Women of MIT: Celebrating Science and Engineering Breakthroughs (Session I)

SIVE: I'd like to welcome you to this first afternoon of this historic symposium. Historic not only because it brings together people who have considered societal issues and considered policy issues, and have changed policy to move women to where they should be as intellectual leaders. But also because it brings together leaders in the fields of science and engineering who are some of our most eminent faculty here at MIT and who have agreed to respond to our invitation and tell you about the truly extraordinary work that they're doing. And it's my honor to introduce three of those to you this afternoon.

The first is Susan Lindquist. Professor Lindquist received her PhD in biology from Harvard University and became a member of the faculty at the University of Chicago later, prior to coming to MIT in 2001. She came to MIT as Director of the Whitehead Institute for Biomedical Research, and continues to be a Member of the Whitehead Institute and a member of the biology department, Professor of Biology at MIT. She is a Howard Hughes Medical Institute investigator whose work addresses fundamental mechanisms regulating protein folding. And as she will tell us, this has profoundly changed our understanding of normal and pathologic conditions.

Her list of honors is extraordinary as her research. She was elected to the American Academy of Arts and Sciences in 1997, the National Academy of Sciences also in '97, and the Institute of Medicine in 2006. In a single stunning month last year, Professor Lindquist received our nation's highest honor, the National Medal of Science, as well as the Max Delbruck Medal, and the Mendel Medal.

The title of her talk is-- "Using Simple Cells to Attack Complex Diseases." Professor Lindquist.

LINDQUIST: Hi there. Thank you. I'm really honored to be here.

Chuck mentioned the double helix today. I'm going to start out by reminding you that the genomes that we all have are made of linear DNA, long linear strings of information. And what they do primarily is code for proteins. So just about everything that makes you you is done by a protein.

And when proteins are first decoded from the linear information of the DNA, they are also linear. They are long strings of amino acids. But they do nothing in those shapes. They have to fold into some very, very complicated shapes, like this. And this.

And the crazy thing is that they have to do that in an absolutely ridiculous environment. They have to do that inside of a living cell where the concentration of other proteins is amazingly high. So if you look at those proteins in their context inside the cell, you can see how incredibly crowded it is. And what you're not really seeing here with this image is that not only is it crowded, but the cells are experiencing a tremendous amount of kinetic energy. So the proteins are moving around like crazy and constantly bumping into each other. So that when you think about proteins going about their business inside of a living cell, it's not like Fred Astaire and Ginger Rogers. It's much more like the characters in a Marx brothers movie, where chaos is poised on the precipice of disaster.

And so in the full protein folding world we call that going off-pathway. And it's happening all the time in living cells. And it's responsible for some of mankind's most devastating diseases. Including these neurodegenerative diseases: frontotemporal dementia and ALS, Parkinson's, dementia with Lewy bodies, multiple systems atrophy, Huntington's, Alzheimer's, prion diseases. These are all characterized by globs of misfolded proteins. So you might have heard of plaques, amyloid plaques, in the brains of Alzheimer's disease victims. Those plaques are plaques of misfolded protein.

And so this is, it turns out, this protein folding problem is as ancient as life itself. Cells are built to be so incredibly crowded because once you get proteins folded you have tremendous efficiencies by having them so close together. You can pass very reliably an electron from one protein to another. But it does create this, and this has created throughout the history of living systems, has created this incredible problem in protein misfolding that gets worse and worse as we get older and older.

So having studied protein folding for a long time now, we got the idea that these devastating diseases-- and this is an example of a normal human brain in the brain of someone who's died of Alzheimer's-- that we might be able to study these devastating diseases in a simpler cell. So we're actually using yeast cells as sort of a living test tube to study this protein folding problem, and specifically to study what goes wrong when those particular types of proteins misfold, and those particular neurons. And so this idea-- this is a yeast cell, by the way, it divides by budding-- and they are very simple little cells. But they're not like bacterial cells.

So our idea was, they are not like bacterial cells, they are complicated cells, and I'll show you a picture again in a moment. But this was the way in which, when we first proposed to use yeast cells to study this problem of these neurodegenerative diseases, this was the general attitude that we were greeted with. And I have to say, it's an attitude that is very familiar to me, it's very much like the attitude that I faced when I decided I was going to get a PhD in biology lo many years ago. And that was talked about this morning.

It still continues in different ways. And one of the things that I've loved about MIT is that you can make some rather crazy ideas and propose them to your colleagues, and they don't actually laugh at you. They think, well, gee, maybe that would be interesting. So I want to thank MIT for being a wonderful environment in which we were able to do of this crazy sort of thing.

So the reason why we decided we were going to do this is that yeast cells, unlike bacterial cells, are really complicated cells. They're single cells, but they have all kinds of complicated machinery that characterizes all of our cells, including our neurons. And they have a very complicated cell cycle. They decide when to grow, and when to divide, and when not to divide in a very complicated way that's a lot like our cells.

They have all kinds of membrane-bounded compartments which traffic various proteins around in the cell. They have lipids and membranes. And they have complex systems for generating energy that are called mitochondria within them. And again, our nerve cells depend upon all of these things very, very intensely.

And this is just to remind you of that picture of a eukaryotic cell. And some of this is going to be featuring in my talk, so I just want to let you know what these green things are here. These green things are membranes. And they divide the cell up into several different compartments. And these membranes will bud off these little vesicles, and traffic those packets of other proteins to a different part of the cell. And neurons are particularly susceptible to problems with that going wrong, because they are huge, gigantic cells that have to traffic things from one end to the other.

So another aspect of yeast that made us decide to do that, is that there is no organism on the planet that we can manipulate as well as we can manipulate yeast. We know more about it. It has a very simple genome. The DNA was the first eukaryotic genome to be decoded. And it has certain kinds of properties-- that were actually first discovered by brewers trying to make better beer many years ago-- that allow it to be genetically manipulated in ways that are just like a dream come true for biology.

So we've got a really unrivaled toolkit for taking advantage of this organism. And because it's cheap to grow, and it grows very, very fast, we can do experiments in this organism on a time frame that is just staggering in comparison to the time frame it takes to do experiments in, for example, a mouse.

So I'm going to tell you, first of all, about some work that we've been doing with the synucleinopathies. And these are diseases like Parkinson's, dementia with Lewy bodies, and multiple systems atrophy, in which you see a particular protein, this alpha synuclein protein, misfolded in these different cell types.

And Parkinson's disease and parkinsonism, it's really part of a spectrum of disorders, and it's really the second most common of the neurodegenerative diseases. It's characterized by the loss of particular neurons dopaminergic neurons, and in particular regions of the brain, and of course the severe motor defects. It's a devastating illness. And there's a lot of bewildering things about this disease. There are some diseases that we know are caused by one particular change in one particular protein. But with this disease, there's a bewildering variety of environmental associations with toxins like manganese and mitochondrial poisons.

There's also clearly a large number of genetic factors that play into these diseases. And these, the loci that have been identified as being mutated in various people with parkinsonism, are called the PARK loci. And in the autopsy of those people, quite often you see in fact blobs of this alpha synuclein protein even when it isn't mutated. But interestingly, the very first parkinsonism gene to be discovered was in fact alpha synuclein. Because the mutation in that protein, or a simple amplification of the gene that is just making a few extra copies of that gene, is sufficient to cause parkinsonism. And it's a very simple little tiny protein that binds lipids and misfolds when it's not bound to lipids. And so we figured it might be a simple place to start in terms of trying to understand the biology of that protein, how it interfaces with different cellular compartments, and what happens when it misfolds.

So we took the alpha synuclein gene, and we put it on the regulation of a control element that we could switch on and off. And we fused the sequence of it to something called green fluorescent protein, which is actually a protein that biologists very broadly have borrowed from jellyfish. And it glows green when you shine blue light on it. So if it's used to tag the sequence of another protein, it allows you to follow that protein around the cell and actually look at it under the microscope.

So when we did that, when we put in two copies of the alpha synuclein gene into yeast cells, the protein did what we hoped it would do. It went to the membrane, the outer membrane of the cell. And that's great because in neurons this protein is found at the ends of the neuron, at the very farthest outer reaches of the nerve cell. And it's associated with membranes. When we put in one extra copy, things started to go wrong. When we put in two extra copies, like the amplification of the human genes in humans, things really went wrong. And in fact, these cells grow normally, these cells grow slowly, and those cells die. So what's great about that, is that gave us the opportunity to do genetic screens to try to find things that would make these cells grow better, rescue them. And we've done that with both this strain and this strain.

I'm going to tell you first about the genetic screen that we did with this strain here. We used the strain that was growing slowly, because what we wanted to do was be able to find genes that made the cells better or worse. And what we can do in yeast is what's called an unbiased genome-wide screen. That is, we have libraries of every single gene encoding every single protein in the yeast genome. And those are little plasmids. And we can put those plasmids, by genetic transformation, into a yeast cell that's also expressing this human protein.

And as I said, the cells are growing slowly. And then we can keep those genes off until we want to start the experiment. And then when we want to start the experiment, we move them from the media where the gene is off to the media where both of them are now on. And we look for things that make cells better or worse. And the way the data looks is sort of like this. So what we have here is, cells that are not expressing that disastrous human protein, and they're growing just fine. When they are induced, they start to-- they slow down. They don't grow as well.

And we have every single gene that we've transformed. And we test in four independent strains so that we don't get things messed up by just sheer error. And so you can see here that these four strains all have one gene in it, and that gene is making the cells better. These four strains all have a different gene in it, that gene is making the cells worse.

Now the identities of those genes were really quite exciting. Because they looked like they might be involved in some of the biology that's been involved in parkinsonism. But that the yeast screen meant absolutely nothing until we actually took those genes, took the homologs, the related genes from other organisms, and tested them actually in a neuronal cell. So in fact, we not only tested them in a neuronal cell, we tested them in dopaminergic neurons, the particular cell type that suffers in parkinsonism.

And I'm just going to show you the results from-- Oh, there we go-- I'm just going to show you the results from our work with rat embryonic midbrain cultures. But I'll tell you that it worked in the nematode, and in the fruit fly, and there's various reasons why these organisms each have their own advantages and disadvantages for doing research. But since this is closer to home-- rats are, believe it or not, really quite similar to you-- I'll just show you the results here.

And I just want to mention that we are now taking this, we hope, to an entirely new level. Because this disease is such a complicated disease, we really believe this is really a kind of a poster child for the kind of disease that you would want to do personalized medicine with. And so we're collaborating with Rudolf Jaenisch's laboratory to make induced pluripotent stem cells from human patients with this disease. The goal being to try to determine how we can help the individual person based upon their underlying risk factors.

So in the midbrain cultures of the embryonic midbrain of rats, we culture those in a dish for a few weeks, and what we get out is about 7% dopaminergic neurons. And when we express alpha synuclein in those cultures using a virus, a lot of the neurons die, but the dopaminergic neurons die first. So there there's a difference and the percentage goes down.

And if we coexpress a random gene with that alpha synuclein, it doesn't help at all. But if we coexpress the gene that was our first strong hit in yeast, it rescues. And moreover, you can see from these little blobby things over here, there's been a defect in fact in the vesicle trafficking in those neurons. Those little globs of membrane that move around the cell and deliver neurotransmitters at the end of a neuron, those, there's a problem their trafficking. And our Rab1 one gene here turns out to be something of a protein that's involved in regulating that trafficking. And when you overexpress it in the presence of alpha synuclein, it corrects the defect that alpha synuclein causes.

So we've now done this with several other genes that were completely diverse in function. You would not have really guessed that they would be related to the vesicle trafficking defects. But these very, very diverse genes, every one that we've tested except for one-- and that's a pretty good ratio, we've tested several of them-- all of the human homologs of those genes actually fix the rat embryonic neurons. Or the nematode homologs fix the nematode brains.

So what we did then was with some biochemistry, and we were able to show that in fact what happens is that if you have vesicles, and you're looking for the budding from one compartment to the other, you can do that with some pretty fairly easy little assays. And you look for the changes in modification that take place in different compartments. And we found that in fact, when we did that with alpha synuclein, what happened was we had a defect in protein trafficking and the vesicles wouldn't fuse, and that's really bad news in a neuron. So let me just tell you about one of the other genes that we-- and that's why that Rab1 actually rescues that effect because it helps to move those vesicles forward.

So let me tell you about another gene that we hit, which at the time we first hit it had a very uninformative name. Its name is Yeast Open Reading frame 291W, because no one had any idea what it did. But it was of great interest to us, because when we looked in the human genome we found a homolog in the human genome that was a very high homology. And then up popped the following story. That people who have mutations in that gene will get early onset parkinsonism.

So our gene, when overexpressed, is rescuing the cells from the problem of alpha synuclein. That ties directly alpha synuclein function now to this other protein function. And we were able to use our yeast cells then to figure out what the function was. Which I think would have taken a very long time in mammalian neurons. And what we basically found was that this gene functions in manganese transport. In fact it functions and detoxifying manganese, which as I mentioned to you earlier is an environmental toxin, exposure which has been associated with parkinsonism.

Another thing we found was that mitochondrial damage, and this is just a genetic signature of the changes that are taking place in the gene expression profile of the cells that tell us that mitochondria, those energy generating factories in the cell, are getting sick. Remember I told you that there is a mitochondrial poison that is associated with environmental exposure to production of things like parkinsonism.

And so, again this nucleon is tying all these things that have been seen in human neurons together in our yeast cells. So that allowed us to do a chemical screen for compounds that would rescue the yeast cell. And we screened 150,000 compounds. And we were able to do this in a very short period of time relative to screening in neurons. We asked for something simple like restoration of growth in that extremely high toxicity strain. Because in that high toxicity strain, that's where all those different things are going wrong. The problem with manganese, the problem with mitochondria, the problems with proteins moving around the cell.

So we want to see if it might be possible to find a compound that would rescue. And we only got a few. It's an extremely rigorous screen. But in fact when we looked at the cells, they fixed the problem in trafficking. They fixed the mitochondrial defect. And they worked in nematode and rat neurons too. And moreover, they not only provide protection in rat neurons from alpha synuclein toxicity. They provide rescue from a mitochondrial poison to which I mentioned that environmental exposure can cause early onset parkinsonism.

And so here's just a simple little snapshot of that. This compound is called rotenone. And these are neurons in a dish that are mostly dying because of the rotenone. And if in the presence of one of our rescuing compounds, these neurons obviously look an awful lot better.

So what we can tell, then, is that this particular protein is causing very specific neuropathology. Particular types of neurons are dying. But the reason it's doing that is not because it's affecting a process that's specific to neurons. It's that it affects a very general basic cellular process that all cells need, but that dopaminergic neurons are simply particularly sensitive to. And so those cells die first. The individual dies. And you don't notice that in fact, there's lots of problems everywhere else.

So why dopaminergic neurons? We think it's partly because neurons are so highly dependent on trafficking. Because they're so long and they have to move things at such long distances. And they communicate with each other with these little vesicles releasing packages of signaling molecules to other neurons.

And we think that the high local concentration of those kind of bowling balls of alpha synuclein aggregation that I showed you is responsible for another feature that that's toxic, which is that the aggregates themselves can be toxic. There's a special vulnerability to metals which we're still unraveling. But it's clearly true for both the neurons and the yeast cells when this synuclein that's misfunctioning. Neurons have particularly high rates of respiration, hence they're particularly dependent upon their mitochondria.

And then finally dopamine is normally made out in the cytosol. And it's a dangerous neurotransmitter. It needs to be packaged into these vesicles right away, or it breaks down the cytoplasm and does bad things. And so if there's a defect in the production of these little membrane-bounded vesicles in the cell, it's going to affect a dopaminergic neuron more than most neurons.

So we're beginning to understand some of this special vulnerability. And beginning to think that there might be a more rapid route to trying to find some therapeutic targets for this disease.

So I want to finish by telling you something we haven't published yet. This is some work we're doing to look at the toxicity of Abeta, a peptide that accumulates in the brains of Alzheimer's victims, which is the number one cause of neurodegeneration in humans. And with all of us living longer, healthier lives, we are all facing the prospect of getting this disease.

Abeta is a little tiny peptide that's actually made in the brain in a rather complicated way. It's made from a much larger precursor protein and there's a couple of different enzymes that are involved in clipping it into this small little peptide here that's prone to misfolding. And so in humans that protein is trafficked, again, through these very complicated pathways.

It starts out in something called the endoplasmic reticulum. Then it moves to something called the Golgi. Then goes to the plasma membrane. It comes back into the cells and something called the endosomal compartments. And it's believed that it's processed into this Abeta peptide in all of those different places. And it's believed that this external stuff out here is perhaps some of the most dangerous stuff when it gets out there, alone and loose by itself, and can form [INAUDIBLE] species.

So how do we ever reproduce this in a yeast cell? Well, we skip that whole processing step to begin with. And what we do is we take a little signal sequence that will get the protein delivered into the endoplasmic reticulum. And we just take the Abeta sequence itself, nothing else. So when we put that gene into a yeast cell, when the protein starts to be made, the protein first gets delivered to the endoplasmic reticulum.

And then there's a cleavage system in that that's very, very highly conserved. And it cleaves that protein and makes just the simple little Abeta peptide. Which then is free to move into the other compartments of the cell with a series of vesicle fusions, which I don't have fully animated on this particular slide. It gets delivered to the outside of the cell.

And what's cool about yeast cells is that they've got a cell wall. So that means, when they make their Abeta it's not going to just to diffuse away. It's going to stick close to the plasma membrane, which is believed to be one of the most dangerous sites for susceptibility to Abeta. And it's going to come back in. And so we set up cells, and we did the screens in exactly the same way that I just showed you for alpha synuclein.

And we've, as I mentioned, we're just getting ready to submit this paper, but I wanted to just tell you about it because I'm really quite excited. We've hit a variety of different genetic modifiers of Abeta toxicity, many of which have homologs in humans. And we found that clathrin mediated endocytosis, three of the genes were all involved in that one pathway. And this gene here, which is involved in that in that endocytosis, that taking things that have hit the plasma membrane and bringing them back into the cell.

One of them is PCALM. And it turns out that in the human genome-wide arrays that have been used to try to find genetic risk factors in human beings for Alzheimer's disease, PCALM is the third highest hit. So it's a very, very well validated protein that's involved somehow in Alzheimer's disease. But there was really nothing to link it directly to Abeta. In fact, the hypothesis of how it was working was not through Abeta. So we think we've uncovered that.

We also have two additional connections to unpublished data that I'm not at liberty to talk about here. I just want to mention that we have set up now a nematode model for this disease process. And again using green fluorescent protein you can visualize the neurons of the nematode here. In particular we're looking at the glutaminergic neurons, which are the most relevant neurons in Alzheimer's disease. Nematodes, lowly as they are, they have these different types of neurons.

And when we express Abeta in those cells, we don't get as many of the neurons. And this is a very dramatic change, believe it or not. Because nematodes, one of the reasons they are such a darling for research, is that every worm always develops with exactly the right number of neurons. But here Abeta is causing some of those neurons to die. And you can see that here is, it's an age-related neuronal death. 40% of the worms are normal at three days, and fewer are normal at seven days. So there's age related neurodegeneration going on.

And if we express two other random genes in those nematodes, it has no effect whatsoever. But if we take our modifiers from the yeast screen, the things involved in clathrin mediated endocytosis rescue. Three other genes with completely different functions rescue. And one of the genes that makes the yeast cells worse makes the nematode neurons worse as well.

So, data just in. We've also done this now with rat primary cortical neuronal cultures. And they are also affected by the Abeta. And they're also protected from Abeta by PCALM.

And finally, what gives us quite a bit of hope is that there's this compound, it's the only compound that's been shown so far to rescue a mouse model of Alzheimer's disease that actually reduces the burden of Abeta and restores cognition and learning and memory. These are yeast cells growing without Abeta. These are yeast cells not growing with Abeta. And this is the rescue of our yeast cells with that chemical compound.

So we have just been given a grant to screen 350,000 compounds at the MIH for compounds that might rescue yeast cells and cross over into neurons. So that's all I have to tell you, except I hope this is the tip of the iceberg. I'm not sure it will be or not. We have a long, long, long way to go. But I want to thank the extraordinary young people in my laboratory who did this work. These are the people who set up the synuclein. These are the people who did the second stage. And these are the people who did the Abeta work, which is as I mentioned not yet been published. And these are our wonderful, wonderful collaborators. Science has become much more collaborative over the years. And in order to make progress we are making, we really use the help of these people.

So that's it. Thank you. And I'm happy to answer questions, or did you want to take questions after, or would do you want to do?

SIVE: Questions... Questions now.

LINDQUIST: Yes.

AUDIENCE: What's the mechanism for the neurotoxicity of [INAUDIBLE] Abeta?

LINDQUIST: Well it's been not well understood. It's quite clear that there's something about the misfolding of that protein. And it's thought to be that the misfolding of that peptide creates a variety of surfaces that have a hydrophobic character to them that interact with membranes and punch holes in membranes. But it hasn't really been entirely clear.

And that's why being able to do genetic screens, genome-wide, without any preassumptions of what this protein is doing, the genetics, the things that we get that correct the defect, tell us about what is going wrong. And so the fact that one of the genes that we're getting is a gene that's involved in grabbing things from the membrane and bringing it inside the cell, and dumping it into a vacuole or compartment for degradation, tells us that that membrane and that trafficking process is part of the actual reason for the toxicity of Abeta protein. Yes.

AUDIENCE: If you're screening compounds, how do you select the compounds to screen?

LINDQUIST: Yeah, so the first library that we've screened was a library of compounds at the Harvard Institute of Chemical Biology. And they've actually gathered compounds from all over the world. All sorts of different places, from failed pharmaceutical companies, and from when the Soviet Union shut down they grabbed a bunch of the compounds their chemists were making, et cetera. Now, more recently what we're going to be doing with NIH, is the National Institutes of Health has put together a library of 350,000 compounds that are new compounds, that are easier to resynthesize, that are cleaner.

And they are setting up screening centers at several different places. We're doing some screens at the Broad. Also doing some screens at NIH itself. And they actually have all kinds of very fancy robots that allow academics to come into those facilities and do things that normally an academic lab could never have done before. So it's a very large collaborative effort.

AUDIENCE: What's rotenone?

LINDQUIST: Rotenone is a toxin that hits a particular enzyme in the mitochondria and causes the breakdown of that energy-generating system.

AUDIENCE: [INAUDIBLE]

LINDQUIST: I don't know, but-- I don't know where it's actually synthesized from. But it's a pretty terrible compound. It's a compound that's used actually, for example, for killing fish in ponds by certain individuals who want to harvest fish rather quickly, rather than using a line and pole. And it's used in many other places in the environment. There are a lot of people who are now really quite alarmed by the fact that it is being used out in the environment.

Any other questions? Thank you all very much.

SIVE: Thank you so much, Sue, for sharing your really groundbreaking research with us. I'm going to take my glasses off so I can read what I have in front of me here.

The theme that we organized this session around spans departments, and spans schools, which is really one of the things about MIT-- research spans so many departments and schools, and life sciences research particularly spans the schools and the departments very broadly at MIT. And we continue with a life sciences theme, and with the theme of faculty who are addressing huge issues in human health with our next speaker, Professor JoAnne Stubbe.

Professor Stubbe is the Novartis Professor of Chemistry and Biology at MIT. She received her BS from the University of Pennsylvania and a PhD in organic chemistry from the University of California at Berkeley. After taking faculty positions at Williams College, Yale, and the University of Wisconsin, she happily joined the MIT faculty in 1987.

Professor Stubbe analyzes the mechanisms of ribonucleotide reductases, enzymes that are pivotal in DNA metabolism. She studies natural product DNA cleaving agents that impact development of new cancer treatments and environmentally friendly polymers.

She's been broadly decorated. She was elected to the National Academy of Sciences in 1992, was the recipient of the American Chemical Society's Nakanishi Prize, the Benjamin Franklin Medal in Chemistry, and in 2009 received our nation's highest honor, the National Medal of Science.

The title of her talk today is: "Radicals: Your Life in Their Hands!" Professor Stubbe.

STUBBE: Okay. Boy, I'm really nervous. Anyhow, I would like to begin by thanking the organizing committee for inviting me to participate in this symposium celebrating the contributions of women in the School of Engineering and the School of Science. I'm really flattered to be here. Second, I would also like to thank MIT for providing me over the last 20-- almost 25 years, with such a stimulating and intense environment to carry out my passion. And to providing-- to allow me to have access to outstanding graduate students who are motivated and enthusiastic. And also finally to have access to colleagues across all disciplines, some of whom are shown here, that have allowed me to push my science forward in ways that I never could have imagined.

Okay, so today I'm going to be talking about radicals. Your life is in their hands. And I hope by the end of my talk you have some idea what that title means. Okay, so what is a radical? Radicals, if you look it up in the dictionary, have many definitions. To a chemist, biochemist, or biologist, a radical is a molecule with an unpaired electron in its outermost shell that's chemically extremely reactive, and it's dying to pick up a second electron and form a covalent bond.

And so radicals, being highly reactive, are also considered to be highly destructive. And so most radicals, when people think about it in biology, are bad radicals. And in fact, if you look in the literature, each month there are literally hundreds of papers published on reactive oxygen species. And reactive oxygen species are reductive metabolites of oxygen gas, an unavoidable consequence of the fact that we live in an aerobic world. Radical species can also be generated by light. These reactive radical species react indiscriminately with whatever they encounter. So they react with small molecules. They react with proteins.

And here is a cartoon of our genetic material, that Susan just talked about, DNA. And when they get encountered DNA, they form a covalent modification, a stable modification, leading to a mutation. And if the mutation is not repaired and you replicate your DNA, these mutations ultimately contribute over a period of time to the aging process and to disease states. So in fact, organisms have evolved many ways to try to remove or annihilate these free radicals. So, for example, my sister goes to the health foods store at least once a month to buy vitamin C and vitamin E. These are antioxidants that can trap these free radicals, preventing them from doing damage to our DNA.

If, in fact, our small molecules that have been evolved to inhibit these free radicals escape then nature has also evolved many proteins or enzymes that can figure out that the DNA is damaged, and then can cut out the damage, and repair the damage. And those enzymes, DNA repairing enzymes then prolong our advances towards the aging process and disease. So in general, when people think about radicals in biology, they think about destruction.

And so today what I want to do is talk about good radicals in biology, and that's my take-home message. That, in fact, nature has figured out how to harness the reactivity of these radicals to do extremely challenging chemistry with exquisite specificity. And so in the next few slides I just want to show you-- I was hoping some of my 507 students would be in the audience, but I haven't seen many students-- that many of the things they learn about in introductory biochemistry that involve more conventional two-electron chemistry also involve radicals.

So for example, many of the metabolic steps that occur inside all cells require cofactors. And these cofactors are derived from vitamins. And so here, vitamin B1 is involved in the pentose phosphate pathway, vitamin B2 is involved in redox chemistry, vitamin B6 is involved in amino acid metabolism. Well, conventionally everybody thinks these cofactors do chemistry two electrons at a time. In fact, they catalyze chemistry as well by one electron, or radical chemistry.

In addition, vitamin B12 is converted into nature's most beautiful cofactor. It's bright pink, adenosylcobalamin. And it has been a paradigm for thinking about radical mediating chemistry to generate this reactive species involved in fatty acid metabolism. I think the most amazing observation that has been made about radicals is that in the last decade it is now known that S-adenosylmethionine-- and so this is, you learn in introductory courses, a universal methylating agent. It methylates small molecules, it methylates proteins, it methylates RNA, it methylates DNA.

We now know from biochemical studies, and also from genomic analysis that SAM can mediate over 3,000 reactions that involve free radical chemistry. And the reactive intermediates are exactly the same as the intermediate that's generated by B12. So I think it's widely underappreciated how important radicals are in normal metabolic pathways.

But even more underappreciated, I think, is the fact that nature uses our proteins a specific amino acid with these proteins, and converts them to radicals which do really important chemistry. So in fact, many people at MIT now, we're in the post-genomic or proteomic era, have studied post-translational modification of proteins that changes their regulatory properties by phosphorylation, methylation, acetylation. Most people do not realize that we have post-translational modification by oxidation. And in fact this oxidation involves metal base cofactors. So here are the normal amino acids: tyrosine, glycine, tryptophan, and cysteine. And all of these radical species are now known to play a role in a number of reactions involved in primary metabolism.

And today I'm going to focus mostly on a tyrosyl radical. Tyrosyl radicals in solution have lifetimes on the order of 10 to the minus 3 to 10 to the minus 6 seconds. Yet in the enzyme that I'm going to be talking to you about today, it can have lifetimes, depending on the organism, from 30 minutes to four days. And tyrosyl radicals play a central role, as you'll see today, in making the building blocks, the deoxynucleotides required for DNA biosynthesis.

But it goes beyond that particular step. In fact, we now know that a major mechanism of oxidation of water to oxygen gas, everybody is interested in that because of the energy problem, involves a manganese calcium machine and transient tyrosyl radicals. And so plants, and Prochlorococcus-- you'll hear a lot about that from Penny Chisholm later on, I think her talk is tomorrow-- can convert water into oxygen using light for the energy of this uphill process, and using transient radicals, tyrosyl radicals. Transient tyrosyl radicals are also involved in the target of aspirin and non-steroidal anti-inflammatories like ibuprofen or Tylenol. And so tyrosyl radicals play much more, and I think underappreciated, roles then people-- normal biochemists actually think about.

So today what I'm going to be doing is talking about tyrosyl radicals and sulfur radicals in the role of the system that we've focused on for over 30 years. So my laboratory has spent a lot of time studying ribonucleotide reductases, RNRs. The genes that code for these enzymes are called nucleotide reductase genes, or the "nerd" genes. And so I'm often reminded that it's appropriate that I'm at MIT.

Anyhow, ribonucleotide reductases in all organisms, from bacteria that grow on methane gas to humans, catalyze the conversion of nucleoside diphosphates, all four of them here, into deoxynucleoside diphosphates. Providing the appropriate ratio required for DNA replication and repair and the appropriate amounts, both of these are essential for the fidelity of DNA replication and repair.

And so today what I want to do is tell you how radicals are involved in this nucleotide reduction process. I also want to mention that the central role in nucleic acid metabolism in general has made these enzymes a successful target now of three drugs that are used clinically.

So this is the actual reaction that the enzymes-- that the ribonucleotide reductases catalyze. This is a nucleotide. This is a precursor to RNA. This is a deoxynucleotide. This is a precursor to DNA. Ribonucleotide reductases catalyze cleavage of a carbon hydroxyl bond and formation of a carbon hydrogen bond. That's a reduction process, and the reducing equivalents are provided by two cysteines that get oxidized to a disulfide.

Now in addition to these two cysteines, we showed in the mid-90s that there was also a thiyl radical involved in this transformation. I think it was unanticipated that a reaction that looks pretty simple would involve complex free radical chemistry. So what happens is this thiyl radical initiates this process by cleaving this bond, removing a hydrogen atom. And I will spare you the details of the mechanism, but we spent many years trying to understand how this process works.

So this raises a question. Where does this thiyl radical, or cysteine radical, come from? And so we now know, we've gone through the age of genomics, proteomics. We have had a revolution in structural biology. We now know by putting all-- and biochemistry-- putting all this information together, that there are three classes of ribonucleotide reductases. They all share an identical active site, shown here, where the nucleotide is reduced to a deoxynucleotide. So here's the substrate, the nucleotide. Here are the two cysteines that provide the reducing equivalents. And in a finger loop through the center of this ten-stranded barrel, you have a cysteine that needs to be oxidized to a thiyl radical to initiate this process.

So then you ask, how do we define these different classes? And the classification is based on this cloud. And this cloud, which I will unveil in a minute, provides an understanding of the mechanism by which this oxidation occurs. So we'll see that nature has probably by divergent evolution made a common machine to convert nucleotides to deoxynucleotides, but the mechanism of oxidation differs.

So in the class II enzymes-- here is nature's most beautiful cofactor again-- she uses B12. Here is the cloud unveiled. That's the structure part of the B12 molecule. Ribonucleotide reductases in the class II are found in both prokaryotic systems and a few eukaryotic systems.

The class III enzyme shows you another molecule I showed you at the very beginning. SAM, S-adenosylmethionine, which generates the same reactive species and generates a protein radical, a glycyl radical. And this glycyl radical then mediates the oxidation. And so here is the glycyl radical sitting right next to the cysteine that needs to be oxidized.

Here is adenosylcobalamin sitting right next to the cysteine that needs to be oxidized. And this chemistry happens over a few angstroms by direct hydrogen atom obstruction. The class III enzymes are found only in organisms that can grow under strictly anaerobic conditions.

But the one most of us who have spent our time on is the one that's found in humans. And this is the class I ribonucleotide reductases. And what you see if you look over here, here is where the cloud was, that in place of the cloud now we see two tyrosines. But tyrosines are unable to carry out an oxidation of a cysteine to a thiyl radical.

So the question is, how can that happen? And the answer is that when you purify this protein-- again, this is common to all ribonucleotide reductases-- you find in the class I enzymes a second subunit. And this second subunit is different in different organisms, although it's structurally identical.

And so we've subdivided the I class into A, B, and C based on the unusual metallocofactor. So the metallocofactor in human organisms has a diiron center. And the function of this iron center, which needs to be made biosynthetically, is to oxidize tyrosine to a tyrosyl radical.

This is the oxidant, remember in solution, and it has a very short lifetime. In the active site of the human enzyme it has a lifetime of 30 minutes. So something is unique about this system. If you reduce this tyrosyl radical back down to the amino acid tyrosine, your enzyme is dead.

Only in this past year, my lab and a laboratory from Germany discovered another ribonucleotide reductase. This one has a dimanganese tyrosyl radical cofactor. And this cofactor appears to be unique to pathogenic organisms.

Finally, the class I C ribonucleotide reductases was discovered by one of my former students in 2005. This structure is found in human intracellular parasite chlamydia. And it has an unprecedented metal cluster, manganese 4 and an iron 3.

So now the question is, how do these tyrosyl radicals in this manganese cluster oxidize a cysteine, which it turns out you will see in a minute these guys are 35 angstroms removed from the cysteine that needs to be oxidized. So the model is the following. This is the common structure of all ribonucleotide reductases. This is the place where nucleotides are reduced to dioxane nucleotides. This is the center where we need to oxidize a cysteine to a thiyl radical.

The second subunit is the one I just showed you on the previous slide. Here these two balls are the iron clusters, and the iron center is involved in the oxidation post-translational modification of tyrosine to a tyrosyl radical. From this model, this is tyrosyl radical is located 35 angstroms away from the cysteine that needs to be oxidized.

So no matter what you do you, you learn from basic biochemistry that electrons and oxidation in reduction centers are usually spaced on the order of 10 to 15 angstroms. If you go much farther, the rate constant [INAUDIBLE] can become so slow that you can't do chemistry. So the question is, how does this oxidation occur?

And so this is the working hypothesis in the field, which I think we've obtained quite a bit of support for in the past few years. So here is the subunit with the iron cluster that generates this tyrosyl radical. And in E.coli, this radical has a lifetime of four days. Over here is the subunit that's common to all ribonucleotide reductases where the nucleoside diphosphates bind, and deoxynucleotides are going to be made.

Here is the cysteine that needs to be oxidized. But we see no metals between the tyrosyl radical and the cysteine. So we're forced to use aromatic amino acids, which are easy to oxidize to catalyze this oxidation in a process that we believe involves proton-coupled electron transfer.