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Discovery of Chaperonin-Assisted Protein Folding

Transcript of Part 1: Discovery of Chaperonin-Assisted Protein Folding

00:00:07;07		Hi, I'm Art Horwich, from Yale School of Medicine and HHMI.
00:00:10;13		And I want to tell you a story about a discovery that has led to a really wonderful
00:00:17;29		what's now a fifteen to twenty year experience of experimentation that followed.
00:00:23;19		This discovery was really somewhat serendipitous,
00:00:27;09		and I think there was a lot of luck involved.
00:00:29;09		It was helped enormously by two wonderful collaborators, who have remained very close friends,
00:00:36;09		and constant contacts, Ming Cheng, who's now in Taiwan,
00:00:41;12		and Ulrich Hartl, who's now in Martinsried.
00:00:44;13		And so I'll just tell you the story as it occurred.
00:00:48;09		So, the story is about information transfer in the cell.
00:00:52;16		As we know, DNA is transcribed into RNA and is in turn translated into protein
00:00:57;12		and the step that's involved here is the final step of information transfer,
00:01:03;08		namely that of protein folding.
00:01:06;01		So, we know about machines that carry out transcription and translation,
00:01:10;05		and have very fine molecular details to those reactions for some years now,
00:01:16;22		increasingly fine.
00:01:19;11		But protein folding has remained somewhat mysterious
00:01:23;14		and it's really only during the last decade or two
00:01:25;18		that we've really started to gain some understanding about how this works in cells.
00:01:31;23		So, for those unfamiliar, I'd just point out that proteins are composed of twenty different amino acids,
00:01:38;17		and these strings of amino acids ultimately fold into characteristic three-dimensional structures
00:01:44;18		that carry out biological activities.
00:01:47;03		For example, they can be enzymes, cytoskeleton, hormones, hemoglobin, channels,
00:01:52;18		receptors, what have you.
00:01:54;15		Proteins are major effectors in the cell
00:01:57;21		and so their proper folding is crucial to them being biologically active,
00:02:03;08		because when they're effectively strands of spaghetti,
00:02:07;00		leaving a ribosome, they don't necessarily have any biological activity at that point.
00:02:13;08		They have to be folded correctly in order to be active.
00:02:16;24		And so the granddaddy experiment of protein folding
00:02:20;00		that I heard about when I was an undergraduate at Brown University
00:02:24;04		in 1972 was carried out by Christian Anfinsen, in fact, in the late 50's,
00:02:30;29		he received his Nobel Prize for this work in 1972,
00:02:35;27		a really astonishing experiment where he started with an enzyme,
00:02:39;23		ribonuclease, 100-odd amino acids with 4 disulfide bonds,
00:02:44;05		and unfolded that protein by treating it with denaturant,  urea,
00:02:48;20		and reductant, mercaptoethanol.
00:02:50;28		That produced essentially a random coil version of the protein,
00:02:54;26		and he asked whether, when he removed the urea and reductant,
00:03:00;14		whether the protein could find its way back, on its on, in a test tube,
00:03:04;21		to the native active form, and astonishingly, it did so.
00:03:09;01		So, this proved that a polypeptide chain contains all of the information
00:03:14;12		for folding into the unique native structure in its primary amino acid sequence structure.
00:03:20;21		He also presumed that in fact this native state would lie at an energetic minimum.
00:03:27;17		And so, this astonishing experiment led people to believe that perhaps inside cells,
00:03:36;19		proteins also fold spontaneously.
00:03:39;13		But increasingly, there were instances where that didn't really happen,
00:03:44;03		both in test tube, where many proteins could also not be spontaneously refolded,
00:03:50;05		as in the Anfinsen experiment, with ribonuclease,
00:03:53;18		but inside cells when the biotech industry began to express mammalian proteins in E. coli,
00:03:58;28		for example, they also saw these terrible looking aggregates with no biological activity
00:04:04;27		and failed to recover their favorite proteins in a native, active form.
00:04:09;13		And this, once again, one saw, as in the test tube,
00:04:13;26		a misfolded, aggregated, inclusion body, as it were.
00:04:18;21		And so this suggested that something more might be required in the cell
00:04:23;24		to deal with these so-called, what would be kinetic problems of folding,
00:04:30;10		where proteins simply get it wrong and go off pathway
00:04:34;07		and misfold and aggregate.
00:04:36;00		And so we never thought we would have anything to do with this,
00:04:40;18		but we were busy in the late 80's studying mitochondrial protein import.
00:04:48;19		So, it turns out that most of the proteins of mitochondria are encoded in the nuclear genome,
00:04:54;22		the messages for these proteins are then translated on cytosolic polyribosomes
00:05:02;17		and an N-terminal targeting peptide in these proteins directs them to mitochondria.
00:05:10;06		And they're then imported through the mitochondrial membranes,
00:05:14;16		and the targeting peptide is then clipped and the mature-sized protein
00:05:19;00		then has to fold into its biologically active form.
00:05:22;29		Well, an important experiment was carried out in 1986 by Gottfried Schatz and his coworkers
00:05:28;18		that showed that a protein had to be unfolded in order to transit the mitochondrial membranes.
00:05:35;19		He used dihydrofolate reductase as a substrate in these experiments,
00:05:41;03		showing that if you lock DHFR into its native state,
00:05:44;22		by adding the ligand methotrexate, the protein couldn't go through the membranes,
00:05:49;19		whereas, if you now removed methotrexate or denatured the protein,
00:05:54;06		it would go through the membranes.
00:05:55;25		So, the question that arose to us, as we were studying mitochondrial protein import,
00:06:01;05		was, do proteins, upon reaching the mitochondrial matrix space,
00:06:07;03		refold spontaneously to their active form, as might be suggested by the Anfinsen experiments,
00:06:13;12		or could there be a machinery that was required to assist the protein to reach its native, active form.
00:06:20;27		And so we were in a position to actually test this
00:06:24;11		because we had a bank of temperature sensitive lethal yeast mutants
00:06:28;28		that contained a galactose regulated version of a mitochondrial protein,
00:06:34;20		ornithine transcarbamalase, a human protein that we had shown in yeast already
00:06:39;10		could be expressed and would go faithfully to its mitochondria, have its signal peptide cleaved,
00:06:44;12		and become biologically active and make OTC enzymatic activity.
00:06:48;05		And so our screen was basically to start with temperature sensitive lethal yeast mutants,
00:06:54;11		the idea being from Schatz and others in the field
00:06:57;03		that if you can't make new mitochondrial proteins and new mitochondria,
00:07:02;10		the cell basically stops its growth and ultimately, it's a lethal.
00:07:07;09		So, we start with a bank of lethal mutants,
00:07:10;12		programmed to express mitochondrial OTC behind a gal operon promoter,
00:07:16;03		and we proceeded to test each mutant individually for the ability to make OTC activity
00:07:21;28		after shift to non-permissive temperature and shift into galactose,
00:07:26;05		to turn on OTC expression under non-permissive conditions.
00:07:30;13		We then analyzed OTC activity; if it wasn't present, we would then Western blot
00:07:36;20		the particular mutant after shift to 37 C and induction of OTC to see what the fate of the OTC subunit was.
00:07:44;09		Was it present as a mature size protein, or was it still a precursor
00:07:48;11		that hadn't been recognized by mitochondria and so our idea was to dissect the mitochondrial import pathway.
00:07:54;26		Well, as we were asking this question about refolding inside the mitochondrial matrix,
00:08:01;28		we knew what we would be looking for in the form of a mutant
00:08:05;05		that affects refolding in the mitochondrial matrix space.
00:08:08;20		We would have mature size OTC subunits because their signal peptide would be cleaved
00:08:13;29		and they'd be inside the matrix space, but we would have no enzymatic activity,
00:08:18;26		because they would have failed to correctly fold, and in the case of OTC,
00:08:22;23		trimerize into the active form of the enzyme.
00:08:26;20		So, we started to look through our bank of mutants
00:08:31;12		and to our astonishment, almost right away, having asked this question,
00:08:35;23		we found a mutant in which exactly that happened.
00:08:38;20		So, OTC was our initial reporter enzyme and what we found was that OTC subunits entered,
00:08:45;20		they reached their mature size,
00:08:48;03		and then there was no biological activity at all.
00:08:50;28		So, this astonished us.
00:08:53;20		It seemed like heresy to have a result like this.
00:08:56;18		So, we though, uh huh, we better start to look at endogenous yeast proteins
00:09:01;27		and see what is happening to them.
00:09:04;04		So, we looked at this particular protein, the F1 beta subunit of ATPase,
00:09:09;05		of the F1 ATPase.
00:09:11;02		And we found that in fact, similarly, this beta subunit had failed to assemble
00:09:17;26		into this stalk like structure and the subunits themselves looked like had misfolded
00:09:24;06		in the mitochondrial matrix space.
00:09:27;11		We were in a bit of a quandary, because, again, it seemed like heresy,
00:09:33;06		and we couldn't really feel comfortable about the data at hand
00:09:38;04		and we were sort of pondering what proteins to look at next
00:09:42;12		when thankfully, the phone rang and it was Ulrich Hartl and Walter Neupert
00:09:47;03		calling from Munich to say, well, we understand that you guys have some mutants of yeast
00:09:52;23		that affect mitochondrial protein import
00:09:55;11		and we would love to get together with you and perhaps help on some of the biochemistry of those mutants,
00:10:01;12		if you'd have some interest.
00:10:03;08		So, at that point in history, my lab had four people in it,
00:10:07;18		and we weren't very well versed at fractionating yeast mitochondria,
00:10:12;29		whereas these were world experts,
00:10:15;09		so we were just delighted to interact and I went to Munich the next week
00:10:20;29		and presented some of the better described mutants that we had
00:10:25;12		that affected the mitochondrial signal peptide processing.
00:10:29;08		But I also, at the end of my talk, described this particular mutant,
00:10:33;13		and Walter and Ulrich were somewhat worried that what was happening in this mutant
00:10:39;07		was that proteins were coming in through this contact site
00:10:43;08		but weren't getting entirely into the mitochondrial matrix,
00:10:47;01		that they were poking their N-terminal signal peptides in and the signals were being cleaved,
00:10:52;04		but then the proteins weren't entirely entering
00:10:54;18		and of course, they wouldn't fold if they couldn't enter through the membranes.
00:10:58;12		And so, I said, well I'd be absolutely delighted to send you the mutant
00:11:02;29		and you can make mitochondria, I am sure, from this mutant, very effectively,
00:11:08;06		and analyze to see what's going on and in fact, as soon as I got back, I sent the mutant to Munich,
00:11:16;10		and two weeks later, Ulrich called up to say, excitedly, you're right,
00:11:22;05		the proteins are entirely in the mitochondrial matrix, I can add exogenous protease,
00:11:27;17		and it doesn't touch them, so it seems like they have gone into the matrix space
00:11:32;10		and they have failed to fold correctly.
00:11:35;06		So, this was a really wonderful time where there was a lot of back and forth,
00:11:39;16		with Ulrich and I crossing the Atlantic to visit each other and discuss the phenotype of this mutant.
00:11:47;07		This is us together at his parents' house,
00:11:51;03		here's his lovely wife Manajit, who is a biochemist herself
00:11:55;13		and has worked some on the GroEL system as well, in recent times,
00:11:59;13		a chaperonin of bacteria.
00:12:02;03		And maybe this is her telling us how this is all going to work out.
00:12:06;00		But in any case, we carried out together a set of further experiments
00:12:13;15		on a number of mitochondrial matrix proteins
00:12:16;12		to see what was happening to them.
00:12:18;18		And one of the most exciting results came with this particular protein,
00:12:22;12		the Rieske iron sulfur protein.
00:12:24;29		So, this is a protein that is imported to the matrix space and has to be cleaved twice
00:12:30;24		as a monomer, before it enters the inner mitochondrial membrane.
00:12:35;18		And when we looked at this iron sulfur protein,
00:12:38;19		we found it in either a completely uncleaved state
00:12:42;24		or in a once-cleaved state, as if the protein had not folded correctly
00:12:47;00		and had not undergone the proper cleavage that enables its correct biogenesis.
00:12:52;01		So, this was the first of a monomeric protein where we saw effects in this particular mutant.
00:12:58;02		We, at this point, we rescued the mutant with a yeast library.
00:13:04;12		So, we put on a bank of yeast clones and the gene that rescued was sequenced
00:13:12;07		by Ming Cheng during, I believe it was summer 1988.
00:13:17;06		And at this point, we knew that there was a modestly heat inducible protein
00:13:23;02		inside mitochondria that had been identified the year before by Richard Hallberg,
00:13:27;26		working in Iowa, in Tetrahymena.
00:13:30;11		And we had a feeling that Hallberg was analyzing the sequence of the corresponding homologue in yeast
00:13:38;06		with a view to doing further experiments in that system.
00:13:41;16		So, on a Saturday afternoon in that summer, Ming Cheng called up Richard Hallberg
00:13:48;12		with, to see whether her sequence of this gene would match the sequence that he had
00:13:54;18		of the homologue he was studying.
00:13:56;22		Well, I had gone to JFK airport to pick up Ulrich,
00:13:59;28		because the two of us were going to a Gordon conference on mitochondria the next day,
00:14:03;28		and my son came with us, it was many hours, but we got back to the, to our little beach house
00:14:10;18		at around 6 o'clock in the evening and there's Ming Cheng and the rest of the lab,
00:14:15;01		standing in our front yard and Ming's just jumping up and down holding a sequencing gel,
00:14:20;00		saying, "it's a dead match, it's a dead match!" and we said, well, what's the dead match?
00:14:25;07		She said, oh, I called up Hallberg, our protein corresponds to this ring assembly that he's studying!
00:14:31;10		And so, two and two came together in a really amazing moment and we had a wonderful evening of celebration,
00:14:38;00		but basically, what it amounted to was what we then called heat shock protein 60,
00:14:44;05		collectively, with Richard and Ulrich and Walter and everybody.
00:14:48;26		This modestly heat inducible protein
00:14:52;29		was actually assisting the folding of all these proteins being imported in the mitochondrial matrix.
00:14:59;16		And in fact, however, Hsp60 is essential at all temperatures,
00:15:06;08		so its assistance to polypeptide chain folding is required under normal conditions,
00:15:12;01		which embody, often, relatively high temperatures,
00:15:15;25		and very high solute concentrations in the mitochondrial matrix,
00:15:19;13		ergo this machine is essential under all conditions.
00:15:22;27		Now, shortly after this, Ulrich carried out a beautiful experiment with Joachim Ostermann
00:15:28;03		where they analyzed another monomeric protein, dihydrofolate reductase.
00:15:33;14		And what they could see was that when they put a signal peptide on DHFR to make it go into mitochondria,
00:15:39;04		much as in the Schatz experiments,
00:15:41;13		letting it go into mitochondria, it became associated with this complex,
00:15:46;24		with this ring assembly that Hallberg had first identified, the Hsp60 complex,
00:15:52;12		as long as ATP was relatively deprived from mitochondria.
00:15:56;15		If they added ATP back to this system, they now saw DHFR depart from this complex,
00:16:02;13		and appear as a native folded protein.
00:16:05;02		While the protein, I should say, was associated with Hsp60,
00:16:09;12		it seemed to be non-native, very protease susceptible,
00:16:12;21		recognizable by antibodies that specifically saw non-native forms of DHFR,
00:16:17;25		and so these experiments, with Rieske iron sulfur protein, with DHFR,
00:16:23;01		convinced us that Hsp60 was some sort of a folding machine in the mitochondrial matrix.
00:16:28;21		And of course, there's some generality to this because when we saw the sequence of Hsp60,
00:16:36;13		we knew already that it was going to be homologous, and is homologous,
00:16:41;14		to the GroEL protein in the bacterial cytoplasm, essential in that context,
00:16:47;05		and had been shown to be involved, or had been implicated
00:16:50;23		early as being involved in lambda phage head assembly,
00:16:53;29		now this idea had to move to lambda phage protein folding.
00:16:58;15		There was also a protein inside of chloroplasts called the rubisco binding protein,
00:17:04;04		identified by John Ellis as being required for assembly of rubisco,
00:17:08;17		which is a CO2 fixing enzyme, in fact, the most abundant protein in the biosphere.
00:17:14;01		This now, had to go to the level of thinking that rubisco binding protein
00:17:19;07		was actually mediating protein folding of rubisco subunits.
00:17:23;29		And finally, shortly thereafter, working together, Ulrich and I, with Jonathan Trent,
00:17:29;18		identified a heat shot protein in thermophilic archaebacteria
00:17:34;02		that we called thermophilic factor 55, another double ring assembly,
00:17:38;24		that is highly induced when you take these normally heat loving bacteria
00:17:43;12		and take them to an even higher temperature,
00:17:45;21		that turns out to look just like a chaperonin, and it also could turn over ATP
00:17:51;21		and behave just like these other ring assemblies.
00:17:54;24		And, in fact, when we analyze this subunit,   we found a homology
00:17:59;08		to a protein in the eukaryotic cytosol called TCP1, or CCT for short,
00:18:05;00		this being a member of the eukaryotic cytosolic chaperonin 1 subunit of the 8 subunit rings
00:18:12;07		that comprise that chaperonin, essential for folding actin, tubulin, beta propeller proteins,
00:18:18;02		and many other proteins.
00:18:19;25		So, the generality of these ring assemblies in mediating protein folding to the native state
00:18:25;00		seemed to be getting established.
00:18:27;17		And so finally, mechanistically,
00:18:30;08		20 years later, we understand at least how the bacterial chaperonin system works
00:18:35;23		in pretty good detail now, with much biochemistry, X-ray crystallography,
00:18:40;28		NMR studies and a lot of EM conducted on this particular machinery, the GroEL machine
00:18:47;19		and its cooperating component, GroES, and the bottom line in terms of mechanism is
00:18:53;25		that an open ring of GroEL with all this hydrophobic surface specifically binds non-native proteins
00:19:00;03		through their exposure of hydrophobic surface that will be buried to the interior
00:19:04;22		in the native state, thus GroEL specifically recognizes non-native proteins
00:19:10;01		and once it's bound to protein, when GroES now encapsulates the same ring
00:19:15;23		that's bound by polypeptide, the polypeptide is released into this hydrophilic folding chamber,
00:19:22;18		all this blue here, signifies electrostatic residues,
00:19:26;21		and the polypeptide now folds in isolation, in a sequestered environment,
00:19:31;17		where it simply can't aggregate with any other protein and it basically uses Anfinson's principles,
00:19:38;14		its primary structure, to find its way to the native state without the fear of aggregation or going off-pathway
00:19:46;19		and being lost. So, in general, molecular chaperones,
00:19:51;23		if one wants to talk about all of the classes of these components,
00:19:55;27		they basically recognize exposed hydrophobic surfaces
00:20:01;18		that are proffered when a protein misfolds, or during biogenesis,
00:20:06;12		and the idea of the chaperone is to bind these surfaces and prevent them
00:20:12;03		from self-associating with each other in multi-molecular aggregates.
00:20:16;13		The idea of all of the chaperones is to prevent aggregation
00:20:20;19		by binding these surfaces in various geometric contexts.
00:20:24;05		The remarkable thing about the chaperonins, however,
00:20:27;00		that's really unique, is that they use nucleotide to release the polypeptide
00:20:32;13		into a sequestered folding chamber where the polypeptide really has no chance of aggregation
00:20:38;08		and has a chance to fold using its own primary structure
00:20:42;11		to the native state, one of nature's really most beautiful machines,
00:20:46;22		used for this final step of information transfer.
00:20:49;23		So, thanks very much for listening to this lecture, I hope you enjoyed it.
00:20:54;05		Thank you.

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. 2122350 and 1 R25 GM139147. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speakers and do not necessarily represent the views of the Science Communication Lab/iBiology, the National Science Foundation, the National Institutes of Health, or other Science Communication Lab funders.

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