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The Surprising World of Prion Biology

Part 1: Prions and Evolution

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00:00:06.12 So I work in a variety of different biological problems.
00:00:09.14 And they all have one thing in common: they have to do with a problem
00:00:15.02 of protein folding. So what's the problem of protein folding?
00:00:18.24 Well, proteins do just about everything in our cells.
00:00:23.23 and for us in our bodies. When you think about seeing an apple on a tree
00:00:29.05 the light that you perceive that apple by
00:00:33.10 is being harnessed by protein receptors in our eyes.
00:00:37.23 And it's the series of actions of various proteins
00:00:40.19 that transmit that signal from our eye
00:00:43.23 into other parts in our brain that can wind up interpreting that image.
00:00:46.28 When we go to pick that apple, our arm is powered by muscle. Muscle is protein.
00:00:51.25 The engine for creating the energy, the mitochondria inside of our cells
00:00:56.07 are made up of thousands of different proteins
00:00:58.13 that are involved in providing that energy for that movement.
00:01:01.07 When we bite the apple and we taste it, the taste is being perceived by
00:01:05.17 receptors on our tongue and in our nose
00:01:09.23 that are made up of proteins. And when we digest the apple
00:01:12.05 the enzymes that are involved in digesting it are proteins.
00:01:15.02 So proteins are us, I guess, is a way to put it.
00:01:19.26 And proteins however, are encoded by DNA. DNA is the code of life, a double helix.
00:01:27.14 And DNA is linear. It is a long, very complicated code.
00:01:33.09 But it's sort of like not really in some ways all that interesting in and of itself.
00:01:39.13 It is the information for life, but rather like a cassette tape,
00:01:46.02 it can play different tunes depending on
00:01:49.12 what part of the DNA is being accessed.
00:01:52.13 But the tape itself is linear.
00:01:54.20 Now the way this DNA plays different tunes
00:01:58.00 basically, is that it is decoded into long linear strands of amino acids
00:02:04.05 based upon the sequence of nucleotides in the DNA.
00:02:07.04 So here are two very different proteins that are being made
00:02:10.18 by the different parts of the DNA.
00:02:12.17 And in order for those proteins to function, they have to
00:02:19.26 fold up into these incredibly intricate, complicated shapes
00:02:24.02 This is hemoglobin here, the protein that is involved in carrying oxygen.
00:02:27.16 around in our blood.
00:02:28.14 And this protein over here is green fluorescent protein.
00:02:31.23 It's a wonderfully useful tool in biology.
00:02:35.13 It actually comes from a jellyfish, but it has this lovely property of
00:02:38.25 glowing green when you shine blue light on it.
00:02:42.15 And we use it as a tool as you will see a little bit later in this talk.
00:02:45.08 But you can imagine that it is kind of complicated for these proteins
00:02:50.29 to fold up into these shapes.
00:02:53.15 But the problem becomes much, much greater inside the crazy environment
00:02:58.07 of a living cell, where the concentration
00:03:00.10 of other proteins is about 300 milligrams per mL.
00:03:05.27 That's about the concentration of protein that crystallographers
00:03:09.20 have in packed crystals.
00:03:11.11 when they are actually exploring the protein structure.
00:03:14.06 but of course it is not like a packed crystal.
00:03:16.29 The cell is very, very dynamic and proteins
00:03:19.13 are moving around and colliding into each other all the time.
00:03:22.18 So when you think about proteins going about their business inside of a living cell
00:03:27.01 it's not like Fred Astaire.
00:03:30.10 and Ginger Rogers.
00:03:39.20 It's more like the characters in a Marx Brothers movie.
00:03:42.25 Where chaos is poised on the precipice of disaster.
00:03:57.28 So that's what we folks in the protein folding field
00:04:02.19 call going off pathway.
00:04:04.11 And proteins are doing that all the time in living cells.
00:04:07.17 They're trying to fold, and they often get it right, and they often don't.
00:04:12.11 And disaster can ensue when they don't get it right.
00:04:16.10 And in fact it is cause of a lot of human diseases.
00:04:20.00 I am going to be telling you about a very interesting
00:04:22.29 disease caused by protein misfolding in just a minute.
00:04:25.28 but first I want to tell you where and why we study this human disease
00:04:31.23 in a very unusual and unexpected place.
00:04:35.07 So this problem is as ancient as life itself.
00:04:38.25 and the solutions to it were actually arrived at very early on
00:04:43.10 in evolution. And they are shared amongst all organisms.
00:04:47.12 They involve things like protein chaperones.
00:04:50.13 osmolytes, protein remodeling factors,
00:04:53.01 protein degradation machinery, and compartmentalization of proteins.
00:04:57.15 And it's a pretty complicated problem,
00:05:00.17 so our motto is "If you've got a really complicated problem
00:05:02.26 that can give rise to a lot of very difficult diseases in human beings
00:05:07.17 for example, it'd be a good place to start someplace simple."
00:05:12.25 A good thing to start is someplace simple, and then move around a lot.
00:05:17.07 So we very often start our experiments with this organism here,
00:05:23.15 This is the budding yeast, known as Saccharomyces cerevisiae.
00:05:29.19 and it's actually been an incredible friend to mankind.
00:05:36.00 Man has been taking advantage of this organism for several thousand years
00:05:41.07 because it likes to take sugars and turn it into alcohol and CO2.
00:05:45.12 And that means that we have used this organism to make bread,
00:05:49.16 and we have used this organism to make beer and wine
00:05:52.10 for thousands of years. Now about a hundred years ago or so,
00:05:57.18 brewers were facing the problem that they'd brew a batch of beer
00:06:01.00 and it would be great, and another time they would
00:06:02.23 brew a batch of beer and it would be terrible.
00:06:04.28 And so they decided to start working on the biology of what was going on
00:06:09.10 in these brewing vats of beer.
00:06:14.09 And they found out that there was this
00:06:17.21 particular organism that was responsible for changing
00:06:20.20 the grain into alcohol and CO2.
00:06:24.01 and they then started to do more and more work was done on this organism.
00:06:27.26 originally with this goal of brewing better beer, but
00:06:31.27 it turns out that as biologists studied this organism more and more,
00:06:36.24 we found out that it has a tremendous number of properties that make it
00:06:40.12 just ideally suited for biological research.
00:06:43.18 For example, it can grow either in the haploid or the diploid state
00:06:48.12 and we are obligate diploids, we have one set of chromosomes
00:06:53.15 from our mother and another set of chromosomes from our father
00:06:55.09 and until that sperm and egg comes together
00:06:58.20 the sperm itself is a haploid, the egg is a haploid.
00:07:01.26 They are not doing anything much.
00:07:03.13 It is only when they come together that you form a new organism.
00:07:07.20 But in yeast they can live and you wouldn't really know
00:07:12.18 much difference between them being the diploid state and the haploid state
00:07:15.24 and for a geneticist, that's a dream.
00:07:17.29 because if you have one bad copy of a gene and once good copy of a gene
00:07:21.22 you can keep the bad copy in a diploid state,
00:07:26.19 so that the good copy takes care of the cell
00:07:28.22 and then you sporulate the cells and you get the haploid
00:07:31.28 progeny out you can see what that mutation does.
00:07:35.11 It's really a wonderful property.
00:07:37.00 Yeast cells also have very small genomes, so it turns out that this was the first
00:07:42.05 higher organism to have its genome sequenced.
00:07:45.14 And you may be wondering why we'd call this a higher organism
00:07:48.06 It doesn't seem like a higher organism.
00:07:51.11 but it actually is. It has much more in common with human cells than bacterial cells do.
00:07:57.15 And the reason is that these yeast cells have a membrane bounded nucleus.
00:08:04.27 They contain mitochondria, which are energy factories for the cell.
00:08:09.04 They contain a whole series of membrane bounded
00:08:12.16 compartments where they put different proteins that are compartmentalized and secreted
00:08:16.24 from the cell, or not secreted from the cell,
00:08:18.00 moved around the cell in different ways.
00:08:19.16 And that's a lot like how our cells work.
00:08:23.15 So even though these cells are separated from us by about a billion years of evolution.
00:08:27.09 they actually do things with a remarkable number of details.
00:08:33.25 And they do them very much like we do.
00:08:35.28 So we often use these cells as living test tubes to study
00:08:40.11 really complicated problems in protein folding biology.
00:08:44.06 And that takes us into problems of evolution and it takes us into problems of human diseases.
00:08:49.23 And so I am going to actually bring two of those problems together today
00:08:53.25 by telling you about some work we initially started to do because
00:08:57.14 we were interested in a very bizarre human disease.
00:08:59.29 that then when we got into yeast and started studying this
00:09:03.03 process, it took us into a very interesting realm of evolutionary biology.
00:09:09.00 So the disease that I am going to be talking to you about it a neurodegenerative disease
00:09:13.16 It's a really dreadful one
00:09:16.13 and it's also a rather mysterious one. It is one that we still don't understand completely
00:09:21.07 although we are making a lot of progress on it.
00:09:23.20 This is a front page from my home town newspaper
00:09:27.17 and as you can see "Imported Cows Killed" and "Brain ailment a mystery"
00:09:31.07 This was from back in the days when Great Britain was staggering under the mad cow epidemic
00:09:39.18 It was very, very frightening because we didn't know...
00:09:43.11 it became apparent that the disease was actually crossing the species barrier
00:09:48.15 and some young people in Great Britain were coming down with the disease.
00:09:52.14 But thank goodness, there is a very strong species barrier, and so really
00:09:57.13 only a small number of people have come down with the disease, so far.
00:10:00.28 And it looks like it is not going to be of outrageous proportions.
00:10:05.13 This is a very, very bizarre disease
00:10:08.04 in many different ways, and I am just going to tell you a couple of them.
00:10:13.00 So most diseases, most infectious diseases that we get
00:10:19.11 are caused either by viruses or by bacteria.
00:10:24.04 So these viruses and bacteria have their own nucleic acids,
00:10:29.11 their coding information of life,
00:10:31.18 that they carry with them and they use that to subvert what...
00:10:34.23 to take advantage of our bodies and to make more of themselves.
00:10:39.24 This particular disease, the disease is known as the prion diseases
00:10:46.14 can be manifested as infectious forms
00:10:50.26 but you can also get the same disease by inheriting a mutation in a particular protein
00:10:57.12 in your body. Moreover, you can also get this disease just by sheer spontaneous chance
00:11:04.04 and that usually happens in the elderly, and thank goodness it doesn't happen very often.
00:11:08.07 But there really isn't any other disease that I know of that has these properties.
00:11:13.00 It's really quite strange, and the really strange part about it is
00:11:16.21 that these spontaneous forms of the disease and the heritable forms of the disease
00:11:20.16 can both give rise to infectious material.
00:11:25.28 Now when Stan Prusiner was studying this
00:11:28.23 disease a couple of decades ago, and trying to purify the infectious material
00:11:33.08 The more and more he purified it, the more it became apparent that it looked
00:11:37.13 like it was just made up of protein without any nucleic acid, without any code, in there.
00:11:42.20 So how could this protein possibly cause a disease
00:11:47.14 and moreover replicate inside our bodies and give rise to more and more infectious material?
00:11:52.01 Well, the solution to this turned out to be...the solution to this problem
00:11:59.15 that this infectious agent might be a protein
00:12:02.08 is that it is one of our own proteins.
00:12:06.00 So it is a protein that we already have in our brain. We are expressing it all the time.
00:12:10.06 and it has a normal conformation that is obviously diagrammed in a very simplified way
00:12:17.01 here. And thank goodness it normally stays in that conformation.
00:12:21.24 Every once in awhile however, it can get into this altered conformation down here
00:12:26.10 which is really a profoundly different conformation,
00:12:28.19 it folds in a very different way.
00:12:30.21 And it has a sort of uncanny ability in that changed folded state
00:12:37.01 to be able to entice the other protein to change its shape too.
00:12:43.04 And it's that process that generates a sort of protein conformational chain reaction
00:12:48.12 that generates more and more infectious material
00:12:50.16 and in the process, in a way that we still don't understand,
00:12:54.16 winds up killing our neurons.
00:12:55.22 Now we think based upon some work that we have been doing in yeast cells actually
00:13:01.01 that it's not that this protein in this shape here interacts with protein in that shape there
00:13:06.13 directly and gets it to change its shape,
00:13:09.20 but rather, we think, that this protein transiently
00:13:12.24 exists in an unfolded state, every once in awhile it folds and unfolds
00:13:16.25 and when it's in this unfolded state that it is susceptible now
00:13:20.00 to being templated by this altered protein.
00:13:22.28 to generate more and more of this altered state.
00:13:30.00 Now this problem with protein folding, as I have been mentioning
00:13:34.06 to you, is very, very deeply rooted in biology.
00:13:36.19 And one of that ways that it has been deeply rooted in biology is
00:13:40.18 the way that I have been talking about the fact that the
00:13:43.02 problems and the solutions are
00:13:45.09 shared by all organisms. Chaperone proteins, protein remodeling factors,
00:13:49.05 protein degradation machinery, etcetera.
00:13:51.11 But the real take home message that I'd like to leave you with today is
00:13:55.24 that it's even more deeply rooted than that.
00:13:59.17 And that is that mother nature is pretty clever,
00:14:02.23 and she's been coping with this problem for a few billion years
00:14:06.29 you might imagine that she would actually find a way to take advantage of this problem.
00:14:12.05 in certain situations.
00:14:13.14 And so the prion biology that I am going to be telling you about,
00:14:16.26 that exists in yeast,
00:14:18.28 which is a really very similar sort of protein folding cascade
00:14:22.10 that occurs in this neurodegenerative disease,
00:14:24.22 can actually in yeast cells can provide a beneficial effect.
00:14:31.12 So these are yeast colonies growing on a Petri dish.
00:14:38.10 And these red guys over here turn out to be genetically identical to these white guys over here.
00:14:47.13 And I am not going to tell you all the different lines
00:14:51.04 of evidence that indicate that they are genetically identical,
00:14:53.00 that is they are genetically identical in terms of the DNA that codes for their properties.
00:15:00.01 There is one genetic property different between them,
00:15:03.26 and obviously you can see that when you streak out
00:15:06.16 the red cells you give rise mostly to red cells,
00:15:08.26 and when you streak out the white cells you give rise almost always to white cells.
00:15:12.03 So that is a heritable property that these cells have,
00:15:16.05 but every once in a while the red cells will change to white,
00:15:21.20 about 1 in 100,000, 1 in a million cells or so will change color.
00:15:24.14 And then when you take those white cells and streak them out over here,
00:15:28.20 those white cells will always give rise to
00:15:32.00 white cells, but every once in awhile they'll change color, and you can pick
00:15:35.21 them up and streak them out and they will give rise to more red cells.
00:15:38.07 And the first person to find this phenomenon was Brian Cox
00:15:42.28 and Brian deserves a tremendous amount of credit
00:15:46.23 because he studied this in a very, very careful way
00:15:51.13 and he discovered the underlying basis for this change in color.
00:15:55.20 The change in color is due to the fact that there is
00:15:58.28 a change in the translation properties, that is, the way that ribosomes
00:16:03.01 are decoding the messenger RNA
00:16:04.27 and there is a particular messenger RNA that is involved in the production of
00:16:08.04 this pigment, and it is decoded in a different way in these cells.
00:16:12.05 The underlying basis for why they would be changing the way they are decoding
00:16:17.04 their messenger RNAs wasn't known at all.
00:16:19.07 And Brian named this…these different states
00:16:26.05 "psi", and I sometimes think that
00:16:29.04 he might have done that because he was really sighing
00:16:32.08 because the inheritance was just plain bizarre,
00:16:34.25 and frustrating, and difficult to understand.
00:16:36.23 So there are brackets around the name psi
00:16:41.13 and the reason for that is really to denote the fact that it is not being inherited the way
00:16:45.07 normal genetic change would be inherited.
00:16:48.02 So when you mate the white cells with the red cells
00:16:52.14 the white state is dominant. So the diploid organism that comes
00:16:57.10 from the mating of those two haploid organisms is white.
00:17:01.00 But then when you sporulate those cells,
00:17:04.02 if this was caused by a change in the nucleic acid in the DNA,
00:17:08.19 in these cells, you would bring one white copy of the DNA and one red copy of the DNA
00:17:12.25 and then when you sporulated them, half of the progeny would be white,
00:17:16.07 and half of the progeny would be red. That's the way it would normally work.
00:17:20.27 But that's not what happened with this strain.
00:17:22.11 What happened with this strain was that when you
00:17:23.20 sporulated the cells, all of the cells were white.
00:17:26.29 Now that is just one of the very weird aspects of the biology of these cells.
00:17:33.00 I don't have time to tell you all the others, but
00:17:35.06 the genetic behavior was just very, very strange.
00:17:37.20 And it was Reed Wickner, about a decade ago,
00:17:41.21 who suggested well, maybe, this is based on some sort of self perpetuating change in protein.
00:17:47.01 And since the mother cell shares some of her proteins with the daughter cell
00:17:52.12 that would be understandable why it would have those properties that the mother cell…
00:17:56.00 all of the progeny would have that white state.
00:18:01.11 And so we began to study this problem
00:18:05.01 because I got a call from Yury Chernoff in Susan Liebman's laboratory
00:18:09.17 and Yury had been studying this bizarre biological problem
00:18:14.05 for quite a long time, and he had been looking for
00:18:17.12 other genes and other factors that influenced this pattern of inheritance.
00:18:22.24 And what he found was a protein that we'd been working on
00:18:28.05 for quite a long time. It had a big influence on whether or not this trait could be inherited.
00:18:33.18 This was a protein called Hsp104, it is a heat shock protein.
00:18:38.29 And it's made in vast quantities when cells are exposed to high temperatures
00:18:43.01 because it copes with the problem of protein folding.
00:18:46.13 So let me just digress here for a few moments.
00:18:49.13 To tell you about what Yury was connecting up with when he made that phone call.
00:18:54.03 He was actually connecting up with a line of research that we had been following
00:18:58.00 in my laboratory for many years.
00:18:59.20 Something called the stress response. And this in fact is a very vivid example of it.
00:19:06.12 What we have done in this experiment is very simple.
00:19:10.27 Cells that were growing at room temperature
00:19:14.25 in a culture swirling around in our laboratory
00:19:17.17 We stopped the culture, took out two aliquots,
00:19:21.08 and then one of those cultures we treated at 37 degrees,
00:19:27.21 sort of a warm summer day kind of temperature.
00:19:29.16 for half an hour. And then after that we took both of the portions
00:19:34.13 of the culture and put them in now at much higher temperatures:
00:19:37.27 fifty degrees for several minutes.
00:19:40.22 And then we simply plated them onto agar plates to see how many cells were still alive.
00:19:44.28 And as you can see over here, these cells that got
00:19:49.15 the mild preheat treatment are doing just great.
00:19:51.00 But these cells that did not have time to adjust
00:19:55.09 to the changing condition are dead. Or most of them are dead.
00:20:00.09 So this mild stress tolerance inducing treatment
00:20:06.10 is actually a universal phenomenon.
00:20:10.06 And here is an example of it in another organism,
00:20:12.00 that we have been working on for quite awhile.
00:20:14.09 It is called Arabidopsis. It is a simple little mustard plant.
00:20:18.11 It grows very fast and is used in research very frequently.
00:20:21.19 And this is basically the same type of experiment,
00:20:24.17 in one case the plants were given a mild preheat treatment to allow them to
00:20:29.29 adjust to the second heat treatment, which was the test condition.
00:20:35.02 And in this other case they were just put directly
00:20:38.02 up at the high temperature, and then both were returned to normal temperature to see how
00:20:43.24 they would survive.
00:20:45.02 And as you can see the plants that got the mild preheat treatment did great.
00:20:49.04 The ones that did not get the preheat treatment died.
00:20:53.25 This is universal, it happens in us too.
00:20:58.10 So when we have a fever for example,
00:21:00.24 we are mounting this mild stress response that is helping to protect us.
00:21:04.23 And it turns out that the major reason, the major thing that is going on in this stress response
00:21:09.27 is that we are making proteins called heat shock proteins.
00:21:12.09 These heat shock proteins cope with the crisis of protein folding.
00:21:17.21 Remember that I told you earlier that it is very, very crowded inside the cell
00:21:21.04 and that the proteins are constantly going off pathway and misfolding
00:21:24.01 and getting into trouble with each other.
00:21:26.01 Well it is bad enough at normal conditions,
00:21:28.23 but when you change the temperature or you add a little ethanol,
00:21:32.08 or you oxidize the proteins,
00:21:34.25 or you expose them to metal ions,
00:21:36.06 many, many different types of stress will cause
00:21:39.00 the problem of protein folding, that's constant all the time,
00:21:43.04 to get a whole lot worse in a hurry.
00:21:45.07 And so this very, very ancient response,
00:21:48.12 that's actually been conserved since probably the dawn of life,
00:21:51.29 this ability to respond and protect proteins from misfolding,
00:21:58.18 has been conserved very, very highly.
00:22:02.17 And we are actually now working on it in a variety of other types of organisms
00:22:06.03 including in mammals, because
00:22:08.16 there is very good evidence that many of the diseases
00:22:11.18 of misfolding, not just Mad Cow Disease, but sickle cell anemia, cystic fibrosis,
00:22:16.25 Alzheimer's disease, and many others, actually it would be better
00:22:21.00 if we were able to get those cells in our brains to be prepared
00:22:26.24 for the stresses of protein misfolding.
00:22:28.23 This protective response also helps cancer cells to survive.
00:22:35.00 They, as part of their natural biology, undergo a wide variety of different stresses
00:22:39.16 They are starved for nutrients. They are exposed to the immune cells.
00:22:44.15 They are trying to live in different environments.
00:22:46.18 And this mild protective stress response actually helps to protect them.
00:22:52.03 So it actually turns out to be a multifaceted arm:
00:22:55.17 very important in many different aspects of human disease.
00:23:02.05 And that is what really connects up the protein folding issues
00:23:06.12 that we have been studying
00:23:06.26 in my laboratory now with this phone call I get from Yury Chernoff
00:23:11.06 asking me if I knew what one of the particular proteins
00:23:14.28 that is induced during the stress response
00:23:17.08 a protein called Hsp104, does.
00:23:21.07 Well we had found that Hsp104 is the single most important protein for
00:23:28.24 yeast cells and actually in Arabidopsis as well,
00:23:32.10 for providing this type of protection against extreme heat.
00:23:36.27 Now many of the other heat shock proteins provide
00:23:39.20 protection against lesser extreme heat
00:23:41.20 and it has turned out over the years as we have learned what they are doing.
00:23:45.20 It turns out that these other heat shock proteins
00:23:47.28 prevent proteins from aggregating.
00:23:50.14 But this protein called Hsp104 did something very, very unexpected.
00:23:55.22 It can actually take proteins that have aggregated, for example under conditions of a sudden
00:24:02.07 and extreme stress and it can take those proteins apart.
00:24:06.24 And return them to circulation so that the organism can live again.
00:24:11.06 Now the reason Yury was asking me if I knew what this protein did
00:24:16.17 is that we had published a genetic experiment showing that if we knocked the gene out
00:24:22.22 for the protein so that the cells could do everything else they do in the heat response
00:24:26.04 but they couldn't make Hsp104,
00:24:29.03 instead of looking like this, they'd look like this.
00:24:33.07 So Hsp104 was vitally important for providing that protection.
00:24:37.17 That had been published, but we hadn't published the molecular mechanism
00:24:41.21 of dis-aggregation because, quite honestly, nobody believed us.
00:24:45.19 It seemed impossible that cells would be able to dis-aggregate a protein aggregate.
00:24:49.26 But let me just give you an example of some of the evidence
00:24:52.12 that that does, in fact, actually take place.
00:24:55.00 So these are simple electron micrographs, and
00:25:00.17 what we are simply doing is cutting across a cell, a yeast cell
00:25:05.20 and you are seeing that there is a general gray coloration of the cytoplasm.
00:25:10.06 The nucleus looks a little bit different.
00:25:12.29 But the cells are really pretty smooth. They maybe contain a little inclusion here or there.
00:25:19.23 But when they have been heat shocked,
00:25:23.10 what you can see here is that there's a tremendous amount
00:25:26.18 of protein aggregation that has taken place in these cells.
00:25:29.17 And you can see these big dark blobs all over the cell.
00:25:34.09 When they are allowed to recover from heat shock,
00:25:37.07 you can see that those blobs disappear. And that is
00:25:43.05 because Hsp104 has taken those aggregates apart.
00:25:46.01 These cells down here are genetically absolutely identical to these cells,
00:25:50.21 except for one small difference. And that is we've knocked out the gene encoding Hsp104.
00:25:57.22 And in these cells when they get the damage of heat shock,
00:26:04.23 and get protein aggregation, and then are allowed to recover at 25 degrees,
00:26:09.16 they don't do so well. They cannot dis-aggregate protein aggregates.
00:26:16.29 Now Yury called me because we had shown and published that this protein
00:26:24.26 factor, Hsp104, was vital for allowing cells to survive high temperatures.
00:26:31.16 But, we hadn't published its molecular mechanism yet,
00:26:34.06 and Yury called me to ask if we knew anything much about it.
00:26:37.03 And sure enough, we did know a lot about it,
00:26:39.13 in fact we had a manuscript sitting on my desk
00:26:42.29 that was just about ready to go back out
00:26:44.27 for review because it had been rejected the first time.
00:26:48.15 What we had shown this protein does
00:26:52.17 is to actually take aggregated proteins
00:26:55.05 and take them apart.
00:26:56.22 It uses a tremendous amount of energy of ATP to take them apart.
00:26:59.18 And that had been something that people didn't believe could happen.
00:27:04.03 Once a protein was an aggregated mess,
00:27:05.03 the only thing you could do was to just get rid of it.
00:27:08.18 But in fact, the cell has a back up mechanism,
00:27:12.06 it's been coping with protein folding problems for a long time.
00:27:14.04 And this was a very, very highly conserved protein,
00:27:16.01 and actually once proteins aggregate you actually can take them apart.
00:27:22.27 So we started working with Yury on this project actually
00:27:27.04 even before we had made that suggestion that it was protein based
00:27:31.00 and of course as soon as he made that suggestion everything made sense.
00:27:34.20 Because our protein takes protein aggregates apart,
00:27:37.15 and this inheritance was due to a protein aggregate,
00:27:42.21 maybe that would explain it.
00:27:44.25 So how could it be that the inheritance of a trait could be due to a protein aggregate?
00:27:49.09 Imagine you have a protein in one state,
00:27:52.25 and it can every once and awhile get to this profoundly different state.
00:27:55.13 just like was true, or was believed to be true for that prion disease.
00:28:01.19 Of course in these different folds, the protein's going to have a very different function.
00:28:07.01 And so, if this particular state, when it does arise every once and awhile in the cell
00:28:12.07 has the property of being able to get the other protein to change shape too
00:28:16.04 it can actually serve as an element of genetic inheritance.
00:28:20.09 Because when the mother cell divides she is going to pass
00:28:24.03 some of that altered protein into her daughter cells.
00:28:26.11 And then when the daughter cells start to make their own proteins,
00:28:30.09 those proteins are going to wind up encountering that template,
00:28:33.12 and changing their shape, and changing their function too.
00:28:36.11 So you actually will inherit an altered function,
00:28:41.11 inherit a different property, a different trait,
00:28:44.23 but you don't inherit that difference because you have a change in nucleic acid,
00:28:49.00 you don't inherit it through mutation,
00:28:50.25 you inherit it because you inherit a protein with a different shape.
00:28:56.14 And that trait is basically carried by that change in shape.
00:29:00.16 Because it is a self perpetuating change in shape.
00:29:03.11 So the fact that Hsp104 was involved in this inheritance,
00:29:08.04 and we knew that what Hsp104 was doing
00:29:10.11 was taking protein aggregates apart, immediately got us started thinking t
00:29:14.17 hat this might all fit together and make sense.
00:29:19.00 And in having this type of a very difficult protein folding problem
00:29:23.09 to study in an organism like yeast, we were very lucky because we could take
00:29:28.18 advantage of the genetic manipulations that I have just been telling you
00:29:30.24 you can do in yeast
00:29:31.24 which you can't begin to do in any other organism.
00:29:34.28 We were lucky in two other ways.
00:29:38.10 First of all we actually knew the identity of the protein
00:29:41.25 that was involved in undergoing this change in shape.
00:29:44.05 And we knew what would happen when that protein changed shape.
00:29:48.04 It would change its function, and this change in color from red to white
00:29:51.26 make complete sense.
00:29:53.00 So unlike the mammalian prion we don't know why when it
00:29:56.24 changes its shape it is toxic to cells, but here we could
00:30:00.27 understand that the change in shape would give rise to these different colors.
00:30:05.18 And I am going to be telling you a little bit more specifically about that
00:30:09.02 mechanism in a bit.
00:30:11.29 We were lucky in another way too,
00:30:14.21 and that is, when we purified the protein,
00:30:16.24 at first it was really hard to work with because it kept aggregating on us.
00:30:21.16 But when we finally figured out how to work with it,
00:30:23.10 we were really lucky that we were able to reproduce this change
00:30:28.11 in folding in a test tube very easily, very reproducibly,
00:30:33.03 all by using just this one isolated protein in the test tube.
00:30:40.06 And so that meant we could study it in a way that again,
00:30:45.01 with the mammalian prion, that has just been accomplished now after decades of trying.
00:30:49.26 And I think maybe that it's not just that we are lucky,
00:30:53.23 I think it may be that the reason why we are able to do this
00:30:57.02 is that this change in the function of this protein,
00:31:01.06 that produces these differences in red and white cells,
00:31:03.23 is actually producing a lot of other differences in their biology as well.
00:31:07.26 A lot of different types of traits.
00:31:10.06 It's a translation termination factor, and as I mentioned it
00:31:13.25 changes the way in which messenger RNAs are decoded
00:31:17.14 when it's in one shape versus the other shape.
00:31:20.14 And that can produce a lot of useful properties for the cells.
00:31:26.00 So this is the protein. It is known as Sup35.
00:31:30.03 And it has an interesting domain structure.
00:31:35.13 This C terminal region over here
00:31:38.11 is the business end of the protein. It is the translation termination factor.
00:31:42.02 And it tells ribosomes to stop, to terminate translation,
00:31:46.25 when it sees a STOP codon, the signal for stopping translation.
00:31:51.20 And in its normal folded functional state, it is responsible for turning the cells red.
00:31:59.14 as I mentioned, I'll show you that in a little bit more detail in a minute.
00:32:02.12 Attached to this translation termination factor, however,
00:32:07.01 there are two additional domains. There's an N terminal domain,
00:32:11.20 which we abbreviate as N, and a middle domain, M.
00:32:16.00 And these are very bizarre looking domains.
00:32:19.06 If you are a person who is used to looking at protein amino acid sequences,
00:32:24.01 you just look at the sequence of amino acids
00:32:26.04 in these proteins, and they just don't look like normal protein domains.
00:32:30.10 The N-terminal domain is extremely rich in glutamine residues
00:32:35.04 and it is very prone to misfolding and going into an aggregate,
00:32:41.00 a special kind of aggregate, as I will mention in a minute.
00:32:43.22 The middle domain is very, very highly charged,
00:32:46.21 so the middle domain has a tendency to keep this protein soluble.
00:32:50.00 It likes water. It likes to be in the soluble state.
00:32:53.22 And it's the interplay of the aggregating domain of the protein and this middle domain
00:32:59.21 that likes to be soluble that allows the protein to exist
00:33:03.15 in this sort of bistable state.
00:33:05.12 Once it gets into an aggregate it will self perpetuate.
00:33:07.19 If it's soluble, it will stay soluble.
00:33:09.25 And only every once and awhile will it change states.
00:33:17.14 So this is the translation termination domain here.
00:33:20.17 And that's the prion determinant.
00:33:24.17 Now one of the things we did to try to figure out what was happening to this protein
00:33:31.06 in these red versus white cells,
00:33:33.14 was simply to put green fluorescent protein on the end of it.
00:33:36.02 We encoded, made a new gene and had the genetic code
00:33:41.01 read right into the code for green fluorescent protein.
00:33:44.05 So that we could look at the cells under the microscope
00:33:48.26 and actually see what was happening to the protein because the protein would glow green.
00:33:54.02 And this is basically what happened.
00:33:57.08 In the red cells the protein was in the soluble state. It was going about the cell doing its business.
00:34:02.29 And in the white cells, the [PSI]+ cells,
00:34:07.19 you could see that a lot of the protein, not all of it, but a very large fraction of the protein
00:34:12.16 is found in these little aggregates here. These little intense foci.
00:34:17.10 So a large portion of the protein is actually not available to serve its normal function in translation.
00:34:24.08 And this was really a great joy to actually be able to see this thing happening
00:34:28.05 in living cells because you could actually watch the inheritance process.
00:34:32.21 You could watch mother cells passing on these little templating dots
00:34:37.20 into their daughter cells and that is why once this process gets started,
00:34:41.23 it keeps on going, keeps on going.
00:34:44.05 And we could take Hsp104, our protein remodeling factor,
00:34:47.16 and when we over-expressed that protein,
00:34:50.19 as Yury had found, that if you put an extra gene into the cells,
00:34:55.12 the cells will turn from white to red.
00:34:58.16 It turns out to be because Hsp104, takes these protein aggregates apart.
00:35:02.15 stops them from being made,
00:35:04.28 and then the daughter cells wind up coming off
00:35:07.20 of the mother cell without the protein aggregates.
00:35:10.02 And so they are cured. They are permanently cured.
00:35:13.01 We can also watch new prions forming in these cells here.
00:35:18.28 If we just take that prion determining domain
00:35:21.28 that I told you about and cause cells to over-express that,
00:35:26.27 it makes it more likely that the proteins will encounter each other
00:35:29.13 in such a way that they will nucleate this altered aggregated form.
00:35:32.27 And once that's started, it is self perpetuating.
00:35:35.14 So a lot could be done just by actually visualizing these cells.
00:35:42.00 So when we purified the protein and looked at what types of
00:35:48.21 structures were formed by the protein and different pieces of the protein
00:35:53.26 we found something really interesting.
00:35:56.10 It looks like blobs in a yeast cell, but at the level of much higher magnification
00:36:02.19 that you can achieve with an electron microscope,
00:36:05.07 what we found was not just an aggregated non-specific blob,
00:36:09.07 but what we found were fibers,
00:36:12.10 highly ordered structures, and in fact these are amyloid fibers.
00:36:16.05 Amyloid fibers are very particular type of fiber,
00:36:20.29 in which there is a beta sheet strand crossing perpendicular
00:36:27.21 to the axis of the fiber, back and forth, back and forth, back and forth.
00:36:33.24 And these amyloids are unusual. Most proteins don't form amyloids.
00:36:39.06 But they've been seen before many times,
00:36:42.10 particularly in the brains of people who have suffered
00:36:45.07 from neurodegenerative diseases.
00:36:46.11 And neurobiologists and neuropathologists
00:36:50.16 have developed some tools to stain those proteins
00:36:54.16 so that they could diagnose what the disease
00:36:56.21 was that this person had died of.
00:36:58.11 And so in Alzheimer's disease
00:36:59.26 you might have heard of amyloid plaques
00:37:02.02 because they are accumulating massive fibrous plaques
00:37:06.17 of this sort of a protein structure.
00:37:08.02 Now it's very important that I tell you, however,
00:37:11.04 that although these proteins that cause this neurodegenerative diseases
00:37:14.04 seem to have a common structure
00:37:16.19 this is a completely different protein than the proteins involved in Alzheimer's disease
00:37:22.14 or even the protein that is involved in the human prion disease
00:37:25.21 that I was just telling you about.
00:37:27.07 So you don't have to worry about drinking beer or wine
00:37:31.28 or eating bread. You are not going to
00:37:32.28 get Mad Beer Disease from these yeasts.
00:37:36.03 Anyway that prion determining domain that was responsible for all that weird inheritance
00:37:43.02 and responsible for allowing the protein
00:37:45.00 to go into the aggregate state
00:37:46.11 forms the basis of the fiber's spine.
00:37:50.24 And this C-terminal domain, the translation termination factor domain,
00:37:54.20 this basically sticks out along the side.
00:37:57.03 If that means that it's stuck, it can't get to the ribosome and do what it is supposed to do
00:38:01.26 so if this sort of thing is happening in the cells that have turned white
00:38:05.22 it would make perfect sense that translation termination factors,
00:38:10.22 a lot of it, is caught up in these aggregates, and that you are not stopping at STOP codons
00:38:14.25 as efficiently as you normally should.
00:38:19.20 In order to understand how this really works however, we had to figure out
00:38:23.04 a way to purify the protein in its soluble form.
00:38:26.05 And once we figured that out, we had some very interesting results.
00:38:34.01 So this is isolated soluble protein
00:38:39.06 sitting in a test tube, and we are monitoring assembly over time.
00:38:44.02 And what you can see is that not much is happening
00:38:47.12 for a very long time. It is a very, very inefficient process of assembly.
00:38:52.14 However, if you take a very small amount of the
00:38:55.21 material that actually has assembled, a very small fraction of it,
00:38:59.18 and add it to a new reaction at time zero here,
00:39:04.22 something very interesting happens.
00:39:06.17 The protein has a hard time getting into that amyloid state
00:39:14.22 but once it is in that state, it can very efficiently recruit
00:39:19.23 other protein to that same state.
00:39:21.22 This is in a test tube, in an isolated protein
00:39:26.13 doing exactly the kind of templating that had been hypothesized
00:39:30.03 to be responsible for the Mad Cow Disease
00:39:33.02 and prion diseases. And in fact,
00:39:37.13 we did a lot of things to establish that this protein folding
00:39:41.08 process was responsible for the change in the biological behavior of these cells.
00:39:50.14 For example we used mutations that would cause the protein
00:39:55.14 to slow its polymerization down,
00:39:57.28 and it turns out that those mutations also made it much less likely
00:40:03.03 that the cells would ever turn from red to white.
00:40:07.05 We did a lot of other things as well. We wanted to know
00:40:11.10 whether or not the fact that it was an amyloid
00:40:14.22 and that amyloids are seen in the brains of people
00:40:18.10 who are suffering from these various diseases including this human prion disease.
00:40:25.15 We wanted to know whether it really was fundamentally a similar process taking place.
00:40:33.09 And so we compared the protein that causes the human disease with the yeast protein
00:40:40.06 and as I mentioned, they are completely different proteins,
00:40:43.00 so there is really nothing in common between them.
00:40:46.11 Except for one simple thing.
00:40:48.21 And that is that there's a little amino acid sequence
00:40:54.08 in the protein that causes this human disease
00:40:57.25 that is repeated several times. And it's an interesting
00:41:03.14 repeat because when people inherit a copy of that gene
00:41:10.11 that contains extra repeats,
00:41:12.04 or the repeat has expanded a little bit
00:41:14.00 they will get this disease in its heritable form.
00:41:18.03 That is, because this repeat makes the protein more likely to misfold
00:41:23.28 into that prion form, and therefore instead of getting the disease spontaneously,
00:41:29.08 as people do, maybe one in a million people will get that
00:41:31.28 disease spontaneously. These people will get the disease later on in life
00:41:36.14 because the protein is just much more prone to misfolding.
00:41:39.25 And when we looked at the yeast protein, we found that the yeast protein
00:41:44.23 although different in every other way,
00:41:47.24 it also had a little sequence of amino acids that was repeated.
00:41:52.00 And so we though well, gee, if these processes really are similar,
00:41:56.26 then if we expanded that repeat sequence in the yeast protein,
00:42:01.06 instead of switching every one in a million cells,
00:42:04.11 every one in a thousand cells,
00:42:05.11 we might expect to see the cells switching into this prion state
00:42:08.24 because the protein is more prone to misfolding much more often.
00:42:12.09 And that is exactly what happened.
00:42:15.09 So as I told you, cells that are white generally give rise to more white cells,
00:42:20.14 cells that are red give rise to more red cells,
00:42:23.10 but if we take these red cells and we replace the normal copy of that sup35 gene
00:42:31.09 with the copy that contains that repeat expansion,
00:42:34.05 what we see is that the prion state keeps popping up all the time.
00:42:40.18 So the very same type of mutation that causes
00:42:45.06 this dreadful disease in humans
00:42:47.06 also causes the protein to change its shape in the yeast cells much more often as well.
00:42:53.12 Now it's not causing the disease in yeast
00:42:55.24 cells, and the reason why think that's happening
00:42:58.07 is that it's not inherently a toxic amyloid.
00:43:01.17 We've been doing a lot of work lately on the actual molecular mechanism
00:43:07.12 by which this protein changes shape.
00:43:09.12 And we've found the sequences that are involved in helping
00:43:13.01 it change shape. In fact we know which sequences need to get
00:43:15.18 together in order to initiate this process.
00:43:18.12 Now we've been doing quite a lot of work on this protein
00:43:22.22 and how it changes shape,
00:43:24.11 and we've actually identified the molecular sequences that are involved
00:43:27.20 in initiating this change in shape
00:43:29.06 and we are making a lot of progress on it.
00:43:32.05 This protein not only changes into one particular
00:43:35.22 altered shape. I showed you one particular type of fiber.
00:43:39.17 But it can also not only form smooth fibers,
00:43:42.23 but also curly fibers, and those actually confer slightly different
00:43:45.10 biological properties so that
00:43:46.27 instead of seeing just red versus white cells,
00:43:50.12 you also get different colors of pink.
00:43:52.17 These are called prion strains.
00:43:55.08 And in the human disease they are also
00:43:58.04 that same property occurs. That protein cannot just fold into
00:44:01.07 one particular type of shape,
00:44:02.16 but it can fold into a suite of related
00:44:05.14 types of misfolded shapes. And those related
00:44:09.14 misfolded shapes are called prion strains in the human disease.
00:44:13.07 An awful lot of the biology and biochemistry
00:44:16.03 of these two proteins is very similar in terms of their folding mechanisms.
00:44:21.13 But in the yeast cells this change in shape is not killing them.
00:44:24.29 And we think the reason for that is
00:44:30.05 that this change in shape is actually quite a complicated process
00:44:33.03 that involves lots of different intermediates.
00:44:35.15 And the yeast cells undergo this change in shape very, very efficiently.
00:44:41.26 The human protein does not.
00:44:44.16 And we think that it is these intermediates that are actually the toxic species.
00:44:48.11 And we are not the only ones to think that.
00:44:50.09 This is actually something that is being thought about in a lot
00:44:54.23 of other human neurodegenerative diseases as well.
00:44:57.26 The diseases that are characterized by these large amyloid fibrous plaques.
00:45:01.14 It is not that the fibrous plaques are the toxic species, but these intermediates.
00:45:05.28 So by studying how these intermediates
00:45:08.21 change shape, we think we can learn a lot
00:45:11.20 about human disease.
00:45:13.00 But it is the efficiency of the process in yeast that prevents
00:45:16.05 this from being out right toxic.
00:45:19.26 But it does something else that's interesting.
00:45:22.01 Before I get to that, I'd like to just thank the people
00:45:25.11 in my laboratory who did most of the work that I have just been telling you about.
00:45:30.01 John Glover did the biochemistry. Maria Patino did a lot of the cell biology
00:45:36.03 and JiaJia Liu did a lot of the genetics.
00:45:38.02 And you'll be seeing some other people as the talk goes along
00:45:41.00 because I've really been very, very fortunate
00:45:43.08 to have a wonderful group of people working with me over the years.
00:45:45.26 And that's actually one of the great joys of doing science:
00:45:48.28 it's the young people that you interact with all the time.

Part 2: Prions and Evolution

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    Part 1: Prions and Evolution
  • Part 2: Prions and Evolution
    Part 2: Prions and Evolution
Total Duration: 1:11:56
Recorded: November 2008

Talk Overview

What do “mad cows”, people with neurodegenerative diseases and yeast cells growing happily on a deadly antibiotic have in common? They are all experiencing the consequences of misfolded proteins. Each organism has thousands of different proteins, which define its nature. Like origami paper, they can take the right path and fold into a swan or take the wrong path and fold into a rapacious hawk. The consequences can be deadly, leading to devastating neurodegenerative diseases in humans. Remarkably, a very similar folding process has been discovered in yeast, where it does no harm and can be studied easily and inexpensively.

Part II: In the case of the yeast prion [PSI], the misfolding of the Sup35 protein results in a simple change in metabolism. When misfolded Sup35 is passed from mother cells to their daughters, this metabolic change is inherited. This unusual genetic mechanism changes the organism in a heritable way due to a self-perpetuating change in protein conformation with no change in its DNA. The mechanism of epigenetic inheritance we have delineated provides a one-step process for the acquisition of complex traits and affords a route to the genetic assimilation of unstable traits that are not yet encoded in the genome.

Related Resources

Alberti S, Halfmann R, King O, Kapila A, Lindquist S, 2009. A systematic survey identifies prions and illuminates sequence features of prionogenic proteins. Cell 137: 146-158.

Shorter J and Lindquist SL, 2005. Prions as adaptive conduits of memory and inheritance. Nat Rev Genet. 6: 435-50.

True HL, Lindquist SL, 2000. A yeast prion provides an exploratory mechanism for genetic variation and phenotypic diversity. Nature 407: 477-83.

Li L and Lindquist S, 2000. Creating a protein-based element of inheritance. Science 287: 661-664.

Glover JR, Kowal AS, Schirmer EC, Patino MM, Liu, J-J, Lindquist S, 1997. Self-seeded fibers formed by Sup35, the protein determinant of [PSI+], a heritable prion-like factor of Saccharomyces cerevisiae. Cell 89: 811-19.

Patino MM, Liu J-J, Glover JR and Lindquist S, 1996. Support for the prion hypothesis for inheritance of a phenotypic trait in yeast. Science 273:622-626.

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