Good afternoon,
I am very happy to be here, I always enjoy talking with young people, students,
so please feel free to interrupt me whenever you don’t understand anything, don’t be shy.
And also I leave plenty of time for discussion because that is the main idea of these meetings,
that we will have interaction, not just a formal lecture.
I understand this might be a mixed audience, I’m not sure how elementary I would have to go,
how many of you are from biology or biomedicine, ok almost everybody, who is not from biomedicine, physics,
ok so I explain to you also, you deserve an explanation.
So I give you a little historical background first
because I think it is important to explain to young students how discoveries are made.
And then I’ll come to our present state of knowledge on the roles of the ubiquitin system in health and in disease.
So I am interested now for a long time in the problem of how proteins are degraded in cells.
And this, when I started about 40 years ago now, not many people were interested in that problem,
even though it was clear even at that time that protein degradation has important roles in biology.
And some properties of intercellular protein degradation that were known at that time when I was about your age,
in 1970 are listed in this slide.
It was known that abnormal proteins are rapidly degraded, so proteins have very delicate structures, 3 dimensional structures.
They can be easily damaged by things like oxygen radicals when we breath,
as a by-product of our breathing we have oxygen radicals that damage different micro-molecules including proteins.
Just our body temperature is not too great for keeping the proteins,
it would be better to keep them in a refrigerator, but that’s not how we live.
So there is constant damage to our proteins.
And proteins that are getting damaged, they lose their normal 3 dimensional structures and they can be toxic to cells.
So they have to be removed and they are removed very efficiently by intercellular protein degradation,
by taking them into their building blocks, a piece to their building blocks, amino acids.
But also it was recognised already in 1970 that completely normal proteins are also degraded
and they are degraded at widely different rates.
So some proteins are degraded very quickly, within minutes, half time, half minutes,
some others are hours and still some others live for days.
So it’s highly selective process.
It was also recognised at that time that levels of specific proteins, which are the machines of our body as you know,
can be regulated, not only by changes in rates of synthesis, but also by changes in rates of degradation.
So for example a protein can be stable at some time and then be rapidly degraded some other time when it is not needed,
completely normal protein.
So this has regulatory functions.
So these rapidly degraded proteins are usually also regulatory proteins.
So all this was known and was summarised in a very nice review by Zimka in 1970.
But in spite of that not many people were interested in protein degradation,
maybe because that was the times after the great genetic revolution,
the understanding of the double helix DNA structure, protein synthesis,
the cracking of the genetic code, so people were more interested in protein synthesis than in protein degradation.
So that was the time when I came to the laboratory of Gordon Tompkins in San Francisco,
in USCF as a young post doctorate fellow.
And Tompkins at that time was interested in the way in which steroid hormones caused the increase in certain enzymes,
a certain enzyme called tyrosine aminotransferase.
And when I got there I saw that it was a very large lab, there were 25 post docs.
They all worked on synthesis, the regulation of synthesis of tyrosine aminotransferase.
So I saw that this was a bit too crowded and I asked Gordon to give me another subject.
So he told me why don’t you work on the degradation of these enzyme,
so that’s how I became involved in protein degradation in which I have been working ever since.
And this slide shows one of the first experiments that I did in the lab of Tompkins,
it was published in 1971 and I found at that time, quite by accident I must say,
the degradation of this enzyme is inhibited by potassium fluoride.
You can see that whenever potassium fluoride was added, the degradation of the enzyme was completely blocked
and potassium fluoride is an inhibitor of energy production.
So this showed that the degradation of the enzyme requires energy
and this was not only for the degradation of this enzyme, the degradation of all proteins required energy.
And I was very much impressed by these results because why would you need energy for the degradation,
for taking the protein to pieces, it was known that energy is needed for the synthesis of peptide bonds in proteins
but not for taking them apart.
And there were proteases known at that time, such as proteases in our digestive tract, that didn’t require any energy,
they actually released energy.
So that energy dependence of the degradation of the protein indicated to me that there is some kind of a new,
unknown mechanism, biochemical mechanism that degrades proteins in dependence of energy
and I assume also that maybe the energy requirement of this process will explain the high selectivity.
To get selectivity you need energy so maybe the energy requirement
and the high selectivity of intercellular protein degradation are somehow linked.
So when I finish my post-doctoral work which was at that time 2 years and not like now 5 or more years,
I went back to Israel and I continued to work on the problem of how proteins are degraded
and why is energy required for this process.
And I saw that the only way to find out how this system works is the classical, the way of classical biochemistry.
And how classical biochemistry approaches or tries to energise the mechanisms of the complete,
here I skip that one, so here again I numerated the reasons for starting to work
on the mechanisms of intercellular protein degradation.
When I went back after my post doctorate fellowship, so as I said protein degradation was known
to be important for the control of cellular proteins so it was a basically important biological process.
But the mechanism, the biochemical pathways responsible for this highly selective degradation was not known.
And the energy dependence of protein degradation that I just mention suggested
that there is some kind of a new biochemical mechanism and the force and maybe one of the important reasons
for working on it that not many were interested.
So I did not have to worry about competition.
So I started to work and I saw that I needed ways of classical biochemistry
and here I show the stages in the biochemical analysis of the complete system.
First one has to make a cell free extract that faithfully reproduces the process in the test tube.
So in order to find out, to analyse by biochemistry how a process works we have to open the cells,
you have to break the cells open and to make a self-resistance, so it’s very easy to break open cells,
it’s not so easy to break them open in a way that will still retain the properties of the process in the cells.
In our case I looked for an energy dependent protein degradation system.
Once one has such a cell free extract, one fraction is the extract,
so usually such system is composed of different enzymes and one has to isolate which are the players.
Once one fractionates and purifies each enzyme component because purification is needed
for the characterisation of the mode of action.
How each peripheral component works.
And the final stage in the biochemical analysis of the complex system is that the constitution
of the activity of the complete system by the addition of all isolated components.
If you can add back all the purified isolated components and get the system to work,
then you really understand how it works.
I like to try to understand how for example a watch, a mechanical watch works.
You open it first, carefully, don’t break it, you take out all the different parts,
the wheels and the springs, you examine them to understand how they work,
you put them back and if the watch works again then you really understood how does it work,
so that is how a complex system is analysed by biochemistry.
And that's what we tried to do, it took us some time and my first step in this direction
was a couple of years later, this experiment was done by Aaron Ciechanover who was then my graduate student
and he shared the Nobel prize with me and tomorrow you will hear him talk about some aspects of the same discovery.
So what we did, we took a cell free system from reticular sites, which are in major red cells.
And others found that when one takes such an extract or biopsy from reticular sites,
one finds that the degradation protein, in this case globin is strongly stimulated by ATP.
ATP for those of you who are not, don’t have a biological background,
is the molecule in the cells that carries energy in cells.
So you can see the degradation was strongly stimulated by ATP,
so that means that in the system we got a faithful reproduction of the energy dependence of protein degradation.
And then we tried to analyse and that was the first step in the right direction, we published a small paper,
not very prestigious, BBRC, it is not Nature or Science, not one of these high impact papers,
but still I am very fond of this small paper because that was the first step in the right direction.
What we did was we fractionated this ligase from reticular site into 2 good fractions by an anion exchange column
and all the protease in this extract were fractionated to 2 fractions,
fraction 1 are all the proteins that did not bind to the column,
fraction 2 are all the proteins that bound to the resine and then the reticulate site.
You can see that in fraction 1 there was no activity, but in fraction 2 where most proteins are
and where we expected to have the activity, also most of the ATP dependent activity was lost.
We found however that when we combined fraction 1 with fraction 2
we could reconstitute completely ATP dependent proteolytic activity.
So these very simple fractionation reconstitution experiment told us that the system
and the degrade proteins in cells is composed of at least 2 components.
Now we know there are many more but at least 2.
So as I said once one fractionates one has to purify and I decided first to purify the active component
from fraction 1 because it seemed to be simpler, it contains mostly haemoglobin
which is presenting these early red blood cells and a couple of other proteins and most of the other proteins were in fraction 2.
And that was a very lucky decision because the purification of the active component from fraction 1
was actually the key to the understanding of the mode of action of this system.
So we purified this component and it turned out to be a small protein composed of only 76 amino acids.
And in this gel, again I am explaining for those who don’t run gels,
proteins are separated by size so the small proteins are running ahead, the large proteins are migrating slower.
And here we had increasing amounts of our purified factor for fraction 1
and as you can see there was only 1 band which means it’s quite pure.
We called it at that time APF1, quite a cumbersome name, factor 1 of the ATP dependent proteolytic system.
Later after we found out its function, it turned out to be similar to a previous neuron protein
that was ubiquitously expressed in all eukaryotic cells but its function was not known so it was called ubiquitin.
It was ubiquitously present protein, unknown function, so that was our APF1.
We didn’t discover ubiquitin, we discovered the ubiquitin system.
But I use now the word ubiquitin because it would be to facilitate the description.
So the active component that is needed for ATP dependent protein degradation in fraction 1
is the small protein and now we wonder how does it work.
It looks smaller than most enzymes, most enzymes have a couple of hundred amino acid residues, it had only 76.
So we thought maybe it is an activator, small protein that activates some other ATP dependent protease in fraction 2.
And for that reason we labelled this ubiquitin with iodine, pure ubiquitin and mixing back we screwed fraction 2
in the absence of ATP and looked for its binding to some component, just to find out, to what protein it does interact.
And then we had our great surprise, it bound not to one protein but to a huge number of proteins
and binding was covalent after incubation with ATP.
So this is a very smeary looking gel, biochemists usually like nice looking gels with sharp bands
like the one I showed you before.
So once I was introduced to the person who used to go to meetings and show very smeary gels.
So this original smeary gel and as you can see what I did here was incubate labelled ubiquitin in this growth fraction 2,
in the absence of ATP, that was the first or in the presence of ATP, that was here.
And then run them on an SDS polyacrylamide gel, which again separates proteins by size
and it’s under very strong denaturing conditions.
So in lane 1 that’s following the incubation result ATP, labelled ubiquitin remains small
and migrated ahead but following incubation with ATP it became linked to a huge number of proteins
that are present in fraction 2 from reticulocytes.
And when I saw this I suddenly understood that ubiquitin is not linked to an enzyme,
it’s linked to the substrate of the system because crude fraction 2 from reticulocytes
contains not only enzymes but also substrates of the system, proteins which degraded by this system.
And that the binding is covalent because this gel was run under conditions,
denaturing conditions that would disrupt all noncovalent interactions.
So there is a kind of a covalent link that is formed between ubiquitin and putative substrates.
And to test this idea now we added lysine which was an artificial but good substrate for the system
including the mouse and as you can see on top of this high background of ubiquitin conjugated covalently
to these endogenous proteins several new bands showed up and these bands contain radio activity
not only from ubiquitin but also from lysosome.
So these were, we analysed them as having increasing numbers of ubiquitin molecules labelled to one molecule of lysosome.
And based on this finding we proposed in 1979, 1980 that ubiquitin is the tag, the tags proteins for degradation.
And our original proposal is shown here in the upper panel and that was the proposal from a paper in 1980,
it was done together with Irwin Rose from Fox Chase Cancer Centre with whom I spent my sabbatical
and with Aaron Ciechanover whom as I said was my graduate student, so both of them shared the Nobel Prize for this discovery.
And our proposal was that the protein in order to get degraded has to be linked to several molecules of APF1
or ubiquitin and that is the place where ATP is needed for protein degradation, it is needed for this tagging reaction.
So that is a tagging reaction that tags that this protein is assigned for degradation
and once it is tagged we propose that it is degraded by some type of protease that recognises
only protease linked or tagged by a ubiquitin or APF1.
And then we propose that ultimately ubiquitin will be recycled and used again for protein degradation.
So that was the original ubiquitin tagging hypothesis from 1980 and in the next 10 years or so I worked on the details,
on the biochemical details of this system and the lower part of this slide summarises our present knowledge.
And this took us about 10 years from 1980 to 1990, it summarises our work in these 10 years
and on the basic biochemistry of the ubiquitin system.
I won’t go through all the details, it can now be found in biologic text books but what we found,
the original hypothesis was essentially correct but we added much more important detail.
There are 3 enzymes that are involved in the linkage of ubiquitin to proteins, ubiquitin activating enzyme, E1,
then several ubiquitin carrier proteins, E2 and the heart of the system are these third type of enzymes,
E3 or ubiquitin protein ligases because they are the enzymes that bind, that recognise specific proteins and bind them.
And they also transfer ubiquitin from the second enzyme to the target protein and form the polyubiquitin chain on these proteins.
So the cell activity and many times also the regulation of protein degradation
is carried out by this great number of E3 ubiquitin ligases now close to one thousand different candidate E3’s
or ubiquitin ligases are known to be present on the human genome.
And we need these huge number of ubiquitin ligases in order to target specific proteins for degradation at the right time.
So that is where the selection is made by these ubiquitin ligases.
And once the proteins are linked to ubiquitin chains they are degraded by a large structure
called the 26S proteasome that was discovered by other people and that degrades the proteins to small fractions.
And then the last step as we proposed was the recycling of ubiquitin by cleaving these isopeptide linkages
between ubiquitins and ubiquitin chains or intermediates.
And thus using again the ubiquitin system for degradation of other substrates.
So now we got by 1990, we had more or less the picture of the basic biochemistry of ubiquitin mediated protein degradation
and by that time I saw that the time has come, until now we work with model protein substrates
and I suppose the time has come to find out how an important cellular protein is degraded by the ubiquitin system.
And this is how I got interested in the roles of the ubiquitin system in the cell division cycle.
By the work of Tim Hunt, by the discovery of Tim Hunt who is now talking I guess in another place but is here in this conference.
He discovered the first cyclin, cyclin B as a protein which is destroyed at the end of each mitosis.
So cyclin B then turned out to be a regulatory subunit of a protein kinase, CDK1, cyclin-dependent kinase 1,
which is a member of a whole family of cyclin-dependent kinases.
So the level of CDK1 is constant throughout the embryonic cell cycle, while the level of cyclin B oscillates.
If cyclin B accumulates during the interface then it binds to CDK1 and forms the active protein kinase complex,
which is also called MPF or mitosis promoting factor.
So this protein kinase, again for those who are not from biology, these are enzymes that link phosphate to proteins
and thereby change their function and they are very important in biology,
again people got the Nobel Prize for the discovery, very important.
And they were known to control the cell divisions cycle, how cells cycle,
and this MPF is a major mitotic protein kinase that is important for entry of cells into mitosis and to the advance in mitosis.
Then to get out of mitosis, this enzyme has to get inactivated and this inactivation
is done by the sudden degradation of cyclin B at the end of mitosis.
So I got interested in this program at around that time, why is cyclin B stable in the interface
and why it is degraded rapidly or how it is degraded rapidly at the end of mitosis.
So this was a fascinating problem and is the ubiquitin system important there.
So again I approach this problem by biochemistry and here I again looked for a cell free system
and here I needed another cell free system because reticulocytes do not have cell cycles,
they are terminally differentiating cells but I found a system in this marine animal,
it is a clam, a big clam which is usually used to make clam chowder.
But in the marine biological laboratory in Woods Hole it is also used to study cell division
because a female clam contains hundreds of millions of eggs and these eggs can be fertilised in sea water
by the addition of sperm and once they are fertilised they begin to divide at a very high synchrony.
So it’s an excellent model system to study cell division and work by other people also showed
that one can make a cell free extract, one can break open these fertilised early embryos
and cell division continues in the test tube provided that the ATP is provided to the test tube.
So again I use this cell free system to study this problem and this time the problem was how is cyclin B degraded,
is the ubiquitin system involved and why it is degraded only at the end of mitosis.
And I show you the conclusion of a couple of years of work and the conclusion was that there is an E3 enzyme,
specific ubiquitin ligase that we call the cyclosome because it was very large and it had important roles in the cell cycle.
And it’s a large complex that targets the ubiquitin ligase complex, that targets cyclin for degradation at the end of mitosis.
And this was done in collaboration with Joan Ruderman, we worked together at the marine biological laboratory.
And the next slide shows a summary from that time, that was from a mini review at 1996.
Which shows how this cyclosome is regulated.
Since then other people called the same complex or ubiquitin ligase complex APC or anaphase promoting complex
and now it is known by these 2 names, anaphase promoting complex cyclosome or APCC.
So at the heart of the system is this ubiquitin ligase, it is in active in the interphase and it gets active
at the end of mitosis and the activation is by phosphorylation.
So the same protein kinases that eventually is inactivated by the ubiquitin ligase,
the CDK1 cyclin B phosphorylates several subunits of these large complex.
There are many more subunits, at that time we didn’t know, there are at least a dozen different subunits
but there are several subunits are phosphorylated and once it’s phosphorylated it can bind a certain activator
that is called CBC20.
And that converts the ubiquitin ligase to its active form.
And the active form of the ubiquitin ligase, ubiquitinates cyclin B, targets it for degradation,
destroys the activated subunit and thereby CDK1 is turned to the inactive form.
And once the protein kinase is inactivated the ubiquitin ligase is also inactivated by the taking
of the phosphates by the action of the phosphotase.
So the idea here is the inter relationship between protein degradation and protein phosphorylation in cycle control,
the protein kinase, the ubiquitin ligase inactivates the protein kinase by the degradation of its regulatory subunit
and then in the next round the protein kinase activates the ubiquitin ligase by phosphorylation.
So now we discover this enzyme in clams but now it is known that this enzyme is highly conserved in evolution
from yeast to man and it has certainly important roles in the cell cycle control.
Work by others showed that not only cyclin B but also some other important cell cycle regulators are degraded by this APCC.
For example a certain protein called securin.
It is an important substrate.
Securin is an inhibitor of a proteolytic enzyme called separase.
Separase is an enzyme that cleaves the linkage between sister chromosomes at the end of mitosis.
So as long as the cleavage, the cohesive complex is not cleaved they cannot separate.
Now separase is not active as long as it’s bound to securin but once the APCC becomes active it targets securin for degradation
so separase becomes free and now it can cleave the linkage and the sister chromatides can separate.
So this regulation or APCC has an important role in starting the anaphase, the separation of chromosomes at the end of mitosis.
And it turns out that APCC is the target for a very important check point system, check point systems are systems
that check that the previous step has been fulfilled and only then the organelle or the cell goes to the next step.
So sister chromatide separate only after they are all correctly aligned to the plate, to what is called the mitotic plate.
Only after they are all linked to the 2 poles of the mitotic spindle.
As long as there is one chromosome that is not linked to the spindle, the APCC is inhibited and it’s very important
to inhibit it because if things started before they are linked then we would have the formation of cells
which do not have the normal number of chromosomes.
So this is called the mitotic check point system and it acts by inhibiting the APCC because sister chromosomes
cannot separate as long as the APCC is inactive.
So only after all the chromosomes are correctly in place APCC is active and the anaphase can start.
So this is a very important check point system and a lot of work was done on it.
And we know some of the players but we don’t know enough about the mechanisms, biochemical mechanisms
and that's what I am working on right now.
But I tell you about that some other time, I am running out of time anyhow.
So you see how ubiquitin ligases are important in control mechanisms.
So I want to leave some time for discussion so I skip over there, that’s another system, another protein
which is important in the cell cycle on which I worked.
And just want to indicate that after we did the initial biochemical work, other people began to bank into the system,
to get interested in the ubiquitin system for protein degradation and now it is know that ubiquitin system
for protein degradation has important roles in the regulation of almost every basic cellular process.
It is very important in the control of cell division as I just told you, in certain types of signal transduction.
In the regulation of the expression of certain genes such as those involved in inflammation and immunity.
In many embryonic developments.
In programs such as apoptosis that we just heard this morning from Bob Horvitz.
The ubiquitin mediated degradation of both apoptotic and anti-apoptotic molecules, I place very important roles and so on.
So ubiquitin system is involved in almost every type of cellular regulation.
And here I summarise how does it work, so it works by degradation of regulatory proteins.
The regulator can be a positively active regulator and these are usually proteins that have to be,
to act only for a very window, narrow window of time.
I can liken the cell to a huge orchestra in which you have thousands of players, thousands of proteins,
they all have to play in concert, in harmony.
Now let’s say one trumpeter gets up and plays a tune and at the right time it’s good but then he has to stop,
if the trumpeter will not stop he will ruin the whole symphony.
So it’s very important not only to start something at the right time but also to stop something and the ubiquitin system stops
the action of proteins that have to act only for a certain time, period of time.
In other cases the ubiquitin system acts on a negative regulator like we saw in the case of securin
so when the negative regulator is destroyed then the break is off and the process can start.
Now sometimes it’s either a positive or negative reacting regulator but it’s all the time turned over
and the ubiquitin system acts by stabilising, it degrades it and the regulation is by stabilisation of this regulator.
For example an important tumour suppressor called P53 is made all the time but is destroyed all the time
but when there is a general toxic stress, when there is a danger to our genome,
then the P53 is activated by inhibiting this degradation.
So it goes up, it inhibits the cell cycle or starts apoptosis.
Now all these types of regulation, especially with the last one are very wasteful, you know these are disposable proteins,
you make a protein, the cell makes the protein, it’s a huge expensive input of energy and then it destroys it.
And the reason maybe to ensure irreversibility, so that is ultimately irreversible way of regulation.
If you pass a bridge and then you burn it, you cannot go backward, you can go only forward.
So I’ll skip this, I want to leave time for discussion so I won’t talk about other functions, ubiquitin-like proteins,
I just want to point out the ubiquitin system is now known to be involved in many diseases, it is heavily involved in cancer,
for example cancer cells are cells that divide without control and the normal control is
because of a balance between oncoproteins that stimulate cell division and tumour suppressor proteins
that inhibit cell division so it’s like a car, you need the gas pedal and you need the brake pedal but you have to use it wisely.
So if the gas pedal is pushed in all the time or the brake pedal is off then the car gets out of control,
that’s what happens in Toyotas I guess.
So you don’t want that to happen.
So in cancer usually the oncoprotein levels of proteins are high and the level of tumour suppressors are low
and it’s important to know that most oncoproteins and tumour suppressors proteins are in a state of constant turnover,
so cancer can be caused by decreased degradation of oncoproteins or by increased degradation of tumour suppressor proteins
and there are many incidences and I won’t go into that, I skip all that.
And I just want to show that all this basic knowledge on the ubiquitin system is now used by the pharmaceutical companies
and now the first generation of drugs is a drug called Velcade, it’s an inhibitor of the proteosome.
It’s not a selective inhibitor but certain cells in which the proteosome is kind of flooded will be selectively apoptosed
or killed by this agent and this agent turned out to be quite efficient
against certain type of bone marrow cancer called multiple myeloma.
But of those this unspecific agent and now other companies are looking for more specific agents
that are targeting specific targets, there are many specific targets, ubiquitin ligases that are involved in many diseases.
So I want to summarise the lessons for this story for you young people.
One is to choose a research subject that is, you think is important but it’s not yet interesting to the mainstream.
So don’t go with the mainstream, if you go with the mainstream the big labs will get ahead there before you.
Try to identify something that you believe is important but not yet in the mainstream of science.
And second, the biochemistry is still very much needed.
What I call good old fashioned biochemistry, fractionation, reconstitution, purification.
You heard about the human genome, now you know all our genes, we can do individual genomes
but still we know the function of about 1/3 of our genes, we don’t know the function of most of our genes
and if we want to know the function of our genes, we need biochemistry.
And you saw the example of the ubiquitin system, micro genetics is very important, genomics is very important
but they have a limit and you couldn’t find such a system like the ubiquitin system by using genetics by itself,
you need biochemistry.
And of course one cannot do all that work myself and here are people from my lab who help me for many years
and among my former students Aaron Ciechanover whom you may hear tomorrow morning,
was a great help in the times of the discovery of the system.
And the collaboration with Irwin Rose was also very important.
So I stop here and I’ll take questions and don’t be bashful.
