Mitochondrial Pyruvate Carrier: Getting Fuel To The Cell's Engine

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MAKING SCIENCE A PART OF EVERYONE’S STORY SIGN UP FOR UPDATES Mitochondrial Pyruvate Carrier: Getting Fuel to the Cell’s Engine – The Importance of Metabolism in Disease
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00:00:13.02 Hi guys. My name is John. 00:00:14.28 I'm an MD/PhD candidate 00:00:16.10 at the University of Utah 00:00:17.29 in the Department of Biochemistry 00:00:19.10 and I'm in Jared Rutter's lab. 00:00:21.00 And really what we like to study 00:00:22.08 is metabolism and how this is important 00:00:24.06 for human disease. 00:00:25.14 So I'm going to tell you a story 00:00:27.00 about how we used yeast 00:00:29.08 as a model organism 00:00:30.12 in order to understand really complex things 00:00:32.10 in human disease and cancer. 00:00:34.26 So, for those of you 00:00:38.02 that might not know what an MD/PhD is, 00:00:39.14 I didn't know when I started this program, 00:00:41.18 really the goal is to get training in medicine, 00:00:44.02 so you can see patients and be, you know, a regular doctor, 00:00:47.08 but also to get very basic training 00:00:49.18 in science, do research, 00:00:51.18 and understand fundamental questions about human health. 00:00:54.20 So, we like to study big diseases, you know, 00:00:56.20 important things like cancer, 00:00:58.12 like heart failure, and diabetes, 00:01:00.01 things that really have impacts on human health, 00:01:01.29 and hopefully by better understanding them 00:01:04.12 maybe we can cure them, 00:01:06.02 or at least help people. 00:01:07.17 That's really the goal. 00:01:09.10 So, when I started in the lab, 00:01:10.25 I was really excited to ask these big questions, 00:01:13.07 to study cancer. 00:01:15.02 But my boss basically told me, 00:01:16.26 you know, I'd be working on this yeast project. 00:01:18.16 So you can imagine my surprise, 00:01:19.29 I was a little bit bummed, 00:01:21.06 and I think this is what most people think of when they think of yeast. 00:01:23.14 They go to the supermarket, 00:01:25.00 they'll buy this bag, 00:01:27.10 it's this powder, you add it to bread, 00:01:29.01 you know, when you're baking something, 00:01:30.20 and it makes the bread rise. 00:01:31.25 And that's really the extent of what people think about. 00:01:34.17 But these are really little microorganisms 00:01:36.20 and their metabolism is important 00:01:38.15 for things like making bread and brewing beer, 00:01:40.28 and this is what I thought. 00:01:42.02 I though it was, you know, bread and beer. 00:01:43.22 And when I went into the lab, 00:01:46.05 one of the most surprising and satisfying things 00:01:47.23 is when you work with yeast, 00:01:49.12 you actually get the smell of bread and the smell of beer. 00:01:50.29 It's great. 00:01:52.10 But the other really cool thing 00:01:54.07 is that when you go to the supermarket, 00:01:55.15 you're actually buying a strain of yeast 00:01:57.08 that we work with in the lab - it's very similar. 00:01:59.09 So you can do science with something 00:02:01.02 that you get at the grocery store. 00:02:03.00 But really understanding yeast 00:02:05.07 was this thing that I didn't understand 00:02:07.18 or care much about when I started, 00:02:10.01 but it was really connecting this 00:02:12.24 up the ladder to a human disease 00:02:14.26 that gave us this context 00:02:16.28 and I really started to understand 00:02:18.15 how important this was for patients. 00:02:20.28 So, I'm going to tell you this story 00:02:22.19 about a little girl. 00:02:24.17 She died before her second birthday. 00:02:26.26 When she was born, she was small 00:02:28.26 - she was only about six pounds. 00:02:30.20 She was very weak - she couldn't lift her arms, 00:02:32.19 she couldn't lift her head. 00:02:34.17 She couldn't even really see. 00:02:36.04 She was kind of, you know, stuck in her own little world 00:02:38.12 and everything was a struggle for her. 00:02:40.13 And you can imagine, you know, this little girl's family, 00:02:42.01 this was their first baby, 00:02:44.11 and the frustration and the heartbreak 00:02:46.07 of your first child having all of these problems 00:02:48.26 that you couldn't do anything about. 00:02:50.09 Doctors tried, 00:02:51.23 they really investigated as much as they could to try 00:02:53.28 to understand what was going on with this little girl, 00:02:57.17 and they did very careful studies 00:02:59.14 looking at how she got energy from food, 00:03:01.27 and there was this very specific defect, 00:03:04.16 but they couldn't do anything about it, 00:03:06.09 because the didn't know the genes to look for. 00:03:08.03 They didn't have an identity 00:03:10.16 for this thing that was so important that caused her disease, 00:03:12.11 and they even wrote about that in their paper 00:03:14.14 that I'll talk about later. 00:03:16.01 It was just impossible to identify. 00:03:18.04 They couldn't find it because they didn't know what they were looking for. 00:03:21.11 And by using yeast in this project, 00:03:23.20 we started very simply, 00:03:26.12 but we actually gave them this answer after 10 years. 00:03:28.12 We contacted them 00:03:30.10 and were able to solve this puzzle, 00:03:31.26 piece it together, and really figure this out. 00:03:34.04 So, I'm going to tell you a little bit of a story about metabolism. 00:03:38.13 This is my dog, this is Gideon. 00:03:40.15 He loves to get excited. 00:03:41.29 He likes to run. 00:03:43.22 He's still kind of a puppy, so, you know, 00:03:45.12 when I come home he's really excited, 00:03:46.26 he spins around and gets really animated. 00:03:48.15 And that's what most people think of 00:03:50.29 when they think of metabolism. 00:03:52.07 They might think of something like calories or carbs or fat, 00:03:55.29 but metabolism is this really important process 00:03:59.02 that underlies all of biology, 00:04:00.27 and it's important to know that metabolism 00:04:03.02 happens even when you're resting, 00:04:04.17 even when you're laying down. 00:04:06.05 It's not just when you're breathing hard, 00:04:07.19 or walking up the stairs and you get tired. 00:04:09.19 It's when you're taking a nap, 00:04:11.12 when you're not doing anything, 00:04:12.22 when you're just sitting and watching this video. 00:04:14.26 Metabolism is still important 00:04:16.19 and it underlies all processes of biology. 00:04:20.28 But I'm really interested 00:04:22.29 in how food is taken in 00:04:24.22 and how this becomes energy 00:04:26.16 to keep your body running. 00:04:28.10 So, at the most basic level, 00:04:30.03 when you eat something, you know, you chew it, 00:04:32.00 you have a good meal... you chew, you swallow, 00:04:34.12 and that's basically the end of what you think about 00:04:37.00 with metabolism and with food. 00:04:38.26 But I'm really interested in what happens 00:04:40.18 in this part, you know, 00:04:42.03 when the food is digested, it gets into your bloodstream, 00:04:43.27 and then it circulates through the blood 00:04:46.07 and is taken up by your cells. 00:04:49.25 But on the structural level, on the basic level, 00:04:52.08 I think we can see evidence of metabolism 00:04:54.12 in other very important areas, 00:04:56.16 and it's shaped a lot of the animals around us. 00:05:00.02 So, like a hummingbird we have here, 00:05:01.25 it has this long beak 00:05:03.24 and this tongue that allows it to access 00:05:07.06 this very long flower 00:05:09.10 that it otherwise wouldn't be able to eat from. 00:05:11.09 The same thing happened with Darwin's finches. 00:05:13.00 So, these birds have different sizes of beaks 00:05:15.27 that allow them to eat different types of seeds, 00:05:18.16 and that's really the first step in metabolism 00:05:20.22 is what you eat and how you get it, 00:05:22.07 and it's driven all of evolution 00:05:24.04 and it's a very important process 00:05:26.05 that I don't think we think about enough, 00:05:28.17 partly because it works so efficiently. 00:05:30.03 It's just always going in the background, 00:05:32.14 so we don't need to think about it. 00:05:34.00 You know, you can think about it and hold your breath, 00:05:36.06 but you can't think about it and stop metabolism. 00:05:37.16 It's always going, 00:05:40.08 and that's an important point. 00:05:42.06 So, like we have here, 00:05:43.29 it's kind of a little bit before lunch, 00:05:45.07 I'm getting kind of hungry, so I'm looking at this. 00:05:47.10 It looks delicious. 00:05:49.03 I could eat any of this on here. 00:05:50.15 Let's say I take a piece of bread, 00:05:52.06 I chew it, I swallow it, 00:05:53.26 and then it's going to start getting digested 00:05:56.03 and those nutrients, that food, 00:05:57.22 is going to be broken down 00:05:59.10 and delivered to my cells. 00:06:01.10 And I'm really interested in the part 00:06:03.15 where the food... the nutrients are in the cell 00:06:05.24 and what happens to that to give it energy. 00:06:09.05 So, once it's in the cell, up here... 00:06:11.05 we have these little organelles 00:06:12.28 that are just these fascinating things called mitochondria, 00:06:15.05 and I remember being kind of like being shocked and horrified 00:06:17.21 when I was in seventh grade 00:06:19.08 and I learned that there were these little things 00:06:21.17 inside my cells that gave me energy. 00:06:23.07 I didn't quite get it, 00:06:24.25 but I just knew they were cool 00:06:26.16 and that it was interesting. 00:06:27.27 So it was really fortuitous that I get to study this, 00:06:29.18 and this is my job now. 00:06:31.04 It's kind of cool. 00:06:32.23 But at the most basic level, 00:06:34.02 if you haven't heard what mitochondria is, 00:06:36.03 you might have heard "the powerhouse of the cell", 00:06:39.09 but it's so much more than that. It's beautiful. 00:06:41.23 It does so many important things. 00:06:43.17 But at the very basic level 00:06:45.01 if we take nutrients like carbs or fat, 00:06:48.03 nutrients will be in the cell 00:06:50.25 and then they have to get into the mitochondria through these channels, 00:06:52.16 these transporters, 00:06:54.05 that really allow these nutrients 00:06:55.28 to go at a very specific rate, 00:06:57.28 because you don't really just want to dump everything 00:07:00.28 on the mitochondria. 00:07:02.12 You want to measure it out very slowly 00:07:04.27 to get the energy as you need it, 00:07:06.14 because you don't want to just blow through all of your energy 00:07:08.18 if you don't have to. 00:07:10.03 And then once it's in the mitochondria 00:07:11.26 it goes through different pathways 00:07:13.20 and you get energy from this. 00:07:15.02 So, I'm really interested in this process, 00:07:16.25 very focused, 00:07:18.08 but keeping in mind this entire context 00:07:19.27 and how important it is. 00:07:22.05 So, the other way I like to think of mitochondria, 00:07:23.24 rather than the powerhouse of the cell, 00:07:26.24 is the mitochondria are like the engine of the car. 00:07:28.17 So, you gas up your car, 00:07:30.10 gas is like food for your car, 00:07:32.09 you know, you kind of like fill up the tank 00:07:33.26 and the gas is delivered to the engine 00:07:35.27 and then the engine runs. 00:07:37.27 But like metabolism, 00:07:40.10 the engine, if you idle it 00:07:42.03 - you know, you keep the engine running, 00:07:43.14 you're not really doing anything - 00:07:45.00 you're still burning gas. 00:07:46.19 And then if you accelerate, 00:07:48.15 if you step on the gas and go really fast, 00:07:50.14 you're going to get lower miles per gallon, 00:07:52.10 and that's like if you're sprinting. 00:07:53.25 You're going to move really fast, 00:07:56.02 but you're going to use a lot of energy to do that, 00:07:58.06 and it's a lot like a car in that way. 00:08:00.20 Another thing that I like to use the car for 00:08:03.07 is the fact that some things on a car 00:08:05.06 haven't really changed very much 00:08:06.24 since the car's been invented. 00:08:08.12 You're always going to have wheels, you're gonna have tires. 00:08:11.04 You're gonna need to stop, so you have brakes. 00:08:12.18 Those things haven't really changed very much, 00:08:14.06 at least in general principles. 00:08:15.26 The subtleties might change, 00:08:17.22 they might be modified a little big, 00:08:19.18 but they haven't fundamentally been any different than before. 00:08:22.14 And this really informed biology... 00:08:24.16 I know talking about cars 00:08:26.07 doesn't really seem important for a science talk, 00:08:28.19 but this premise really is important 00:08:31.13 for how we decided to ask a very central question in biology, 00:08:34.24 which is, if something has stayed the say, 00:08:36.24 like in a car since it was invented, 00:08:39.05 if it's the exact say almost, 00:08:40.20 it must be important, 00:08:42.09 there must not be many ways that you could get around this. 00:08:45.01 It must have a very important role. 00:08:47.19 So, in our system, we use this premise of conservation. 00:08:52.15 So, when I say conservation 00:08:54.01 you might think I'm, you know, conserving a species 00:08:56.22 or I want to conserve shark species 00:08:58.28 - I'm out there trying to maintain populations. 00:09:02.08 And it's really a similar process that I'm talking about. 00:09:04.18 It's... you want to keep something around. 00:09:06.17 If you want to keep something around 00:09:08.05 and, you know, have it stay the same 00:09:10.18 it's important. 00:09:12.05 Like, you know, you want to conserve tigers, 00:09:13.20 you want to conserve elephants. 00:09:15.03 Like, it's the same premise. 00:09:17.23 But really what we wanted to do 00:09:20.00 is take yeast genes and compare them to human genes 00:09:22.09 and really see what has stayed the same 00:09:24.17 over this gulf of evolution. 00:09:26.05 What has not really been tweaked on too much 00:09:29.17 by random mutation that might, you know, 00:09:31.12 muddle this effect? 00:09:32.25 And this is what we call the conserved overlap here. 00:09:35.08 So, this green is our conserved proteins 00:09:37.28 in the mitochondria, 00:09:39.28 and most of these are known. 00:09:41.13 We know a lot about what the mitochondria does. 00:09:43.08 We've been studying it for quite a long time, 00:09:45.00 so there are very basic processes 00:09:47.08 that we've known about. 00:09:48.17 But then there are these things down here, 00:09:50.03 this question mark, these uncharacterized proteins, 00:09:52.26 and that just means we knew nothing about what they did. 00:09:54.17 We had no clue. 00:09:56.14 So we really wanted to study this 00:09:58.28 and we got lucky, and we found some really interesting things, 00:10:01.16 and we've studied multiple uncharacterized proteins here 00:10:04.18 and identified their function, 00:10:06.18 but I'm going to tell you about one very specifically 00:10:09.06 that has relevance to that little girl 00:10:11.06 that I told you about before, 00:10:12.24 but also really fits with this car analogy 00:10:15.29 that you need to get gas into the engine. 00:10:17.24 You need to fuel this engine in order to move, 00:10:20.26 in order to survive. 00:10:23.05 So, at a very basic level, 00:10:24.17 why would we study yeast to do this? 00:10:26.20 And yeast is a great system. 00:10:28.12 It's very flexible; it's adaptable. 00:10:30.05 You can do a lot of things with yeast 00:10:31.24 that you couldn't do in other systems, 00:10:33.28 and I think the main thing about yeast is... 00:10:37.08 we want to take out genes and put genes back in, 00:10:39.18 and other cells might die from this, 00:10:41.08 but yeast are so adaptable that they can tolerate it, 00:10:44.04 so you can really understand what's happening. 00:10:46.16 It's also very fast, it's very cheap, 00:10:48.20 and they smell good and they make beer. 00:10:50.13 It's fun to work around. 00:10:52.25 So, at the basic level, 00:10:54.24 we started very simply with this. 00:10:56.19 We wanted to start with an observation, nothing more. 00:10:58.17 Just happens when we do something, right? 00:11:01.17 The basis of science. 00:11:03.04 So, do to this, we used a technique 00:11:05.14 called the spot test, 00:11:06.22 where we'll take different dilutions of yeast 00:11:08.08 at different concentrations, 00:11:09.24 so you know, more concentrated to less concentrated, 00:11:12.12 spot them out on plates of different nutrients, 00:11:15.14 and then just grow them to see if there are 00:11:17.08 any differences under different nutrient conditions. 00:11:19.10 And that can tell us about where this defect might be. 00:11:22.24 So, we'll see here... 00:11:24.18 we have a wildtype, normal strain of yeast, 00:11:26.14 like you'd get at the grocery store, 00:11:28.08 no modifications done to it at all. 00:11:31.07 It grows really healthily, really normally. 00:11:33.22 It has these white colonies, those are the yeast. 00:11:36.13 And then if we knock out a gene, 00:11:38.03 say we take away gene X, 00:11:40.26 these yeast grow much worse 00:11:43.19 and there's an observation there, 00:11:45.22 that in this very specific case the yeast are growing worse. 00:11:48.17 But that's all the information we had, 00:11:50.18 we knew nothing else about what this gene did. 00:11:52.10 We took it away and they grow differently, 00:11:54.04 but we have no idea what's going on here. 00:11:56.16 So what do we do next? 00:11:58.06 I mean, it's like having a flashlight 00:12:00.06 at the Grand Canyon, you know, 00:12:01.26 you don't really know what you're looking for. 00:12:03.09 So what are you gonna do next to really figure this out? 00:12:06.08 And we started, again, very simply. 00:12:08.00 We just ground up these yeast 00:12:09.23 and looked at their different nutrient levels, 00:12:11.17 and that actually gave us a very important clue. 00:12:15.14 So, I'm just going to introduce pyruvate. 00:12:17.26 We talked about the mitochondrial pyruvate carrier, 00:12:20.02 but you might not know what pyruvate is. 00:12:22.12 It's basically this breakdown product 00:12:24.24 of these nutrients... 00:12:26.14 so, carbohydrates are broken down into smaller pieces, 00:12:29.06 and those smaller pieces are called pyruvate. 00:12:32.04 Pyruvate has to go into the mitochondria 00:12:34.10 via those transporters that I talked about before, 00:12:36.18 in order to make energy. 00:12:38.10 So, this was really the step in the pathway 00:12:40.18 that we looked at when we were crushing up these yeast. 00:12:43.28 So, when we did this with our gene X deleted, 00:12:46.25 that's now called the MPC, 00:12:48.26 we saw in normal cells that pyruvate levels 00:12:51.02 were pretty normal, you know, 00:12:53.08 it's there but it's not huge. 00:12:55.06 But then when we take away the MPC 00:12:57.10 we actually see this increase in pyruvate, 00:12:59.26 and that's very specific. 00:13:01.06 It's like you've blocked this pathway, 00:13:02.18 you've kind of dammed up this channel, 00:13:03.28 and everything upstream of that is building up, 00:13:05.18 but everything downstream slows to a trickle. 00:13:08.09 So, like I said, downstream we have acetyl-CoA. 00:13:10.24 Acetyl-CoA is this immediate step downstream 00:13:13.12 once pyruvate gets into the mitochondria. 00:13:15.24 This was actually reduced when we knocked out the MPC. 00:13:18.21 So, we could isolate basically 00:13:20.21 where this block was, 00:13:22.11 you know, if I give this pipe analogy, 00:13:24.20 you know, you have this block here. 00:13:26.03 Upstream, pyruvate builds up, 00:13:27.20 and then everywhere upstream of that 00:13:29.12 you also have a buildup 00:13:30.24 and that's exactly what we saw. 00:13:32.09 And then everywhere downstream you also see a reduction, 00:13:34.18 everywhere - acetyl-CoA I showed, 00:13:36.04 but everything downstream was reduced. 00:13:38.24 And then you can imagine if you remove this block 00:13:40.26 you can actually restore the flow 00:13:42.14 and that's, you know, that's the normal condition. 00:13:45.11 So we had a very specific defect 00:13:46.27 and we got really lucky here, 00:13:48.29 because it was so narrowed in for us 00:13:50.20 that we really only had two possible options. 00:13:53.12 It could either be the metabolism 00:13:55.04 -- once pyruvate has gotten into the mitochondria, 00:13:58.05 to make acetyl-CoA, the breakdown by pyruvate hydrogenase -- 00:14:03.04 or it could be the ability of pyruvate to get through. 00:14:05.08 So, there were only two possibilities, 00:14:08.10 and we started with possibility number one. 00:14:09.27 Very simply, what we did was basically 00:14:14.00 punch holes in the membrane of the mitochondria 00:14:15.18 and just allow the pyruvate to get through. 00:14:17.19 So, it didn't need any kind of channel, 00:14:19.09 it didn't need a transporter, 00:14:20.17 and it could get access to this enzyme PDH. 00:14:24.08 So, when we look at normal cells, 00:14:25.18 if we look at the activity of this enzyme, 00:14:27.22 we saw that normal cells have good activity, 00:14:30.18 our MPC deletion, this gene X, 00:14:34.06 has normally activity 00:14:35.18 -- it's still there, it can still metabolize 00:14:37.14 perfectly fine if you give it pyruvate -- 00:14:39.29 but then this PDH mutant, 00:14:41.23 if we take the enzyme away we see that it's totally gone, 00:14:44.04 which is a nice control, 00:14:45.28 so we know that, you know, when you take the gene away 00:14:47.19 or there's anything wrong with it, 00:14:48.29 you don't get activity, which is just a good control. 00:14:52.04 So, the next step was really to see what happened 00:14:55.05 in terms of the transport of pyruvate. 00:14:57.07 So, we knew that the metabolism was fine 00:14:59.05 if it could get in, 00:15:01.07 if you could get gas into the engine. 00:15:02.25 But can it get there, 00:15:05.00 it is like the fuel line is cut or something? 00:15:07.02 So, to do this, we used radiolabeled pyruvate 00:15:10.14 - so, these little green dots on the end of pyruvate 00:15:12.16 are just radioactivity that we can look at 00:15:15.02 and really tell where pyruvate's going. 00:15:17.17 So, we isolated the mitochondria 00:15:19.12 and then just incubated them with pyruvate, 00:15:22.02 either with our gene absent 00:15:24.02 or with the gene present, 00:15:25.24 and then we can compare this and look 00:15:27.22 if pyruvate's getting in or not. 00:15:29.16 And this confirmed exactly what we would expect 00:15:32.05 - that in normal cells you get pyruvate taken up, 00:15:34.10 the radioactivity increases, 00:15:35.29 and we can see that very well. 00:15:37.18 In the MPC knockouts, 00:15:39.20 we see that you can't get pyruvate in, 00:15:41.02 so it's totally blocked. 00:15:42.17 This channel [can't let anything in], 00:15:43.28 you've cut this gas line from the engine. 00:15:46.05 And then when you put MPC back in, 00:15:47.25 after you've taken it away... 00:15:49.08 kind of a take it away and give it back... 00:15:52.07 you see that this fully restores the ability of pyruvate to get in, 00:15:55.06 suggesting that it's very specific. 00:15:57.08 It's just this channel that's defective 00:15:59.12 in this entire process. 00:16:01.04 The mitochondria are fine, 00:16:02.24 they can metabolize it normally, 00:16:04.14 it's just this one step at that membrane 00:16:06.18 that's blocked. 00:16:08.10 So, we have this system now, 00:16:09.26 this is kind of what we've discovered. 00:16:11.25 We have glucose -- carbs -- 00:16:13.09 going to pyruvate, 00:16:15.08 and then it needs to cross this barrier 00:16:17.10 in the inner mitochondrial membrane, 00:16:18.23 so it's kind of allowed through at a very specific rate, 00:16:22.22 as you need it... 00:16:24.25 otherwise, you don't wanna just dump everything on the mitochondria. 00:16:27.16 And then once it's inside, 00:16:29.05 pyruvate is broken down by PDH to acetyl-CoA, 00:16:32.04 and then it makes the energy that you need to move. 00:16:36.10 So, I know I talked about the yeast stuff 00:16:38.06 and all of our experiments, 00:16:39.18 but I want to get back to that little girl 00:16:41.00 and why this was so important, 00:16:42.17 and why I was excited about this entire project. 00:16:45.19 This little girl actually had the observations we made in yeast, 00:16:49.18 where I showed the one of two possibilities. 00:16:51.04 Her doctors did the exact same thing. 00:16:53.14 They punched holes in the membrane 00:16:55.04 and showed that once pyruvate could get in 00:16:57.14 it could be metabolized totally fine. 00:16:59.10 It was just the fact that it couldn't get through this membrane. 00:17:02.00 So, they showed the exact same thing 00:17:04.13 that we did in yeast 00:17:05.29 10 years earlier in this patient, 00:17:07.11 but they didn't know the gene that they were looking for, 00:17:09.22 so they couldn't do anything about it. 00:17:11.05 And this is really... 00:17:13.04 we came in and read this observation, 00:17:14.18 and it fit with everything that we'd studied so far, 00:17:17.05 and it was this perfect combination 00:17:19.12 to really make us contact these people 00:17:21.10 and, you know, offer to help, offer our assistance, 00:17:24.02 because they were asking for help in this case report. 00:17:26.04 They didn't know what to do 00:17:27.20 and they were really frustrated by this. 00:17:30.08 So, what I'm showing here 00:17:31.29 is just the amino acid sequence, 00:17:33.14 so the protein of the MPC 00:17:35.18 across all of these species in a little chunk, 00:17:38.14 just a little frame of this. 00:17:40.00 But going from yeast to fruit fry 00:17:41.27 to zebrafish to human, 00:17:43.12 and you can see in black they have the residues, 00:17:45.13 the amino acids that are most similar 00:17:48.17 across all of these species. 00:17:50.06 So, you can see here, this one that I've indicated 00:17:53.01 by the red star, this arginine, this R. 00:17:55.27 It's a very important amino acid, 00:17:58.05 and the significance of this is basically, 00:18:00.14 when I looked in the patient, 00:18:02.13 I solved this kind of three-dimensionally 00:18:04.23 in a three-layered puzzle. 00:18:06.04 So, what we started with was the DNA. 00:18:08.06 We were actually able to get fibroblasts from this patient 00:18:11.06 that they'd saved, 00:18:12.16 so the skin cells from this little girl. 00:18:13.29 They sent them to us, 00:18:15.20 and we were able to look at what was going on 00:18:17.13 in these cells. 00:18:18.27 So, while the doctors had done a really thorough metabolic characterization, 00:18:22.22 we kind of suspected that we knew the gene 00:18:24.21 we were looking for, 00:18:25.25 so we wanted to see if that was defective in this little girl. 00:18:28.05 So we started with the DNA, 00:18:29.16 which is the first level of this three-layered puzzle, 00:18:32.09 and then the second layer is the mRNA, 00:18:34.01 and the third layer is the protein 00:18:36.01 that actually has the function. 00:18:37.10 And, you know, a defect in one of these 00:18:40.00 or a problem in one of these 00:18:41.08 doesn't necessarily go all the way to the protein level. 00:18:43.24 So it's actually kind of rare that a DNA mutation 00:18:47.14 will have an effect on the protein function, 00:18:50.04 and it might even be totally silent. 00:18:51.26 It might not change the protein at all. 00:18:54.27 So, late one night, I was in lab, 00:18:57.16 we'd gotten the data back from the patient, 00:18:59.06 we'd gotten the results. 00:19:00.21 We'd only looked at the very important parts of this gene, 00:19:04.11 and when I was putting them together 00:19:06.05 it was kind of like, you know, 00:19:08.09 writing down... highlighting different pieces of paper, 00:19:09.26 like, very hectic, 00:19:11.19 like, I didn't know quite what I was doing, 00:19:13.06 but I was really excited about it, 00:19:14.28 probably overexcited 00:19:16.22 and so I took longer than I probably needed to, 00:19:19.06 but I basically pieced this together, 00:19:21.22 the fact that the DNA mutation 00:19:23.21 led to an RNA mutation, 00:19:25.03 which led to a protein mutation. 00:19:26.24 And carrying that all the way through in this patient, 00:19:29.08 it actually looked like in this conserved residue 00:19:31.17 it would have function 00:19:33.23 on the mitochondrial pyruvate carrier, 00:19:35.10 it would have function in terms of 00:19:37.11 getting gas into the engine, 00:19:38.19 and this might be why she had this horrible problem 00:19:40.25 and why she died. 00:19:42.16 And for me as an MD/PhD, 00:19:44.04 that was the single most salient part 00:19:46.18 of anything I've done so far in my life, 00:19:48.29 was identifying this human disease 00:19:51.00 that actually caused this little girl to die, 00:19:53.20 and now we know more information about this, 00:19:55.15 and hopefully we can do genetic counseling and screening, 00:19:58.21 and the hope is that her parents got to hear this. 00:20:01.14 I've never heard if they've actually 00:20:04.02 been contacted to understand this, 00:20:05.28 all I heard is they were lost to follow-up. 00:20:07.11 But this was by far the most significant thing I've done 00:20:10.24 and it was like winning the lottery. 00:20:12.10 If someone offered me the chance 00:20:14.18 to win the lottery or do this experiment, 00:20:16.06 I'd rather do this experiment, 00:20:17.20 even though it was late and night 00:20:19.22 and, you know, no one was around 00:20:21.11 and I was just kind of like, "Oh, there it is". 00:20:23.08 But it was so cool. 00:20:25.14 So, the metabolism of this little girl 00:20:27.29 really mirrored what happens in cancer. 00:20:30.22 It's very similar 00:20:32.13 and it's the same thing with yeast. 00:20:33.26 So, the yeast and the patient 00:20:35.14 looked almost exactly like this phenomenon in cancer, 00:20:37.23 first described here by Otto Warburg, 00:20:40.01 a German scientist who won the Nobel prize, 00:20:42.05 a very eminent biochemist 00:20:44.16 who did a lot of very basic studies. 00:20:47.14 Well, what he said was that mitochondria in cancer 00:20:50.02 were defective, 00:20:51.13 and that's why you have this buildup of acid, 00:20:54.02 and why cancer grows in a different way. 00:20:56.10 And ultimately he was proven wrong 00:20:58.00 -- it was shown that the mitochondria are perfect fine -- 00:20:59.23 it's just that they don't utilize energy 00:21:01.23 in the same way, 00:21:02.29 which is really important for us 00:21:05.14 because, like in the yeast, when I put the MPC back, 00:21:07.07 the mitochondria were totally fine, 00:21:09.06 they just needed this door in order to get the pyruvate in. 00:21:14.02 So, this is the very general aspect of this. 00:21:16.20 In cancer, whether there is oxygen or not, 00:21:19.28 cancer cells just want to grow, 00:21:21.12 they want to get as much energy as possible 00:21:23.29 as quickly as they can, 00:21:25.12 to become the worst cancer they can be, basically. 00:21:28.07 And then pyruvate will get shunted out here 00:21:30.17 into another metabolite called lactate, 00:21:32.24 but also you'll have a buildup upstream 00:21:34.20 of all these biosynthetic intermediates 00:21:36.28 that just help the cancer make more cells. 00:21:39.07 And what's important about that 00:21:41.04 is that it's been thought that if you could kind of relieve this, 00:21:44.00 relieve that block, 00:21:46.04 you could actually start to help cancer patients. 00:21:50.08 So, very initially, 00:21:52.00 we wanted to start by just looking at, 00:21:53.24 does the MPC have a role in the Warburg effect. 00:21:55.29 Could this explain part of way 00:21:58.28 the pyruvate is getting shunted 00:22:00.27 and doesn't get into the mitochondria? 00:22:02.20 It totally makes sense, right? 00:22:04.07 You have this inability in these patients 00:22:05.27 and the yeast to get pyruvate in. 00:22:07.25 That would totally explain the Warburg effect. 00:22:10.02 And that's actually exactly what we saw. 00:22:11.21 We saw in colon cancer 00:22:13.20 that the transcript level was significantly reduced 00:22:15.24 - this was in the top 100 most underexpressed genes 00:22:18.26 in this type of cancer. 00:22:20.06 And across many different cancers 00:22:21.22 it's very underexpressed or even mutated. 00:22:24.19 And then at the protein level, 00:22:26.10 we also wanted to compare... 00:22:27.25 does the normal tissue compared 00:22:29.18 to the cancerous tissue 00:22:31.08 in the same patient have a difference in the MPC level 00:22:33.16 that might explain this? 00:22:34.26 And that's exactly what we saw. 00:22:36.03 So, in these two patients, 00:22:38.14 we actually saw the normal tissue has high MPC [protein levels] 00:22:41.22 and the cancer tissue just adjacent to that 00:22:43.29 has low MPC expression. 00:22:45.29 So, you know, a scientist, 00:22:47.18 if something's low, 00:22:49.18 we want to put it back, right? 00:22:50.27 And we want to do the opposite of what's going 00:22:52.12 and see if it's important in that way, 00:22:54.04 and that's exactly what we did. 00:22:55.08 We just decided to reexpress 00:22:57.14 or overexpress the MPC 00:22:58.21 in this exact cancer type, 00:23:00.13 colon cancer, 00:23:02.01 and see if that changed anything about how the cancer behaved. 00:23:05.14 So, that's basically just relieving this block here. 00:23:07.23 If we go from this case, 00:23:09.16 where you can't get fuel to the engine, 00:23:11.12 to the case where you, you know, 00:23:13.20 allow the engine to function normally, 00:23:15.21 can we bring it back to normal? 00:23:17.00 Can we slow cancer growth, 00:23:18.14 and can we deplete these biosynthetic precursors, 00:23:20.24 these building blocks of the cell, 00:23:22.26 which is what I mean by biosynthetic. 00:23:25.20 And that's exactly what we saw. 00:23:27.03 We saw very strikingly, very conclusively, 00:23:30.06 time after time, 00:23:32.08 that when we had the MPC back in in these cancer cells 00:23:34.26 we could change their metabolism, 00:23:36.15 which I'm not showing, 00:23:37.29 and then we could also change their growth. 00:23:39.19 So, the spheroid assay that I'm talking about here, 00:23:41.24 these little cancer balls are called spheroids... 00:23:44.25 and what we do is basically grow these 00:23:46.14 and they'll basically form little cancer tumors 00:23:48.01 and we can count these and 00:23:49.20 see how fast they grow, see how big they grow. 00:23:51.27 And what we found is when the MPC is back, 00:23:53.22 as you can see really clearly here, 00:23:55.17 not only does the number decrease, 00:23:57.16 so the number of little cancers that forms is lower, 00:24:00.12 but the size is lower too, 00:24:01.27 so they're small tumors, which is great. 00:24:04.08 I mean, this is phenomenal. 00:24:06.27 The next part we wanted to do... 00:24:08.15 that was an in vitro assay, 00:24:10.00 so kind of artificial. We wanted to do this in mice. 00:24:12.08 Get a little bit closer to maybe 00:24:14.25 what a cancer patient would be like, 00:24:16.10 and in order to do this, 00:24:18.02 we took those same cells, 00:24:19.14 we injected them into the side of mice, 00:24:21.14 with and without MPC, 00:24:22.28 the normal cancer and the cancer 00:24:24.22 where we changed MPC levels, 00:24:26.10 and then we just looked at the tumor growth rate 00:24:28.12 over time to see how fast 00:24:30.06 and how aggressive these tumors were. 00:24:31.27 And like you would expect, 00:24:33.27 since I'm talking about this, 00:24:35.11 it was exactly the same as the spheroids. 00:24:37.21 When you have the MPC put back in 00:24:39.24 there's this slower growth rate, 00:24:41.06 but it also plateaus out here, 00:24:42.27 it just stops growing. 00:24:44.05 So there's something about changing the metabolism 00:24:45.25 that we're continuing to study, 00:24:48.06 that actually slows cancer growth, 00:24:50.24 which is awesome. 00:24:52.08 And then you can see the tumor over here, 00:24:53.26 that when you modulate this, 00:24:55.14 when you change metabolism, 00:24:56.26 you actually change the cancer 00:24:58.25 and it behaves more normally. 00:25:01.04 So, I just want to conclude 00:25:02.26 by talking about how great yeast is as a model system, 00:25:05.10 how great and important basic science is 00:25:07.29 to lead us to all of these observations, 00:25:10.05 and it was really the way that my boss 00:25:12.29 decided to ask this question, 00:25:14.10 it was the way that we pursued this, 00:25:16.01 that really allowed all of these benefits 00:25:17.25 to come from this. 00:25:19.09 We started in a basic system, 00:25:20.25 but the way the question was asked 00:25:23.19 it was directly translatable to human health. 00:25:25.10 Even if that wasn't the overall goal, 00:25:27.12 that's what we've been able to accomplish here. 00:25:29.24 So, basic science is important, 00:25:31.02 yeast is a great model organism, 00:25:32.20 even for studying something like, 00:25:34.12 you know, metabolic diseases, 00:25:36.10 development, cancer, other things... 00:25:39.22 And then it's really important 00:25:41.16 in how you think about metabolism. 00:25:43.18 It's important to think about 00:25:46.11 how you're getting the gas to the engine, 00:25:47.27 and just a subtle defect in that 00:25:50.16 can have devastating consequences, 00:25:52.09 like I talked about for this family. 00:25:54.22 The other aspect is... 00:25:57.10 cancer is very complex, obviously. 00:25:58.26 Cancer is not just one disease, it's many diseases. 00:26:01.16 And there are some cancers with high MPC, 00:26:03.25 some cancers with low MPC, 00:26:05.10 different situations where this will be important, 00:26:07.18 but the really critical thing here 00:26:10.08 is that we've identified this gene 00:26:11.20 and now we have the ability to study it. 00:26:13.16 We can understand it better. 00:26:14.23 We can do all of these different things 00:26:16.18 in cancer, and in heart failure, 00:26:18.06 and in diabetes 00:26:20.02 that I set out to do when I started my MD/PhD, 00:26:22.00 and that was all because of yeast. 00:26:23.13 But in cancer, 00:26:25.11 understanding this really allows for therapeutic opportunities. 00:26:29.04 So, now that we know what the gene is, 00:26:30.16 we know how we can target it, 00:26:32.08 maybe we can activate it, 00:26:33.19 we can do different things with it. 00:26:34.29 If we know that it's low, 00:26:36.10 maybe that's, you know, one weakness of cancer 00:26:38.13 that if we inhibit something else 00:26:40.04 we can kill the cancer faster, 00:26:42.04 which is a really exciting potential 00:26:44.06 and that's what I'm excited in pursuing. 00:26:46.29 So, I just want to stop with: 00:26:48.29 metabolism is always happening. 00:26:50.23 If nothing else, 00:26:52.27 metabolism is a very important part of biology. 00:26:55.05 It's interesting 00:26:57.20 and it's not just calories or carbs or diet. 00:26:59.17 It's everything beyond that. 00:27:02.17 So, I need to stop by thanking the people, 00:27:04.25 the teams of people that made all of this work possible. 00:27:07.01 They could just as easily be standing up here, 00:27:09.02 but I'm lucky enough to get this opportunity. 00:27:11.27 Dan Bricker 00:27:13.22 was a grad student in Carl Thummel's lab 00:27:15.04 at the University of Utah. 00:27:16.22 He used Drosophila as the first system 00:27:18.13 to knock out to the MPC 00:27:19.27 and really understand, in an organism, 00:27:21.14 what was going on in a, 00:27:23.21 you know, larger multicellular system. 00:27:26.08 He made that first observation 00:27:28.03 that there was this block in pyruvate. 00:27:30.05 Thomas Orsak was a grad student 00:27:32.01 that I overlapped with very briefly 00:27:33.26 that kind of taught me everything I know 00:27:35.11 about yeast in Jared's lab. 00:27:37.14 He started this yeast project 00:27:38.29 and really spearheaded the knockout, 00:27:41.02 and characterization, 00:27:42.10 and all of this stuff. 00:27:43.24 Eric Taylor was a postdoc, now he has his own lab, 00:27:46.26 and he did a lot of work on, actually, 00:27:48.24 the patient fibroblasts 00:27:50.15 and showing when we put the MPC back in, 00:27:51.27 it totally rescues these fibroblasts. 00:27:54.18 And while we couldn't do anything for this little girl, 00:27:56.12 that was an important thing to show, 00:27:58.02 that we could rescue this 00:27:59.28 by just putting the gene back. 00:28:01.12 That was the only thing that was wrong. 00:28:03.08 This next group... 00:28:06.05 our most recent paper working on the cancer stuff 00:28:08.13 and how the MPC is important... 00:28:10.12 Kris Olson is another MD/PhD student 00:28:12.09 with me at Utah in the program, 00:28:14.19 and we really spent a lot of time 00:28:16.18 spitballing ideas and kind of coming up with crazy ideas 00:28:18.29 on how to study this and understand cancer better, 00:28:21.12 so it's been great to work with him... 00:28:23.02 Jared, obviously... 00:28:24.24 the doctors in France 00:28:26.14 who identified and really thoroughly described this patient. 00:28:28.22 Without their description, 00:28:30.23 we wouldn't have known how important this was 00:28:33.06 for human health, 00:28:34.15 and it was really their call for help 00:28:36.23 and their really basic scientific insight 00:28:39.15 that allowed us to understand this better. 00:28:42.12 Other than that, I have to thank the core facilities 00:28:44.03 at the University of Utah 00:28:45.22 -- Metabolomics, Flow Cytometry, Imaging -- 00:28:48.14 all of these people have been phenomenal 00:28:50.18 in making this work progress at the fast rate that it has. 00:28:54.00 For funding, I'm funded by the Developmental Biology program 00:28:56.26 at the University of Utah 00:28:58.14 on an NIH training grant. 00:28:59.26 My MD/PhD program, 00:29:01.12 for all of their support 00:29:03.07 and kind of talking me into, you know, 00:29:05.21 melding both of these worlds together, 00:29:07.05 and using science to understand medicine 00:29:09.15 and medicine to understand science. 00:29:11.06 It's been a lot of fun. 00:29:12.12 The Nora Eccles Treadwell Foundation 00:29:14.06 for some basic support 00:29:16.05 on how the MPC is important in different diseases. 00:29:19.06 And then Jared's R01. 00:29:21.10 But last, I really have to thank 00:29:23.08 the people that have made this opportunity possible, 00:29:25.12 why I'm getting to talk to you right now. 00:29:27.26 The team at iBiology is absolutely phenomenal. 00:29:30.18 Even though we've only worked together for two days, 00:29:32.14 I'm going to go out to San Francisco 00:29:34.18 and I'll have a place to crash, which I great. 00:29:36.27 Shannon, Sarah, Monica... 00:29:39.27 all of them have been just absolutely phenomenal. 00:29:42.18 The Lasker Foundation, 00:29:44.10 for making this all possible. 00:29:45.25 Without their generous support, 00:29:47.03 we wouldn't be able to do this, 00:29:48.13 and it's been a great experience, 00:29:49.25 and it's really changed 00:29:52.04 how I'm going to communicate about science. 00:29:53.16 And then especially the Alda Center 00:29:55.06 for Communicating Science, 00:29:56.20 especially Val and Louisa 00:29:58.16 for making me completely comfortable 00:30:00.02 with everything that was going on here, 00:30:01.21 even when I was a little bit hesistant 00:30:03.10 they could see that and kind of be like, 00:30:04.12 "No, it'll be okay, don't worry about, it's fine". 00:30:06.26 So, they've been phenomenal to work with, 00:30:09.19 an absolutely great group of people. 00:30:11.16 And the other young scientists have also been a lot of fun to learn from 00:30:14.11 and talk about these ideas with. 00:30:16.26 So, thank you. 00:30:18.08 My email will be on the website. 00:30:19.15 If you have any questions about this... 00:30:20.26 I simplified a lot, so if you want to talk about 00:30:22.27 any more complex things, 00:30:24.18 please feel free to contact me. 00:30:26.01 Thank you.

Speaker: John SchellAudience:
  • General Public
  • Educators of H. School / Intro Undergrad
  • Student
  • Educators of Adv. Undergrad / Grad
  • Researcher
  • Educators
Recorded: May 2015
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Talk Overview

John Schell tells the story of a little girl that died over a decade ago from an unknown metabolic disease. Confounded, her doctors published her case in a scientific paper hoping that the mystery would eventually be solved. Schell read this paper soon after making a big discovery: He and his colleagues, using yeast and Drosophila genetics, found a new gene involved in the cellular breakdown of nutrients, the mitochondrial pyruvate carrier. The mitochondrial pyruvate carrier is responsible for transporting pyruvate (a nutrient) from the cytoplasm into the mitochondria, where it can be broken down to release energy. Schell and his team predicted that a defective mitochondrial pyruvate carrier was the cause of the little girl’s disease, so they asked the doctors for a sample of her skin cells to sequence her mitochondrial pyruvate carrier gene. They discovered that she indeed had a mutation in her mitochondrial pyruvate carrier gene. Further analysis showed that the mutation was likely damaging to the protein, supporting their prediction. Looking at other patients with other metabolic-related diseases, Schell also discovered that mitochondrial pyruvate carrier function is altered in certain cancers. Increasing the expression of mitochondrial pyruvate carrier in mice slowed tumor growth over time until the tumors stopped growing altogether, which suggests a new possible mechanism for cancer treatment.

This talk is part of the Young Scientist Seminars, a video series produced that features young scientists giving talks about their research and discoveries.

Speaker Bio

John Schell

John Schell

John Schell is an MD/PhD candidate in Jared Rutter’s lab at the University of Utah. Schell’s research has focused on how cells get energy for growth and the role mitochondria play in this process. He helped discover the identity of the mitochondrial pyruvate carrier, an important gene involved in cellular metabolism that was long overlooked…. Continue Reading

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Related Resources

Bricker DK, Taylor EB, Schell JC, Orsak T, Boutron A, Chen YC, Cox JE, Cardon CM, Van Vranken JG, Dephoure N, Redin C, Boudina S, Gygi SP, Brivet M, Thummel CS, Rutter J. (2012) A mitochondrial pyruvate carrier required for pyruvate uptake in yeast, Drosophila, and humans. Science. 337(6090):96-100

Schell JC, Olson KA, Jiang L, Hawkins AJ, Van Vranken JG, Xie J, Egnatchik RA, Earl EG, DeBerardinis RJ, Rutter J. (2014) A role for the mitochondrial pyruvate carrier as a repressor of the Warburg effect and colon cancer cell growth. Molecular Cell. 56(3):400-13

 

Reader Interactions

Comments

  1. Excellent work and well presented. I just want to comment that cancer mitochondria aren’t healthy as you mentioned in your presentation, if they were not dysfunctional, cancerous cells would stop fermenting when you upregulate MPC. Yes, you got smaller tumors, but it is still not a cure because the pathology of cancer comes from mitochondria dysfunction and alteration in the epigenetic landscape comes as a downstream effect of the disease. Random epimutations cause nuclear plasticity which leads to progressive aggressive tumor phenotypes (survival of the fittest between the tumor heterologous cells to cope with the tumor microenvironment).

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Tag » How Does Pyruvate Enter The Mitochondrion