Xiaodong Wang (U Texas Southwestern/HHMI) Part 3: Extrinsic Pathway of Apoptosis

October 3, 2019

I’m also going to use a few slides to introduce the extrinsic pathway of apoptosis. I mentioned to you the mitochondrial pathway of apoptosis, also called intrinsic pathway to apoptosis, and because the apoptotic signals initiated from the inside of the cell, mitochondria. And the extrinsic pathway is apoptotic signal generated from outside of a cell. In this case, by a group of death-inducing cytokines, such as CD95 ligand, also called Fas ligand. And these cytokines will bind specific receptors on the cell membrane and activate these receptors by trimerizing them. And these receptors have these unique features at their C-terminus, that they have at their C-terminus they have this domain that’s called a death domain, and these are also protein-protein interaction domains. And these domains, once bound to ligand, is able to recruit these adapter proteins, for example like this FADD, “fad” protein. This FADD protein has basically a protein with two domains, linked by a linker region, and one domain is also a death domain. This death domain is very similar to these CARD domains, as they form the secret handshake to find its partner. For example, the FADD protein now will get recruited to the receptor, death inducing receptor, and this FADD protein, in addition to the death domain, also has this, demonstrated in blue, the domain called death effector domain, also DED domain. I mentioned a little bit earlier that for the caspase-8 and caspase-9, which are two caspases that have two death effector domains, DED domains, at their N-terminus. And with the FADD recruited to the receptor, the pro-caspase-8 and -10 will also be able to recruit to the receptor through the interactions between the death effector domain and once the caspase-8 gets recruited to this receptor complex, also called a DISC, for death inducing signaling complex, and the caspase-8 and -10 will become activated, again in a process that may be similar to the activation of caspase-9 by the apoptosome, that results in the activated caspase-8 and -10, which will subsequently cleave caspase-3 and -7 and cause apoptosis. So, in this way, the caspase activation is initiated from a receptor, cell surface, and that’s called extrinsic pathway, and the caspase activation unique from mitochondria is called intrinsic pathway. And there is one interesting regulator for this extrinsic pathway. It’s called Flip and this Flip looks like caspase-8, it has, also has this death effector domain at the N-terminus, and it has homology to caspase-8, but the critical enzymatic site, a cysteine, is missing. So, this Flip can be recruited to the DISC, but because it does not have active sites, it cannot function as a caspase-8. So, if Flip is overexpressed in cells then they can block caspase-8 activation. So, the previous slides is a signal caused by CD95, it’s relatively simple, and another important death-inducing cytokine is tumor necrosis factor alpha, TNF alpha, and this cytokine, the signal transduction pathway is a little more complex, that in addition to forming a caspase-8 activation complex, they’re also able to form a signaling complex that signals to activation of NFkappaB. And activation of NFkappaB is actually good for cell survival because the target of NFkappaB 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. And also there are two proteins that were initially identified in the signaling complex in the TNF receptor, it’s called the cIAP1 and 2, initially isolated by David Goeddel’s group, as well as David Vaux’s group. The cIAP 1 and 2 are the proteins, these proteins, one of the proteins in that receptor complex. And how this TNF receptor complex switches from NFkappaB signaling complex, which is anti-apoptosis, pro-survival to a caspase-8 activation complex, which is pro-death, is still a great mystery and understanding this particular process should be very important for our understanding of this TNF signaling. The TNF alpha is very much relevant to many human diseases, for example, neutralizing antibodies or soluble receptors for TNF-alpha is widely used for treatment of autoimmune disease, like rheumatoid arthritis. So, that gives me a good opportunity to introduce this IAP family of proteins. So, the cIAP1 and 2 in here is first isolated in the TNF receptor complex and in human genome, now we know there are again, a whole family of IAP proteins and the reason that we know they are IAPs is because they all, here, have the signature domain called BIR domains, for baculovirus IAP repeat domains. Because this IAP proteins, the function of IAP, was first described in a baculovirus insect cell system by Lois Miller’s group at University of Georgia a few years ago. And these BIR domains are again signature domains. They’re often present in multiple repeats in the IAPs, for example, both XIAP, cIAP1, cIAP2, NAIP have three BIR domains at their N-terminus. And there are also IAPs, such as ML-IAP, which is highly expressed in melanoma cells, that’s how it gets its name, only has a single BIR domain. And in several IAP molecules, at the C-terminus, they also have a ring finger domain. As you may know, the ring finger domain is one of the three described ubiquitin E3 ligase that can ubiquinate substrates and cause its turnover. And several IAPs also have this CARD domain in the middle of these proteins, and the exact function of these CARD domains are still not well understood. The best studied IAPs is actually this so-called XIAP, and now we know the XIAP, the reason they called it XIAP is because they are encoded by the X-chromosome, the BIR domains of XIAP, particularly BIR3, which is the third BIR domain, and BIR2, the second BIR domain, is able to directly bind caspase-9 and caspase-3, respectively, and inhibit its activity. The other BIRs in other IAPs are still not very well characterized. But the function of IAPs and the regulation of the IAPs, was, the initial insight actually comes from genetic study in fruit flies. So, a few years ago Hermann Steller’s group at MIT, at that time, isolated a mutant fly they called H99. And in these mutant flies, if you irradiate these fly embryos, and in wild-type flies, you will see this massive apoptosis, as demonstrated by this staining, but in these H99 embryos, you don’t see this apoptosis, because they are missing an apoptosis inducing protein that is deleted from this H99 region. So, they found, there are three proteins, transcripts, that get deleted in this H99 mutant, and they called these proteins reaper, grim and hid. But what was interesting about these three proteins is, first of all, that the three proteins doesn’t show much homology, except at its very N-terminus, the significance will become clear later. But the rest of the protein is not homologous to each other. And also, for quite a while, there’s no mammalian homologous protein, to either reaper, grim and hit. So, it’s not clear whether the function of these proteins, three proteins, although they are critically important for apoptosis in C. elegans, was conserved in higher organisms, like mammals as well. But the function of these reaper, grim and hid is known that they can counter the IAP proteins, they can counter the function of IAPs. So, a few years ago, in an assay for caspase activation, we actually identified another protein that we called Smac, and this protein also was independently discovered by David Vaux’s group, and they called it Diablo, and this Smac protein, the sequence is listed here, and it’s a, we realized early on that it’s a mitochondrial protein, and it’s a nuclearly-encoded mitochondrial protein, so at its N-terminal, is had this typical mitochondrial targeting sequence. And this sequence will be responsible for taking this protein, that is made in the cytosol, to mitochondria. After they are getting in the mitochondria, the mitochondrial targeting sequence will be cleaved. So they generate this new N-terminus so the mature Smac protein will have started with this ADP-Pi. And we isolated this protein as an activity that will promote caspase-3 activation and independently, David Vaux’s group identified this protein as one of the proteins that interacts with XIAP. So, putting this together, it’s become quite clear that this protein is just like Reaper, Grim and Hid in Drosophila, it’s mammalian IAP antagonists and it’s a mitochondrial protein. So, the molecular mechanism of this protein has become clear by biochemical as well as structure analysis, and here is a model generated by Yigong Shi’s group at Princeton that’s showing how these Smac counters the IAPs. And the model is based on a co-crystal structure of the BIR3 domain of XIAP with the Smac. As you see here, the Smac protein is a homodimer with each monomer having three alpha helix bundles. The N-terminal part of Smac is unstructured before it’s bound to the BIR domains of XIAP. But once it’s bound, they can, they show this structure. And what is really interesting and surprising was the interaction is limited to the first four amino acids of the IAP molecule. And the rest of the molecule seems only to function as a structure motif; the functional motif is limited to the four amino acids, with this N-terminal alanine playing the most critical role in this interaction because, you see here, this is the BIR3 domain is right inside XIAP. The alanine sticks into this hole and provides the most of the binding energy. And the hole is only big enough for alanine and glycine to get in; the other amino acids are too big. And the alanine also provides the critical additional hydrogen binding that glycine couldn’t. So, the alanine turns out to be the only amino acid that’s able to mediate this critical protein-protein interaction. And this alanine, as you remember, is hidden from, is hidden in the protein when it’s first translated in the cytosol, it’s not able to bind IAP, but only when the protein gets transported into the mitochondria was this mitochondrial targeting sequence cleaved, then this alanine gets exposed, but now, this Smac protein gets trapped inside the mitochondria, just like cytochrome C, and all the IAP is in the cytosol, so they are not able to interact. So, only when the cell is receiving apoptotic stimuli and this Smac protein comes out of the mitochondria, they can encounter the IAP proteins. Another, there are a couple of interesting implications for this finding that the interaction between Smac and IAP is limited to the first few amino acids. Remember I told you that for the longest time, we couldn’t find homologous proteins to Reaper, Grim and Hid. It was all a function of these Smac and reaper, grim and hid seem to be very similar. And also, there is not much sequence homology between reaper, grim and hid themselves. So, after knowing that the function of these proteins is only limited to the few amino acids at the N-terminus, the rest of the protein only play a structural role, now everything seems to be rather clear. Again, when we line up the N-terminus of Smac with the N-terminus of this reaper, grim and hid, and you can see indeed, they are actually quite homologous at this N-terminus. The difference is, for the Smac, because it’s a mitochondrial protein, the alanine is hidden by the mitochondrial targeting sequence, but for reaper and grim and hid, the alanine follows immediately after the initiation methionine. And as you know, in many proteins, the first methionine gets processed, gets cleaved off when a protein gets finally translated. And these proteins are often only transcribed when the cell is destined to die. And also, based on these homology, at the very N-terminus, now, the fourth protein, which is also localized in this, close to this H99 region, but never been, hadn’t been identified before, just simply based on this homology, now this protein, Sickle, was also identified, that also plays a functional redundant and similar role as reaper, grim and hid. And it’s also based on this similarity with AVPI, another mammalian mitochondrial protein called Omi or HtrA2, 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. So, this is my final slides, and today we discussed two pathways, from multiple organisms, that play, that execute apoptosis. The intrinsic pathway started from mitochondria and is regulated by the Bcl-2 family of proteins, and there are also extrinsic pathways started by the death inducing cytokines, such as Fas or CD95. There are also two others, DR4 and DR5, death receptor 4 or 5, which are the receptor for another death inducing cytokine called TRAIL. Also, TNF receptor. So, for the intrinsic pathway, that can be activated by radiation, as well as transforming oncogenes and many of the currently used chemotherapeutic drugs, and I give you this deoxynucleotide analogue as an example that also functions at the apoptosome formation. So, at least there are, at three different positions of these pathways, that the new, anti-specific apoptosis inducing drugs for potential chemotherapy is being developed. I already mentioned to you that the deoxynucleotide analogues that were developed by Dennis Carson at UCSD that were used to substitute, that can substitute deoxy-ATP to promote apoptosome formation efficiently. And also, this BH3-only protein, such as BAD interaction with BAX and these interaction, this interaction is mediated by this BH3 peptide and there are small molecules that are being developed, with the leading group in Abbott laboratory, led by Steve Fesik, they have a small molecule that can disrupt the interaction between BAX and BAD, so that will make the Bax active and promote apoptosis and this is also being tried for cancer therapy. And the third example is Smac mimetics. And I showed you that the Smac protein, the functional motif is only four amino acids. And now there are many groups trying to develop small molecules, chemicals, that mimic the four amino acids of Smac, AVPI, for cancer therapy. And why is this so important? Because IAP molecules, like XIAP, and cIAP 1 and 2, are able to block both the extrinsic and intrinsic pathway at multiple sites. And the Smac protein, all Smac mimetics, will be able to eliminate this inhibition. And for the native Smac protein, it’s normally inside the mitochondria, and it’s subject to the regulation of Bcl-2 family proteins and in many cancer cells, the Bcl-2 family proteins are overexpressed, and the mitochondria is very well protected. But, if you have Smac mimetics, you add to the cell, you can pass the cell membrane directly, and they will bypass the mitochondrial regulation of Bcl-2 and they will make, they will take out live inhibitions on both receptors, as well as the downstream caspases. That will make these cancer cells very sensitive, super sensitive, to these apoptotic stimuli, and potentially make the therapies, by either death receptor ligand, as well as other chemotherapeutic drugs, much, much more effective. And hopefully in the near future, we will see drugs that are based on our understanding of apoptotic pathway, and make it through the clinic and benefits patients. Thank you.

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