes apoptosis, gives us the known morphological and biochemical markers of apoptosis
00:22:55.11 so the activation of the caspase must play a very important role in this whole apoptotic process,
00:23:06.08 and we are going to spend the next few minutes talking about the activation process of the caspase,
00:23:18.23 and how much we know about it.
00:23:21.27 So, why is the caspase important? And here, I'm going to give you one example,
00:23:29.24 how the caspase activity will generate the typical biochemical changes that we associate with apoptosis.
00:23:38.26 And in this case, we have caspase 3 and 7, these are two of the major caspases in a dying cell,
00:23:49.07 and we call these executioner caspases because its function is executing apoptosis.
00:23:55.25 And in the inside nucleus of our cells, we have a heterodimeric protein that we called DFF for DNA fragmentation factor.
00:24:08.08 And the same protein also gets called ICAD and CAD, for CAD is for the caspase activated DNase,
00:24:16.21 also CPAN, and they are independently discovered by three groups.
00:24:23.15 That's how they got three different names.
00:24:24.26 And in this ICAD or DFF45, which is 45 kilo Daltons, has two cleavage sites for caspase-3 and -7.
00:24:37.01 So, in an apoptotic cell, the caspase-3 and -7 will cleave this DFF45,
00:24:43.07 and the cleavage will dissociate the fragments from another subunit, which is 40 kilo Daltons in size,
00:24:51.11 it's called DFF-40 or CAD, and it's these subunits, once the inhibitor subunits get cleaved off,
00:25:00.21 they will form active enzyme, in this case, a DNase,
00:25:06.27 and this DNase will come to the chromatin-nucleosome linker region and cleave the DNA at this region.
00:25:20.20 And that's why when you take the DNA out of apoptotic cells, and you will see this characteristic DNA ladder,
00:25:28.14 and each ladder was an interval of a DNA wrapped-up nucleus.
00:25:34.06 And this cleavage will also cause the chromatin to come back to collapse and condense,
00:25:39.13 and thus give you this typical half-moon staining when you stain the nuclei of apoptotic cells.
00:25:49.20 So, as you can see, once the caspase is activated, they will trigger this chain of reactions,
00:25:56.10 leading to the fragmentation of DNA into pieces.
00:26:02.15 So, the activation of these caspases is quite important for apoptosis.

Part 2: Factors Involved in the Intrinsic Pathway of Apoptosis

00:00:09.00 We started our career, now 12 years ago, that we wanted to study the biochemistry of apoptosis,
00:00:19.21 and particularly how caspase-3, as an example, becomes activated during apoptosis.
00:00:27.00 And we actually took a relatively new approach at that time,
00:00:33.10 that is trying to set up the biochemical reaction, to study the biochemical reactions,
00:00:39.11 that will activate caspase-3.
00:00:43.00 And in this case, and we made cell extracts, so-called S100 extracts, basically you break the cell in hypertonic buffer,
00:00:55.12 and then you pellet all the organelles and the membranes with 100,000 g force spin,
00:01:02.13 and you take the supernatant, which is called S100, that usually people use that as a cytosolic fraction,
00:01:13.23 and we take this cytosolic fraction, and at that time we made this S100 from large cultures of HeLa cells,
00:01:22.28 and one interesting part in here is we made these S100 from living cells, a cell that's not, apoptosis is not activated.
00:01:34.26 So if we incubate, in this case an in vitro translated, S35 labeled pro-caspase-3, with S100 from HeLa cells,
00:01:48.23 and you see that it will, the pro-caspase-3 is not going to be cleaved and activated.
00:01:56.09 But in one of these experiments, and we add small nucleotides into the S100 fraction, and we found, actually quite surprisingly,
00:02:08.24 that either ATP or even better deoxy-ATP is able to accelerate this caspase reactivation.
00:02:18.05 And in the cell extracts that's made from the living cells,
00:02:23.09 meaning that with addition of the dATP, we can trigger apoptosis basically in vitro, in a test tube.
00:02:32.20 And this gave us an assay, so it allowed us to fractionate the cell extracts and reconstitute this pro-caspase reactivation reaction,
00:02:45.27 so in this way, we are able to identify the components, as well as the reaction that gives the caspase reactivation.
00:02:54.26 So, here, is a general scheme for how we perform that experiment.
00:03:02.20 So, we take S100 from HeLa cells and we separate these S100 by different chromatography steps.
00:03:15.28 And in this case, we can separate S100 into three different fractions,
00:03:23.01 and we called apoptotic protease activating factors, APAPs, and we know that in S100, before we fractionate,
00:03:34.07 we can add dATP to activate caspase-3.
00:03:38.09 And after we separate into these different fractions and we show in here, that we can put all three fractions together,
00:03:48.14 we can reconstitute this particular dATP-dependent caspase reactivation,
00:03:53.20 meaning that within S100, there are three protein factors that are necessary and sufficient to activate caspase-3.
00:04:04.18 So, we used a strategy that we can fix two of the factors as constant
00:04:11.03 and further fractionate the third factor until we get that factor purified, and identified as what it is.
00:04:20.08 And using this strategy, we are able to identify all these three factors required for the caspase reactivation.
00:04:31.28 And here is the first factor we purified and this is the last column step we used
00:04:40.00 and here is just basically a demonstration of what we saw.
00:04:44.24 That these numbers are the fractions that collected from this particular column and eluted with the linear salt gradient.
00:04:54.26 And we measured the activity of this factor in these fractions by adding radiolabeled pro-caspase-3,
00:05:05.25 as well as the two other fractions, containing the two other factors,
00:05:11.10 together with dATP, and we saw a nice activity peak around fraction 2 to 4.
00:05:18.07 And then we ran these fractions on SDS-PAGE and we stained with silver, and we saw this small protein down there,
00:05:27.28 that appeared to be purified to homogeneity and also that correlates perfectly with the functional activity.
00:05:39.01 So, this is the first protein that we purified.
00:05:43.09 And at that time, we also noticed after we purified this protein the fractions containing this protein also looks pink,
00:05:52.05 which was quite surprising to us because at that time, none of the proteins known involved in apoptosis are known to have a pigment,
00:06:03.22 and, but with the color, we can put in the spectromer and read its absorbance profile.
00:06:16.21 And this is exactly what we did and here is absorption profile, and you can see they have these three peaks of absorption,
00:06:26.21 and after getting this absorption profile, and comparing to the textbook,
00:06:32.10 and we realized very quickly that the protein we purified is actually cytochrome C.
00:06:39.03 Which was a very big surprise at that time,
00:06:45.11 it took us a long time to even convince ourselves that cytochrome C is indeed something we are after as a caspase activating factor,
00:06:56.12 because cytochrome C is such a well-studied protein,
00:07:00.27 and even in our introductory biochemical textbook, that we know cytochrome C, which is this little pink guy,
00:07:10.10 is part of the electron transport chain in the mitochondria.
00:07:15.02 In living cells, the cytochrome C is exclusively localized in the mitochondria.
00:07:21.26 It has a heme cofactor in it and it's part of the electron transport chain
00:07:28.22 and what's interesting is they take electrons from cytochrome C reductase and they donate electrons for cytochrome C oxidase.
00:07:39.02 And we all know this electron transport chain, which is happening at the inner membrane of mitochondria,
00:07:47.03 is the major energy source that provides, by oxidative phosphorylation, ATP to our cells.
00:07:57.28 An interesting part is the cytochrome C is the only water-soluble component of the electron transport chain,
00:08:06.23 and they are trapped inside the mitochondria by the outer membrane of the mitochondria.
00:08:13.05 Although the cytochrome C is encoded by the nuclear genes, they are made, the pro-protein is made in the cytosol,
00:08:22.05 but they can, the heme ligand is only added inside the mitochondria because the heme ligase is inside the mitochondria,
00:08:33.07 and only when heme is added, the protein is folded properly, but they are trapped in the mitochondria.
00:08:39.26 And we later on realized that the reason that we are able to get cytochrome C from in our S100 fraction,
00:08:49.00 which presumably is a cytosolic fraction, is when we make the cell extracts in hypotonic buffer,
00:08:56.18 we not only break outer membrane of cells, we also break the outer membrane of mitochondria.
00:09:02.20 So, in this way the cytochrome C actually leaked out to the cytosolic fraction.
00:09:10.03 And we are actually, in this case, extremely lucky because the cytochrome C release from the mitochondria,
00:09:19.22 out of the, from the mitochondria, is actually one of the key initiating events for apoptosis.
00:09:27.00 So, we basically, by breaking, in the process of making cell extracts, we initiated, unknowingly, mimicked the apoptotic initiation process.
00:09:41.24 So, here, just to give you a visual demonstration that the protein like cytochrome C,
00:09:51.02 which normally localizes in the mitochondria, if you stain these proteins in a living cell,
00:09:58.10 you see they have usually this punctate stain, indicating mitochondrial localization.
00:10:05.11 If you give them apoptotic stimulus, such as UV radiation, and you wait for a few hours, for apoptosis to happen,
00:10:16.12 and you can see the protein, the mitochondrial proteins, like cytochrome C, start to come out of the mitochondria,
00:10:24.12 and in the late stages of apoptosis, they are diffused staining, they are everywhere.
00:10:31.03 So, release from the mitochondria to the cytosolic compartment is a critical step for apoptosis to happen.
00:10:42.08 But the one reason that we are so surprised about cytochrome C
00:10:49.16 is the previous electron transport activity has nothing to do with this ability to activate caspase.
00:10:58.15 And we don't know at that time how the cytochrome C was able to activate caspase,
00:11:04.00 so to understand that process required purification and identification of the other two proteins,
00:11:11.29 and one of them is Apaf-1. Again, we used the same strategy and by fixing the two other factors
00:11:20.01 and purify the third factor to homogeneity, and in this case, Apaf-1, which runs about 140 kilo Daltons,
00:11:28.14 and when we get a protein from Apaf-1, we realized that we are dealing, at that time, with a novel protein,
00:11:37.17 that protein had never been characterized before.
00:11:40.27 And using the protein sequence, we eventually were able to clone the full-length protein,
00:11:46.18 which is diagrammed here.
00:11:51.00 And this protein is a big protein, it's about 140 kilo Daltons long, but once we get the protein sequence,
00:12:01.06 and we quickly recognized that this protein has three distinct domains.
00:12:06.17 At N-terminal part, it has a domain called CARD domain, and I will talk a little more about this domain in the next few slides.
00:12:16.08 The CARD stands for caspase recruitment domain.
00:12:20.24 So, its function is to recruit caspase.
00:12:23.23 And in the middle part of this protein, has homology with the CED-4 protein of C. elegans.
00:12:32.11 And if you still remember, CED-4 is an integral part of the apoptotic pathway in C. elegans,
00:12:41.13 and CED-4 functions upstream of CED-3, which is C. elegans caspase,
00:12:48.02 and this function is critical for the apoptosis in that organism.
00:12:53.14 So, this region is homologous to CED-4 and it has two unique features about this domain.
00:13:02.27 It's called Walker's A and B box and this Walker's A and B box are known nucleotide binding sequences,
00:13:12.18 which again, not very surprising because the initial observation about the function of this system
00:13:22.02 is we need to add nucleotide, and particularly deoxy-ATP to S100 in order to trigger this caspase activation.
00:13:32.22 And with Apaf-1, has nucleotide binding region, it's truly not surprising.
00:13:38.13 What was a little bit surprising was the C-terminus has 13 WD-40 repeats.
00:13:46.00 These WD-40 repeats are these protein motifs that usually have 40 amino acids long
00:13:52.22 and they often ended with tryptophan or aspartic acid, this is how we call them WD-40 repeats.
00:14:00.21 And these are the protein motifs often mediating protein-protein interactions
00:14:05.05 and Apaf-1 has 13 of them. What was a little bit surprising was the worm C. elegans does not have these WD-40 repeats.
00:14:18.10 But CED-4 protein literally ended right before that.
00:14:23.22 So, which is telling us that apoptotic program of human and C. elegans,
00:14:30.04 although here the homologous protein, that the mode of action is quite different.
00:14:36.07 For example, now we know the function of cytochrome C is probably not conserved in C. elegans in activation pathways.
00:14:52.22 So, this Apaf-1, when you do immunostaining, and it's all cytosolic location in a living cell.
00:15:07.03 Again, which tells you that in a living cell, the Apaf-1 and the cytochrome C are separated by the outer membrane of mitochondria.
00:15:18.29 And only during apoptosis, cytochrome C comes out of the mitochondria,
00:15:23.04 they are able to trigger apoptosis.
00:15:27.25 And the third factor which we initially called Apaf-3, here is again, we purified this protein,
00:15:38.02 and identified this Apaf-3 protein as another caspase, which is caspase 9.
00:15:46.06 So, this told us that the caspase-3 activation is actually through a cascade of caspase,
00:15:54.26 with caspase-9 as the upstream caspase, which are required for the activation of caspase-3,
00:16:01.26 which is a downstream caspase.
00:16:03.23 So, what's the difference between caspase-9 and caspase-3 that makes caspase-9 an initiator caspase
00:16:14.07 and caspase-3 a downstream caspase?
00:16:17.10 Here, I aligned these caspases together and you see all these numbers,
00:16:24.00 these are the caspases encoded in human genome, now we know there are at least 12 of them,
00:16:32.22 but the function, the caspase function in apoptosis, the well characterized ones are all listed here.
00:16:44.07 And the reason that we recognize they are caspases is they are, their active site, the sequence around their active site,
00:16:52.22 this cysteine, QACGG, is conserved between all caspases.
00:17:00.01 The difference between caspase-9, caspase-3 and -7 is the caspase-3 and -7 are smaller
00:17:10.03 and they're homologous to caspase-9 at the C-terminus, but caspase-9 has additional domains at the N-terminus.
00:17:20.04 And this domain is also homologous to CARD, caspase recruitment domain.
00:17:28.07 So, remember, Apaf-1 protein also has a CARD at their N-terminus,
00:17:33.20 and these CARDs are actually the protein-protein interaction domains that basically like secret handshake
00:17:42.18 that's how they find their partner.
00:17:46.04 Another pair of caspases are caspase-8 and 10.
00:17:52.26 And also, unlike caspase-3 and -7, caspase-8 and 10 also has additional domains at this N-terminus.
00:18:03.06 But, instead of CARDs, they are called DED domains, for death effector domains.
00:18:10.25 Their function I'm also going to discuss in the next few slides.
00:18:15.22 So, after we identified these three factors required for apoptotic activation of caspase-3,
00:18:24.10 so, after, again, many years of work, now we can summarize these reactions into this particular pathway.
00:18:34.24 We called apoptotic, mitochondrial apoptotic pathway, or also called intrinsic apoptotic pathway.
00:18:46.02 And the reason why we called them intrinsic versus extrinsic will become clear in later part of my talk.
00:18:54.12 So, we know that apoptotic stimuli, now, such as radiation by UV light,
00:19:01.18 will exert their effects on mitochondria. And the signal is interpreted by the BCL2 family of proteins.
00:19:14.25 The BCL2 family proteins regulate the apoptotic response by mitochondria.
00:19:22.05 As a result, the protein, such as cytochrome C, that normally localizes in the intermembrane space of mitochondria
00:19:30.27 will come out of the mitochondria and find its binding partner, Apaf-1.
00:19:38.24 And Apaf-1 has a good affinity for cytochrome-C, so when cytochrome-C comes out to the cytosol, it will meet Apaf-1,
00:19:50.03 it will specifically bind Apaf-1, at the WD-40 repeat region.
00:19:56.03 And in the presence of the nucleotide, preferably deoxynucleotide,
00:20:03.03 dATP, and this cytochrome C and Apaf-1 will form this large heptamer protein complex,
00:20:12.00 now we call it 'apoptosome.'
00:20:15.01 The CARD domain of Apaf-1, before it meets cytochrome C, before it's formed this apoptosome,
00:20:23.24 is folded in and is not accessible, so after the formation of the apoptosome, which is like this wind wheel-like structure,
00:20:36.09 the CARD domains form the central ring of this wheel-like structure
00:20:42.12 and the CARD domains now become exposed and they are able to recruit, through the CARD-CARD interaction of pro-caspase-9.
00:20:52.20 And once pro-caspase-9 gets recruited to the apoptosome,
00:20:57.23 this undergoes this auto activation process, the molecular details of which we still do not understand,
00:21:04.00 but as a result, the capase-9/Apaf-1/cytochrome-C complex now has this enzymatic activity
00:21:11.16 that's able to cleave the caspase-3 and the 7 precursor, and results in its activation,
00:21:18.08 which subsequently cleaves many important substrates, such as DFF-45 or ICAD,
00:21:24.10 leading to the characteristic changes associated with apoptosis.
00:21:29.01 I also want to point out that some of the deoxynucleotide analogues, such as 2CdA,
00:21:38.12 also called Cladribine, another one called, Fludarabine,
00:21:44.23 these nucleotide analogues are actually clinically used drugs for treating leukemia
00:21:57.01 and these nucleotides can substitute dATP to drive the formation of the apoptosome
00:22:06.20 and in certain cases, for example, fludarabine, is even better than deoxy-ATP to cause the apoptosome.
00:22:14.21 So, here is, just to give you a brief visual demonstration of what does the apoptosome look like?
00:22:26.01 And here is an early cryo-EM structure of the apoptosome, generated in Chris Akey's group,
00:22:32.12 in Boston University, as you see here, it's a beautiful structure, it's just like a wind-wheel, with a central ring,
00:22:40.04 and also the spikes come out of the central ring. The cytochrome-C is somewhere here, this hump,
00:22:48.12 although we couldn't actually see it with 27 Angstrom resolution,
00:22:54.00 but recently a higher resolution structure unequivocally demonstrated the cytochrome C is in there,
00:23:00.17 it's bound at a 1 to 1 ratio. It's a flat structure, just like a flying saucer,
00:23:07.20 and with this central ring and the hole.
00:23:13.07 When the cytochrome C, no, when the caspase 9 is bound to the apoptosome, the hole in the central ring was covered,
00:23:24.00 it's like it's covered with a dome, and this dome is formed by the caspase-9
00:23:30.08 and here it's demonstrated that this large protein complex, cytochrome-C, Apaf-1, caspase-9, are able to cleave pro-caspase-3,
00:23:44.00 and this is the active form of the enzyme caspase-9.
00:23:49.20 So, upstream of mitochondria, I introduced to you the Bcl-2 family of proteins.
00:24:03.06 And these Bcl-2 family proteins have three categories, the anti-apoptotic proteins such as Bcl-2,
00:24:10.23 and Bax/Bac, pro-apoptotic proteins, as well as BH3 only pro-apoptotic protein.
00:24:18.04 So, here, I'm just going to summarize with you what we have known now, how these proteins interact with each other
00:24:27.08 to control the permeability of the mitochondrial outer membrane.
00:24:32.16 So, apoptotic stimuli, again, such as UV, will activate through the BH3 only protein first.
00:24:47.02 And these proteins are normally not associated with mitochondria,
00:24:52.23 it's stored in other cellular compartments, or it may be transcription targets, for p53, for example,
00:25:02.07 Puma and Noxa are transcription targets for p53. As you know, the p53 level will increase in response to radiation.
00:25:11.23 And these BH3 only proteins will transduce your signals to Bax and Bak.
00:25:20.27 And the anti-apoptotic proteins, such as Bcl-2, Bcl-XL or Mcl-1,
00:25:27.19 will be able to bind to the BH3 only protein and neutralize its activity.
00:25:33.23 The activation of Bax and Bak by the BH3 only protein can cause Bax and Bak to oligomerize
00:25:46.12 on the outer membrane of the mitochondria. And in case of Bax, it also translocates from the cytosol to the mitochondria as well.
00:25:56.10 And the exact molecular mechanism of this activation of Bax/Bak, and its oligomerization, is still not very well understood.
00:26:07.07 The anti-apoptotic Bcl-2 or Bcl-XL will also be able to directly bind Bax or Bak, and inhibits its oligomerization.
00:26:22.14 So, as you can see, the anti-, as well as pro-death members of these families interacts with each other to form this complex
00:26:35.19 signaling complex to finally leading to oligomerization of Bax and Bak,
00:26:43.12 and this oligomerized Bax and Bak are believed to be protein channels
00:26:48.20 that allow the protein such as cytochrome C to come out of the mitochondria to the cytosol and cause apoptosis.
00:26:57.02 And in addition to cytochrome C, there are now quite a few other proteins that also come out of mitochondria in the membrane space,
00:27:06.16 such as EndoG, endonuclease G, AIF, for apoptosis inducing factor,
00:27:12.28 as well as Smac/Diablo, or HtrA2 or Omi, also come out of mitochondria and they play a role in apoptosis.
00:27:23.13 And I'm going to discuss a little more about Smac/Diablo in a later part of my talk because we understand how it functions quite well.

Part 3: Extrinsic Pathway and Regulation of Apoptosis

00:00:10.09 I'm also going to use a few slides to introduce the extrinsic pathway of apoptosis.
00:00:18.03 I mentioned to you the mitochondrial pathway of apoptosis, also called intrinsic pathway to apoptosis,
00:00:25.00 and because the apoptotic signals initiated from the inside of the cell,
00:00:32.15 mitochondria. And the extrinsic pathway is apoptotic signal generated from outside of a cell.
00:00:40.20 In this case, by a group of death-inducing cytokines, such as CD95 ligand,
00:00:49.22 also called Fas ligand.
00:00:52.13 And these cytokines will bind specific receptors on the cell membrane and activate these receptors by trimerizing them.
00:01:03.21 And these receptors have these unique features at their C-terminus, that they have at their C-terminus they have this domain
00:01:13.00 that's called a death domain, and these are also protein-protein interaction domains.
00:01:19.14 And these domains, once bound to ligand, is able to recruit these adapter proteins,
00:01:27.15 for example like this FADD, "fad" protein. This FADD protein has basically a protein with two domains, linked by a linker region,
00:01:38.22 and one domain is also a death domain. This death domain is very similar to these CARD domains,
00:01:46.25 as they form the secret handshake to find its partner.
00:01:50.21 For example, the FADD protein now will get recruited to the receptor, death inducing receptor,
00:02:01.02 and this FADD protein, in addition to the death domain, also has this, demonstrated in blue, the domain called death effector domain,
00:02:12.17 also DED domain. I mentioned a little bit earlier that for the caspase-8 and caspase-9,
00:02:20.16 which are two caspases that have two death effector domains, DED domains, at their N-terminus.
00:02:26.27 And with the FADD recruited to the receptor,
00:02:31.07 the pro-caspase-8 and -10 will also be able to recruit to the receptor through the interactions between the death effector domain
00:02:39.29 and once the caspase-8 gets recruited to this receptor complex, also called a DISC, for death inducing signaling complex,
00:02:50.17 and the caspase-8 and -10 will become activated,
00:02:53.29 again in a process that may be similar to the activation of caspase-9 by the apoptosome,
00:03:01.06 that results in the activated caspase-8 and -10, which will subsequently cleave caspase-3 and -7 and cause apoptosis.
00:03:12.29 So, in this way, the caspase activation is initiated from a receptor, cell surface,
00:03:19.18 and that's called extrinsic pathway, and the caspase activation unique from mitochondria is called intrinsic pathway.
00:03:28.29 And there is one interesting regulator for this extrinsic pathway.
00:03:34.18 It's called Flip and this Flip looks like caspase-8, it has, also has this death effector domain at the N-terminus,
00:03:43.28 and it has homology to caspase-8, but the critical enzymatic site, a cysteine, is missing.
00:03:52.09 So, this Flip can be recruited to the DISC, but because it does not have active sites,
00:04:02.21 it cannot function as a caspase-8.
00:04:05.17 So, if Flip is overexpressed in cells then they can block caspase-8 activation.
00:04:13.23 So, the previous slides is a signal caused by CD95, it's relatively simple,
00:04:29.11 and another important death-inducing cytokine is tumor necrosis factor alpha, TNF alpha,
00:04:39.05 and this cytokine, the signal transduction pathway is a little more complex,
00:04:44.23 that in addition to forming a caspase-8 activation complex,
00:04:52.17 they're also able to form a signaling complex that signals to activation of NFkappaB.
00:05:02.17 And activation of NFkappaB is actually good for cell survival because the target of NFkappaB
00:05:11.27 also includes Flip, and I showed you before that Flip is able to, if the level of Flip is high, it's able to antagonize the caspase-8 activation.
00:05:24.05 And also there are two proteins that were initially identified in the signaling complex in the TNF receptor,
00:05:35.23 it's called the cIAP1 and 2, initially isolated by David Goeddel's group, as well as David Vaux's group.
00:05:45.00 The cIAP 1 and 2 are the proteins, these proteins, one of the proteins in that receptor complex.
00:05:53.21 And how this TNF receptor complex switches from NFkappaB signaling complex, which is anti-apoptosis,
00:06:06.04 pro-survival to a caspase-8 activation complex, which is pro-death, is still a great mystery
00:06:15.29 and understanding this particular process should be very important for our understanding of this TNF signaling.
00:06:25.20 The TNF alpha is very much relevant to many human diseases,
00:06:32.10 for example, neutralizing antibodies or soluble receptors for TNF-alpha
00:06:39.12 is widely used for treatment of autoimmune disease, like rheumatoid arthritis.
00:06:46.01 So, that gives me a good opportunity to introduce this IAP family of proteins.
00:06:56.22 So, the cIAP1 and 2 in here is first isolated in the TNF receptor complex
00:07:04.26 and in human genome, now we know there are again, a whole family of IAP proteins
00:07:12.10 and the reason that we know they are IAPs is because they all, here, have the signature domain called BIR domains,
00:07:19.18 for baculovirus IAP repeat domains. Because this IAP proteins, the function of IAP,
00:07:26.25 was first described in a baculovirus insect cell system by Lois Miller's group at University of Georgia a few years ago.
00:07:37.11 And these BIR domains are again signature domains. They're often present in multiple repeats in the IAPs,
00:07:47.29 for example, both XIAP, cIAP1, cIAP2, NAIP have three BIR domains at their N-terminus.
00:07:57.03 And there are also IAPs, such as ML-IAP, which is highly expressed in melanoma cells, that's how it gets its name,
00:08:06.04 only has a single BIR domain.
00:08:08.18 And in several IAP molecules, at the C-terminus, they also have a ring finger domain.
00:08:14.14 As you may know, the ring finger domain is one of the three described ubiquitin E3 ligase
00:08:23.12 that can ubiquinate substrates and cause its turnover.
00:08:29.17 And several IAPs also have this CARD domain in the middle of these proteins,
00:08:37.17 and the exact function of these CARD domains are still not well understood.
00:08:43.06 The best studied IAPs is actually this so-called XIAP, and now we know the XIAP,
00:08:53.20 the reason they called it XIAP is because they are encoded by the X-chromosome,
00:08:58.04 the BIR domains of XIAP, particularly BIR3, which is the third BIR domain, and BIR2, the second BIR domain,
00:09:06.12 is able to directly bind caspase-9 and caspase-3, respectively, and inhibit its activity.
00:09:15.01 The other BIRs in other IAPs are still not very well characterized.
00:09:22.06 But the function of IAPs and the regulation of the IAPs, was, the initial insight actually comes from genetic study in fruit flies.
00:09:38.28 So, a few years ago Hermann Steller's group at MIT, at that time, isolated a mutant fly they called H99.
00:09:54.07 And in these mutant flies, if you irradiate these fly embryos,
00:10:01.11 and in wild-type flies, you will see this massive apoptosis, as demonstrated by this staining,
00:10:13.19 but in these H99 embryos, you don't see this apoptosis,
00:10:18.22 because they are missing an apoptosis inducing protein that is deleted from this H99 region.
00:10:27.28 So, they found, there are three proteins, transcripts, that get deleted in this H99 mutant,
00:10:35.27 and they called these proteins reaper, grim and hid.
00:10:42.00 But what was interesting about these three proteins is, first of all, that the three proteins doesn't show much homology,
00:10:50.07 except at its very N-terminus, the significance will become clear later.
00:10:55.27 But the rest of the protein is not homologous to each other.
00:10:59.26 And also, for quite a while, there's no mammalian homologous protein, to either reaper, grim and hit.
00:11:10.19 So, it's not clear whether the function of these proteins, three proteins,
00:11:17.12 although they are critically important for apoptosis in C. elegans, was conserved in higher organisms, like mammals as well.
00:11:26.16 But the function of these reaper, grim and hid is known that they can counter the IAP proteins,
00:11:37.12 they can counter the function of IAPs.
00:11:39.28 So, a few years ago, in an assay for caspase activation, we actually identified another protein that we called Smac,
00:11:54.08 and this protein also was independently discovered by David Vaux's group, and they called it Diablo,
00:12:00.29 and this Smac protein, the sequence is listed here, and it's a, we realized early on that it's a mitochondrial protein,
00:12:11.07 and it's a nuclearly-encoded mitochondrial protein, so at its N-terminal, is had this typical mitochondrial targeting sequence.
00:12:19.29 And this sequence will be responsible for taking this protein, that is made in the cytosol, to mitochondria.
00:12:28.28 After they are getting in the mitochondria, the mitochondrial targeting sequence will be cleaved.
00:12:35.18 So they generate this new N-terminus so the mature Smac protein will have started with this ADP-Pi.
00:12:45.14 And we isolated this protein as an activity that will promote caspase-3 activation
00:12:57.21 and independently, David Vaux's group identified this protein as one of the proteins that interacts with XIAP.
00:13:06.07 So, putting this together, it's become quite clear that this protein is just like Reaper, Grim and Hid in Drosophila,
00:13:16.04 it's mammalian IAP antagonists and it's a mitochondrial protein.
00:13:23.06 So, the molecular mechanism of this protein has become clear by biochemical as well as structure analysis,
00:13:36.14 and here is a model generated by Yigong Shi's group at Princeton
00:13:43.06 that's showing how these Smac counters the IAPs.
00:13:47.27 And the model is based on a co-crystal structure of the BIR3 domain of XIAP with the Smac.
00:13:56.28 As you see here, the Smac protein is a homodimer with each monomer having three alpha helix bundles.
00:14:06.26 The N-terminal part of Smac is unstructured before it's bound to the BIR domains of XIAP.
00:14:17.13 But once it's bound, they can, they show this structure.
00:14:25.04 And what is really interesting and surprising was the interaction is limited to the first four amino acids of the IAP molecule.
00:14:37.09 And the rest of the molecule seems only to function as a structure motif;
00:14:42.12 the functional motif is limited to the four amino acids, with this N-terminal alanine playing the most critical role in this interaction
00:14:51.20 because, you see here, this is the BIR3 domain is right inside XIAP.
00:14:59.15 The alanine sticks into this hole and provides the most of the binding energy.
00:15:05.18 And the hole is only big enough for alanine and glycine to get in; the other amino acids are too big.
00:15:13.19 And the alanine also provides the critical additional hydrogen binding that glycine couldn't.
00:15:20.26 So, the alanine turns out to be the only amino acid that's able to mediate this critical protein-protein interaction.
00:15:28.23 And this alanine, as you remember, is hidden from, is hidden in the protein when it's first translated in the cytosol,
00:15:41.24 it's not able to bind IAP, but only when the protein gets transported into the mitochondria
00:15:47.23 was this mitochondrial targeting sequence cleaved, then this alanine gets exposed,
00:15:53.05 but now, this Smac protein gets trapped inside the mitochondria, just like cytochrome C,
00:15:58.09 and all the IAP is in the cytosol, so they are not able to interact.
00:16:02.12 So, only when the cell is receiving apoptotic stimuli and this Smac protein comes out of the mitochondria,
00:16:11.05 they can encounter the IAP proteins.
00:16:15.07 Another, there are a couple of interesting implications for this finding
00:16:21.13 that the interaction between Smac and IAP is limited to the first few amino acids.
00:16:28.22 Remember I told you that for the longest time, we couldn't find homologous proteins to Reaper, Grim and Hid.
00:16:37.15 It was all a function of these Smac and reaper, grim and hid seem to be very similar.
00:16:44.04 And also, there is not much sequence homology between reaper, grim and hid themselves.
00:16:48.24 So, after knowing that the function of these proteins is only limited to the few amino acids at the N-terminus,
00:16:57.28 the rest of the protein only play a structural role, now everything seems to be rather clear.
00:17:04.09 Again, when we line up the N-terminus of Smac with the N-terminus of this reaper, grim and hid,
00:17:14.08 and you can see indeed, they are actually quite homologous at this N-terminus.
00:17:20.09 The difference is, for the Smac, because it's a mitochondrial protein,
00:17:27.10 the alanine is hidden by the mitochondrial targeting sequence,
00:17:32.07 but for reaper and grim and hid, the alanine follows immediately after the initiation methionine.
00:17:38.23 And as you know, in many proteins, the first methionine gets processed,
00:17:44.11 gets cleaved off when a protein gets finally translated.
00:17:47.23 And these proteins are often only transcribed when the cell is destined to die.
00:17:54.14 And also, based on these homology, at the very N-terminus, now, the fourth protein,
00:18:02.04 which is also localized in this, close to this H99 region, but never been, hadn't been identified before,
00:18:10.22 just simply based on this homology, now this protein, Sickle, was also identified,
00:18:15.22 that also plays a functional redundant and similar role as reaper, grim and hid.
00:18:23.08 And it's also based on this similarity with AVPI, another mammalian mitochondrial protein called Omi or HtrA2,
00:18:37.06 also has this N-terminus that is similar to this, has N-terminal this sort of AVPS, which may also play a role in countering IAPs.
00:18:47.22 So, this is my final slides, and today we discussed two pathways, from multiple organisms,
00:19:04.02 that play, that execute apoptosis.
00:19:07.16 The intrinsic pathway started from mitochondria and is regulated by the Bcl-2 family of proteins,
00:19:17.05 and there are also extrinsic pathways started by the death inducing cytokines, such as Fas or CD95.
00:19:26.23 There are also two others, DR4 and DR5, death receptor 4 or 5,
00:19:32.26 which are the receptor for another death inducing cytokine called TRAIL.
00:19:37.20 Also, TNF receptor.
00:19:40.04 So, for the intrinsic pathway, that can be activated by radiation,
00:19:48.06 as well as transforming oncogenes and many of the currently used chemotherapeutic drugs,
00:19:56.26 and I give you this deoxynucleotide analogue as an example that also functions at the apoptosome formation.
00:20:07.29 So, at least there are, at three different positions of these pathways,
00:20:18.29 that the new, anti-specific apoptosis inducing drugs for potential chemotherapy is being developed.
00:20:31.03 I already mentioned to you that the deoxynucleotide analogues that were developed by Dennis Carson at UCSD
00:20:41.00 that were used to substitute, that can substitute deoxy-ATP to promote apoptosome formation efficiently.
00:20:51.18 And also, this BH3-only protein, such as BAD interaction with BAX and these interaction, this interaction
00:21:04.05 is mediated by this BH3 peptide and there are small molecules that are being developed,
00:21:11.28 with the leading group in Abbott laboratory, led by Steve Fesik,
00:21:16.28 they have a small molecule that can disrupt the interaction between BAX and BAD,
00:21:27.13 so that will make the Bax active and promote apoptosis and this is also being tried for cancer therapy.
00:21:43.13 And the third example is Smac mimetics.
00:21:49.14 And I showed you that the Smac protein, the functional motif is only four amino acids.
00:21:56.03 And now there are many groups trying to develop small molecules, chemicals, that mimic the four amino acids of Smac,
00:22:07.17 AVPI, for cancer therapy. And why is this so important?
00:22:17.29 Because IAP molecules, like XIAP, and cIAP 1 and 2, are able to block both the extrinsic and intrinsic pathway
00:22:30.05 at multiple sites. And the Smac protein, all Smac mimetics, will be able to eliminate this inhibition.
00:22:40.02 And for the native Smac protein, it's normally inside the mitochondria,
00:22:45.18 and it's subject to the regulation of Bcl-2 family proteins and in many cancer cells,
00:22:51.22 the Bcl-2 family proteins are overexpressed, and the mitochondria is very well protected.
00:22:59.02 But, if you have Smac mimetics, you add to the cell, you can pass the cell membrane directly,
00:23:06.13 and they will bypass the mitochondrial regulation of Bcl-2 and they will make,
00:23:11.16 they will take out live inhibitions on both receptors, as well as the downstream caspases.
00:23:19.00 That will make these cancer cells very sensitive, super sensitive, to these apoptotic stimuli,
00:23:25.27 and potentially make the therapies, by either death receptor ligand, as well as other chemotherapeutic drugs, much, much more effective.
00:23:40.04 And hopefully in the near future, we will see drugs that are based on our understanding of apoptotic pathway,
00:23:50.00 and make it through the clinic and benefits patients. Thank you.

Videos in this Talk

Part 1: Introduction to Apoptosis
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad
Part 2: Factors Involved in the Intrinsic Pathway of Apoptosis
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad
Part 3: Extrinsic Pathway and Regulation of Apoptosis
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad

Speaker: Xiaodong Wang

Total Duration: 1:17:53

Recorded: June 2007

All Talks in Cell Biology

Talk Overview

Dr. Xiaodong Wang gives an introduction to apoptosis. Apoptosis is a form of programmed cell death that plays important roles during animal development, immune response, elimination of damaged cells, and maintenance of tissue homeostasis. Apoptosis is executed by intracellular proteases named caspases that are activated during the onset of apoptosis by extrinsic and intrinsic pathways.

The intrinsic pathway is triggered by the release of proteins such as cytochrome c from mitochondria to cytosol and the extrinsic pathway is activated by the binding of death-inducing cytokines such as Tumor Necrosis Factor to its receptor at the cell surface. Both pathways are regulated at multiple steps to ensure proper apoptosis.

Speaker Bio

Xiaodong Wang

Xiaodong Wang is the Director and an Investigator of the National Institute for Biological Sciences, Beijing. Previously, Dr. Wang was a professor of biochemistry at The University of Texas Southwestern Medical Center at Dallas and an investigator of the Howard Hughes Medical Institute from 1997-2010. Wang received a bachelor’s degree in biology from Beijing Normal… Continue Reading

Part 1: Introduction to Apoptosis

Part 2: Factors Involved in the Intrinsic Pathway of Apoptosis

Part 3: Extrinsic Pathway and Regulation of Apoptosis

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Speaker Bio

Xiaodong Wang

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