Lecture 13 - Mechanisms of Protein Action

[Auto-generated transcript. Edits may have been applied for clarity.] I don't. Know. How? You're feeling. It's. Okay. Everyone will make starts. I think so, um, my name is Matt Coleman. I'm the head of Department of Cancer Genomic Sciences, which is part of our School of Medical Sciences...

[Auto-generated transcript. Edits may have been applied for clarity.] I don't. Know. How? You're feeling. It's. Okay. Everyone will make starts. I think so, um, my name is Matt Coleman. I'm the head of Department of Cancer Genomic Sciences, which is part of our School of Medical Sciences. And I'm here today to talk to you about, uh, molecular basis of protein action. So the first thing I wanted to do is kind of give a few introductory slides and put things into context and the importance of why we need to think about protein action. Um, and in terms of context, this is quite a good starting place. So this visual here is actually an online resource that you can access through the link at the bottom. Um, that's provided by a, um, a life sciences company called Cell Signalling. And it's an interactive map of the cell. So, um, imagine that we're looking down a cross-section of the cell here. And, uh, so hopefully you can recognise that we've got, um, the cytoplasm. The nucleus, uh, this nuclear pore here, uh, the endoplasmic reticulum. And each of these coloured blobs is a different protein. And when you access the website and you find this, you can hover over each of those, um, coloured blobs. And it will tell you what the protein is. Um, you can also select different types of cellular processes and pathways. And it will highlight, um, proteins in groups. But what I like this for um, other than the fact that it's interactive and you can um, click on some resources, there is I think it nicely kind of summarises visually, um, this uh, contextual problem of how do you control proteins, um, in a cell in this kind of environment where there's this huge complexity? Um, it's literally like a very heterogeneous, um, mix of different proteins, all undertaking different roles, all mixed in together. Um, how on earth does anything, uh, happen in a kind of organised and efficient way? And essentially the answer is, um, in, um, much of the content that we're gonna talk about today. So. Although I, you know, visually, uh, hopefully captured that complexity when you think about it from a sort of, um, numbers perspective. Um, you might think it's not actually, uh, that, that complex. So, yes, we have a lot of genes in our genome, but there's only around 20,000, um, which doesn't really highlight this complexity on this slide here. So you might think very simply, what if there's 20,000 genes and there's 20,000 proteins? That seems okay. Surely we can handle that. Um, but when you think, uh, that each gene, each RNA can be decoded into a variety of different protein species because of changes in RNA splicing, or maybe the mature protein can be played in different ways by proteomics. Then we can go from 20,000, um, proteins to around 50 to 500,000 different species in the cell, just from this extra level of control here. And then when we layered on the fact that each of those proteins that arise from those processes could be modified in different ways, we can talk about protein modifications, um, a lot more. And, um, we can get up to around 1 million protein species. But of course, there isn't going to be just one copy of each modified, um, altered protein in the cell is going to be multiple copies that it spread around in different parts. So within each cell, we can think that probably roughly around 1 billion protein molecules of different types, uh, in the cell at any one time. So I think that probably more accurately reflects that diagram underneath, um, in terms of the complexity in the cellular environment and how, uh, that's a problem that needs to be, um, addressed and how you get there through control. So. That's the main topic of the of the lecture is how proteins are controlled. Um, before we get there and give specific examples. One thing I like to do is to again think about, um, context and think about why that's important. Uh, other than the complexity in the cell in that kind of very heterogeneous environment, what happens, um, when it goes wrong. And so you can't really get better examples of the importance of control, um, than human disease, um, diseases. Human diseases are generally where a protein, uh, a protein or a cellular pathway or process has been, um, disrupted and that control has been lost. So to highlight the importance, we'll give a few, um, examples. Uh, I'm a cancer biologist by heart, so, um, I'm going to focus that start with, but then we'll give some other examples. So the first example I'm going to give a cancer, um, which is invariably, um, a disease caused by loss of um, protein control. I'm going to give this example called Braf. And Braf is an enzyme. It's a kind of enzyme called a kinase. And we're going to talk more about kinases later. So come back to that. This up here is uh, what's called a signal sequencing trace. So this is the genetic sequence, um, of the Braf gene, um, in an actual, uh, uh, patients. Um, so this is the sequence from some of their normal tissue, and this is the sequence from that tumour, um, genome. So you can see here that there's this change. And what we have is that one copy has been mutated. Uh, and it's a what's called a missense substitution. So, um, it's a single nucleotide change, and that results in a single amino acid change, um, from a, uh, a v, uh, position 600 to an eight. That single nucleotide change that results in a single amino acid change is sufficient to disrupt VRE control. And we're going to talk more about that in a second. And that is sufficient to initiate gene agenesis. So just one tiny change can cause absolutely havoc in the cell. So if everything's working, I'm going to first show you this little video that just, um, hopefully highlights, um, what RAF is and what it doesn't sell. And then we'll talk a bit more, uh, once that gets. Way. Normally, the raft raft pathway is activated by growth factors binding to receptors on the cell surface. This starts. Sorry. Try to make it louder. Signal is passed from one protein to another. Ultimately, the signal ends up in the cell's nucleus where the DNA is found here. Genes that make the cell grow or survive are turned on. And the rats raft pathway. The rats Braf, MEK, and Erk proteins carry out this chain of events after a signal is communicated from the outside of the cell. Through this pathway and into the nucleus. The proteins in the pathway are each turned off. If the pathway fails to turn off, the signal is continuously transmitted and the cells may multiply uncontrollably. The second intracellular protein in the rats pathway is called Braf. Advances in genetic sequencing have revealed that the gene encoding this protein is mutated in some cancers. Often, these mutations result in a Braf protein that is locked into a constantly active state. These activating mutations tend to occur in one specific region of the gene and are called these 600 mutations. Mutant Braf proteins that are locked into an active state are believed to lead to constant activation of proteins downstream of Braf. This may lead to uncontrollable cell growth and potentially contribute to cancer. Overall, scientists estimate that Braf v 600 mutations are found in about 8% of all solid cancers. Okay. So this is just a cartoon representation of what you just saw in the video there. On the left hand side, we've got a normal cell. So in this context, um, Braf is wildtype. So uh, similar to what's here. And it's functioning normally. It's controlling, um, the transmission of growth factors from the outside of the cell into the nucleus, uh, to control normal cell growth. So everything's fine there. When you have this V 608 mutation, you lose control. Braf no longer needs any signals from outside the cell. It's always active. It's always signalling the nuclear nucleus and driving those growth signals. And so you get a cell growth that's out of control and starts to lead to a tumour. Of, you know, visually striking representation of what can happen when that, uh, happens in cancer. Um, is the, uh, cancer type melanoma. So skin cancer, um, which is very frequently mutated, uh, in the breath gene. And so just this very simple, um, nucleotide change, very simple mutation can initiate a very devastating, um, type of cancer. So moving away from cancer. Here's another example. This is polycystic kidney disease. So this disease is driven by mutations in the genes that encode these two proteins. So PKD one and two. And these are normally required for um normal renal tubule development. So kidney function and these are both um transmembrane proteins. So um what we mean by that is that that protein sequence spans um, to and fro, uh, across the cell membrane. So you have some of that protein in the outside of the space, some in the cytoplasm. Uh, and these proteins are really critical for, um, regulating calcium signalling and regulating how different cells in the kidney interact with each other. And again, um, when these proteins are functioning normally and they're well controlled, the kidney is healthy in the normal size. Uh, but as the name of the disease suggests, when, uh, these genes are mutated, um, you get complete loss of, um, the function of those PKD proteins, and you end up with these highly cystic, uh, and poorly functioning, um, kidneys, of course. Um, kidney disease. Here's another example. Haemophilia. So symptoms with haemophilia is, um spontaneous bleeding. So even small cuts um won't promptly uh, and you'll get prolonged, uh, bleeding tightness in the joints and internal bleeding. And so, you know, this is obviously, um, a high risk, uh, disease, um, where you can get haemorrhages and it can be quite devastating. And haemophilia A, um, is a genetic deficiency in one of the clotting factors. Eight um, and, uh, it causes increased risk of dementia usually affects males. So again, another small genetic change leading to um loss of control in this case of a clotting factor and having quite devastating disease implications. So to avoid that, of course, um, cells, um, uh, organisms have evolved ways of very, uh, tightly and uh, sophisticated controlling protein, um, function. And when we think about what kinds of, um, control examples there are, um, it's important to think, uh, often about the context in which they act. So different proteins will function in different parts of the cell, different processes. Um, so the context in which they act is can be different to another protein. And because of that contextual difference, um, the way that they're controlled might be markedly different. So I'm just going to give a few kind of high level examples of that. So here's an example of a um linear enzyme pathway. So here we have enzyme one. And that might signal to enzyme 234 and five for example. So very simple very linear one talking to the next. And the way that this pathway is controlled might be quite different um to other types of pathways. So for example here's a pathway that's amplifying where one enzyme will signal to two ends on twos. And then each of those um signal to two more different enzymes. And so you get is amplifying effect. So the way that this pathway these proteins are controlled might be quite different to the previous one. But in this pathway, each enzyme, each protein may act in isolation. And that might be different to, for example, this protein complex here. So this is a um, a structural, um, figure of the ribosome, um, which is involved in making proteins and protein synthesis. So there's around 80 different proteins in the ribosome. They're all bound up together with RNA. And so this is what we would call a multi protein complex or a multi protein complex machine. So the way that the uh control happens here could be very different to the way it happens for a protein in isolation. So we always have to think about the contexts when we're thinking and studying these things. Okay, so that's enough. Um, background and, um, important. So now we can get on with, um, some of the, uh, examples of parts of control. And there are many, many examples of many, many ways. And we're going to touch on, um, just a few of those, some of the key ones. And these are the, the headlines. So we're going to touch, uh, on all of these. And I'm going to take you through some examples of those. So a major way that proteins are controlled in cells is simply by regulating how much of them there is. Uh, and so, um, we might talk about their expression. What we mean by that is, is just that how much there is in the cell. So we might boost expression or we might reduce it by, um, degradation. So, um, if you think of that example that we used, uh, a second ago of a linear enzyme pathway, there might be, uh, an enzyme in here that's rate limiting, um, for the, for the functional output of that pathway. So we might say that this is a really critical enzyme. And the amount of that enzyme in the pathway is what determines what comes out at the end. And so you can control that whole pathway simply by controlling how much of this, um, enzyme is present in the cell at any one time. And so you could increase the output of that pathway. So that's indicated by the thicker arrows simply by making more of the enzyme E3. And the way that we can do that, of course, is, um, the basic, um, cell of molecular biology. Uh, we could drive transcription from the gene to increase the amount of mini production, um, of that A3, and then that would correspondingly, uh, result in more, um, E3 protein being synthesised from that mRNA. So ultimately, more transcription, more protein synthesis, more E3. Conversely, we might want to shut down the output of that pathway. So we might, um, down regulate the amount of that E3 enzyme and reduce the flow through the pathway from here. And the way that that could happen, it could be a reverse of the example I gave before. So we could simply dampen transcription and protein synthesis, or that might not change. And what might happen is that the, um, E3 protein might be degraded. Um, so that's where proteases or protease complexes in the cell will, um, actually chew up and cut up the protein. So we would call that Pam post transcriptional control. And so here's a classical example of how that can happen. And um, if you haven't already had examples of this you will. Uh, later in the course. So here's an example of a protein that needs to be dampened and downregulated in cell. And so um, it gets tagged with this flag. It's another protein called ubiquitin. And that's like a signal that sends it to this large um proteolytic um complex called the protein. So and then it's degraded in the cell. So that's a way of um, down regulating a protein and dampening a pathway. So we're also going to talk about um here effector ligand binding. So binding of a um and a of an effector uh molecule can induce a conformation conformational change in a in a protein. And that can result in a change in its activity that activates or inhibitory um effectors can be a range of things. So they could be tiny, um, like, uh, something as simple as a proton. And that could be the result of a change in. And the pH in cell or effector could be massive and it could actually be another protein itself. So, you know, one large protein binding to another large protein could change, um, the, the confirmation of that target protein and alter its function in some way. These effectors can bind to a range of different parts on their target protein. But one way that they can bind and regulate is through, say, the active site of an enzyme. And we're going to talk about that in a second. A little bit more about, um, this idea of competitive inhibitors and feedback innovation. But they can also bind to other regions of the protein. So not the active site of an enzyme, but somewhere else on the surface. Um, and that can regulate conformation activity. And if that happens we call these allosteric proteins. So a protein that's regulated by an effector binding somewhere on the surface, somewhere else on the surface, um, what we called an steric protein. And we call that outside regulation. Some allostatic proteins can have binding sites for one effector um such as one binding site. But they could also have multiple um binding sites. Um, and we're going to touch on one example of that in a minute. So here's just a visual example of that. Our state regulation. So you might have a protein um with a kind of open conformation here. And then one of these effectors might find in his pocket. And that draws the protein around itself. And so you can see I've switched here the conformation. And that might lead to some sort of change in activity and function. Here's an example of, um, how allostatic regulation might function in one of those linear enzyme pathways that we touched on earlier. So here we have an example of, for example, enzyme one um, that generates a product. Um, and then that product interacts with an enzyme two and then that generates a new product. So it's a classical example of something like this would be a metabolic pathway. And then the output of that pathway is the end product. But here's where the allostatic regulation comes in. So the end product, which is what we call the effector, can come back and bind to another part of that enzyme one and change its conformation. And now it can no longer accept substrates because the conformations changed. So you can see that this is a way of switching off the activity of enzyme one and shutting down the pathway. And we call this product inhibition or feedback control because you've got this kind of negative feedback loop. So here's a specific example of that I mentioned metabolism. So here's an example of um feedback inhibition of this enzyme called training diamonds. Um so this is the enzyme um the action in this step here, um, that takes training the substrate and then acts on the first step in this process to make our solution in this, um, metabolic pathway. Now, when the activity of that pathway is high and generate a lot of my solution as a product, it gets to a certain threshold where it then is able to bind effectively to this enzyme training disseminates and to change its conformation and to ultimately regulate it and to um, uh, competitively inhibit it and reduce the activity. So it's a way of just fine tuning, um, the pathways activity and getting that level of control. It's essential. Here's another example. This is a protein kinase. Again we're going to touch on that in more detail later. So don't worry too much about what is a kinase if you're not too familiar yet. Um, but that the importance of this is to highlight that, um, this um, protein kinase called pKa, um, is regulated. It's it's a um, a protein that's regulated by this effector, um, called C and P. And so in a resting cell you have this inactive kinase, pKa, where the um, catalytic subunits of the kinase catalytic subunit is held inactive by these two regulatory subunits. And these are the ones, um, that are the target of the CCM camp. So if this the levels of this effect are rising. So, um, the concentration gets to a certain threshold where they then able to bind to the regulatory subunit and to cause, um, a conformation change. So this subunit is being allosteric regulated by this effector. So you can see that the shape has changed from here to here. And so that's no longer that's not compatible anymore with binding to the um the catalytic subunit. And so it's kicked off and it's free in the cell. Uh, and in in that context, it also then undergoes this shape change, um, being itself, uh, stable, deregulated, um, and in this form it's been active. So the kinase subunit is activated. Is an example of an elastic protein that's regulated by a very small effect of oxygen and not just one molecule, but multiple molecules of oxygen. So I'm sure you'll be familiar with this example. But this is haemoglobin. Uh, and haemoglobin has um four different um subunits. So, so um attachment. And each of the subunits has a binding site for oxygen. And as each oxygen binds it causes a conformational change in the other subunits that increases their affinity for action. So oxygen effector uh, and uh when it binds you've got this steric regulation that communicates to the other subunits. And so increases the affinity for oxygen in a stepwise manner. And so when you look at oxygen saturation curves you get this kind of sigmoidal curve. We get a sudden burst of um oxygen binding to things like. As I mentioned earlier, being, um. Uh, an opportunity uh, itself for um, effector, um, binding of, of maybe just a proton and how that can regulate, um, conformational changes in protein. So out of state regulation by a tiny, tiny effective uh, and this is an example here. Um, so remember that the pH of the cytosol is about neutral. Um, uh, and if proteins are in excess protons, sorry. Or in excess, uh, the pH, acidic and ionised groups, um, proteins can become, um, protonated so they can change their charge. And a really good example of this is a, um, type of protease. We mentioned protease aliases, enzymes that chop up other proteins. A good example of this is um, a catalytic, um, aspartate residue in a protease. And a specific example of that is this enzyme called uh steps in D. Which undergoes this effect change when the changes. So in this conformation here and the enzyme has this N-terminal tail that sort of folds around on itself, uh, and it sort of excludes the active site of the protease. It blocks it. As that decreases so I as it becomes more acidic. Um, protons, um, can bind to this tail. Protonate. Um, uh, in this, um, sorry, this, um, uh, catalytic aspartate in the pocket. And that can, um, cause a release of this N-terminal tail and so that the catalytic pocket is no longer, uh, blocked by the enzymes of the enzyme, and it's then free to interact with substrates of the protease to go about its business. So this is a really great way of, um, controlling, uh, protease activity in response to a pH change. And the reason that that's important in this context is this enzyme, um, works, uh, it needs to have its activity controlled and restricted only to the end zone, which is this acidic pH compartment where um, proteins can be, um, altered and uh, uh, cut out by proteases. And so in that enzyme or compartment with a pH is low. Um, these, uh, the cassette cassettes and D is active through this mechanism. And what this ensures is that if for any, for any reason, some of that capacity was accidentally kind of leached out or lost into the cytoplasm where the pH is higher, the control would revert back to this, um, and the enzyme would be shut down. And so you wouldn't get accidental kind of, um, release of, uh, proteases into the cytosol would run around in a kind of rampant way and just degrade all kinds of proteins. Um, that they shouldn't be. So, um, here's an example of, uh, interaction domains. So many important proteins consist of like what we call a modular architecture, um, where they might have, um, you know, you don't you wouldn't end up with a spherical blob for most proteins, right? They might have different, uh, modular domains that fold up in specific ways. And then lots of those modular domains come together to form the overall structure of the protein. And lots of, um, modular domains and proteins that work on protein protein interaction domains. So the example show here, um, of this um protein in blue, the surface here colours in yellow is is what we would call the interaction domain. And so it's able to form a kind of scaffolding site um where another protein so the protein green can come in and bind to that region. And then you get these two proteins interacting with each other. And these can have all kinds of, um, roles in cells. Um, so as I said here, that can be involved in localisation. Um, they can be involved in creating these scaffolds that lots of proteins can come together. Um, and these can create sort of signalling hubs and cell. It can also regulate activity. So that could feature in a state regulation like we were just talking about, or even simply if the catalytic domain of the enzyme is in the vicinity of this interaction domain. You know, this other protein binding could simply, um, block the activity by blocking, um, the active site. So there's a lot of different interaction domains in cells that have evolved. And, um, it's worth just thinking about, um, the, the variety, uh, and highlighting that just to sort of give you a sense of, um, the again, the complexity and the ways that biology cells have evolved, uh, creates a number of different interaction molecules for controlling protein action. So I'm not going to go through these. Um, but I just wanted to show you, um, these are crystal structures of structural, um, structures that have been solved, uh, different protein interaction domains. So hopefully you can see, um, so each of these is a different type of interaction name. And hopefully you can see that the variety that's that's out there very, very different uh, overall shapes, topologies and structures. And each of these will have um, a different type of function, uh, in the cell or different types of proteins or other molecules that they might bind to. It's another way of controlling, um, protein action is through localisation. So simply, um, dictating where it is in. So. So it might be, for example, that um, a protein has a really critical function in the nucleus like a transcription factor. So if you, um, restrict it in the cytoplasm, if you, if you kind of anchor it in the cytoplasm for part of its life, that it's not going to be doing its job. And so that's a really effective way of controlling something simply by pulling it into the wrong place. And then that step can be regulated. So you can switch that on. And uh, you can then control cells can control where the protein is in deciding whether or not it's functioning. So um, here's a really good example, um, of that in this video. Nuclear import and export can be directly visualised in living cells that express the green fluorescent protein GFP, fused to the gene regulatory protein Infatti. Nrf2 is normally localised in the cytosol and excluded from the nucleus, but when the cytosolic calcium concentration is raised enough, fat migrates to the nucleus. This is done here experimentally by adding an iron A that allows calcium to enter the cells from the medium. Upon removal of the ionosphere, calcium levels return to normal and infatti is exported from the nucleus. Re addition of the iron of four triggers re-import of 80. So hopefully you can see that how dynamic that process is. And what we mean by that is that it can it responds to a signal. Um, and then um, it can, uh, translocated the nucleus. Um, it can switch back out. You can go backwards and forwards depending on whether the signal there. So in this case it was an eye on the for the regulator calcium. Often with these switches that regulate localisation the the actual molecular switch um is a modification of the protein. Um and again we're going to talk about that in a bit more detail. Um later. Another way of controlling how a protein functions, and is thinking about whether that protein might have a switch like function. It might be, um, on or off. And, um, protein switch is a very. Classical way of, um, controlling the pathway, the activity of a pathway, particularly, for example, pathways that are involved in transmitting signals from the, um, excited, uh, space of the cell to the nucleus, for example, like that example of like that pathway we talked about earlier around RAF, um, signalling. So some proteins enzymes are switched. And a classical way of switching and enzymes function is to um regulate um uh, the hydrolysis of a nucleotide is specifically a nucleotide triphosphate. And so there are two main types of these kind of switching enzymes that rely on this kind of biology. So what we call a GTPase. So this is an enzyme that hydrolyse this the nucleotide triphosphate called GTP. And these are often involved in these cellular processes. And then there's another group of enzymes um, that uh undertake this kind of uh, switching uh, in response to hydrolysis, um, of a different type of type triphosphate called ATP, which I'm sure you would have heard of. So here's a graphical representation of what that looks like. So this is um, a, uh, an enzyme here that binds to GTP. So it's one of these GTPase. So just to orientate you, the coloured portions here, uh, the protein in question, um, so this is the protein that switches and it binds to GTP. So um, this stands for um, uh triphosphate. And the triphosphate is um, indicated here. Um, so we have uh, this is the phosphate group. So that's terminal, um, third phosphate. And this is the um di phosphate version. So GDP. So when collectively together this is called um GTP. So the triphosphate form. So when this GTP is bound to the protein it either side. So up here um one and two fold in. And these two residues interact with and bind to the GTP. So specifically um to the phosphate this terminal phosphate here. And this pulls in these two regions and causes this conformation change. And so in this form it's in a particular active state. But then when it hydrolyse this, um, the GTP and removes that phosphate as shown here with this idea. Right. Once you that no longer have that terminal phosphate across, of course there's nothing there for it to bind to these parts. These switch functionally switch areas. Appointments are opens up. And so you get this conformation change and that is the switch, um, that allows the um, uh, the protein to function in the pathway on or off. It's a very, um, famous example of this, um, is in that same pathway that we talked about earlier where we talked about, um, that RAF, uh, enzyme suppressed acts, uh, upstream of RAF in that signalling enzyme. And um, this is, uh, what we call a GTPase. Uh, and it's, uh, cycle is shown here. So this is, uh, this ret, um, bound to GTP down here. We recall that, um, this is, um, and it's a switch like function where it's all an activated, it's able to bind to its downstream signalling molecule effectors and that then activate actually that kinase RAF in signal to cell growth. So this is the only state this can be um, uh, switched off, uh, so we can turn it into its off state here simply by hydrolyse that GTP and releasing a phosphate. And that can be stimulated by a different class of enzymes, which I won't go into detail here. After the brass has been turned off, it can be turned back on again. This dynamic process. It can be loaded with fresh GTP, uh, to be activated again. One thing that's important to point out here is that GTP binding, um, down here, uh, and, um, the addition of GTP to refs, that's not a covalent modification. So it's not chemically coupled, it's just associated. And that's something. And often students will, um, kind of get wrong here, uh, thinking that it's a type of protein modification. Um, it's not it's actually just associated rather than being correctly bound. So the importance of that control for rest is that if it's, uh, if that control is lost, just like the example we talked about earlier of the Braf kinase being mutated and driving melanoma when Rex's mutated and that switch like function is lost. Um, uh, an example of an enzyme, um, of Ras family could cross when that happens. Um, you get um, uh, colorectal cancers become much more frequent and, uh, crashes frequently mutated in, um, many cancers, including colorectal cancer. So I've been promising all the way through that we were going to talk about different protein modifications, um, which we're gonna do next. So. These are examples, um, where the protein actually is covalently modified. So where it's where a modification is chemically stitched onto the the protein. Around 50% of proteins are then modified in the cell. Although I think that was probably an estimate um, based on um, research done several decades ago when the technology probably wasn't, um, uh, accurate and sensitive enough to detect modifications, I think probably it's likely that all proteins are modified at some point in their, in their lifetime. That would be my guess. Um, protein modification, uh, allows increased complexity. So we touched on that right at the very beginning of the lecture, uh, lecture. So essentially it allows you to go from 20 to 25,000 genes and genome to that kind of, um, massive number. Um, uh, that we talked about before in the millions and billions, um, simply by, um, creating many more types of protein species, just by kind of attaching these different flags to them through these, um, protein modifications. And modifications can regulate a whole range of different types of control and activities as listed here, and they can be dynamic. And by that I mean they can be, um, reversible. So they can go on, they can be taken off again. Uh, and in some cases they can be, um, less dynamic, so irreversible. There's a lot of different types of protein modifications that happen. And this is just one, uh, this is just a figure here highlighting a few of the more famous ones. Um, I like to show this one because it's one of the few, uh, figures that highlights the modification of mind like work, which most people still haven't heard of. Um, but we're going to hopefully this time I'll touch on an example of our work that highlights the importance of that, the end of lecture, but number of really, um, uh, important and, um, common protein modifications, um, that happen in the cell. So each one of these, as you could see if this is your protein here, um, each one of these is, um, chemically tagged physically to the protein. So it's a covalent modification, um, that's chemically, uh, joined to the target protein. So here's a first example is um phosphorylation um and phosphorylation um or actually take a step back to highlight the the possibility that these modifications are dynamic. Uh, so this is where modification can be added. And then it can be removed by a different class of enzymes. Is it really clear example of the importance and prevalence of dynamic modifications in epigenetics. Specifically in histones. These are the proteins that will reference the DNA or wrap the DNA around histones in, um, the cell in the genome. And the N-terminal tail of these proteins is very heavily, um, uh, regulated by modifications in a whole range of different types of modifications. So M stands for methylation. So um, we can get methylation for example, of specific lysine. That's what K stands for here. And the methylation can be added by one set of enzymes um called um histone methyltransferase. This also it can be removed by a different class of enzymes. So demethylation. And the same is true um a phosphorylation as shown down here that can be added by kinases removed by phosphatase. And here's another um modification called acetylation. So added by this study centre transferase is and removed by deacetylase is. So they can be highly dynamic. They can go on. They can be laid on within seconds and they can be removed within seconds of minutes. So this slide just summarises, um, uh, the role of protein modifications and what they can, um, be used for to, to control, um, protein function in cells. I won't go through each of these. I'll just pick out, um, a couple of examples of how they might function. So here's an example, um, of a protein, uh, that is targeted by a kinase. The phosphorylation is added to this task group. And that creates uh, um kind of adaptor sites. So it creates a binding site for another protein, um, a protein that has one of these so-called s two domains. And so this can recruit a protein after it's been modified by phosphorylation. This is a nice example here where for example, you might have a protein over here on this side where as we said it might be phosphorylated. So that's P here on this tyrosine. It recruits a protein that has this SH2 domain. And this protein is called simple. And then this enzyme catalyses a different type of protein modification to a neighbouring residue. So the addition of ubiquitin to this lysine and then has another function. So it's this sequential way of acting in stepwise way. And there's a number of different ways, um, that I won't go through as represented here. If you're interested, you can um, read this paper which lays out very nicely. So just to touch on, um, protein, um, phosphorylation in a bit more detail. So this is um, as I said, covalent addition of a phosphate to sidechain. It can happen on um, serine. Uh, here shown here to this hydroxyl group. The addition of this phosphate similarly can happen on threonine again because it has a hydroxyl that it can add a phosphate to. And it can happen on tyrosine shown here. The addition of the the phosphates is catalysed by kinase, is like virus and removed by a different class of enzymes. But phosphatase is, as I mentioned, and phosphorylation because it has this double negative charge that actually changes the chemical properties of the side chain of that residues. So this is how it can very dramatically change the, um, the activity, uh, of a protein can be used for control. So it can change how the protein is folded. It can change enzyme activity, protein protein interactions, um, localisation in cell, the whole kind of, um, uh, spectrum of potential regulation. Here's a just a real life example of a thanks that touches on a few things we discussed already. So this is a cartoon of a protein, um, a type of enzyme called a kinase, as I mentioned, that are involved in phosphorylation. So this is called Sarc. It's a it's sort kinase. It catalyses the phosphorylation of other proteins but it's itself regulated by Salt. I. And often you find this in biology where you get this cascades of modification. So this is one end of SoC kinase. And this is a tasking list. You. And in this sort of standing state in the cell it's phosphorylated. And when it's phosphorylated the C-terminal end of the protein is able to look back round and bind to the phosphorylated Tasya. And so it sort of loops around. One end binds to the other and holds it in a closed conformation. If that residue is de phosphorylated, it releases um, the the other end of the kinase. And that changes the confirmation of the catalytic domain here and allows the kinase to become active. Um, and so you can see here how, um, uh, modifications like phosphorylation not only be dynamic, but how they can regulate enzyme activity. Uh, I won't go through this in detail in the interest of time, because I'm running over a bit. But just to point out that phosphorylation and kinase is, uh, really heavily used as a way of protein control in how cells, um, grow and divide. So the cell cycle, how cells, um, are regulated as they pass through, for example, mitosis and how one cell becomes two. Um, and the example I've just pulled out here is an example of the cyclin dependent kinases. Um, and for those that are interested, you can um, look in a bit more detail into some, um, reviews and these links here to find out more about that. So lastly, I just wanted to touch on what my own group, uh, research. And it's this modification of here called hydroxylase. So what is hydroxylase and why do we study it. So this is a just as, um, carbon hydrogen bond on the site side chain of an amino acid. Um, and the hydroxyl action is essentially this. It's the one of the smallest, um, protein modifications. It's the chemical addition of a single oxygen atom. So we've gone from a kind of, uh, carbon hydrogen bond to a carbon oxygen bond to create this kind of alcohol, this hydroxyl group here. And this tiny change, um, can have really profound consequences on protein control. This hydroxyl chain reaction is catalysed by an enzyme family. So this is just what we call a sort of family tree. Um, at the hydroxylase enzyme family. There's lots of different types. They do lots of different things in the cell, some really fascinating biology. Um, and they're very heavily involved in disease. Um, a lot of, um, evidence to say that they're important in cancer. They're a novel type of drug target, for example. And there are new therapies being developed for disease and cancer, biological time. Um, and some of that work we're doing in my own group here. Um, but I want to draw your attention to this little subfamily down here and this particular hydroxylase enzyme called gem jelly four. Now, when we started working on this enzyme, nothing was known about it in, uh, in biology, it's never been studied before. Um, so it looked like a great one. A very exciting project to look at in more detail. As I mentioned, my lab is a cancer research group. And so we we were really interested in whether this enzyme had a role in tumour biology. Was it deregulated during tumorigenesis? Did it contribute in some way to, um, cancers? And could it be targeted as uh, as a new therapeutic opportunity? And I won't go into the detail, but we did have genetic evidence that this enzyme was probably, um, an important player in cancer that have been overlooked before. And so with that hypothesis, we were able to go into the lab and test that, um, using kind of some of our standard approach. So. Well, first thing we did is we took a range of different tumour cell types of cell lines and grew them in lab. And then we, um, kind of artificially inhibited this enzyme, um, using some standard techniques in the lab where we. So this example is one called RNA interference, which you'll hear more about in other lectures. So this is our way of inhibiting, um, and, and blocking the function of jam jelly for the tumour cells. And when we do this, um, we completely block the growth of those cancer cells in the lab. So these are the cells that either our control cells that grow normally and very, very rapidly, as you would expect for tumour cells. Um, but if we inhibit this hydroxylase enzyme, um, we really cut down or block that growth. So that's indicating that protein hydroxylase and this modification catalysed by this enzyme probably has some role in the growth and viability of these tumour cells. So the next question for us was, well, how does that happen? How does this hydroxylase in hydroxylase control cancer cell growth? Um, so the next question was, well, what's the substrate? What does it target. What does this enzyme and uh control. What does it hydroxylase. And again, I won't go through a lot of work, but essentially we identified that this hydroxylase targets very specific proteins. So far we only have one substrate in the cell. And it's this protein called release factor one. So I have to tell you just a couple of minutes on what, um, respect to one is um, essentially it's involved in, um, protein synthesis specifically controlling the end of protein synthesis. So you'll be familiar from your, um, previous learning, um, that the genetic code has a stock code on different codons that encode different amino acids and then a stop signal. So stock code on um, that blocks um and prevents any further translation in protein synthesis. And the way this works is this protein really spoke to one. So er if one recognises the stock code on when it appears in the ribosome and it shuts down protein synthesis. And what we found in my lab is that this er if one protein is hydroxylase it's by Jane jelly for a very specific part of the the protein. And then that's critical for controlling er one's function and controlling the recognition of the stock code on the website. And that's why we think this enzyme so potent in cancer. We think it supports normal protein synthesis and normal growth. And if hydroxyl action is blocked and lost, then the ribosome goes through the stock code on um and generates these kind of Frankenstein proteins, which are toxic for the cell. So in the interest of time, I won't go through these bullet points that I have for you just to use as a summary. Um. Uh, that we've reached, uh, hopefully giving you an overview of the grounding of how proteins are controlled, the impulse of. Thank you.